Trichohyalin and Transglutaminase-3 and Methods of Using Same

Field of the Invention The present invention relates to the discovery of the sequences of several proteins which

* 5 are involved in forming structural components in epidermal tissue: human trichohyalin, human transglutaminase-3, and mouse transglutaminase-3. Human trichohyalin is cross-linked to other proteins (including other trichohyalin proteins) by transglutaminase-3. Human and mouse transglutaminase-3 can be used to form gels and perform other useful functions.

Background of the Invention 10 I. Trichohyalin

One of the major differentiation products of the inner root sheath and medullary cells of the developing hair follicle. Upon terminal differentiation in these tissues, the granules disperse, but the final fate and structure of TRHY appears to be site dependent: in the inner root sheath, the TRHY protein becomes enmeshed with the keratin intermediate filaments ("KIF") 15 of the cells with an apparent periodicity of about 200 nm (range 100-400 nm) or 400 nm (range

200-500 nm); in the medulla, the protein forms amorphous deposits that are not organized in any specific way.

TRHY undergoes a series of calcium-dependent postsynthetic enzymic modifications.

For example, it becomes highly cross-linked to the KIF by way of N ^c -(γ-glutamyl)lysine

20 isodipeptide crosslinks which may be formed by the action of transglutaminases of the hair follicle cells. In addition, many of the arginine residues are desimidated to citrullines by the action of the enzyme peptidylarginine deiminase.

More recently, it has become clear that the expression of TRHY is not confined to the hair follicle. There is evidence showing that TRHY is expressed in the filiform papillae of 25 dorsal tongue epithelium (Lynch, M.H., et al., J. Cell Biol. 103:2593-2606(1986)), a region that undergoes a course of "hard" keratin differentiation related to that in the hair follicle. In addition, indirect immunofluorescence data indicate that TRHY is also expressed in modest amounts in the granular layer of newborn human foreskin epidermis, although whether it is expressed in interfollicular trunk epidermis is not yet clear.

* 30 Current physico-chemical data suggests that human, sheep and pig TRHYs are large ( proteins of apparent molecular weight of about 200 kDa (Fietz, M.J., et al., J. Cell Biol.

110:427-436 (1990); O'Guin, W.M., et J. Invest. Dermatol. 98:24-32 (1992); Hamilton, E.H., et al., J. Invest. Dermatol. 98:881-889 (1992)). For pig TRHY there is evidence that two components of about 220 and 200 kDa exist. Shadowed electron micrographs of native pig

tongue TRHY reveal an elongated particle of about 85 nm with a small bead on one end. II. Transglutaminase-3

Transglutaminases (TGases) are calcium- and thiol-dependent enzymes that modify proteins by catalyzing the formation of an isodipeptide crosslink between an ε-NH ₂ of a lysine and the γ-amide of a glutamine residue (1-4). In mammals, five distinct TGases are known to exist: a membrane-associated activity first discovered in keratinocytes of about 92 kDa, TGasel, which is now known to be widely expressed; an ubiquitous "soluble" or "tissue" activity of about 80 kDa termed TGase2; a soluble pro-enzyme activity of about 77 kDa, known as the "epidermal" or "hair follicle" TGase3 (see, e.g, Kim, H.-C, et al., J. Biol. Chem. 265:29171-21978 (1990)); an inactive TGase-like protein of about 75 kDa, band 4.2, which is an ubiquitous constituent of the subplasma membrane of most eukaryotic cells (see Sung, I.A., et al., Proc. Natl. Acad. Sci. U.S.A. 87:955-959 (1990)); and the catalytic subunit of the blood clotting factor XIII of about 77 kDa (see, e.g, Takahashi, N., et al., Proc. Natl. Acad. Sci. U.S.A. 83:8019-8023 (1986)). Curiously, all but the latter member of this family are expressed in terminally differentiating epidermis.

Several early studies reported a soluble protein of about 50 kDa from both epidermal and hair follicle tissues of the guinea pig (see, e.g., Chung, S.-I. and Folk, J.E., Proc. Natl. Acad. Sci. U.S.A. 69:303-308 (1972)), but more rigorous biochemical and cell biological analyses revealed that it is in fact a proenzyme, of molecular weight about 77 kDa, which becomes active upon proteolytic cleavage into a 50 kDa (amino terminal) and 27 kDa species

(Negi, M., Colbert, M.C. and Goldsmith, L.A., J. Invest. Dermatol. 85:75-78 (1985)). While newer work has shown that these fragments are not normally separated upon activation (Kim, H.-C, et al., J. Biol. Chem. 265:29171-21978 (1990)), the fact that the isolated 50 kDa fragment can retain catalytic activity was the source of confusion in earlier studies. Furthermore, despite earlier work, it is now generally agreed that the epidermal and hair follicle pro-enzyme species are the same (Lichti, V., Ann. N.Y. Acad. Sci. 642:82-99 (1991)).

Summary of the Invention In one aspect, the present invention comprises a purified molecule of DNA which having 20 or more consecutive nucleotides from SEQ ID NO:93, including a sequence that is homologous to SEQ ID NO:93 or complementary to SEQ ID NO:93, wherein SEQ ID NO:93 codes for the human trichohyalin gene. This DNA molecule can, in one embodiment, comprise the complete coding sequence of SEQ ID NO:93. Such a DNA molecule can also comprise a probe or primer selected from the group consisting of molecules having the sequences of SEQ ID NO:l to SEQ ID NO: 10. In yet another embodiment, the DNA molecule according to this

aspect of the invention is present in a recombinant DNA vector, such as a plasmid. Such a vector can in turn be placed into a cell line which does not naturally contain the molecule of DNA. In another embodiment, the present invention comprises a molecule of RNA which can be translated in vitro or in vivo into the human trichohyalin protein. Such an RNA molecule comprises the coding sequence of SEQ ID NO:93, except that the thymine molecules of SEQ

ID NO:93 are replaced by uracil molecules, including an RNA molecule having a sequence that is homologous or complementary to this sequence. Molecules of RNA which comprises 20 or more consecutive nucleic acids from such an RNA molecule are also included in the invention. In yet another embodiment, the invention comprises a purified protein molecule comprising 20 or more consecutive amino acids of the amino acid sequence of SEQ ID NO:94, including a protein molecule that is homologous to SEQ ID NO:94. In one embodiment, the protein molecule comprises the sequence of the human trichohyalin protein and contains the sequence of SEQ ID NO:94. Antibodies, such as monoclonal antibodies, having binding affinity for human trichohyalin and not for trichohyalin derived from other species are also included in this aspect of the invention.

In another aspect, the present invention comprises another purified molecule of DNA which contains sequences coding for human transglutaminase-3. Such a molecule can comprise 20 or more consecutive nucleotides from SEQ ID NO: 109, including a sequence that is homologous to SEQ ID NO: 109 or complementary to SEQ ID NO: 109. Alternatively, such a molecule can comprises the sequence of SEQ ID NO: 109. A purified molecule of DNA according to this aspect of the invention can be placed in a recombinant DNA vector, such as a plasmid. Such a vector can then be placed in a cell line which does not naturally contain the molecule of DNA. Also included in this aspect of the invention is a purified molecule of DNA for use as a probe or primer, wherein the molecule is selected from the group consisting of molecules having the sequences of SEQ ID NO:47 to SEQ ID NO:54.

Another embodiment of this aspect of the invention includes a purified molecule of RNA which can be translated in vitro or in vivo into the human transglutaminase-3 protein and which comprises the coding sequence of SEQ ID NO: 109, wherein the thymine molecules of SEQ ID NO: 109 are replaced by uracil molecules. An RNA molecule having a sequence that is homologous or complementary to this sequence is also included. A purified molecule of

RNA which comprises 20 or more consecutive nucleic acids from these RNA molecules is included as well. In another embodiment, the invention includes a purified protein molecule comprising 20 or more consecutive amino acids of the amino acid sequence of SEQ ID NO:l 12, including a protein molecule that is homologous to SEQ ID NO: 112. Such a protein molecule

can comprise the sequence of the human transglutaminase-3 protein, wherein the molecule comprises the sequence of SEQ ID NO: 112. In a further embodiment, the invention also includes an antibody, such as a monoclonal antibody, having binding affinity for human transglutaminase-3 and not for transglutaminase-3 derived from other species. In a further aspect of the present invention, the invention includes a purified molecule of DNA which comprises 20 or more consecutive nucleotides from SEQ ID NO: 110, including a sequence that is homologous to SEQ ID NO: 110 or complementary to SEQ ID NO: 110, wherein SEQ ID NO: 110 codes for the mouse transglutaminase-3 gene. Such a purified molecule of DNA can, in one embodiment, comprise the sequence of SEQ ID NO: 110. Such a molecule of DNA can also be selected from the group consisting of molecules having the sequences of SEQ ID NO:33 to SEQ ID NO.-46. In another embodiment, the DNA molecules according to this aspect of the invention are present in a recombinant DNA vector, such as in a plasmid. Such a vector can futher be present in a cell line which does not naturally contain the molecule of DNA. In a further embodiment, this aspect of the present invention includes a purified molecule of RNA which can be translated in vitro or in vivo into the mouse transglutaminase-3 protein and which comprises the coding sequence of SEQ ID NO: 110, wherein the thymine molecules of SEQ ID NO: 110 are replaced by uracil molecules, including an RNA molecule having a sequence that is homologous or complementary to this sequence. Also included is a purified molecule of RNA which comprises 20 or more consecutive nucleic acids from such an

RNA molecule. In another embodiment, the present invention comprises a purified protein molecule comprising 20 or more consecutive amino acids of the amino acid sequence of SEQ ID NO: 111, including a protein molecule that is homologous to SEQ ID NO:l l l. Such a protein molecule can comprise the sequence of the mouse transglutaminase-3 protein, wherein the molecule comprises the sequence of SEQ ID NO: 111. Also included in this aspect of the invention is an antibody, such as a monoclonal antibody, having binding affinity for mouse transglutaminase-3 and not for transglutaminase-3 derived from other species.

In yet another aspect, the present invention comprises a method of forming a proteinaceous gel, comprising the steps of: providing a gel forming substrate, the substrate comprising the human trichohyalin protein; adding to the substrate a gel-forming amount of an enzyme capable of cross-linking the human trichohyalin protein; and thereby forming a gel. This method can include the step of adding to the substrate a gel forming amount of human or mouse transglutaminase-3. In another embodiment, a food or cosmetic substance is mixed with the substrate.

Another aspect of the present invention comprises an aqueous gel composition, wherein the gel comprises cross-linked human trichohyalin molecules. This gel is preferably formed in a mold into a desired shape.

Yet another aspect of the present invention comprises a method of facilitating the healing of a wound, whereby tissue which has been torn to form the wound can be bound together, comprising the steps of: providing a solution containing human trichohyalin protein, wherein the concentration of the trichohyalin protein is between .01% and 5.0%; providing a solution containing an enzyme capable of cross-linking the trichohyalin protein; mixing the solution containing human trichohyalin with the solution containing the enzyme; and applying the mixture of solutions to a wound, whereby the enzyme in the mixture cross-links the human trichohyalin protein in the mixture and causes the mixture to solidify, thereby covering and protecting the wound. In this method, the enzyme used can be human transglutaminase-3, which is preferably present in an amount of between approximately 2% to 5% of the weight of the trichohyalin protein. Brief Description of the Figures

FIG. IA is a picture of an x-ray film exposure of a northern gel (25 μg of sample per well) probed with a 504 bp cDNA clone encoding the carboxyl-terminal end of human trichohyalin. The samples probed represent one batch of total cellular RNA from each of: a sample of human foreskin epidermis (lane 1); a sample of mouse epidermis (lane 2); and a sample of mouse hair follicles (lane 3). Positions of size markers (Gibco-BRL) are shown.

FIG. IB is a picture of an x-ray film exposure of a slot blot experiment which was performed to estimate the relative amounts of specific epidermal mRNAs in 10 μg of total cellular RNA from the sources shown. This figure shows an exposure of 6 days. The abbreviations use in this figure are: K10=keratin 10; FIL=profilaggrin; LOR=loricrin; TRH=trichohyalin.

FIG. 2. is a strategy map for the sequencing of human TRHY cDNA and genomic clones. The upper line designates the location of the genomic clone λH-TRHY-18 in relation to the sequencing information. The abbreviations for restriction enzyme sites are: R=EcoRI; S=Sac I; X=Xho I. The nested arrows denote sequences determined by deletion subcloning. The second line illustrates the structure of TRHY gene. Exon I of 63 nt consists entirely on 5'- non-coding sequences; intron 1 is 1275 nt long; exon II of 169 nt contains the likely initiation codon and encodes the first EF-hand motif; intron 2 is 864 nt long; and the large exon III (6609 nt) consists of an additional 5553 nt of coding sequences and 1056 nt of 3 '-non-coding sequences to the polyadenylation signal sequence. The positions of the two EF-hand calcium

binding domains are shown in black; the remainder of the coding sequences are hatched; 5'- and 3 '-non-coding sequences are open boxes. The third line designates mRNA structure.

Dotted lines connect the exon sequences to the mRNA structure. Below this are shown the locations of the 504 bp cDNA clone referred to above and of the various cDNA clones constructed by primer extension and anchored PCR methods. The numbered spots 1+ (SEQ ID

NO: 1), 1- (SEQ ID NO: 2) denote the primers used to amplify the 504 bp cDNA clone; primers 2- (SEQ ID NO: 3), 3- (SEQ ID NO: 4), etc. refer to the primers used for extension and PCR. The sequences and numbered locations of these primers are listed in Table 2 below.

FIG. 3 shows the DNA nucleotide sequence (SEQ ID NO:93) and predicted amino acid sequence (SEQ ID NO:94) of human TRHY. These data were accumulated from both the

RNA-mediated anchored PCR cDNA clones and the genomic clone λH-TRHY-18. The nucleotide sequence is numbered from the extent of our available sequence data above the likely

CAAT box. Intron sequences are shown in lower case letters. The likely CAAT, TATA, capsite, initiation, termination and polyadenylation signal sequences are underlined. The single letter code for amino acids is used, and residues are numbered from the codon following the initiation codon. Comparisons of the cDNA sequences and the available genomic sequences revealed a number of polymorphisms: the genomic clone contained an additional glutamic acid at position 459; and a number of silent nucleotide substitutions in the following codons: 15

(AAT or AAC), 1 13 (CGC or CGG), 460 (AGA or AGG), 842 (CGG or CGC), 1024 (CGC or CGG), 1199 (TGC or TGT), 1361 (GAA or GAG), 1362 (CAA or CAG), 1516 (GAG or

GAA), 1559 (CTC or CTG) and 1766 (CTC or CTG).

FIG. 4 depicts the predicted secondary structure features of human TRHY. This is set out in linear form for three structural motifs based on Chou-Fasman (CF), Robson-Garnier (RG) or consensus (CfRg) analyses of the WGCG package of analyses (Devereux, J., et al., Nucleic Acids Res. 12:387-395 (1984)). The molecule is mostly α-helical, configured in a series of long segments, interspersed by less regular regions matching the occasional proline residues, and it contains three potential sheet structures.

FIG. 5 is a graph of the circular dichroism spectrum of pig tongue TRHY showing that this protein is a highly α-helical molecule. The pig tongue TRHY, prepared under non- denaturing conditions, was equilibrated into 20 mM sodium phosphate (pH 7.0) containing 1 M NaCl, and its circular dichroism spectrum was measured in a Jasco J600 spectropolarimeter. FIG. 6 is a dot matrix profile which reveals that human TRHY evolved assembled from multiple repeating peptide sequences. The homology scoring method of Pearson, W.R. and Lipman, D.J., Proc. Natl. Acad. Sci. U.S.A., 85:2444-2448 (1988) was used with a window size

of 18 residues and homology of 50%. Several major and some minor blocks of repeating sequences are evident, suggesting that the TRHY molecule was formed during evolution from blocks of peptide repeats that were joined together by non-conserved sequences.

FIG. 7 depicts a model of the structure of human TRHY. This model consists of 9 domains. Domain 1, shown as a circle, contains two EF-hand calcium binding domains.

Domains 2, 3, and 4 are largely α-helical and delineated by varying peptide repeats (see Table

3). Domain 5 contains several short and one longer stretch of α-helix interspersed by turn, coil or possibly sheet regions. Domain 6 also adopts an elongated configuration and constitutes the most regular portion of the molecule, to which KIF may associate through periodic ionic interactions. Domain 7 is also likely to be folded. The long domain 8 consists of peptide repeats which adopt an elongated α-helical configuration. Domain 9 contains the carboxyl- terminus, apparently conserved among TRHY molecules of different species. The lengths (in nm) of the more regular domains are shown. Human TRHY appears to either: (a) fold in half around domains 5 and 7 so as to produce an elongated configuration about 100 nm long with a large bend 15-20 nm in diameter corresponding to domain 6; or (b) remain extended and is

>215 nm.

FIG. 8 is a chart which aligns the amino acid sequences of human TRHY with a selection of human SlOO-like calcium binding proteins which contain two homologous EF-hand motifs. The arrow after residue 45 delineates the point at which intron 2 splices coding sequences between the two EF-hand motifs. The helix-tum-helix sequences which define each motif are shown. Relative amino acid deletions are denoted by -. The amino acid sequences of the non-TRHY proteins are derived from: Markova, N., et al., Mol. Cell Biol. 13:167-182 (1993) (profilaggrin); Kligman, D. and Hilt, R.H., Novel Calcium-Binding Proteins, Heizman, C.W., ed., Springer-Verlag, Berlin, 65-103 (1991) (SlOOα, pl l, calcylin and cystic fibrosis antigen); Becker, T., et al., FEBS Lett. 207:541-547 (1992) (SI OOP).

FIG. 9A is a picture of pig TRHY which as been electrophoresed on a polyacrylamide gel. This picture reveals two bands of about 220 and 200 kDa that can be stained with coomassie blue (lane 1), and can also be detected by ⁵ Ca binding (lane 2) or by use of a specific carboxyl-terminal epitope antibody (lane 3). FIG. 9B is a dot blot for quantitating ⁵ Ca binding. The indicated proteins (1-10 μg) were applied and incubated with ⁴⁵ CaCl ₂ . The first lane of numbers describes a quantitative value determined by scanning densitometry. In the second lane, these values have been scaled in relation to calmodulin (1.00) as ⁴⁵ Ca binding/mol.

FIG. 10 illustrates the strategy employed in generating the nucleotide information of the

mouse TGase3 enzyme (SEQ ID NO:lll) and human TGase3 enzyme (SEQ ID NO: 112). in each case, the upper line represents the full-length sequence showing the initiation, termination and polyadenylation signal sequences. Below are shown bars displaying the primers used (primer sequences are listed in Table 5) and the extent of sequence information obtained with each PCR step.

FIGS. 11A-11K show the nucleotide sequences of human TGase3 (SEQ ID NO:109, 11A-11F) and mouse TGase3 (SEQ ID NO:110, FIGS. 11G-11K) and the deduced amino acid sequences of the mouse TGase3 enzyme (SEQ ID NO:lll, FIGS. 11G-11K) and the human TGase3 enzyme (SEQ ID NO: 112, 11A-11F). For ease of description, these figures shall be referred to collectively as "FIG. 11". The initiation, termination and polyadenylation signal sequences are underlined. Nucleotide sequences are numbered following the initiation codon. The amino acid sequences are shown using the single letter code. In the mouse amino acid sequence, only variations from human are shown.

FIG. 12A is a picture of an X-ray film exposure of Northern blots depicting the sizes of human and mouse TGase3 mRNAs. Aliquots of 25 μg of total cellular RNA from human foreskin epidermis (lanes 1-4), newborn mouse epidermis (lane 5) or five day old mouse hair follicles (lane 6) were probed with: lane 1, a 58 nt antisense degenerate oligonucleotide encoding active site sequences (see Kim, H.-C, et al., J. Biol. Chem. 266:536-539 (1991) and Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)); lane 2, a 175 bp 3 '-non-coding probe of a TGasel cDNA clone; lane 3, a 0.7 kbp 3'-non-coding portion of TGase2 generated by PCR (see Table 5); lanes 4-6, a 1 kbp cDNA probe encoding 3 '-non-coding sequences of human TGase3 (see FIG. 10). The individual strips were exposed for: lane 1, 14 d; lane 2, 2d; lane 3, 4d; lanes 4-6, 23d. Positions of migration of RNA size markers are shown to the left of the lanes.

FIG. 12B is a picture of an X-ray film exposure of Northern slot blots in which aliquots of 10 μg of RNA from the sources shown (TGasel, TGase2, and TGase3) were probed with the above TGase-specific probes. For quantitation purposes (see Table 6), the X-ray films were exposed for several different times; this figure shows one exposure (for 6d) only. FIG. 13 is a chart aligning portions of the amino acid sequences of human TGase-like proteins. Alignments of the amino-terminal sequences are arbitrary. The arrowhead marks the presumed site of proteolytic cleavage required for activation of TGase3. Homology and identity scores (see Table 8) were calculated for sequences bounded by the closed dots (which correspond to the positions of known intron boundaries conserved in the genes of TGasel,

factor Xllla and band 4.2).

FIG. 14 shows three charts which show the predicted structural features of human TGase3. The charts were produced using the IB1 Pustell and Intelligenics Geneworks software packages and are based on known analytical methods (see Devereux, J., Haeberli, P. and Smithies, O., Nucleic Acids Res. 12:387-394 (1984); Chou, P.Y. and Fasman, G.D.,

Biochemistry 13:222-245 (1974); Gamier, J., et al., J. Mol. Biol. 120:97-118 (1978); and Pearson, W.R. and Lipman, D.J., Proc. Natl. Acad. Sci. U.S.A. 85:2444-2448 (1988)). The predicted secondary structure, flexibility and hydrophilicity profiles are shown. The arrows centered at residue 475 demarcate the point of cleavage activation of the zymogen and denote a prominent turn region of highest flexibility and hydrophilicity in the entire protein.

Detailed Description of the Invention Among the discoveries of the present invention is the determination of the sequences of three mammalian epidermal proteins, human trichohyalin, human transglutaminase-3, and mouse transglutaminase-3. These proteins are all found in terminally differentiating epidermal tissue and are involved in forming the structural architecture of such tissue. The structure- forming properties of these proteins are exploited in one aspect of the present invention by using the proteins to form gels, which can be used to form food and other useful products. Also included in the present invention is a novel method of facilitating the healing of wounds with the proteins described herein. To facilitate the understanding of the present disclosure, the following terms are hereby defined. The term "coding for" as used herein, when applied to DNA molecules, refers to DNA molecules which contain the coding portions of a particular DNA sequence, that is, the portions making up the exons of such sequences. The exon sequences of these DNA molecules can be transcribed into RNA molecules which can in turn be translated into molecules of protein. RNA molecules which can be translated into such molecules of protein are also said to "code for" their corresponding proteins.

As further used herein, the terms "homologous" and "homology", when applied to proteins or amino acid sequences, describe amino acid sequences in which one amino acid has been substituted for by an amino acid with similar properties. An example of such a substitution is the exchange of an aspartic acid molecule for a molecule of glutamic acid. Other such similar pairs of amino acids are well known to those of skill in the art.

With regard to nucleic acid sequences, however, the terms "homology" and "homologous" carry the meaning of being able to hybridize to nucleic acids with complementary sequences under standard hybridization conditions for Northern hybridizations (when RNA is

being hybridized to a target nucleic acid) or Southern hybridizations (when DNA is being hybridized to a target nucleic acid). Such standard hybridization conditions are discussed in Sambrook, J.. Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Press, Cold Spring Harbor, NY (1989). A nucleic acid with a "complementary" sequence is one which can hybridize to a target nucleic acid sequence under such standard hybridization conditions.

As used herein, the term TGase3, by itself, shall refer to both mouse and human TGase3, unless the context indicates to the contrary. In addition, for the sake of clarity the following list of some of the abbreviations used herein is set forth immediately below: TRHY = trichohyalin

TGase = transglutaminase

TGase3 = transglutaminase-3 (also transglutaminase E)

CE = cornified cell envelope

FFT = Fast Fourier Transform IF = intermediate filaments

IFAP = intermediate filament associated protein

KIF = keratin intermediate filaments

PCR = polymerase chain reaction nt = nucleotide(s) knt = kilonucleotides bp = base pair(s) kbp = kilobase pairs kDa = kilodalton μg = micrograms ng = nanograms fmol = femtomoles pmol = picomoles d = day h = hour Other terms and abbreviations used herein are defined below.

I. Human Trichohyalin

We have now discovered the full-length sequence of human TRHY, deduced from the sequences of PCR-derived cDNA clones and of a genomic clone. Analyses of its secondary structure suggest that it adopts a flexible single-stranded α-helical rod-like conformation. In

this way, TRHY is remarkably similar to but about four times longer than involucrin, a known protein constituent of the cornified cell envelope of the epidermis. However, unlike involucrin, TRHY possesses functional calcium-binding motifs of the EF-hand type at its amino terminus as does profilaggrin, the precursor of a known interfϊlamentous matrix protein of the epidermis. The potential significance of these several structural motifs suggests TRHY may have multiple functions in the epidermis and hair follicle cells.

Trichohyalin is an intermediate filament associated protein that associates in regular arrays with keratin filaments (KIF) of the inner root sheath cells of the hair follicle and the granular layer of the epidermis, and is a substrate of transglutaminases. We have determined the full-length sequence of human trichohyalin by use of RNA-mediated anchored PCR methods and from a genomic clone, and have analyzed its potential secondary structure. We show here that trichohyalin may have at least three important functions in these cells. The protein of 248 kDa is unusual in that it contains one of the highest contents of charged residues of any protein. Of several defined domains (shown in FIG. 6), domains 2, 3, 4, 6 and 8 are almost entirely α- helical, configured as a series of peptide repeats of varying regularity, and are thought to form a single stranded α-helical rod stabilized by ionic interactions between successive turns of the α-helix. Domain 6 is the most regular and may bind KIF directly by ionic interactions. Domains 5 and 7 are less well organized and may introduce folds in the molecule. Thus, human trichohyalin is predicted to be an elongated flexible rod at least 215 nm long, and to function as a KIF associated protein by crosslinking the filaments in loose networks.

A. Procedures Used to Isolate and Sequence the Trichohyalin Gene

I. Procedures for the Isolation and Sequencing of cDNA a,ιd Genomic

Clones A large portion of the cDNA sequences encoding human TRHY was determined by RNA-mediated anchored PCR (as taught in Frohman, M.A., PCR Protocols: A Guide to

Methods and Applications Innis, M.A., et al., eds., Academic Press Inc., New York, 28-38 (1990)) and by characterization of the resulting sequence information. The carboxyl-terminal portion of the TRHY sequence was first identified by probing human genomic DNA with primers which coded for portions of the carboxyl-terminal portion of sheep TRHY (Fietz, M.J., et al., J. Cell Biol. 110:427-436 (1990)). In this way, a 504 bp cDNA clone (later determined to code for the carboxyl-terminal end of human trichohyalin) was identified (see Lee, S.-C, et al., J. Invest. Dermatol. 98:626 (1992)). This probe was then used to reverse transcribe an aliquot of 200 ng of DNasel -treated total foreskin epidermal RNA (Steinert, P.M., et al., J. Biol. Chem. 260:7142-7149 (1985)) at 70°C, resulting in the minus strand marked as 2- in FIG. 2

(SEQ ID NO:3). Approximately 20 picomoles of a primer of about 20 nt having a unique sequence found in the 2- strand was then reverse transcribed at 70° to produce the 3- strand (SEQ ID NO:4). A series of minus strand primers (listed in Table 2 below) were designed in this way and used to determine the full-length sequence of human trichohyalin. The PCR reactions using the specific primers in Table 2 were performed with a commercial DNA amplification reagent kit (from Perkin-Elmer Cetus, Norwalk, CT) by following the manufacturer's specifications, using 25 pmol of an adaptor dG oligonucleotide as the plus primer and 25 pmol of one of the specific primers shown in Table 2 as the minus primer. The conditions of PCR were: 95°C (5 min); and cycled for 30 cycles at denaturation of 95°C (0.5 min); annealing at 42°C (0.5 min); and elongation at 72°C (1.5 min). In some cases where the yield was low (due to difficulties of amplifying the multiple exact repeat regions), a portion of the PCR reaction mixture was then diluted 1 :1000 with buffer and 1 μl was reamplified in a second round of PCR. After each round of PCR, the primer was removed using Chroma spin 100 columns (made by Clontech, Palo Alto, CA), and the cDNAs thus produced were tailed in the presence of 200 μM dCTP with 25 units of terminal deoxytransferase (supplied by Gibco-BRL, Gaithersburg, MD) for 1 h at 37°C

The cDNA products of these PCR procedures were fractionated on low-melting agarose gels and the largest fragments containing the most extended products were excised. Following purification through Chroma spin columns, the ends of the amplified cDNAs were filled in with Klenow DNA polymerase (as taught in Sambrook, J., Fritsch, E.F. & Maniatis, T., Molecular

Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)), subcloned into the pGEM-3z vector (supplied by Promega, Madison, WI), and then sequenced by the didexoy chain-termination method with Sequenase 2.0 (sold by United States Biochemical Corp., Cleveland, OH). Following analysis of the sequence information of the amplified cDNAs from each round of PCR, new primers with unique (non-repeating) sequences were designed. For example, after analyzing the sequence of the 2- strand, a 2- primer was selected which had a unique sequence and this primer was then used to generate the 3- strand. This process was continued as far as possible in a total of nine steps.

Because of uncertainties with the location of the likely initiation codon and 5 '-end of the mRNA, the 504 bp cDNA clone referred to above was used as a probe to screen a human placental genomic DNA library (provided by Clontech, Palo Alto, CA). Using this 504 bp probe, a 14 kbp genomic clone, termed λH-TRHY-18, was isolated and plaque-purified. Following Southern blotting analyses of this genomic clone, 6.5 kbp Sac 1 and 8.0 kbp Xho 1 fragments of the clone containing the entire coding region of the human TRHY gene were

cloned into pGEM-3z. Sequencing of these clones was done following creation of a nested set of deletion subclones by use of the Erase-a-Base kit system (available from Promega, Madison, WI) with the T7 and SP6 vector primers.

2. Northern Blot Analyses Using established methods, total cellular RNA was prepared from human foreskins (see

Steinert, P.M., et al., J. Biol. Chem. 260:7142-7149 (1985)), newborn mouse epidermis (see Roop, D.R., et al., Proc. Natl. Acad. Sci. U.S.A. 80:716-720 (1983)), and human and mouse epidermal keratinocytes grown to confluency in the presence of low (0.1 mM) or high (0.6 mM) Ca ² + (see Yuspa, S.H., et al., J. Cell Biol. 109:1207-121 (1989) and Hohl, D., et al., J. Invest. Dermatol. 96:414-418 (1991 )). RNA was also prepared from hair follicles purified from 5 day old mice. Northern gels loaded with 25 μg of RNA were performed by established procedures (Hamilton, E.H., et al., J. Invest. Dermatol. 98:881-889 (1992)), and calibrated with standard RNA size markers (available from Gibco-BRL, Gaithersburg, MD).

Northern slot blotting was done as described in Sambrook, j., Fritsch, E.F. & Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor,

NY (1989). In this case, the blots were calibrated with 0.01 to 10 femtomole amounts of cloned probes for human and mouse keratin 10 (see Zhou, X.-M., et al., J. Biol. Chem. 263:15584-15589 (1988) and Steinert, P.M., et al., Nature (London) 302:794-800 (1983)); human (33) and mouse (34) filaggrin (see McKinley-Grant, L.G., et al., Proc. Natl. Acad. Sci. U.S.A. 86:4848-4852 (1989) and Rothnagel, J.A. and Steinert, P.M., J. Biol. Chem.

265:1862-1865 (1990)); loricrin (see Hohl, D., et al., J. Biol. Chem. 266:6626-6636 (1991)); and a 6.5 kbp human TRHY genomic clone described below. Slots of samples containing 10 μg of total cellular RNA were then tested with specific 3 '-non-coding probes to each of the above. All Northern filters were washed with a final stringency of 0.5 X SSC at 65°C for 30 min. The resulting X-ray films were exposed for varying amounts of time in order to facilitate quantitation of the abundance of the specific mRNA species by scanning densitometry.

3. Protein Secondary Structure Analyses

Protein sequence homologies and secondary structure predictions were performed using the AASAP (Amino Acid Sequence Analysis Program (obtained from Dr. David Parry in New Zealand), the University of Wisconsin sequence analysis software packages (UWGCG) compiled by the Wisconsin Genetics Computer Group (Devereux, j., et al., Nucleic Acids Res. 12:387-395 (1984)), the International Biotechnologies Inc. Pustell sequence software (version 3.5) (from IBI, New Haven, CT) and Intelligenics Geneworks software (Pearson, W.R. and Lipman, D.J., Proc. Natl. Acad. Sci. U.S.A. 85:2444-2448 (1988)). Dot matrix comparisons were performed

using the COMPARE and DOTPLOT program on UWGCG running on the Massey University VAX or Geneworks. Fast Fourier Transform (FFT) analyses to determine the periodic distributions of residues or residue types were carried out as described by McLachlan, A.D. and Stewart, M., J. Mol Biol. 103:271-298 (1976). General structural principles pertaining to α- helic-rich proteins (Cohen, C and Parry, D.A.D., Proteins: Structure, Function and Genetics

7:1-15 (1990)) were used in preliminary analyses.

Numbers of potential intrachain ionic interactions between oppositely-charged residues four apart (that is, i→i+4) in a likely α-helical structure were calculated and placed on a per heptad (seven residue) basis. These values, designated here as 14, represent a measure the number of charged to uncharged amino acids in a peptide. They allow direct comparisons with the interchain ionic interactions made between the chains of multi-stranded α-fibrous proteins. Such values typically lie in the range 0.2-0.8 (Conway, J.F. and Parry, D.A.D., Int. J. Biol. Macromol. 12:328-334 (1988)). Ionic interactions are known to stabilize an α-helical structure through the formation of salt links between residues on adjacent turns of the α-helix. B. Results of Experiments to Isolate and Sequence the Trichohyalin Gene

1. Northern Blot Analyses

The previously described 504 bp cDNA clone representing the carboxyl-terminal portion of TRHY was used as a probe to estimate the size, relative abundance and expression characteristics of human TRHY mRNA. On Northern gels, human and mouse TRHY mRNAs are approximately 6.7 kb in size (Fig. IA). This estimate is about 10% larger than for sheep

TRHY mRNA. In slot blotting assays, we estimated the abundance of human TRHY mRNA in relation to a number of major epidermal mRNA species, including keratin 10, profilaggrin and loricrin (Fig. IB). As shown in Table 1 below, densitometric scanning of slot blots, including that depicted in FIG. IB, revealed that TRHY is <0.4% and <0.6% as abundant as keratin 10 or profilaggrin, respectively, in both human and mouse epidermis. The results of

Table 1 were determined by exposing X-ray films with the blots for several different time periods, ranging from 4 hours to 46 days.

Since the keratin 10 mRNA is thought to constitute about 25% of epidermal mRNA, this means that TRHY represents about 0.1% of total epidermal mRNA. While we have no information on the rates of turnover or the efficiency of translation of the TRHY mRNA, these data nevertheless confirm that TRHY protein is a minor but significant component of the terminal differentiation pathway of human epidermis. Similar experiments have revealed that the mRNA for involucrin is about five times more abundant than TRHY. In addition, the expression of TRHY mRNA is down-regulated in submerged liquid cultures of both mouse and

human epidermal cells grown in low calcium (0.1 M medium, and its expression is somewhat elevated by raising the calcium concentration to near optimal levels (0.6 mM) (see Fig. IB and Table 1 ). Thus, the expression of TRHY mRNA is closely coordinated with that of other late differentiation products of the epidermis such as keratin 10, profilaggrin and loricrin (Table 1). 2. The Deduced Amino Acid Sequence of Human TRHY

We have used a combination of two strategies to obtain the full-length coding sequence of human TRHY. In the first, human TRHY-specific oligonucleotide primers (listed in Table 2) were constructed and used to prepare cDNAs by primer extension. In this way, it was possible to "walk" up most of the length of the mRNA (as shown in Fig. 2). The primer 9- (SEQ ID NO: 10) extended only an additional 230 bp, indicating that we had extended close to the 5'-end of the mRNA. The sequences of the oligo-dG-tailed product of primer 9- included a potential initiation codon that conformed to a Kozak initiation site (Steinert, P.M. and Steven, A.C, J. Invest. Dermatol. 98:559 (1992)), but we were uncertain whether this represented the true initiation codon, largely because we found these TRHY sequences shared a high degree of homology to those of the much more abundant profilaggrin mRNA (see FIG. 7 below).

Accordingly, in the second approach, the 504 bp cDNA clone was used to isolate a 14 kbp genomic clone, λH-TRHY-18. By Southern blotting techniques using the aforementioned 504 bp clone and several of the primer-extended cD A clones described above, λH-TRHY-18 was found to extend several kbp upstream and thus contains the entire coding region of human TRHY (FIG. 2). Following subcloning, a 4.1 kbp portion overlapping the cDNA information was sequenced using Erase-a-Base methods (Promega, Madison, WI).

Comparisons of the sequences of the available cDNA clones and λH-TRHY-18 revealed the presence of two introns toward the 5'-end of the TRHY gene. One intron of 1275 bp splices sequences 54 bp and a second intron of 864 bp splices sequences 223 bp from the 5'-end of our cDNA sequence information. These introns define an exon 1 of at least 54 bp and an exon II of 169 bp. Exon II contains the in-frame initiation codon described above (FIG. 2, 3). Because we were unable to further primer-extend TRH mRNA sequences, we conclude that the primer extension experiments had reached very close to the cap-site for the TRHY mRNA. Indeed, searches for consensus sequences revealed a likely capsite just 9 bp upstream, at position 139 of FIG. 3.

Potential TATA and CAT boxes reside 23-33 bp and about 100 bp above this capsite. Thus, the 5'-end of the TRHY gene is remarkably similar to the 5'-end of the profilaggrin gene (Markova, N., et al., Mol Cell Biol. 13:167-182 (1993)) and to several genes encoding small calcium-binding proteins of the SI 00 family that contain EF-hand motifs (Kligman, D. and Hilt,

R.H., Novel Calcium-Binding Proteins, Heizman, C.W., ed., Springer-Verlag, Berlin, 65-103 (1991)). The TRHY gene, like all of these other genes, contains an exon I of 50-70 nt in 5'- non-coding sequences, an intron of 1 of 1-10 knt, an exon II of 150-170 nt containing the initiation codon and the first EF-hand motif, a short intron 2, and exon III which contains the second EF-hand motif and the entire remainder of the coding and 3 '-non-coding sequences. In the case of TRHY, exon III 6609 nt (to a consensus polyadenylation signal sequence) including 5553 nt of coding and 1056 nt of 3 '-non-coding sequences. The human TRHY mRNA is likely to be about 6.9 kb in length (including a polyA tail), in good agreement with the size estimate of 6.7 kbp estimated by Northern blotting (FIG. IA). The nucleotide sequence from the likely initiation codon defines a single open reading coding frame of 5691 nt, and thus the deduced amino acid sequence for human TRHY contains 1897 amino acids (excluding initiating methionine) of calculated molecular weight of 248 kDa and pl (isoelectric point) of 5.4 (FIG. 3). Thus, the molecular weight of human TRHY is about 25% and 15% higher than has been reported by SDS polyacrylamide gel electrophoresis for sheep or pig TRHY.

The net calculated pl is lower than predicted previously from histochemical staining methods for arginines (Rogers, G.E., Expt. Cell Res., 14:378-387 (1958)). Only about 3 of the 45 serines + threonines are potential targets for phosphorylation by known protein kinases, and none of the 8 asparagines are likely candidates for glycosylation. The human TRHY sequence contains an extraordinarily high number (59%) of charged residues (aspartic acid D, glutamic acid E, histidine H, lysine K and arginine R), as well as many glutamines (Q). Comparisons with the GenBank and NBRF databases reveal that only one other described protein, involucrin, has similar high content of charged residues (49%). TRHY is also homologous to members of the S100 class of small calcium binding proteins. 3. Secondary Structure Features of Human Trichohyalin

Secondary structure analyses suggest that about 75% of the human TRHY protein will adopt an α-helical conformation. Two pairs of short α-helical segments are predicted to occur in the first 90 residues, followed by a series α-helical segments of 50 to 600 residues in length which encompass all the protein except for a short non-α-helical carboxyl-terminal domain about 40-50 residues long (FIG. 4). The α-helical segments are interrupted by occasional short β-turn sequences containing proline residues (FIG. 4). These algorithms predict only small sections of sheet structure near the amino-terminus, and three other sections along the protein between residues 720-730, 780-790, 885-910, where an (apolar-polar) ₃ ^ periodically occurs.

The presence of the high α-helical content is supported by direct physical measurement

with circular dichroism of pig TRHY (FIG. 5). Although human TRHY had not been isolated from any tissue before the present invention, it has been recently shown that pig tongue TRHY can be isolated in bulk using non-denaturing conditions (Hamilton, E.H., et al., J. Invest. Dermatol. 98:881-889 (1992)). Based on poly(L)lysine standards (Kligman, D. and Hilt, R.H., Novel Calcium-Binding Proteins, Heizman, C.W., ed., Springer-Verlag, Berlin, 65-103 (1991)), the mean molar ellipticity value of -21,400 deg.cm /dmol suggests that pig TRHY has an α- hfelical content of 65-70%, β-sheet content of 10-15% and with 10-20% random coil These values are in good agreement with the computer predictions of human TRHY shown in Figure 4. Analysis of the human TRHY sequence by dot matrix plots using the homology scoring system of Pearson and Lipman (see Pearson, W.R. and Lipman, D.J., Proc. Natl. Acad. Sci. U.S.A. 85:2444-2448 (1988)) reveals the presence of several regions of peptide repeats (see FIG. 6 and Table 3). These repeating regions are interspersed by regions of various lengths that lack the degree of regularity in the primary structure that is characteristic of most of the molecule. These observations indicate that the TRHY molecule consists of multiple domains, as set forth in detail below. Fast Fourier Transform (FFT) analyses (described in McLachlan, A.D. and Stewart, M., J. Mol Biol. 103:271-298 (1976)) were used to evaluate these regions in detail and the results are summarized in Table 3. Repeating peptide sequence motifs are shown. A model based on these analyses is shown in FIG. 7. This model contains the following motifs: (1) The first 94 residues, which are predicted to be about 50% α-helical, contain two calcium binding domains of the EF-hand type which have 60-70% sequence homology in this region with members of the SI 00 class of small calcium binding proteins (described in Kligman, D. and Hilt, R.H., Novel Calcium-Binding Proteins, Heizman, C.W., ed., Springer-Verlag, Berlin, 65-103 (1991)), and 70% homology to human profilaggrin (see Markova, N., et al., Mol. Cell Biol. 13:167-182 (1993) and see also FIG. 8). Each domain is composed of an ordered series of polar residues that are flanked by hydrophobic sequences and which adopt the helix-turn-helix conformation required to bind a single Ca ²⁺ ion. The two EF- hand motifs are separated and immediately flanked by sequences that have not been well conserved between members of the Si 00 class of proteins. (2) Residues 95-312 are predicted to be largely α-helical (>75%) but will suffer disruptions at the two proline residues. There are no developed peptide repeats in this region. Nonetheless, charged residues of opposite sign frequently interface each other on alternate turns of the α-helix, thereby stabilizing a single-stranded α-helix through intrachain ionic interactions of the type i -> i + 4 (Marquesee, S. and Baldwin, R.L., Proc. Natl. Acad. Sci. U.S.A.

84:8898-8902 (1987)). The remarkably high charged/apolar ratio of 4.4 and 14 value of 1.54 (Table 3) are suggestive of an elongated α-helical rod structure about 32 nm long.

(3) Residues 313-443 can be subdivided into two sections, 313-389 and 390-443, on the basis of two well-developed types of repeats of 13 and 6 residues, respectively (Table 3). These are predicted to be entirely α-helical, and again, adjacent turns of the α-helix are likely to be stabilized by favorable ionic interactions between interfaced oppositely charged residues. Thus, the high 14 value and the high charged/apolar ratio together favor the formation of an elongated α-helical rod structure about 19 nm in length.

(4) Residues 444-702 consist of an irregular repeat dominated by numerous net deletions, but with a consensus repeat of 28 residues (Table 3). It is likely to be almost entirely α-helical, though disrupted once by a single proline. It may possess additional flexible regions around multiple adjacent glutamines, the tryptophan and/or serines, threonines. The 14 value of 2.17 is the highest in the entire molecule, indicating the potential for a very stable α-helical structure also stabilized by intrachain ionic interactions on alternate turns of the α-helix. This rod-like domain would be about 38 nm long.

(5) Although residues 703-922 are predicted to have a significant amount of α- helical structure, there are likely to be numerous breaks due to multiple prolines. Also, a semi- conserved repeat containing a characteristic pair of tryptophan (Table 3) is less clearly α-helical than most of this segment and may even favor the formation of intrachain or interchain sheet structures about the (polar-apolar^ environment. No other clear-cut repeat is evident and the

14 value of 1.05 is one of the lowest in the entire molecule. Because of the predicted turns and the tryptophan-rich quasi-repeats, this region may adopt a more folded configuration of indeterminate net length.

(6) Residues 923-1163 consist of eight almost perfect repeats of 30 residues (as evident from the matrix plot of FIG. 6) that are almost entirely α-helical, save for two potential kinks about the prolines. The very high 14 value of 2.03 and the charged/apolar ratio of 4.85 indicate a highly stabilized elongated rod structure of about 36 nm, common in segments 2-4 above. Interestingly, the 30 residue repeat also contains significant subrepeats (Table 3), especially one of length 7.5 residues. This occurs for glutamic acid (scaled Fourier intensity 43.61 and probability of occurring by chance 1.1 s IO" ¹⁹ ), leucine, and for arginine (Table 3) and shows that the true period is approximately quartered. It also has the effect of placing an arginine or glutamate on almost every other turn of one face of the α-helix. Since this is slightly out of phase with 7.2 residues per two turns of an α-helix, it will result in positively- and negatively-charged stripes winding around the axis of the α-helix with a pitch length of

about 14 nm.

(7) Residues 1 164-1249 have similarities to those in segment 5 in that they are predicted to have significant α-helical content, but are nonetheless likely to be folded at least in part through the presence of predicted turns. Also present is a reasonably well defined tryptophan-containing quasi-repeat previously noted in segment 5. There are no other evident repeats and the relatively low 14 value of 0.73 may be insufficient to stabilize a single-stranded α-helix. A folded rather than extended conformation of indeterminate net length may result. In contrast, however, the charged/apolar ratio is still high and is more compatible with a conformation with an appreciable axial ratio. Thus, there is some difficulty in assigning a likely structure to this segment. Different conformations are likely to result under different conditions.

(8) Residues 1250-1849 consist of an almost uninterrupted stretch of α-helix configured as an irregular consensus 26 residue repeat (Table 3); many of the repeats are actually 24 residues long and about half are much shorter, containing only about 16 residues. The sequence RQERDRKFREEEQ (SEQ ID NO: 19) is the common conserved element. Again, these repeats are characterized by long stretches in which oppositely charged residues would interface each other on alternate turns of an α-helix. Interestingly, elements of a 7.7-7.9 residue repeat of very high probability are evident in glutamate and lysine + arginine residues (Table 3), suggestive of a spiral of charged residues about the α-helix of the general type described above for segment 6. The very high 14 value of 1.86 and the high charged/apolar ratio (3.72) favor the formation of an elongated single-chain α-helical rod of length about 90 nm.

(9) Residues 1850-1897: The carboxyl-terminal sequences are likely to adopt a folded or random coil conformation, due to the presence of prolines and glycines. Interestingly, the terminal 20 residues have been precisely conserved between sheep (5) and human, and have afforded the manufacture of a TRHY-specific antibody (Hamilton, E.H., et al., J. Invest. Dermatol. 98:881-889 (1992)).

The net overall length of human TRHY is thus estimated to be at least 215 nm, arising from several distinct elongated rods. Segments 1 and 9 represent the globular amino- and carboxyl-terminal domains respectively whereas segments 5 and 7 occur within the rod domain but have α-helix-rich structure of indeterminate net length. It is possible that the human TRHY molecule in vitro (and possibly in vivo as well) folds about domains 5 and 7, and forms a rod of about 100 nm with a knob of 15-20 nm (that is, half of the length of domain 6) at the bend. This value compares with the approximate 85 nm long rod with a 12 nm bead on one end as visualized for native pig TRHY by shadowing electron microscopy (Hamilton, E.H., et al., J. Invest. Dermatol. 98:881-889 (1992)). Such a folded structure, consisting of antiparallel α-

helices, conceivably could be stabilized by ionic interactions between the many charged residues along the equal-length segments 2 + 3 + 4 and 8. It remains unclear, however, whether human TRHY is folded in half in vivo (as seen in the in vitro preparations studied by electron microscopy) or whether it is a single α-helix at least 215 nm long. 4. Trichohyalin is a Functional Calcium Binding Protein

The sequence data of FIG. 3 and sequence homology data of FIG. 8 indicate the presence of two well-defined calcium binding domains of the EF-hand type. Prior to the present invention, methods were not described for the isolation of human epidermal or hair follicle TRHY. However, we show in FIG. 9A that pig tongue TRHY is capable of binding ⁴⁵ Ca in vitro (lane 2). Interestingly, unlike human, pig TRHY appears as two bands of about 220 and

200 kDa (lane 1), both of which bind calcium (lane 2). In addition, a Western blot using a new TRHY antibody (Hamilton, E.H., et al., J. Invest. Dermatol. 98:881-889 (1992)) elicited against the carboxyl-terminal 18 amino acids, which have been precisely conserved between human and sheep and presumably in pig TRHY as well, also reveals two bands of the same sizes (FIG. 9A, lane 3). Since these data indicate that the amino- and carboxyl-terminal ends have been conserved, this means that pig TRHY is expressed as two distinct protein products. By slot blotting (FIG. 9B), we show that pig TRHY (about 210 kDa, 2 EF-hands/mol) binds ⁴⁵ CaCl ₂ as effectively as calmodulin (14 kDa, 4 EF-hands/mol). Profilaggrin binds calcium somewhat more efficiently (Kozak, M. (1989), J. Cell Biol. 108, 229-241). Most of the calcium binding in the total epidermal extract is presumably due to the profilaggrin.

C Description of Human Trichohyalin

I. Human Trichohyalin is a Long Segmented Rod-Shaped Molecule That

Has the Potential to Interact with Keratin Intermediate Filaments The human TRHY protein is unique in possessing the highest known content of charged residues. By use of secondary structure prediction and FFT analyses, we show that it consists of 9 well-defined domains. The bulk of the sequences, defined by domains 2, 3, 4, 6 and 8, are very highly charged, configured as a series of peptide repeats of varying degrees of regularity, which adopt an α-helical configuration. A point of great significance here is that the α-helix- rich segments do not have a heptad substructure characteristic of all α-fibrous proteins that form a two- or three-stranded coiled-coil conformation (Cohen, C and Parry, D.A.D., Proteins:

Structure. Function and Genetics 7:1-15 (1990)). In each of the α-helical domains, there are well-defined regularities in the disposition of charged residues so that oppositely-charged residues frequently lie on alternate turns of the α-helix, thereby stabilizing the α-helix by intrachain ionic salt bonds. Indeed the very high ratio of charged to apolar residues and the

number of intrachain ionic interactions per seven residues (14 values, see Table 3), are characteristic of a stable single-stranded α-helical configuration (see, e.g., Kligman, D. and Hilt, R.H., Novel Calcium-Binding Proteins, Heizman, C.W., ed., Springer-Verlag, Berlin, 65-103 (1991)). In addition, the very large numbers of polar glutamine residues are to be expected to further contribute to this α-helical structure by H-bonding. Nevertheless, each of these domains is interrupted by an occasional proline residue which is therefore likely to introduce bends or kinks along their length (FIG. 7). More importantly, domains 5 and 7 have distinct and unusual features. While they still contain important elements of the other domains with respect to high α-helix content and high charged/apolar residue ratios, they are likely to adopt a more complex conformation due not only to the presence of multiple prolines, but also to multiple tryptophan and other residues that favor the introduction of turns and even limited sheet structures. Our conclusion is that these regions promote folds in the human TRHY structure. Domains 2+3+4 (total length about 89 nm) and 8 (length about 90 nm), could fold back on each other, hinged about domains 5 and 7, and stabilized by the potential to form many ionic salt bonds across the two arms of the molecule. This would create a molecule about 100 nm long with a knob of 15-

20 nm comprising segments 5, 6 and 7. This model is generally consistent with existing data. Native pig TRHY is about 85-90 nm long and possesses a 12-15 nm bead on one end. Pig TRHY is about 15% smaller than human TRHY, but possesses functional calcium binding domains (FIG. 9) and a conserved carboxyl-terminal domain 9. Our calculations show that unfolded human TRHY is at least 215 nm long in toto, and perhaps as much as 260 nm (including length contributions of domains 5 and 7, but not 1 and 9). This length is the same as the range of the periodicities of interaction of TRHY with KIF in inner root sheath cells (see, e.g., O'Guin, W.M., et al., J. Invest. Dermatol. 98:24-32 (1992)). Thus, it appears that TRHY constitutes an elongated, somewhat flexible crosslinking IFAP in these cells. Based on these analyses, it is clear that the likely secondary structure of human

TRH is different from an intermediate filament (IF) chain. Such proteins are characterized by a well-defined central α-helical rod domain, the sequences of which form a two-chain segmented coiled-coil motif.

Of the α-helical domain segments, domain 6 is the most regular with eight near-exact 30 residue repeats (see FIG. 6) but it nonetheless possesses unusual features. The glutamic acid, arginine and leucine residues are each configured as a quasi-repeat of about 7.5 residues (that is, 30/4) (Table 3), corresponding to slightly more than two turns of the α-helix (3.6 residues/turn). A similar repeat is also evident to some extent in domain 8. Thus while many of the positive and negative charges will form stable ionic interactions, the net result will be a

slow spiral of charged residues around the axis of the α-helix. While the pitch length of this spiral is critically dependent on the number of residues per turn in the α-helix, a length of 14 nm seems likely.

The IB and 2 rod domain segments of intermediate filament chains possess a 9.8 residue periodicity in the linear distributions of charged residues. This is equivalent to a linear rise of the coiled-coil of approximately 1.4 nm, or one-tenth of the periodicity of domain 6. Accordingly, by formation of periodic ionic interactions of the charged residues on the IF rod domain segments with the highly ordered domain 6 (and perhaps also with domain 8), human TRHY could function as an IFAP in the epidermis and inner root sheath cells. Human TRHY has 332 glutamine and 104 lysine residues, which are potential targets for crosslinking by transglutaminases. Earlier peptide sequencing data suggested the presence of numerous isodipeptide crosslinks in both inner root sheath and medulla proteins. In the case of the inner root sheath, many of these involved the non-α-helical end domain sequences of the KIF of the cells, perhaps because of accessibility, and are likely to involve interchain links between the TRHY and KIF.

Early amino acid composition and sequencing data showed that in the mature inner root sheath and medulla proteins of the guinea pig hair follicle, approximately 25% and 40-50%, respectively, of the arginines are converted to citrullines by desimidation. We estimate that conversion of 200 or more arginines to citrullines will lower the pl of the intact human TRHY protein to about 4. Similarly, a significant although unknown number of lysines will be effectively discharged by the formation of the isodipeptide crosslinks. Since the arginines and lysines lie on the periphery of the α-helix, they will be readily accessible by the peptidyl arginine desimidase and transglutaminase(s) enzymes. This discharging of many basic residues will likely interfere with the formation of ionic salt bonds responsible for stabilizing the single- stranded α-helix. Conversion of 200 arginines will lower the charged/apolar ratio substantially, effectively destabilizing the structure. Accordingly, we predict that TRHY becomes a much less regularly-organized molecule upon postsynthetic modification.

2. Trichohyalin is a Functional Calcium Binding Protein

The amino acid sequence information provided by the nucleic acid sequence of trichohyalin has revealed the surprising finding of a pair of calcium binding domains on the

TRHY molecule. These domains are of the EF-hand type, typically found in small SlOO-like calcium binding proteins (FIG. 3,8,9). TRHY and the SI 00 proteins share significant homology with each other at the level of gene structure: their transcribed sequences consist of three exons, of which the first consists of 5 '-non-coding sequences; the second contains the initiation codon

and first EF-hand motif; and the third exon contains the second EF-hand motif, as well as the remainder of the coding sequences. Moreover, the locations of the exon/intron boundaries of human TRHY and the SI 00 proteins have been precisely conserved. In addition, we have recently discovered that human profilaggrin also contains two EF-hand motifs at its amino terminus that are organized at the protein and gene levels in an identical fashion to the SI 00 class and to the human TRHY gene . The experiments of FIG. 9 for pig TRHY and other experiments with human profilaggrin (Markova, N., et al., Mol. Cell Biol. 13:167-182 (1993)), have revealed that these EF-hand motifs in the two proteins are in fact functional in binding calcium in vitro. Therefore, it seems likely that human TRHY is a functional Ca ² + binding protein in vivo.

The most notable difference between TRHY and the SI 00 class of proteins is the size and nature of the amino acid sequences beyond the EF-hand motifs. Most members of the SI 00 class of proteins possess only short sequences flanking the second EF-hand motif and share little overt sequence homology with one another. These sequences are thought to be involved in Ca ² +-mediated interactions with different target effector molecules. In contrast, the human

TRHY sequences extend for more than 1700 residues, largely configured in a series of quasi- repeating peptides. Moreover, as discussed, these sequences are subjected to at least two different types of postsynthetic modifications that are calcium dependent: certain lysin donor and/or glutamine acceptor residues become involved in the formation of N ^c -(λglutamyl)lysine isodipeptide crosslinks catalyzed by transglutaminases (6,10,1 1 ); and many arginines are converted to citrullines by the enzyme peptidylarginine deiminase (12-16). It seems likely, therefore, that the calcium binding properties of the EF-hand motifs are involved in these post- translational reactions.

II. Human Transglutaminase-3 Another discovery of the present invention is the structure and sequence of human and mouse TGase3. As described herein, the mouse and human protransglutaminase-3 enzymes contain 692 amino acids of calculated molecular weight about 77 kDa. While these proteins share 38-53% identity to other members of the transglutaminase family, the mouse, human, and guinea pig enzymes surprisingly have not been highly conserved and show only 50-75% identity to each other. Much of the sequence variation occurs in the vicinity of the proteolytic activation site which lies at the most flexible and hydrophilic region of the molecule and is flanked by a sequence of 12 residues that are absent from all other transglutaminases. Cleavage of this exposed flexible hinge region promotes a conformational change in the protein to a more compact form resulting in greatly increased enzymic activity. Expression of mouse and human

transglutaminase-3 mRNA is regulated by calcium, as with other late differentiation products of the epidermis, suggesting that this enzyme is responsible for the later stages of cell envelope formation in the epidermis and hair follicle.

A. Methods Used to Determine the Sequences of Human and Mouse TGase3 I. Determination of the Amino Acid Sequences of Selected Peptides of

Guinea Pig TGase3 The 50 kDa amino-terminal and 27 kDa carboxyl-terminal fragments of guinea pig TGase3, derived by dispase treatment, were fractionated and purified as described by Kim, H.-C, et al., J. Biol. Chem. 265:29171-21978 (1990). Each portion was cleaved with trypsin (Boehringer sequencing grade, Boehringer Mannheim Biochemicals, Indianapolis, IN) at 10 mg/ml in 0.1 M NH,HC0 ₃ with a final enzyme to protein ratio of 1 :50 and digested for a total of 4 h at 37°C Following drying, the peptides were redissolved in 0.1% aqueous trifluoroacetate, fractionated by HPLC (High Pressure Liquid Chromatography). Well-resolved peaks with absorbances at both 210 nm and 350 nm were then selected for sequence analysis. Absorbance at 350 nm was taken as an indication of cysteine residues alkylated with 5-N

[(iodoacetidoethyl)amino]naphthalene-l sulfonic acid, possibly corresponding to active-site peptides. Sequence analysis of selected peptides was then performed on an Applied Biosystems 470A protein sequenator (Applied Biosystems Inc., Foster City, CA) using the automated Edman degradation method (see Kim, H.-C, et al., J. Biol. Chem. 265:29171-21978 (1990); Hohl, D., et al., J. Biol. Chem. 266:6626-6636 (1991); and Hewick, R.M., et al., J. Biol. Chem.

256:7990-7997 (1981)).

2. Anchored-PCR Cloning Strategies

Once the sequences of the selected guinea pig peptides were obtained, we set out to determine the sequence of mouse TGase3. The strategy for obtaining mouse TGase3 is thus set forth below. However, a very similar strategy was then carried out to identify human

TGase3. Rather than repeat the common steps of the protocols for obtaining mouse and human TGase3, the protocol for obtaining mouse TGase3 will be set forth in detail below, and the steps taken to obtain the sequence of human TGase3 which differ from those taken to obtain mouse TGase3 will be pointed out. Initially, we constructed a series of degenerate oligonucleotide primers based on the available guinea pig TGase3 peptide sequences (see Table 4 for lists of sequences from which primers were prepared) and used these to amplify DNA obtained from a random-primed cDNA library prepared from mouse epidermal mRNA (Roop, D.R., et al., Proc. Natl. Acad. Sci. U.S.A. 80:716-720 (1983)). In obtaining human TGase3, a cDNA library prepared from human

epidermal mRNA was prepared by the same method.

PCR was performed with a commercial DNA amplification reagent kit (from Perkin- Elmer Cetus, Norwalk, CT) by following the manufacturer's specifications, using 25 pmol of primers and with conditions of: 95°C (5 min), and 35 cycles of denaturation at 94°C (0.5 min), annealing at 42°C (0.5 min) and elongation at 72°C (1.5 min). The PCR products were fractionated through low-melting agarose, excised, and purified through Chroma spin 100 columns (Clontech, Palo Alto, CA). The ends of the amplified DNA were filled in with Klenow DNA polymerase (see Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)), subcloned into the pGEM 3z vector (Promega Corp., Madison, Wl), and then sequenced by the dideoxy chain termination method with Sequenase 2.0 (United States Biochemical Corp., Cleveland, OH). Although most sets of degenerate primers did not work, apparently because of the substantial nucleotide sequence differences between guinea pig and mouse TGase3 mRNAs (see Table 9), four were found sufficiently useful to proceed. Subsequently, RNA mediated anchored PCR was used to "walk" in both directions along the mouse TGase3 mRNA by using specific mouse TGase3 nucleotide sequences as primers and by using additional degenerate primers. Aliquots of 200 ng of DNase 1 -treated total newborn mouse epidermal RNA (Roop, D.R., et al., Proc. Natl. Acad. Sci. U.S.A. 80:716-720 (1983)) were reverse transcribed at 42°C In obtaining human TGase3, a lambda-gtl 1 cDNA library prepared from newborn human foreskin was similarly reverse transcribed.

Following removal of the dNTPs through Chroma spin columns, the cDNAs so produced were tailed in the presence of 200 μM dGTP with 25 units of terminal deoxytransferase (Gibco-Bethesda Research Laboratories Inc., Gaithersburg, MD) for 1 h at 37°C (Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)). PCR was then done in two steps.

The conditions for the first round were exactly as described above, with 25 pmol of the primer used as the minus primer, and either a degenerate primer, a specific mouse TGase3 primer, or oligo-dC as the plus primer (when identifying human TGase3, a specific human primer was used instead of the specific mouse primer). These specific primers are listed in Table 5 below. A portion of the PCR reaction mixture was diluted 1 :1000 with buffer and 1 μl was reamplified in a second round of PCR using the more stringent conditions of: denaturation at 94°C (0.5 min), annealing at 55°C (0.5 min) and elongation at 72°C (1.5 min), and using primers on one end that were nested inside those used in the first PCR reaction (see Table 5, FIG. 10). The further subcloning and sequencing procedures were performed as above. In this

way, it was possible to "walk" along the entire length of the mouse TGase3 mRNA in both directions in six steps. The human TGase3 cDNA sequence was generated in essentially the same way using the nested primers listed in Table 5 and using the adduced mouse sequence data. The result of these procedures was the discovery of the DNA coding sequences for human TGase3 (SEQ ID NO:LLL) and mouse TGase3 (SEQ ID NO:QQQ). As is known to those of skill in the art, a purified molecule of DNA containing the mouse or human TGase3 coding sequence can also be synthesized by probing mRNA from mouse or human epidermal tissue, respectively, with a probe specific to either mouse or human TGase3 (see Table 5), extending that probe with a DNA polymerase such as the Klenow fragment of E. coli, and then isolating the resulting DNA strands produced. Desirably, such strands are then subcloned into a vector such as a plasmid reproducible in E. coli.

Once the coding sequences of human and mouse TGase3 were known, it also became possible to produce further probes for these sequences. Such probes are designed by selecting 20 consecutive nucleic acids from the coding sequences of either SEQ ID NO:LLL or SEQ ID

NO:QQQ and can be synthesized by various means known to the art, including the use of automated DNA synthesizers. These probes can be used, for example, to identify TGase3 in the mRNA or genomic DNA of cells or cell cultures. 3. Northern Blotting Procedures Total cellular RNA was prepared from human foreskin epidermis (Steinert, P.M., et al.,

J. Biol. Chem. 260:7142-7149 (1985)), newborn BALB/c mouse epidermis (Roop, D.R., et al., Proc. Natl. Acad. Sci. U.S.A. 80:716-720 (1983)), and human and mouse keratinocytes grown to confluence in the presence of low (0.1 mM) or high (0.6 mM) Ca + (see Yuspa, S.H., et al., J. Cell Biol. 109:1207-1217 (1989) and Hohl, H., et al.,J. Invest. Dermatol. 96:414-418 (1991)). RNA from the hair follicles of 5 day old mice was also isolated. Northern gels using denaturing conditions were loaded with 25 μg of total cellular RNA, performed as described in Yaminishi, K., et al., J. Biol. Chem. 267:17858-17863 (1992), and calibrated with standard RNA size markers (Gibco-BRL, Gaithersburg, MD).

Northern slot blots were then prepared as described in Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring

Harbor, NY (1989). In this case, the blots were calibrated with 10, 1.0, 0.1, 0.01 ftnol amounts of probes encoding the full-length TGasel (Kim, H.-C, et al., J. Biol. Chem. 266:536-539 (1991)), a 0.9 kbp PCR fragment of 3 '-non-coding region for TGase3 (SEQ ID NO:53) and a 0.7 kbp 3 '-non-coding region of the published sequences of TGase2 (Gentile, V., et al., J. Biol.

Chem. 266:478-483 (1991)) (see Table 5 for the two primers used). Aliquots of 10 μg of the several RNA samples were tested separately with the three TGase specific probes. All Northern filters were washed with a final stringency of 0.5 x SSC at 65°C for 30 min. The resulting X- ray films were exposed for varying amounts of time in order to facilitate quantitation of the abundance of the specific mRNAs by densitometry.

4. Computer Analyses of Sequences

Nucleic acid and protein sequence homologies were performed using the University of Wisconsin software packages compiled by the Wisconsin Genetics Computer Group (Devereux, J., et al. Nucleic Acids Res. 12:387-394 (1984)), the IBI Pustell sequence software (version 3.5, International Biotechnologies Inc.) and Geneworks sequence software (Intelligenics Inc.), based on published algorithms (see Chou, P.Y. and Fasman, G.D., Biochemistry 13:222-245 (1974); Gamier, J., et al., J. Mol. Biol. 120:97-118 (1978); and Pearson, W.R. and Lipman, D.J., Proc. Natl. Acad. Sci. U.S.A. 85:2444-2448 (1988)).

B. Results of Search for Human and Mouse TGase3 Our initial attempts to locate clones for either mouse or human TGase3 in available λgtl 1 libraries using low-stringency hybridizations with TGasel or TGase2 probes or active-site probes (Kim, H.-C, et al., J. Biol. Chem. 266:536-539 (1991)) were unsuccessful, perhaps because of low mRNA levels. Accordingly, we made TGase3-specific degenerate oligonucleotide probes derived from the amino acid sequences of tryptic peptides of TGase3 isolated from guinea pig epidermis. The implicit assumption was that the guinea pig, mouse and human TGase3 proteins would share high degrees of sequence homology, as found for the TGasel and TGase2 systems.

I. Amino Acid Sequences of Guinea Pig TGase3 Tryptic Peptides

Although the 27 kDa fragment resulting from dispose treatment yielded a clean amino acid sequence for 28 cycles corresponding to its amino-terminus and the activation site of proteolytic cleavage, no useful information on the larger catalytic 50 kDa portion was obtained. Accordingly, using larger quantities, both peptide portions were cleaved to completion with trypsin. Selected well-resolved peptides, especially those containing cysteine residues, were chosen for sequencing. In this way, sequences from a total of 12 tryptic peptides (six from each of the 50 kDa and 27 kDa portions) and the amino-terminus of the 27 kDa portion were obtained (Table 4). These represented 180 sequenced residues, or about 25% of the total protein. Peptides 1 and 3 (order 3-1) are recognizable as constituting the active site region, based on comparisons with the known sequences of the TGase family members. The amino acid substitutions in this active site region in relation to the other family members are diagnostic

for the TGase3 system (see Table 7 below).

2. Cloning by Anchored-PCR and Deduced Amino Acid Sequences of

Mouse and Human TGase3 Proteins

Degenerate oligonucleotide probes based on the above amino acid sequences of guinea pig TGase3 failed to identify positive-clones in available mouse or human λgtl l cDNA libraries. However, when such oligonucleotides were employed in primer extension experiments with poly(A)-enriched RNA from newborn mouse or human foreskin epidermis, weak signals corresponding to the presumed size of the TGase3 mRNAs (Kim, H.-C, et al., J. Biol. Chem.

266:536-539 (1991)) were found. Therefore, we used the oligonucleotide primers to amplify by PCR the DNA extended by primer P3-(Table 5). One pair of primers (P1+P3-; FIG. 10,

Table 5) yielded a product of 292 bp, and was subcloned into pGEM-3z. About 5% of such clones contained TGase-like sequences, including the active site region, which were identical to peptides 3+1 of the available guinea pig tryptic peptides (Table 4). This finding afforded confidence that we were indeed amplifying the mouse TGase3 mRNA system. Accordingly, we used this exact sequence data to extend the mouse TGase3 sequence by use of RNA- mediated anchored PCR as described above. First, we used one set of specific nested primers and another degenerate primer from the guinea pig peptide information (la+/P10- and lb+/P10-; P5+/2a- and P5+/2b-) (FIG. 10, Table 5). The remainder of the 5'-end up to the capsite was recovered by primer extension, tailing with dG, and PCR amplification in two steps with nested primers (3a-/oligo-dC; then 3b-/oligo-dC) (FIG. 10). The 3'-end sequence information was recovered in two steps by use of primer extension with a random hexamer, followed by tailing with dG. The cDNA products were amplified by PCR in two steps with two sets of nested primers (4a+/oligo-dC; then 4b+/oligo-dC) and (5a+/oligo-dC; then 5b+/oligo-dC) (FIG. 10, Table 5). The human TGase3 sequence was generated in essentially the same manner in three steps, except that an oligo-dT primer was used to generate the full-length 3 '-non-coding information. The primers (see Table 5) and strategy used are outlined in FIG. 10.

A series of further RNA-mediated anchored PCR experiments was performed using primers that crossed over those shown in FIG. 10 and in Table 5 in order to confirm and check the sequences for PCR-induced sequence mutations (lists of primers used are not shown). The natures of 7 ambiguous nt were resolved in additional PCR experiments.

The available nucleotide sequence information consists of 2297 nt for mouse, including the entire 5'-non-coding information, but incomplete 3'-non-coding sequences (FIG. 10). The human data extends for 2645 nt, and is assumed to be near full-length because of the inclusion

of the polyadenylation signal sequence (FIG. 1 1); thus, its estimated mRNA size is about 2.8 kb. In both cases, there is an open reading frame of 2079 bp, so that both proteins contain 692 amino acids of calculated molecular weight of 77.1 kDa (mouse) and 76.6 kDa (human), which are very close to the values adduced for guinea pig TGase3 by analytical ultracentrifugation and SDS-polyacrylamide gel electrophoresis experiments (see Negi, M., et al., J. Invest. Dermatol.

85:75-78 (1985) and Kim, H.-C, et al., J. Biol. Chem. 265:29171-21978 (1990)). Interestingly, mouse TGase3 is near neutral in charge (pl 6.5) compared to human TGase3 (pl 5.6), findings that are also consistent with earlier chromatographic observations.

3. Abundance and Expression of Mouse and Human TGase3 RNAs A series of cDNA probes containing specific 3 '-non-coding sequence information for human TGasel (Kim, H.-C, et al., J. Biol. Chem. 266:536-539 (1991)), TGase2 (generated by PCR; see Table 5 and Gentile, V., et al., J. Biol. Chem. 266:478-483 (1991)), and TGase3 (generated with PCR primers 6a+/6b, Table 5), were used to separately test human foreskin RNA on Northern blots (FIG. 12A). Four distinct bands are seen with a degenerate oligonucleotide probe (Kim, H.-C, et al., J. Biol. Chem. 266:536-539 (1991)) for active site sequences (lane 1), which correspond to the four known TGase-like activities expressed in the epidermis. The TGase3 probe SEQ ID NO:53 identified only the central mRNA species of about 2.9 kb (lane 4). This is consistent with the size of the TGase3 mRNA adduced from the above sequencing data. Furthermore, it is now known that the mRNA encoding TGase 2 is the largest (about 3.4 kb, lane 3) and that encoding TGasel is smaller (about 2.7 kbp, lane 2). The fourth and smallest band of about 2.4 kb corresponds to the mRNA for band 4.2 (Korsgren, C, et al., Proc. Natl. Acad. Sci. U.S.A. 87:613-617 (1990)). Mouse epidermis and hair follicles also express a TGase3 mRNA species of the same size as human TGase3 mRNA (FIG. 12a, lanes 5,6), consistent with the biochemical data which suggests that the epidermal and hair follicle TGase3 pro-enzymes are in fact the same gene product (Kim, H.-C, et al., J. Biol. Chem.

265:29171-21978 (1990)). These highly specific probes displayed almost no cross- hybridization. The data therefore confirm the identity of the TGase3 probes.

Using slot blotting techniques, we also examined the expression characteristics of these mRNA species (FIG. 12B). By using specific cloned probes as calibration standards for each TGase species to account for variations in hybridization and labeling efficiencies, we could estimate the amounts of each species expressed in intact epidermis, hair follicles or cultured cells (Table 6). Whereas the TGasel and TGase2 mRNAs are unregulated in submerged liquid cultures, TGase3 mRNA is greatly diminished and essentially absent in low Ca ² + medium conditions. Furthermore, TGase3 expression is modestly up-regulated in media containing near-

opti al levels of Ca ² +, whereas the former two species are down-regulated. Thus, the TGase3 system is regulated differently from the TGasel and TGase2 enzymes. These data establish that the TGase3 system is regulated in the same general way as other late epidermal differentiation products such as loricrin, profilaggrin, and keratins 1 and 10. These data also support the view that the TGase3 enzyme is involved in a later stage of CE formation or assembly than the

TGasel enzyme.

The data of Table 6 also show that in intact epidermis, the level of TGasel mRNA is about 5-7 times greater than that of TGase3. While little information is currently available on the turnover rates or rates of translation of these mRNAs (compare Michel, S., et al., J. Invest. Dermatol. 98:373-378 (1992) with Schroeder, W.T., et al., J. Invest. Dermatol 99:27-34 (1992) for TGasel ), our present data imply that TGasel is a more abundant enzyme in epidermis than TGase3. Nevertheless, activated TGase3 appears to constitute about 75% of total epidermal TGase enzymic activity (Kim, H.-C, et al., J. Biol. Chem. 265:29171-21978 (1990)). Therefore, it seems possible that the specific activity of TGase3 enzyme is higher than TGasel . 4. Amino Acid Sequences of the Human, Mouse and Guinea Pig TGase3

Proteins Are Not Highly Conserved In Table 7 are listed the several tryptic peptides generated for guinea pig TGase3 that were found in the mouse and human TGase3 sequences. The comparisons further extend confidence for the correct identity of these sequences. In addition, the availability of the amino- terminal information of the 27 kDa fragment formed on proteolytic cleavage activation enabled identification of the activation region in the mouse and human TGase3 proteins as well (Table

7).

Previous studies have shown that the sequences of human and mouse TGasel and TGase2 enzymes have been very highly conserved: sequences show identities of about 93% and homologies of about 97%. In contrast, the data of FIG. 1 1 reveal that mouse and human

TGase3 sequences have deviated more widely (Table 8). Overall, the sequences show 75% identity and 84% homology, with the 27 kDa fragment generated following proteolytic activation somewhat less conserved: 71% identity and 81% homology. Interestingly, the amino acid sequences of the available tryptic peptides of the guinea pig TGase3 show far more variation from mouse and human such that the 27 kDa fragment displays as little as 45% sequence identity in available comparable sequences. Most of the variations have occurred in the vicinity of the proteolytic activation site, which may mean that the different species have evolved alternate mechanisms for proteolytic activation of the TGase3 pro-enzyme. These sequence variations can account for the difficulties we initially encountered in generating mouse

and human sequence information using the guinea pig data.

5. Comparisons Show that Human TGase3 is Distantly Related to Other Members of the TGase Family

Human and mouse TGase3 proteins are notably different from the other four TGase-like proteins by the net insertion of approximately 12 highly polar residues at the side of proteolytic activation. Overall homology and identity scores between the five TGases are shown in Table 8. The sequences were aligned to maximize homologies according to the protocol of Pearson and Lipman (Pearson, W.R. and Lipman, D.J., Proc. Natl. Acad. Sci. U.S.A. 85:2444-2448 (1988)). We have chosen to analyze only sequences bounded by conserved intron locations identified previously (Kim, I.-G., et al., J. Biol. Chem. 267:7710-7717 (1992)), which presumably delineate the conserved structural regions of the TGases. Each TGase chain deviates widely as its termini in both sequence and length, which thus does not admit meaningful comparisons. The human TGase3 protein is most closely related to TGasel and TGase2, and more similar to band 4.2 than factor Xllla, although band 4.2 is least related to the other TGases.

6. The TGase3 Proteins Consist of Two Globular Domains Separated by a Flexible Hinge at the Site of Activation

Secondary structural analyses (see Devereux, J., et al. Nucleic Acids Res. 12:387-394 (1984); Chou, P.Y. and Fasman, G.D., Biochemistry 13:222-245 (1974); Gamier, J., et al., J. Mol. Biol. 120:97-118 (1978); and Pearson, W.R. and Lipman, D.J., Proc. Natl. Acad. Sci.

U.S.A. 85:2444-2448 (1988)) of the human and mouse TGase3 proteins reveal multiple interspersed regions of turns, sheet structures and α-helix, in both the 50 kDa amino-terminal and 27 kDa carboxy-terminal portions (FIG. 14). In general, these features suggest a folded compact configuration. However, the 12 residue insertion immediately following the cleavage site required for activation of the pro-enzyme describes a prominent protein turn that is surrounded by sequences that are the most hydrophilic and flexible in the entire protein (FIG. 14). Thus, this sequence describes a flexible hinge region and is likely to be located near the surface of the molecule. From these observations, we can infer that intact TGase3 molecules adopt an elongated shape consisting of two globular domains, a larger amino-terminal and a smaller carboxyl-terminal, that are separated by a flexible hinge corresponding to the activation site. This is flanked by highly polar residues, which are predicted to lie near the surface of the protein, that may be involved in recognition by and accessible to the activating protease(s). Following cleavage, the hinge region appears to collapse, promoting a more compact configuration that greatly enhances catalytic activity of the TGase3 molecule. No other

members of the TGase family possess a flexible hinge region (FIG. 14) and all are predicted to adopt a compact globular form.

III. DNA Sequences of the Present Invention

A. Coding Sequences As described earlier, the entire coding sequence of human TRHY (SEQ ID NO:93), human TGase3 (SEQ ID NO: 109), and mouse TGase3 (SEQ ID NO:l 10) have been discovered. In most applications, it is anticipated that the portion of these sequences corresponding to the exons of the sequence, that is, the coding portions, will be most useful. A purified molecule of DNA corresponding to any of these coding sequences can be produced through various means known to the art, such as by using an automated DNA synthesizer.

As is known to those of skill in the art, a purified molecule of DNA containing the coding sequence of one of SEQ ID NO:93, SEQ ID NO: 109, or SEQ ID NO:l 10 can also be produced by probing a cDNA library made from mRNA from human epidermal tissue (for human trichohyalin or TGase3) or mouse epidermal tissue (for mouse TGase3) with a probe specific to the desired sequence (such as one of those shown in Tables 2 and 5). Once a cDNA clone has been identified, it can be purified and at least partially sequenced in order to determine whether it contains the entire coding sequence.

It is advantageous that the DNA sequences used in the methods referred to herein be in purified form. The term "purified" does not require absolute purity; rather, it is intended as a relative definition. For example, individual clones isolated from a cDNA library, as described above, can be conventionally purified to homogeneity by running the DNA from such clones on an electrophoresis gel. Such cDNA clones can be said to be purified because they do not naturally occur as such, but rather are obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The conversion of mRNA into a cDNA library involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection. Thus, creating a cDNA library from messenger RNA and subsequently isolating individual clones from that library results in an approximately 10 ⁶ -fold purification of the native message. Purification of starting material or natural material (such as mRNA or genomic DNA) to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.

B. DNA Probes

Since the coding sequences of TRHY, human TGase3, and mouse TGase3 are known, further probes for these sequences as well as primers for amplifying the sequences via PCR can be produced. The sequences falling within the scope of the present invention are not limited

to the specific sequences described, such as those in Table 2, but include human allelic and species variations thereof and portions of SEQ ID NO:93, SEQ ID NO: 109, and SEQ ID NO:l 10 of at least 15-18 consecutive nucleic acids. Such probes can be synthesized by various means known to the art, including the use of automated DNA synthesizers. These probes can be used, for example, to identify TRHY in the mRNA or genomic DNA of cells or cell cultures, or to amplify TRHY sequences using PCR.

C. DNA Vectors

The DNA sequences identified and purified as described above can further be cloned into any of a variety of vectors which are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example:

Bacterial: pBs, phagescript, φX174, pBluescript SK, pBs KS, pNH8a, pNHlόa, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5

(Pharmacia); and

Eukarvotic: pWLneo, ρSV2cat, pOG44, pXTl, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia).

Bacteriophage vectors, such as phage lambda can, of course, also be used.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P _R , and trc. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-l. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

D. Cell Lines Containing Vectors

In order to express vectors containing the DNA sequences of the present invention, such vectors can be placed in appropriate cell lines. A wide variety of cell lines, including bacterial, insect, yeast, mammalian, and other cell lines, are available and known to those of skill in the art. The choice of which cell line to use with which vector is also within the knowledge of one of skill in the art. Introduction of a vector into a host cell line can be effected by calcium phosphate transfection, DEAE dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).

IV. RNA Sequences of the Present Invention

According to a further embodiment of the present invention, RNA can be produced from the DNA sequences of the present invention. The RNA molecules of the present invention are homologous or complementary to SEQ ID NO:93, SEQ ID NO: 109, or SEQ ID NO:l 10 except

that the thymine molecules are replaced by uracil molecules. Included in the invention are RNA molecules which comprise 20 or more consecutive nucleic acids of such RNA molecules homologous or complementary to SEQ ID NO:93, SEQ ID NO: 109, or SEQ ID NO: l 10.

The RNA molecules of the present invention can be produced from the DNA molecules of the present invention by methods known to the art. For example, such molecules can be produced by inserting a DNA molecule having the sequence of SEQ ID NO:93 into a plasmid that has a bacteriophage promoter such as SP6, T7, or T3 upstream of the inserted DNA sequence. The appropriate RNA polymerase (SP6, T7, or T3) can then be used to generate RNA molecules having sequences which can be translated into TRHY (see Short Protocols In Molecular Biology. Ausbel, et al. eds., John Wiley & Sons (1989) and see also Sambrook, J.,

Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)).

Example 1 : Production of Trichohyalin RNA A DNA molecule having the sequence of SEQ ID NO:93 IS subcloned into a pGEM-3z plasmid vector. This plasmid is transfected into E. coli or other suitable host, and the host is cultured in order to increase the amount of plasmid material available to be transcribed into RNA (see Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)). Once sufficient material is available, the plasmid material can be isolated, purified, and transcribed in vitro with T7 RNA polymerase into an RNA molecule which has the sequence of SEQ ID NO:93 except that molecules of uracil are substituted for the thymine molecules. RNA molecules so produced can then be purified, such as by treating the in vitro reaction mixture with a DNase enzyme and then electrophoresing the mixture on an agarose gel. V. Protein Molecules of the Present Invention A. Expression of Protein Molecules

Another aspect of the present invention involves the production of protein molecules from the DNA and RNA molecules previously described. Such protein molecules will be homologous to at least a portion of SEQ ID NO:94, SEQ ID NO:l 11, and SEQ ID NO:l 12 and can be produced by methods known to those of skill in the art. At the simplest level, the amino acid sequence encoded by the foregoing polynucleotide sequences can be synthesized using commercially available peptide synthesizers. This is particularly useful in producing small peptides and fragments of larger polypeptides. (Fragments are useful, for example, in generating antibodies against the native polypeptide.) Alternatively, the DNA encoding the desired polypeptide can be inserted into a host

organism and expressed. The organism can be a bacterium, yeast, cell line, or multicellular plant or animal. The literature is replete with examples of suitable host organisms and expression techniques. For example, naked polynucleotide (DNA or mRNA) can be injected directly into muscle tissue of mammals, where it is expressed. This methodology can be used to deliver the polypeptide to the animal, or to generate an immune response against a foreign polypeptide. Wolff, et al., Science 247:1465 (1990); Feigner, et al., Nature 349:351 (1991). A DNA molecule of the present invention coding for all or part of any of SEQ ID NO:94, SEQ ID NO:l 11, or SEQ ID NO:l 12 can also be introduced into an expression vector in order to express one of these proteins. Techniques to transfer cloned sequences into expression vectors that direct protein translation in mammalian, yeast, insect or bacterial expression systems are well known in the art. Commercially available vectors and expression systems are available from a variety of suppliers including Stratagene (La Jolla, California), Promega (Madison, Wisconsin), and Invitrogen (San Diego, California). If desired, to enhance expression and facilitate proper protein folding, the codon context and codon pairing of the sequence may be optimized for the particular expression organism, as explained by Hatfield, et al., U.S. Patent No. 5,082,767.

Example 2: Gene Expression from DNA Sequences of the Present Invention The methionine initiation codon for a DNA molecule of the present invention and the poly A sequence of this molecule are first identified. If the molecule lacks a poly A sequence, this sequence can be added to the molecule by, for example, splicing out the Poly A sequence from pSG5 (Stratagene) using Bgll and Sail restriction endonuclease enzymes and incorporating it into the mammalian expression vector pXTl (Stratagene). pXTl contains the LTRs and a portion of the gag gene from Moloney Murine Leukemia Vims. The position of the LTRs in the constmct allow efficient stable transfection. The vector includes the Herpes Simplex Thymidine Kinase promoter and the selectable neomycin gene. cDNA is obtained by PCR from the bacterial vector using oligonucleotide primers complementary to the cDNA and containing restriction endonuclease sequences for Pst I incorporated into the 5 'primer and Bglll at the 5' end of the corresponding cDNA 3' primer, taking care to ensure that the cDNA molecule is positioned inframe with the poly A sequence. The purified DNA molecule obtained from the resulting PCR reaction is digested with Pstl, blunt ended with an exonuclease, digested with Bgl II, purified and ligated to pXTl, now containing a poly A sequence and digested Bglll.

The ligated product is transfected into mouse NIH 3T3 cells using Lipofectin (Life

Technologies, Inc., Grand Island, New York) under conditions outlined in the product specification. Positive transfectants are selected after growing the transfected cells in 600ug/ml

G418 (Sigma, St. Louis, Missouri). The protein is preferrably released into the supernatant. However if the protein has membrane binding domains, the protein may additionally be retained within the cell or expression may be restricted to the cell surface.

Since it may be necessary to purify and locate the transfected product, synthetic 15-mer peptides synthesized from the predicted cDNA sequence are injected into mice to generate antibody to the polypeptide encoded by the cDNA. The antibody can then be used to identify and purify the protein of interest by known methods.

If antibody production is not possible, the cDNA sequence is additionally incorporated into eukaryotic expression vectors and expressed as a chimeric with, for example, β-globin. Antibody to β-globin is used to purify the chimeric. Corresponding protease cleavage sites engineered between the β-globin gene and the cDNA are then used to separate the two polypeptide fragments from one another after translation. One useful expression vector for generating β-globin chimerics is pSG5 (Stratagene). This vector encodes rabbit β-globin. Intron II of the rabbit β-globin gene facilitates splicing of the expressed transcript, and the polyadenylation signal incorporated into the construct increases the level of expression.

These techniques as described are well known to those skilled in the art of molecular biology. Standard methods are published in methods texts such as Davis et al. and many of the methods are available from the technical assistance representatives from Stratagene, Life Technologies, Inc., or Promega. Polypeptide may additionally be produced from either constmct using in vitro translation systems such as In vitro Express™ Translation Kit

(Stratagene).

B. Production of Antibodies

Another aspect of the present invention comprises producing antibodies to the proteins of the present invention. Such antibodies can be used, for example, in assays for the detection of the proteins of the present invention.

Example 3: Producing Antibodies to the Proteins of the Present Invention

Substantially pure protein or polypeptide is isolated from the transfected or transformed cells as described above in Example 2. The concentration of protein in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms/ml. Monoclonal or polyclonal antibody to the protein can then be prepared as follows:

(I) Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibody to epitopes of any of the peptides identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler,

G. and Milstein, C, Nature 256:495 (1975) or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody- producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as Elisa, as originally described by Engvall, E., Meth. Enzy ol. 70:419 (1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis, L. et al. Basic Methods in Molecular Biology Elsevier, New York. Section 21-2.

(2) Polyclonal Antibody Production by Immunization Polyclonal antiserum containing antibodies to heterogenous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein described above, which can be unmodified or modified to enhance immunogenicity. Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than other and may require the use of carriers and adjuvant. Also, host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable. An effective immunization protocol for rabbits can be found in Vaitukaitis, J. et al. J. Clin. Endocrinol. Metab. 33:988-991 (1971). Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony, O. et al., Chap. 19 in: Handbook of Experimental Immunology D. Wier (ed) Blackwell (1973). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of semm (about 12 μM). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, D., Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman, eds.) Amer. Soc. For Microbiol., Washington, D.C (1980).

Antibody preparations prepared according to either protocol are useful in quantitative

immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.

VI. Uses of Trichohyalin and Transglutaminase-3 A. Gel Formation

Human TRHY has 332 glutamine and 104 lysine residues, which are potential targets for crosslinking by transglutaminases. Since the arginines and lysines lie on the periphery of the α-helix, they will be readily accessible by the peptidyl arginine desimidase and transglutaminase(s) enzymes. Thus, when TRHY is present in sufficient concentration in solution and an active TGase3 enzyme is added, the TRHY will become cross-linked and form a gel. Another embodiment of the present invention therefore involves the formation of gels with TRHY and TGase3 or other suitable enzymes.

The appropriate salt concentration, pH, and other solution variables for such a solution can be determined by one of skill in the art through routine experimentation. Such variables are determined by exposing solutions of TRHY having different salt concentrations, etc. to

TGase3 (or another enzyme capable of cross-linking TRHY) and then determining the speed and extent to which TRHY becomes cross-linked. Physiological conditions, i.e. those found in mammalian epidermal tissue, are preferred for gel formation, however.

Concentrations of TRHY from approximately 0.01% to approximately 5% by weight of the solution can be used, depending on the rigidity desired in the resulting gel. Preferably,

TRHY is present in concentrations of between 0.05% to 1.0%, and more preferably in a concentration of about 0.1%. Thus, 1 gram of TRHY in one liter of aqueous solution can be used to form a gel. In general, higher concentrations of TRHY will result in more rigid gels. This correlates to the finding that more TRHY is found in hard, terminally differentiated epidermal tissue than in softer, developing epidermal tissue. The desired physical characteristics of a gel for a particular use can, of course, be determined through routine skill in the art.

Concentrations of TGase3 which can be used to form gels with TRHY range from approximately 0.5% by weight of the TRHY to approximately 5% by weight. As is known to those of skill in the art, increasing the amount of enzyme used will, in general, both speed the cross-linking reaction and cause the reaction to proceed further (cause more cross-links to be made). Again, the proper amount of TGase3 to be used can be determined through the application of routine skill.

It is preferred that when the protein to be cross-linked is human TRHY, the enzyme used should be human TGase3. Mouse TGase3 can also be used, however, unless

contraindicated by the use to which the gel is to be put. For example, a gel used in a cosmetic or food preparation might preferably be cross-linked by human TGase3 rather than mouse TGase3, since mouse TGase3 might cause an allergic reaction in some individuals exposed to the cosmetic or food preparation. Other cross-linking agents, such as human TGasel, can also be used in place of TGase3.

The gels formed according to this aspect of the invention can contain any number of other ingredients, as long as the presence or amount of such ingredients does not substantially interfere with gel formation. Ingredients used in the food and cosmetic industries can, for example, be added to form food and cosmetic compositions. It is a matter of routine experimentation to determine the range of possible ingredients which can be used in the gels produced according to the present invention. If one desires to know whether a particular ingredient can be included in such a gel, the ingredient can be added along with a gel-forming amount of TGase3 or other cross-linking agent to a solution of TRHY. If a gel is formed and the gel has the characteristics necessary to act as a food or cosmetic preparation, then the ingredient is one which can be used in the present invention to produce a food or cosmetic gel in combination with TRHY.

Example 4: Gel-Containing Food Preparation Approximately 50 mg of human TGase3 is added to a 1 liter aqueous solution containing the following ingredients: 1 gram of TRHY, enough food coloring (such as a dye approved by the F.D.A. for use in human food products) to impart color to the resulting foodstuff, a preservative, and sufficient aspartame to sweeten the resulting foodstuff. Such a food preparation can be served as a dessert.

Example 5: Gel-Containing Breast Implant A gel-containing breast implant is prepared by first lining a sterile mold with a sterile biocompatible plastic, one which will not cause adverse reactions when implanted into a human body. The mold is shaped and sized so as to produce an implant of suitable size and shape for implantation into a human patient. A sterile solution containing TRHY is poured into the plastic-lined mold, after which a sterile solution containing human TGase3 is poured into the mold. If necessary, another aqueous solution which does not carry either of these components is added to the mold to fill the mold. Sufficient TRHY should be used so that TRHY is present in the filled mold in an amount between 0.01% and 5% by weight of the solution, and preferably in an amount of about 0.1%. Human TGase3 should be present in an amount between about 1% and 5% by weight of the TRHY in solution. After a sufficient period of time to allow gel formation, a gel is formed in the mold surrounded by the plastic. The plastic is

sealed, and any excess plastic not surrounding the gel is removed. A breast implant is thus formed which can then be implanted in a human patient. B. Tissue Glue

In a further embodiment of the present invention, TRHY and TGase3 can be used to form structurally rigid gels for use in promoting the healing of wounds. In this embodiment,

TRHY and an agent capable of cross-linking TRHY proteins such as TGase3 are mixed and applied to an open wound. The gel which forms then acts as a "scaffold" over which new tissue, such as skin, can grow. The gel can thus act not only to cover and protect damaged tissue but also to promote the healing and regrowth of tissue. In treating humans, the use of human TRHY and human TGase3 is preferred so as to avoid the possibility of an allergic reaction. Other non-allergenic cross-linking enzymes or agents, such as a tmncated mouse TGase3 protein which does not present mouse allergens but still retains cross-linking activity, for example, can also be used.

The amount of TRHY and enzyme used on a particular wound will depend on the desired characteristics of the gel to be formed. Preferably, TRHY is present in an amount between 0.01% and 5.0% by weight of the solution, and more preferably in an amount of about 0.1%. When treating wounds to soft, internal tissues, a less rigid gel may be called for, meaning that a solution having a lower concentration of TRFIY should be used. Conversely, when the wound being treated is a skin tear that is open to the environment, a tougher, relatively more rigid composition may be called for, and a higher concentration of TRHY can be used. In this embodiment, the gel can supplement or replace a bandage to cover the exposed tissue. In general, the properties of the wound-healing composition according to the present invention can be varied as described above for controlling the rigidity of TRHY gels, that is, by varying the amount of TRHY and the amount of enzyme used to form the composition. Example 6: Use of TRHY to Form a Tissue Glue

A sterile solution of approximately 1% by weight TRHY is prepared. Just prior to the application of the solution to a tear in the skin, a concentrated, sterile solution containing human TGase3 is mixed with the TRHY solution such that the resulting solution contains approximately 3% human TGase3 by weight of the TRHY in solution. Before the gel completely solidifies, the mixture of TGase3 and TRHY is applied to the skin wound so as to fill and cover the wound. After the gel solidifies, the wound is protected from the environment by the gel. In addition, the gel helps to bind the torn edges of the wound, thus further promoting healing.

Although the present invention has been described in terms of certain preferred

embodiments, such embodiments are provided herein as examples and are not meant to limit the scope of the present invention. The disclosures of the references referred to herein are, in addition, hereby incoφorated by reference.

Tabl e 1

Abundances of sel ected mRNAs in mouse and human epidermi s and cultured cel l s (fmol /10 μg of total cel l ular RNA)

Keratin 10 Profi laggrin Loricnn Trichohyal in

Human foreskin 635 ± 55 423 ± 37 394 ± 38 028 ± 003 epidermis

Human cultured 025 ± 033 0 17 ± 022 007 ± 011 001 ± 002 low Ca ^2*

Human cultured 175 ± 024 073 ± 011 1 05 ± 017 005 ± 004 high Ca ^2*

Mouse epidermis 436 ± 34 298 ± 41 274 ± 25 (033 ± 046)

Mouse cultured 023 ± 033 011 ± 013 009 ± 010 (000 ± 002) low Ca ^2*

Mouse cultured 235 ± 022 028 ± 019 016 ± 009 (005 ± 011) high Ca ^?*

Mouse hair 047 058 097 (885) follicles

The data are the average (± s.d.) of four different experiments, except for mouse hair follicles (one batch, gift of Dr. Ulrike Lichti). These values were calculated from the Northern slot blots (FIG. lb) based on calibrations of 10, 1, 0.1 and 0.01 fmol of known cloned probes. Because we do not have a specific cloned probe of mouse TRH, the numbers in parenthesis adduced here are based on the hybridization signal obtained with the human TRHY probe.

Table 2 Synthetic ol igonucleotides used as primers for anchored PCR primer extensi on of human TRHY mRNA

Primer Sequence SEQ ID NO Location (numbered as in FIG 3)

1- 5 -AGGGCGGTATTGAGATCTCTGCTCT 1 8067-8044

1+ 5 -GACAGAAAATTCCGCGAGGAGGAGG 2 7546-7569

2- 5 -CCTCCTCCTCGCGGAATTTTCTGTC 3 7569-7546

3- 5 -CCTGACGCCGCTGTTGGCCGCGCTC 4 6895-6871

4 5 -TCAGC CTGCTTTTCCTCTTGGGA 5 6211 -6187

5- 5 -GπGCCACCTCCATTTTTGGTC 6 5055-5035

6- 5 -CTTTGCCGTGCGTCGGCCTC 7 4580-4560

7 5 -GCTCGCGTCTTAGTTGTTGCTCGCT 8 4054-4029

8- 5 -GTCGATCπGTAACAGGCTTTCCTT 9 2742-2719

9 5 -CTACCGTCTTAGGGTCATGTGGTC 10 2536-2513

Table 3

Fast Fourier Transforms of segments of the human TRH sequence

Domain Segment Charged/apolar* 14 value" Residue Period Intensity ¹ Comments Ratio

1-94 0.6 two well-defined EF-hand calcium binding domains

95-312 4.4 1.54 no organized repeating element: largely α-helical: elongated rod. ca 32 nm

313-389 10.4 1.91 repeat of 13 residues:

RREQEEERREQQL (SEQ ID NO:11)

390-443 3.0 1.02 repeat of 6 residues:

RREQQL (SEQ ID NO:12) both form highly stabilized α-helix: elongated rod. ca 19 nm ι

R 6.40 (12.8/2) 24.15

Q 6.38 (12.8/2) 18.11

E 12.64 (12.8/1) 7.88

444-702 6.25 2.17 irregular consensus repeat of 28 residues LKREQEERREQRLKREEEEREQERREQR (SEQ ID NO:13) or

[(REQRLKRE' EER EQER] (SEQ ID NO:14) highly stabilized α-helix: elongated flexible rod. ca 38 nm

R + K 6.07 18.76

R 6.08 14.60

Q 6.08 8.19

Q 5.90 9.36

E 10.19 9.92

Domain Segment Charged/apolar ^a 14 value" Residue Period Intensity ⁰ Comments Ratio

703-922 2.62 1.05 potential regularity around residues QEQARE I SRIPK QWQLESEADAR (SEQ ID NO:15) contains α-helix but also several turns: potential for β-turns or even 0-sheet: folded or globular

923-1163 4.85 2.03 highly regular 30-residue repeat QEEEQLLREEREKRRRQERERQYR ^E / _K EEELQ (SEQ ID NO:16) highly stabilized α-helix: elongated rod ca 36 nm

29.90 (30/1) 8.69 10.06 (30/3) 14.47 7.50 (30/4) 18.34 6.01 (30/5) 11.11 I 4.29 (30/7) 9.71 I

R + K 29.90 (30/1) 22.26 R 29.90 (30/1) 22.05 4.29 (30/7) 9.84

E 7.52 (30/4) 43.61 6.00 (30/5) 10.60

Q 15.06 (30/2) 11.12 5.99 (30/6) 12.89 4.28 (30/7) 23.12

1164-1249 3.15 0.73 no apparent regularity: contains α- helix but also several turns: folded or globular

1250-1849 3.72 1.86 well defined but irregular repeat with consensus of 26 residues, but several are shorter

RQERDRKFREEEQQLRRQEREEQQLR (SEQ ID

NO: 17) or [(EEQQLRRQER) _? DRKFRE (SEQ ID

NO: 18) most common element of :

RQERDRKFREEEQ (SEQ ID NO: 19) highly stabi l i zed α-hel i x : elongated rod ca 90 nm

Domain Segment Charged/apolar* 14 value Residue Period Intensity ¹¹ Comments Ratio

L 7.85 13.28 4.91 908

R + K 7.89 9.25

E + D 7.88 9.25 7.73 39.90

E 7.88 39.98 7.73 22.68

Q 4.00 10 19

1850-1897 no repeats: likely to be folded or globular due to frequent turns

³ Charged/apolar = DEKHR/LIVMFYA

" Number ofpossible intrachain ionic interactions arising from residues four apart (48). The result (14) is presented Ul here on a per seven residue basis for ease of comparison with the interchain ionic interactions that stabilize coiled-coil I ropes in α-fibrous proteins (39.40). The higher the 14 value, the more stable is an α-helix: typical values are: four- or more-stranded α-nelical bundled, charged/apolar <0.5.14 ca 0: three-stranded α-helical coiled-coil. charged/apolar ca 0.814=0.5: two-strandedα-helical coiled-coil. charged/apolar ca 1.0.14=0.8: overall charged/apolar ratio of TRHY is 4.1. 14-1.7. This supports the idea that TRHY forms a single-stranded α-helical rod stabilized by ionic salt bridges between oppositely-charged residues which lie four residues apart on adjacent turns of the α-helix. ^c The major scaled intensities >9 corresponding to periods >4 residues are given. The probability of intensity (/) occurring by chance is exp(-/).

Table 4

Amino Acid Sequences of Tryptic Peptides of Guinea Pig TGase3

SEQ ID NO PEPTIDE ORDER

50 kDa fragment: 20 CLGVRSR" 21 2 VSQGVFQCGPASVIAV 22 3 FGQCWVFAGTLNTVL* 23 4 E ^G / _H DVDLNFVMPFIY 24 5 GSDSVWNFHVWNVAWFVR 25 6 LGTFVLLFNPWLQADDVFMS

27 kDa fragment: 26 7 AQRSPGREQAPSISGRFKVNGVLAVGQE" 27 8 TTAICK 28 9 ITYAQYEK 29 10 FEILPTR 30 11 D/LVLDFEGSCLLR 31 12 DV1LDNPTLTEVLD 32 13 KP7 _G NVQCLFSNPLDG

By comparisonswith other TGase sequences, these two peptides constitute the active site (order: 3-1): sequence alignments show that they are unique to and thus diagnostic for the TGase3 system.

This is the amino-terminus of the 27 kDa fragment (21) and thus represents the cleavage activation site of guinea pig TGase3.

Table 5

Sequences of Oligonucleotide Primers Used

For Anchored-PCR Experiments

Primer SEQ ID NO: How Used

Mouse primers:

P1+ 5 ^' -TGGGTITT ^T /,GCIGGNACN7,TIAA ^T /,AC 33 PCR P1+.P3-

P3- 5 ^' -GCIGGICC ^ε / _A CA ^τ / _c TG7^NACICC7 _c TG 34 la+ 5"-ACTGACCTAGGCCCCACATACA 35 lb+ 5'-AGAAGCCAAGGCGTATTCCAA 36 PCR la+.P10-: then lb+.PlO- P10- 5 ^' -ACNCC7 _G TTV _G TA7 _C TT ^A / _G AAIC ^T / _G CC 37

P5+ 5 ^' -CA'/sGC GAVrGAVrGTITTVcATG 38 2a - 5 ^• -TGTATGTGGGGCCTAGGTCAGT 39 PCR P5+.2a-: then P5+.2b- 2b- 5 ^' -CATGGCATCGTAGTACACATCCAC 40

3a- 5 ^' -GTCACGACGGAAATTCAGACTCCT 41 primer extension with

3a-. dG tail

3b- 5 ^' -TTGTCTTTCGGCGTGGTTACT 42 PCR oligo-dC.3b-

4a+ 5'-GGCTTTGGACAAACTCAAACC 43 primer extension with random hexamer

4b+ 5 ^• -GACAAGGAGCCCAGCATTTCT 44 dG tail. PCR 4a+. oligo-dC: then 4b+. oligo-dC

5a+ 5'-GAGAGATACCTGAAGACAGAGAC 45 primer extension with random hexamer

5b+ 5 ^' -AACATGATCCGGATCTCAGCC 46 dG tail. PCR 5a+. oligo-dC: then 5b+. oligo-dC

Human primers:

6a+ 5 -TCCTCTCTGAAACTTGGCTπ 47

6b+ 5'-CAAGCGGATGATGTCTπATG 48 PCR 6a+.7a-: then 6b+.7b-

7a- 5'-GAAAATCATCTGCACGTTCAC 49

7b- 5'-GTCCAGGGGGTTGGAGGAAAAT 50

8a- 5 ^' -ACGGCGGAAATTCAGACTCCT 51 primer extension with 8a-. dG tail.

8b- 5'-CATGCCAATTCGGTTTGTGCT 52 PCR oligo-dC. 8b-

9a+ δ'-GAACATCCCATAAAGATCTCG 53 pri er extensi on wi th ol igo-dT

9b+ 5'-GTACGCTCAGTATGAGAGGTA 54 PCR 9a+ . ol i go-dA; then

9b+ . ol igo-dA

Primer SEQ ID NO: How Used

TGaSe2+ 5 ^' -CCAGCCTGCTGAGAGCCC 55 PCR primers to amplify

0.7 kbp TGase2- 5 ^' -CAGTGGACTCAGCGTCAG 56 3 ^' -non-coding region. using /.gtll cDNA library (45) DNA as template

"P" oligonucleotides were derived from numbered peptide sequences (see Table 4).

"+" means plus (left) primer: "-" means minus (right) primer. The "a" primers were used in the first PCR reaction. The "b" primers were nested inside the "a" primers and used for the second round of PCR on a diluted sample of the first reaction. I - inosine: N - all 4 nt. Note, the primers 6a+. 6b+. 7b-. 7a- are from corresponding mouse TGase sequences.

Table 6

Abundances of Mouse and Human TGase mRNAs

(fmol/10 μq of Total Cellular RNA) ^"

TGasel TGase2 TGase3

Human foreskin epidermis 0.45±0.06 0.071+0.023 0.078+0.022 Human, cultured, low Ca ² + 0.91±0.08 0.135+0.015 <0.002 Human, cultured, high Ca ² + 1.05+0.11 0.112±0.021 0.008+0.002 Mouse epidermis 0.57±0.01 0.053+0.017 0.081+0.019 Mouse, cultured, low Ca ² + 1.37+0.16 0.121±0.028 <0.002 Mouse hair follicles 0.27 0.029 0.087

The data are the average (±s.d.) of four different experiments, except for mouse hair follicles (one x£> batch, a gift of Dr. Ulrike Lichti). These values were calculated from the Northern slot blots (FIG. I 12b). based on calibrations of 10. 1. 0.1. 0.01 fmol of known cloned probes.

Table 7

Comparisons of Amino Acid Sequences of Human, Mouse and Guinea Pig TGase3 Tryptic Peptides

50 kDa fragment: SEQ ID NO: human YGQCWVFAGTLNTALRSLGIPSR 57 mouse F V..C..VR.. 58 guinea pig F V..C..VR.. 59 human AHDTDRNLSVDVYYD 60 mouse 61 guinea pig EG.V.L.F-.MPFIY 62

I human RSQGVFQCGPASVIGV 63 σ mouse NAI 64 I guinea pig V A. 65 human GSDSVWNFHVWNEGWFVR 66 mouse 67 guinea pig VA.... 68 human LGTFILLFNPWLNVDSVFMG 69 mouse ....V QA.D...S 70 guinea pig ....V QA.D...S 71

27 kDa fragment: human SMGLETEEQEPSIIGKLKVAGMLAVGKE 72 mouse .RNP.6.DK S..F..T.I 73 guinea pig AQRSPGR..A...S.RF..N.V Q.* 74 human ISYAQYER 75 mouse • -rt.• o • • ----- -» 76 guinea pig .T K 77 human ITAVCK 78 mouse • o • ----- ---- ---> 79 guinea pig T..1.. 80 I cn human FDILPSR 81 mouse .E.F.T. 82 guinea pig •E...X---> 83 human KPVNVQMLFSNPLDE 84 mouse I Q 85 guinea pig C G 86 human DIILDNPTLTLEVLN 87 mouse .V A E 88 guinea pig .V D 89

human DCVLMVEGSGLLL 90 mouse N...L CSV 91 guinea pig .L..DF...C..R 92

* This peptide is the amino terminus of the 27 kDa fragment and thus represents the cleavage activation site of TGase3.

I ι-π

N I

Table 8 Homology Scores Between Different TGase-like Proteins

A. Inter-species comparisons human TGase3: mouse TGase3 75% identity. 84% homology human TGase3: guinea pig TGase3 ^a 50% identity. 80% homology. overall

60% identity. 84% homology. 50 kDa portion

45% identity. 78% homology. 27 kDa portion human TGasel: mouse TGasel 93% identity. 97% homology human TGase2: mouse TGase2 90% identity. 95% homology

B. Inter-chain comparisons" human TGase3: human TGasel I

53% identity. 68% homology υi human TGase3: human TGase2 48% identity. 64% homology I human TGase3: human band 4.2 46% identity. 62% homology human TGase3: human Xllla 38% identity. 56% homology human TGasel: human TGase2 42% identity. 57% homology human TGasel: human Xllla 47% identity. 61% homology human TGasel: human band 4.2 28% identity. 47% homology human XIIla: human band 4.2 43% identity. 64% homology

^a includes only information from available 180 sequenced amino acids of guinea pig TGase3 tryptic peptides (Table 4). " includes only regions bounded by conserved intron junctions.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Steinert, Peter M. Lee, Seung-Chul Kim, In-Gyu Chung, Soo-Il Park, Sang-Chul

(ii) TITLE OF INVENTION: Trichohyalin and Transglutaminase-3 and Mehods of Using Same (iii) NUMBER OF SEQUENCES: 1 17

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Knobbe, Martens, Olson & Bear

(B) STREET: 620 Newport Center Drive, Sixteenth Floor (C) CITY: Newport Beach

(D) STATE: CA

(E) COUNTRY: U.S.A.

(F) ZIP: 92660 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/056,200

(B) FILING DATE: 30-APR-1993

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Fedrick, Michael F.

(B) REGISTRATION NUMBER: 36,799

(C) REFERENCE/DOCKET NUMBER: NIH054.001A

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (714) 760-0404

(B) TELEFAX: (714) 760-9502

(2) INFORMATION FOR SEQ ID NO:l :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l : AGGGCGGTAT TGAGATCTCT GCTCT 25

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:2: GACAGAAAAT TCCGCGAGGA GGAGG 25

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CCTCCTCCTC GCGGAATTTT CTGTC 25 (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

CCTGACGCCG CTGTTGGCCG CGCTC 25 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:5:

TCAGCAACTG CTTTTCCTCT TGGGA 25 (2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

GTTGCCACCT CCATTrTTGG TC 22 (2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:7:

CTTTGCCGTG CGTCGGCCTC 20 (2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GCTCGCGTCT TAGTTGTTGC TCGCT 25 (2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:9:

GTCGATCTTG TAACAGGCTT TCCTT 25 (2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

CTACCGTCTT AGGGTCATGT GGTC 24 (2) INFORMATION FOR SEQ ID NO: 11 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:l l:

Arg Arg Glu Gin Glu Glu Glu Arg Arg Glu Gin Gin Leu 1 5 10

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:12:

Arg Arg Glu Gin Gin Leu

1 5

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Leu Lys Arg Glu Gin Glu Glu Arg Arg Glu Gin Arg Leu Lys Arg Glu 1 5 10 15

Glu Glu Glu Arg Glu Gin Glu Arg Arg Glu Gin Arg

20 25 (2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTER! STICS:

(A) LENGTH: 28 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO: 14: Arg Glu Gin Arg Leu Lys Arg Glu Xaa Glu Glu Arg Arg Glu Gin Arg

1 5 10 15

Leu Lys Arg Glu Xaa Glu Glu Arg Glu Gin Glu Arg

20 25

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

Gin Glu Gin Ala Arg Glu Arg He Lys Ser Arg He Pro Lys Tφ Gin 1 5 10 15

Tφ Gin Leu Glu Ser Glu Ala Asp Ala Arg 20 25

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO: 16:

Gin Glu Glu Glu Gin Leu Leu Arg Glu Glu Arg Glu Lys Arg Arg Arg 1 5 10 15 Gin Glu Arg Glu Arg Gin Tyr Arg Xaa Glu Glu Glu Leu Gin

20 25 30

(2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

Arg Gin Glu Arg Asp Arg Lys Phe Arg Glu Glu Glu Gin Gin Leu Arg 1 5 10 15

Arg Gin Glu Arg Glu Glu Gin Gin Leu Arg 20 25

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

Glu Glu Gin Gin Leu Arg Arg Gin Glu Arg Glu Glu Gin Gin Leu Arg 1 5 10 15

Arg Gin Glu Arg Asp Arg Lys Phe Arg Glu

20 25 (2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Arg Gin Glu Arg Asp Arg Lys Phe Arg Glu Glu Glu Gin

1 5 10

(2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:20:

Cys Leu Gly Val Arg Ser Arg

1 5

(2) INFORMATION FOR SEQ ID NO:21 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:21 :

Val Ser Gin Gly Val Phe Gin Cys Gly Pro Ala Ser Val He Ala Val 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Phe Gly Gin Cys Tφ Val Phe Ala Gly Thr Leu Asn Thr Val Leu

1 5 10 15

(2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

Glu Xaa Asp Val Asp Leu Asn Phe Val Met Pro Phe He Tyr 1 5 10 (2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Gly Ser Asp Ser Val Tφ Asn Phe His Val Tφ Asn Val Ala Tφ Phe

1 5 10 15

Val Arg

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

Leu Gly Thr Phe Val Leu Leu Phe Asn Pro Tφ Leu Gin Ala Asp Asp 1 5 10 15

Val Phe Met Ser 20

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

Ala Gin Arg Ser Pro Gly Arg Glu Gin Ala Pro Ser He Ser Gly Arg 1 5 10 15

Phe Lys Val Asn Gly Val Leu Ala Val Gly Gin Glu 20 25

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:

Thr Thr Ala He Cys Lys

1 5 (2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:28: He Thr Tyr Ala Gin Tyr Glu Lys

1 5

(2) INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

Phe Glu He Leu Pro Thr Arg 1 5

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

Xaa Leu Val Leu Asp Phe Glu Gly Ser Cys Leu Leu Arg 1 5 10

(2) INFORMATION FOR SEQ ID NO:31 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31 :

Asp Val He Leu Asp Asn Pro Thr Leu Thr Glu Val Leu Asp 1 5 10

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

Lys Pro Xaa Asn Val Gin Cys Leu Phe Ser Asn Pro Leu Asp Gly 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:33:

TGGGTNTTYG CNGGNACNYT NAAYAC 26

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

GCNGGNCCRC AYTGRAANAC NCCYTG 26

(2) INFORMATION FOR SEQ ID NO:35: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

ACTGACCTAG GCCCCACATA CA 22

(2) INFORMATION FOR SEQ ID NO:36: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

AGAAGCCAAG GCGTATTCCA A 21

(2) INFORMATION FOR SEQ ID N0:37: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:37:

ACNCCRTTRT AYTTRAANCK CC 22

(2) INFORMATION FOR SEQ ID NO:38: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO '

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

CARGCNGAYG AYGTNTTYAT G 21

(2) INFORMATION FOR SEQ ID NO:39: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

TGTATGTGGG GCCTAGGTCA GT 22

(2) INFORMATION FOR SEQ ID NO:40: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

CATGGCATCG TAGTACACAT CCAC 24

(2) INFORMATION FOR SEQ ID NO:41 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41 :

GTCACGACGG AAATTCAGAC TCCT 24

(2) INFORMATION FOR SEQ ID NO:42: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:

TTGTCTTTCG GCGTGGTTAC T 21

(2) INFORMATION FOR SEQ ID NO:43: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:43:

GGCTTTGGAC AAACTCAAAC C 21

(2) INFORMATION FOR SEQ ID NO:44: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

GACAAGGAGC CCAGCATTTC T 21

(2) INFORMATION FOR SEQ ID NO:45: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:45:

GAGAGATACC TGAAGACAGA GAC 23

(2) INFORMATION FOR SEQ ID NO:46: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:

AACATGATCC GGATCTCAGC C 21

(2) INFORMATION FOR SEQ ID NO:47: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:47:

TCCTCTCTGA AACTTGGCTT T 21

(2) INFORMATION FOR SEQ ID NO:48: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:48:

CAAGCGGATG ATGTCTTTAT G 21

(2) INFORMATION FOR SEQ ID NO:49: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

GAAAATCATC TGCACGTTCA C 21

(2) INFORMATION FOR SEQ ID NO:50: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:

GTCCAGGGGG TTGGAGGAAA AT 22

(2) INFORMATION FOR SEQ ID NO:51 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:51 : ACGGCGGAAA TTCAGACTCC T 21

(2) INFORMATION FOR SEQ ID NO:52:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: CATGCCAATT CGGTTTGTGC T 21

(2) INFORMATION FOR SEQ ID NO:53:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:53: GAACATCCCA TAAAGATCTC G 21

(2) INFORMATION FOR SEQ ID NO:54:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:54: GTACGCTCAG TATGAGAGGT A 21

(2) INFORMATION FOR SEQ ID NO:55:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:55: CCAGCCTGCT GAGAGCCC 18

(2) INFORMATION FOR SEQ ID NO:56:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: CAGTGGACTC AGCGTCAG 18

(2) INFORMATION FOR SEQ ID NO:57:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:

Tyr Gly Gin Cys Tφ Val Phe Ala Gly Thr Leu Asn Thr Ala Leu Arg 1 5 10 15

Ser Leu Gly He Pro Ser Arg 20

(2) INFORMATION FOR SEQ ID NO:58:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:

Phe Gly Gin Cys Tφ Val Phe Ala Gly Thr Leu Asn Thr Val Leu Arg 1 5 10 15

Cys Leu Gly Val Arg Ser Arg

20 (2) INFORMATION FOR SEQ ID NO:59: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:

Phe Gly Gin Cys Tφ Val Phe Ala Gly Thr Leu Asn Thr Val Leu Arg 1 5 10 15

Cys Leu Gly Val Arg Ser Arg 20

(2) INFORMATION FOR SEQ ID NO:60:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:

Ala His Asp Thr Asp Arg Asn Leu Ser Val Asp Val Tyr Tyr Asp 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:61 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61 :

Ala His Asp Thr Asp Arg Asn Leu Ser Val Asp Val Tyr Tyr Asp 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:62:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:62:

Glu Gly Asp Val Asp Leu Asn Phe Xaa Val Met Pro Phe He Tyr 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:63:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:

Arg Ser Gin Gly Val Phe Gin Cys Gly Pro Ala Ser Val He Gly Val 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:64:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:

Arg Ser Gin Gly Val Phe Gin Cys Gly Pro Ala Ser Val Asn Ala He 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:65:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:65: Val Ser Gin Gly Val Phe Gin Cys Gly Pro Ala Ser Val He Ala Val

1 5 10 15

(2) INFORMATION FOR SEQ ID NO:66:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:66:

Gly Ser Asp Ser Val Tφ Asn Phe His Val Tφ Asn Glu Gly Tφ Phe 1 5 10 15

Val Arg

(2) INFORMATION FOR SEQ ID NO:67:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: Gly Ser Asp Ser Val Tφ Asn Phe His Val Tφ Asn Glu Gly Tφ Phe

1 ^' 5 10 15

Val Arg

(2) INFORMATION FOR SEQ ID NO:68:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:68:

Gly Ser Asp Ser Val Tφ Asn Phe His Val Tφ Asn Val Ala Tφ Phe 1 5 10 15

Val Arg

(2) INFORMATION FOR SEQ ID NO:69:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:

Leu Gly Thr Phe He Leu Leu Phe Asn Pro Tφ Leu Asn Val Asp Ser 1 5 10 15 Val Phe Met Gly

20

(2) INFORMATION FOR SEQ ID NO:70: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:70:

Leu Gly Thr Phe Val Leu Leu Phe Asn Pro Tφ Leu Gin Ala Asp Asp 1 5 10 15

Val Phe Met Ser 20

(2) INFORMATION FOR SEQ ID NO:71 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:71 :

Leu Gly Thr Phe Val Leu Leu Phe Asn Pro Tφ Leu Gin Ala Asp Asp 1 5 10 15

Val Phe Met Ser 20 (2) INFORMATION FOR SEQ ID NO:72:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: Ser Met Gly Leu Glu Thr Glu Glu Gin Glu Pro Ser He He Gly Lys

1 5 10 15

Leu Lys Val Ala Gly Met Leu Ala Val Gly Lys Glu 20 25

(2) INFORMATION FOR SEQ ID NO:73:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:

Ser Arg Asn Pro Glu Gly Glu Asp Lys Glu Pro Ser He Ser Gly Lys 1 5 10 15

Phe Lys Val Thr Gly He Leu Ala Val Gly Lys Glu 20 25

(2) INFORMATION FOR SEQ ID NO:74:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:

Ala Gin Arg Ser Pro Gly Arg Glu Gin Ala Pro Ser He Ser Gly Arg 1 5 10 15 Phe Lys Val Asn Gly Val Leu Ala Val Gly Gin Glu

20 25

(2) INFORMATION FOR SEQ ID NO:75: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:

He Ser Tyr Ala Gin Tyr Glu Arg

1 5

(2) INFORMATION FOR SEQ ID NO:76:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:76:

He Ala Tyr Ser Gin Tyr Glu Arg

1 5

(2) INFORMATION FOR SEQ ID NO:77:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:

He Thr Tyr Ala Gin Tyr Glu Lys 1 5

(2) INFORMATION FOR SEQ ID NO:78:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:78:

He Thr Ala Val Cys Lys

1 5 (2) INFORMATION FOR SEQ ID NO:79:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: lie Ser Ala Val Cys Lys

1 5

(2) INFORMATION FOR SEQ ID NO:80: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:80:

Thr Thr Ala He Cys Lys

1 5

(2) INFORMATION FOR SEQ ID NO:81:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81 :

Phe Asp He Leu Pro Ser Arg

1 5

(2) INFORMATION FOR SEQ ID NO:82:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:82:

Phe Glu He Phe Pro Thr Arg 1 5

(2) INFORMATION FOR SEQ ID NO:83:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:

Phe Glu He Leu Pro Thr Arg

1 5 (2) INFORMATION FOR SEQ ID NO:84:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:84: Lys Pro Val Asn Val Gin Met Leu Phe Ser Asn Pro Leu Asp Glu

1 5 10 15

(2) INFORMATION FOR SEQ ID NO:85: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:

Lys Pro Val Asn Val Gin Met He Phe Ser Asn Pro Leu Asp Gin 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:86:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:

Lys Pro Val Asn Val Gin Cys Leu Phe Ser Asn Pro Leu Asp Gly 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:87:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:87:

Asp He He Leu Asp Asn Pro Thr Leu Thr Leu Glu Val Leu Asn 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:88:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:

Asp Val He Leu Asp Asn Pro Thr Leu Thr Leu Glu Val Leu Asp 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:89:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:89:

Asp Val He Leu Asp Asn Pro Thr Leu Thr Leu Glu Val Leu Asp 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:90:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:

Asp Cys Val Leu Met Val Glu Gly Ser Gly Leu Leu Leu 1 5 10 (2) INFORMATION FOR SEQ ID NO:91 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:91 : Asn Cys Val Leu Leu Val Glu Gly Ser Gly Cys Ser Val

1 5 10

(2) INFORMATION FOR SEQ ID NO:92: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:92:

Asp Leu Val Leu Asp Phe Glu Gly Ser Cys Leu Leu Arg 1 5 10

(2) INFORMATION FOR SEQ ID NO:93:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9551 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1507..1644 (ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1645..2511

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION: 2512..8070

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:93:

AACAAGCCAT TTGTGGAGAC AGAGGTGGAG CTGGGCTTGG TTAGGAATGA ATCAGGCCAT 60

TCCACAGAGT GGGTGTCTCC TTCCCAAGTT GCTTTCCAGG GCACAATTAA AACCCCTATA 120

AAAGGCCCAG CTCCCAGTTA CCCAGTACAC TTGCCTGTGG TGTCAGCAAG CACTGTCGAC 180

TTCTTCCTCT GGTGAAGTGG GTAAGTCCCA TTCTGTGGGA TCGTGGTCTT CTTTATGATT 240

CTCCATTTTT ATAGCTATTT CAGATGTTGG GATATGGGGG GAGGTTCCAT GTGCCAGAAG 300 GTATCAGTAT TGCAGGGATA AATAAACTAT CACTAACTCT ATCCCATCTT

CTTATGGTTG 360

GAGCCATCAC TTGAACTGAA GCATGACCCT TCTCCTTGGG CTCTGAACTC TATACTTCTG 420

CACATCAAGG ATGATCATGT GTGGCTCTGA TAGGGTTCAT CTTCCTAAAA ACTGCTATCT 480

CAAAAGTTTG CCAGCCTTCT GTTCTCTTTT ACATTGGTTC TACCTAATAT GGGCCATATT 540

CATACAGTCA CAGCATTTAA GGTACTGGAG TTGAGAAGTA CATAAAGAAG

TCAGCTAGAT 600 GAACGACTAC CTTATCCCAC CAGCAAAGCC ATTCCATGTA TTCTTATA-AC

ATTGATCTAC 660

TGCTGGCTAA TGTTTTATAA AAAGCCAAGA TTCCAATGAT GCATTTGGGT

TTAACAAAAC 720

CAATATCATT CACAGTTTTC TGGATTCCGT TTGGTTTAGA AAGGACCTCT CAGAAGCTTT 780

CAATACTTCA ATATCCGAAT ATCTTACTAT ATCTGAGTTG AGGCAGGTAA TATACAGTCT 840

CTGTTTTCTG CATTTGTGTA TCTGAATGTT ACATGCCATC TTTTGACTAG

GAAGAAGTAC 900 TATTTAATCT TAGAATTGCT GACTTACAAA TTATATTCTA TAAGAGTTCC

TAAATCCCTT 960

TATGGACTGT AATGTTGAGG AATCATTCAT ATTCCCTTTT CATTGTTCTA

TTTTCTACCA 1020

ATCGTTTTGC TGACATGGCC TCTATCCACT TTAAGATACT CTCAAGTCTT CCTTCACCTT 1080

TTGGCTTTAC CTGTCCTCTT CGCTCAACTT TAAAGGAAGG TGTGTAGCCA TATATAAAAT 1 140

TTTAATTTCT GCACTCTTCT CTTAATTTTC TACTCTGAAA TACGGTGGAG

AGCTGGAAGA 1200

AAGACAGAAG AAAAGGGCAT AGATAATCCA CATTGGGTGG ACAATCAAAA GCTGACAACA 1260

GGATAGTCTG AAGATGATTC CCTGGCTTGG AATTTCTCAG GATCGCTCTT

TCTCTTTCTG 1320 ATACAATATT CAAATATTAA AGTGCTCTGA AAGTCCAGGT TGAAATTACC

GCTATAAATT 1380

CAAATTATTT AGGGATCTGC CTGAAATAGT GTGAATGAAG CCTTCCCAAA

AGCAGAAACG 1440

GGATTTTGAT TCTGGATCTT ATTTTATTGT TCTAGGTTTA CTTGAACTTG

AAGGAAAGAA 1500

AAAAAA ATG TCT CCA CTT CTG AGA AGC ATC TGT GAC ATC ACT GAA ATT 1548

Met Ser Pro Leu Leu Arg Ser He Cys Asp He Thr Glu He 1 5 10

TTC AAT CAG TAT GTC TCT CAT GAT TGT GAT GGA GCA GCA TTA ACT AAG 1596

Phe Asn Gin Tyr Val Ser His Asp Cys Asp Gly Ala Ala Leu Thr Lys 15 20 25 30

AAA GAC CTG AAG AAC CTC CTT GAA AGG GAA TTT GGA GCT GTG CTT CGG 1644

Lys Asp Leu Lys Asn Leu Leu Glu Arg Glu Phe Gly Ala Val Leu Arg 35 40 45

GTAAGAACTA ACAAGAAAAT GAGATCTATT GACTTGAGGC TATGAGATTT ATTCTCAGAG 1704

GAGACCAGAG CAAGGAATGG TGGTTTTATA TTCATTTTAC ACCACAACAG GTCTACACTA 1764 CATCCCCCAT TCATTTCTGA GTCAAAAGGT ACTTACTTGA CATTGTAGTC

TGAATAATAA 1824

AGTATTTCAT GTACTTGATG GCATGGCATG TGAATGAGCT CTTCATGGGA CATTACTACA 1884

AAAGATGTCA AATCACACTA GACTTGGAGG AACTTGGAGG AACTTAAATT TGTTTCCAAA 1944

TTTCAAAACT GAGATCAGCC TGACTCTATT AAATGGTGCT ACCCGTAAAT GTTTTGTTCT 2004

GTTTTCTAAT ATGGAATAGA AACCAAATCA AGAATACTGG CTGCTTCAGA CAGAAATGGC 2064

TACTGCAAAT CCTCATAAAT TTCTATTGTA TCTCTCTCAA GGATGAGTTC ATTCTTTCTC 2124

AATTAAAGCG AACTTGTGTT ATTCTTTCTT GATGTTGAGT AGCTTTGTTA ATTTACACAC 2184 AAGTTCACGA TGCTGTTTTG AATCTTCACC TCAGGCTCTG CTCTAAGGTG

CGTAGGCTTA 2244

CCTGCTATCT ACTTGTGTCT CTCTTCCTGC TTCCTTAGGT TTGATCAGCA CTAAATTACG 2304

AGATGTAAAA ATTTCAAACG AATATATGCT TTAAAGTGAG GGTTCACATT TTACATGGGG 2364

ACAAAACTTG ATACACACTG GACATTTTTC TAATTGCTCT GAATGTCTCT TGAATGTCAG 2424

CATAGCATAA AATATATCAT GTGTGAATAT AATTTTACCA CCTGTAAATA GTGCATTGTA 2484 AAATTTTTGT TTTTCACCAT TTTATAG AGA CCA CAT GAC CCT AAG ACG GTA

2535

Arg Pro His Asp Pro Lys Thr Val

1 5 GAT CTG ATC CTG GAA CTT CTG GAT CGT GAC AGT AAT GGG CGT GTC GAT

2583 Asp Leu He Leu Glu Leu Leu Asp Arg Asp Ser Asn Gly Arg Val Asp 10 15 20 TTC AAC GAA TTC CTC CTA TTT ATT TTC AAA GTG GCT CAA GCT TGT TAC

2631

Phe Asn Glu Phe Leu Leu Phe He Phe Lys Val Ala Gin Ala Cys Tyr 25 30 35 40 TAT GCT CTC GGC CAG GCC ACG GGA CTG GAT GAG GAG AAG CGA GCC CGG

2679 Tyr Ala Leu Gly Gin Ala Thr Gly Leu Asp Glu Glu Lys Arg Ala Arg

45 50 55 TGT GAC GGA AAG GAG AGC CTG TTA CAA GAT CGA CGG ACA GAA GAA GAC

2727 Cys Asp Gly Lys Glu Ser Leu Leu Gin Asp Arg Arg Thr Glu Glu Asp 60 65 70 CAA AGG AGA TTC GAG CCC CGG GAC AGA CAA CTG GAA GAA GAA CCT GGG

2775 Gin Arg Arg Phe Glu Pro Arg Asp Arg Gin Leu Glu Glu Glu Pro Gly 75 80 85 CAA CGA CGC AGG CAG AAG AGG CAG GAA CAG GAG AGG GAG CTA GCT GAG

2823 Gin Arg Arg Arg Gin Lys Arg Gin Glu Gin Glu Arg Glu Leu Ala Glu 90 95 100 GGA GAG GAG CAA AGT GAG AAA CAA GAG CGA CTT GAA CAG CGC GAC AGG

2871 Gly Glu Glu Gin Ser Glu Lys Gin Glu Arg Leu Glu Gin Arg Asp Arg 105 1 10 1 15 120 CAG CGC CGC GAC GAG GAG CTG TGG CGG CAA AGG CAA GAA TGG CAA GAA

2919 Gin Arg Arg Asp Glu Glu Leu Tφ Arg Gin Arg Gin Glu Tφ Gin Glu 125 130 135 CGG GAA GAG CGC CGT GCA GAG GAA GAG CAG CTG CAG AGT TGC AAA GGT

2967 Arg Glu Glu Arg Arg Ala Glu Glu Glu Gin Leu Gin Ser Cys Lys Gly 140 145 150 CAC GAA ACT GAG GAG TTT CCA GAC GAA GAG CAA CTG CGA AGG CGG GAG

3015 His Glu Thr Glu Glu Phe Pro Asp Glu Glu Gin Leu Arg Arg Arg Glu 155 160 165 CTG CTG GAG CTG AGG AGG AAG GGC CGC GAG GAG AAA CAG CAG CAA AGG

3063 Leu Leu Glu Leu Arg Arg Lys Gly Arg Glu Glu Lys Gin Gin Gin Arg 170 175 180 CGA GAG CGC CAA GAC AGA GTG TTC CAG GAG GAA GAA GAG AAA GAG TGG

311 1 Arg Glu Arg Gin Asp Arg Val Phe Gin Glu Glu Glu Glu Lys Glu Tφ 185 190 195 200 AGG AAG CGC GAG ACA GTG CTC CGG AAG GAA GAA GAG AAG TTG CAG GAA

3159 Arg Lys Arg Glu Thr Val Leu Arg Lys Glu Glu Glu Lys Leu Gin Glu 205 210 215 GAG GAG CCG CAG CGG CAA AGA GAG CTC CAG GAG GAA GAA GAG CAG CTA

3207 Glu Glu Pro Gin Arg Gin Arg Glu Leu Gin Glu Glu Glu Glu Gin Leu

220 225 230 CGG AAG CTG GAG CGG CAAGAG CTG AGG AGGGAG CGCCAG GAG GAA GAG

3255 Arg Lys Leu Glu Arg Gin Glu Leu Arg Arg Glu Arg Gin Glu Glu Glu

235 240 245 CAG CAG CAG CAA AGG CTG AGG CGC GAG CAG CAA CTA AGG CGC AAG CAG

3303 Gin Gin Gin Gin Arg Leu Arg Arg Glu Gin Gin Leu Arg Arg Lys Gin 250 255 260 GAG GAG GAG AGG CGC GAG CAG CAG GAG GAG AGG CGC GAG CAG CAG GAG

3351 Glu Glu Glu Arg Arg Glu Gin Gin Glu Glu Arg Arg Glu Gin Gin Glu

265 270 275 280 AGG CGC GAG CAG CAG GAG GAG AGG CGC GAG CAG CAG CTG AGG CGC GAG

3399 Arg Arg Glu Gin Gin Glu Glu Arg Arg Glu Gin Gin Leu Arg Arg Glu 285 290 295 CAG GAG GAG AGG CGC GAG CAG CAG CTG AGG CGC GAG CAG GAG GAG GAG

3447 Gin Glu Glu Arg Arg Glu Gin Gin Leu Arg Arg Glu Gin Glu Glu Glu 300 305 310 AGG CGC GAG CAG CAG CTG AGG CGC GAG CAG GAG GAG GAG AGG CGC GAG

3495 Arg Arg Glu Gin Gin Leu Arg Arg Glu Gin Glu Glu Glu Arg Arg Glu

315 320 325 CAG CAG CTG AGG CGC GAG CAG GAG GAG GAG AGG CGC GAG CAG CAG CTG

3543 Gin Gin Leu Arg Arg Glu Gin Glu Glu Glu Arg Arg Glu Gin Gin Leu 330 335 340 AGG CGC GAG CAG CAG CTG AGG CGC GAG CAG CAG CTG AGG CGC GAG CAG

3591 Arg Arg Glu Gin Gin Leu Arg Arg Glu Gin Gin Leu Arg Arg Glu Gin 345 350 355 360 CAG CTG AGG CGC GAG CAG CAG CTG AGG CGC GAG CAG CAG CTG AGG CGC

3639 Gin Leu Arg Arg Glu Gin Gin Leu Arg Arg Glu Gin Gin Leu Arg Arg 365 370 375 GAG CAG CAG CTG AGG CGC GAG CAG CAG CTG AGG CGC GAG CAG CAG CTG

3687 Glu Gin Gin Leu Arg Arg Glu Gin Gin Leu Arg Arg Glu Gin Gin Leu 380 385 390 AGG CGC GAG CAG CAG CTG AGG CGC GAG CAG GAG GAG GAG AGG CAC GAG

3735 Arg Arg Glu Gin Gin Leu Arg Arg Glu Gin Glu Glu Glu Arg His Glu 395 400 405 CAG AAG CAC GAG CAG GAG AGG CGC GAG CAG CGG CTG AAG CGC GAG CAG

3783 Gin Lys His Glu Gin Glu Arg Arg Glu Gin Arg Leu Lys Arg Glu Gin 410 415 420 GAG GAG AGG CGC GAT TGG CTG AAG CGC GAG GAG GAG ACG GAG AGG CAC

3831 Glu Glu Arg Arg Asp Tφ Leu Lys Arg Glu Glu Glu Thr Glu Arg His

425 430 435 440 GAG CAG GAG AGG CGC AAG CAG CAG CTG AAG CGC GAC CAG GAG GAG GAG

3879 Glu Gin Glu Arg Arg Lys Gin Gin Leu Lys Arg Asp Gin Glu Glu Glu

445 450 455 AGG CGC GAA CGT TGG CTG AAG CTC GAG GAG GAG GAG AGG CGC GAG CAG

3927 Arg Arg Glu Arg Tφ Leu Lys Leu Glu Glu Glu Glu Arg Arg Glu Gin 460 465 470 CAG GAG AGG CGC GAG CAG CAA CTA AGG CGG GAG CAA GAG GAG AGG CGC

3975 Gin Glu Arg Arg Glu Gin Gin Leu Arg Arg Glu Gin Glu Glu Arg Arg

475 480 485 GAG CAG CGG CTG AAG CGC CAG GAG GAG GAA GAG AGG CTC CAG CAG CGG

4023 Glu Gin Arg Leu Lys Arg Gin Glu Glu Glu Glu Arg Leu Gin Gin Arg 490 495 500 TTG AGG AGC GAG CAA CAA CTA AGA CGC GAG CAG GAG GAG AGG CTC GAG

4071 Leu Arg Ser Glu Gin Gin Leu Arg Arg Glu Gin Glu Glu Arg Leu Glu 505 510 515 520 CAG CTG CTG AAG CGC GAG GAG GAG AAG AGG CTC GAG CAG GAG AGG CGA

41 19 Gin Leu Leu Lys Arg Glu Glu Glu Lys Arg Leu Glu Gin Glu Arg Arg

525 530 535 GAG CAG CGG CTG AAG CGC GAG CAG GAG GAG AGG CGC GAT CAG CTG CTG

4167 Glu Gin Arg Leu Lys Arg Glu Gin Glu Glu Arg Arg Asp Gin Leu Leu 540 545 550 AAG CGC GAG GAG GAG AGG CGC CAG CAG CGG CTG AAG CGC GAG CAG GAA

4215 Lys Arg Glu Glu Glu Arg Arg Gin Gin Arg Leu Lys Arg Glu Gin Glu 555 560 565 GAG AGG CTC GAG CAG CGA CTG AAG CGC GAG GAG GTG GAG AGA CTC GAG

4263 Glu Arg Leu Glu Gin Arg Leu Lys Arg Glu Glu Val Glu Arg Leu Glu 570 575 580 CAG GAG GAG AGG CGC GAC GAG CGG CTG AAG CGC GAG GAG CCG GAG GAA

4311 Gin Glu Glu Arg Arg Asp Glu Arg Leu Lys Arg Glu Glu Pro Glu Glu 585 590 595 600 GAG AGG CGC CAC GAG CTG CTG AAG AGC GAG GAG CAG GAG GAG AGG CGC

4359 Glu Arg Arg His Glu Leu Leu Lys Ser Glu Glu Gin Glu Glu Arg Arg 605 610 615 CAC GAG CAA CTG AGG CGC GAG CAG CAG GAA AGG CGC GAG CAG CGG CTG

4407 His Glu Gin Leu Arg Arg Glu Gin Gin Glu Arg Arg Glu Gin Arg Leu 620 625 630 AAG CGC GAG GAG GAG GAA GAG AGG CTC GAG CAG CGG CTG AAG CGC GAG

4455 Lys Arg Glu Glu Glu Glu Glu Arg Leu Glu Gin Arg Leu Lys Arg Glu 635 640 645 CAT GAG GAA GAG AGG CGC GAG CAG GAG CTA GCT GAG GAG GAG CAG GAA

4503 His Glu Glu Glu Arg Arg Glu Gin Glu Leu Ala Glu Glu Glu Gin Glu 650 655 660 CAG GCC CGG GAG CGG ATT AAG AGC CGC ATC CCG AAG TGG CAG TGG CAG

4551 Gin Ala Arg Glu Arg He Lys Ser Arg He Pro Lys Tφ Gin Tφ Gin 665 670 675 680 CTA GAA AGC GAG GCC GAC GCA CGG CAA AGC AAA GTC TTA CTC GAG GCC

4599 Leu Glu Ser Glu Ala Asp Ala Arg Gin Ser Lys Val Leu Leu Glu Ala 685 690 695 CCG CAA GCA GGA AGG GCA GAG GCG CCG CAA GAG CAG GAG GAA AAG AGG

4647 Pro Gin Ala Gly Arg Ala Glu Ala Pro Gin Glu Gin Glu Glu Lys Arg 700 705 710 CGG CGC GAG AGT GAG CTG CAA TGG CAG GAG GAG GAA CGG GCT CAC CGG

4695 Arg Arg Glu Ser Glu Leu Gin Tφ Gin Glu Glu Glu Arg Ala His Arg

715 720 725 CAG CAG CAG GAA GAG GAG CAG CGC CGG GAC TTC ACA TGG CAG TGG CAG

4743 Gin Gin Gin Glu Glu Glu Gin Arg Arg Asp Phe Thr Tφ Gin Tφ Gin 730 735 740 GCG GAG GAA AAG AGC GAG AGG GGC CGT CAG AGG CTG TCG GCC AGG CCC

4791 Ala Glu Glu Lys Ser Glu Arg Gly Arg Gin Arg Leu Ser Ala Arg Pro

745 750 755 760 CCA TTG CGG GAG CAG CGG GAG AGG CAG CTG AGG GCC GAG GAG CGC CAG

4839 Pro Leu Arg Glu Gin Arg Glu Arg Gin Leu Arg Ala Glu Glu Arg Gin

765 770 775 CAG CGG GAA CAA CGG TTT CTC CCG GAG GAG GAG GAG AAG GAG CAG CGC

4887 Gin Arg Glu Gin Arg Phe Leu Pro Glu Glu Glu Glu Lys Glu Gin Arg 780 785 790 GGC CGC CAG CGA CGC GAG AGG GAG AAA GAG CTG CAG TTC CTG GAG GAA

4935 Gly Arg Gin Arg Arg Glu Arg Glu Lys Glu Leu Gin Phe Leu Glu Glu 795 800 805 GAG GAG CAG CTC CAG CGG CGG GAG CGT GCC CAA CAG CTC CAG GAG GAG

4983 Glu Glu Gin Leu Gin Arg Arg Glu Arg Ala Gin Gin Leu Gin Glu Glu 810 815 820 GAG GAC GGC CTC CAG GAG GAT CAG GAG AGG AGG CGA CAG GAG CAG CGC

5031 Glu Asp Gly Leu Gin Glu Asp Gin Glu Arg Arg Arg Gin Glu Gin Arg 825 830 835 840 CGC GAC CAA AAA TGG AGG TGG CAA CTA GAA GAA GAA AGG AAG AGA CGC

5079 Arg Asp Gin Lys Tφ Arg Tφ Gin Leu Glu Glu Glu Arg Lys Arg Arg 845 850 855 CGC CAC ACG CTG TAC GCC AAG CCA GCC CTA CAA GAG CAG CTG AGG AAG

5127 Arg His Thr Leu Tyr Ala Lys Pro Ala Leu Gin Glu Gin Leu Arg Lys 860 865 870 GAA CAG CAG CTG CTG CAG GAG GAG GAG GAG GAG CTA CAG AGA GAG GAG

5175 Glu Gin Gin Leu Leu Gin Glu Glu Glu Glu Glu Leu Gin Arg Glu Glu 875 880 885 CGC GAG AAG AGA AGG CGC CAA GAA CAG GAG AGA CAA TAC CGC GAG GAA

5223 Arg Glu Lys Arg Arg Arg Gin Glu Gin Glu Arg Gin Tyr Arg Glu Glu 890 895 900 GAG CAG CTG CAG CAG GAG GAA GAG CAG CTG CTG AGA GAG GAA CGG GAG

5271 Glu Gin Leu Gin Gin Glu Glu Glu Gin Leu Leu Arg Glu Glu Arg Glu 905 910 915 920 AAA AGA AGA CGC CAG GAG CGG GAA AGG CAA TAT CGG AAG GAT AAG AAG

5319 Lys Arg Arg Arg Gin Glu Arg Glu Arg Gin Tyr Arg Lys Asp Lys Lys 925 930 935 CTG CAG CAG AAG GAA GAG CAG CTG CTG GGA GAG GAA CCG GAG AAG AGA

5367 Leu Gin Gin Lys Glu Glu Gin Leu Leu Gly Glu Glu Pro Glu Lys Arg 940 945 950 AGG CGC CAG GAG CGG GAG AAA AAA TAC CGC GAG GAA GAG GAG TTG CAG

5415 Arg Arg Gin Glu Arg Glu Lys Lys Tyr Arg Glu Glu Glu Glu Leu Gin 955 960 965 CAG GAG GAA GAG CAG CTG CTG AGA GAG GAA CGG GAG AAG AGA AGG CGC

5463 Gin Glu Glu Glu Gin Leu Leu Arg Glu Glu Arg Glu Lys Arg Arg Arg 970 975 980 CAG GAG TGG GAG AGG CAG TAC CGC AAA AAA GAC GAG CTG CAG CAG GAA

551 1 Gin Glu Tφ Glu Arg Gin Tyr Arg Lys Lys Asp Glu Leu Gin Gin Glu 985 990 995 1000 GAA GAG CAG CTG CTG AGA GAG GAA CGG GAG AAA AGA AGA CTC CAG GAG

5559 Glu Glu Gin Leu Leu Arg Glu Glu Arg Glu Lys Arg Arg Leu Gin Glu 1005 1010 1015 CGG GAG AGG CAA TAT CGG GAG GAA GAG GAG CTG CAG CAG GAG GAA GAG

5607 Arg Glu Arg Gin Tyr Arg Glu Glu Glu Glu Leu Gin Gin Glu Glu Glu 1020 1025 1030 CAG CTG CTG GGA GAG GAA CGG GAG ACG AGA AGG CGC CAG GAG CTG GAG

5655 Gin Leu Leu Gly Glu Glu Arg Glu Thr Arg Arg Arg Gin Glu Leu Glu 1035 1040 1045 AGG CAA TAT CGG AAG GAA GAG GAG CTG CAG CAG GAG GAA GAG CAG CTG

5703 Arg Gin Tyr Arg Lys Glu Glu Glu Leu Gin Gin Glu Glu Glu Gin Leu 1050 1055 1060 CTG AGA GAG GAA CCG GAG AAG AGA AGG CGC CAG GAG CGG GAG AGG CAA

5751 Leu Arg Glu Glu Pro Glu Lys Arg Arg Arg Gin Glu Arg Glu Arg Gin 1065 1070 1075 1080 TGT CGG GAG GAA GAG GAG CTG CAG CAG GAG GAA GAG CAG CTG CTG AGA

5799 Cys Arg Glu Glu Glu Glu Leu Gin Gin Glu Glu Glu Gin Leu Leu Arg 1085 1090 1095 GAG GAA CGG GAG AAG AGA AGG CGC CAG GAG CTG GAG AGG CAA TAT CGG

5847 Glu Glu Arg Glu Lys Arg Arg Arg Gin Glu Leu Glu Arg Gin Tyr Arg 1100 1105 1110 GAG GAG GAA GAG CTT CAG CGC CAG AAA AGG AAA CAG CGA TAC CGG GAT

5895 Glu Glu Glu Glu Leu Gin Arg Gin Lys Arg Lys Gin Arg Tyr Arg Asp 1115 1120 1125 GAG GAT CAG CGC AGT GAT CTG AAA TGG CAG TGG GAA CCA GAA AAA GAA

5943 Glu Asp Gin Arg Ser Asp Leu Lys Tφ Gin Tφ Glu Pro Glu Lys Glu 1130 1135 1140 AAT GCA GTT CGT GAT AAC AAG GTT TAC TGC AAA GGC AGA GAG AAT GAA

5991 Asn Ala Val Arg Asp Asn Lys Val Tyr Cys Lys Gly Arg Glu Asn Glu 1145 1150 1155 1160 CAG TTC CGG CAG TTG GAA GAT TCC CAG GTG CGC GAC AGA CAA TCC CAG

6039 Gin Phe Arg Gin Leu Glu Asp Ser Gin Val Arg Asp Arg Gin Ser Gin 1165 1170 1175 CAA GAT CTG CAG CAC CTG CTG GGT GAA CAG CAA GAG AGA GAT CGT GAG

6087 Gin Asp Leu Gin His Leu Leu Gly Glu Gin Gin Glu Arg Asp Arg Glu 1180 1185 1190 CAA GAG AGG AGG CGC TGG CAG CAG GCC AAC AGG CAT TTC CCA GAG GAA

6135 Gin Glu Arg Arg Arg Tφ Gin Gin Ala Asn Arg His Phe Pro Glu Glu 1195 1200 1205 GAA CAG CTG GAG CGA GAA GAG CAA AAG GAA GCC AAA AGG CGC GAC AGG

6183 Glu Gin Leu Glu Arg Glu Glu Gin Lys Glu Ala Lys Arg Arg Asp Arg 1210 1215 1220 AAG TCC CAA GAG GAA AAG CAG TTG CTG AGA GAG GAA AGA GAA GAG AAG

6231 Lys Ser Gin Glu Glu Lys Gin Leu Leu Arg Glu Glu Arg Glu Glu Lys 1225 1230 1235 1240 AGA CGC CGT CAA GAG ACA GAC AGA AAA TTC CGC GAG GAG GAA CAG CTG

6279 Arg Arg Arg Gin Glu Thr Asp Arg Lys Phe Arg Glu Glu Glu Gin Leu 1245 1250 1255 CTC CAG GAA AGG GAG GAA CAG CCG CTG CTC CGC CAA GAG CGT GAC AGA

6327 Leu Gin Glu Arg Glu Glu Gin Pro Leu Leu Arg Gin Glu Arg Asp Arg 1260 1265 1270 AAA TTC CGC GAA GAG GAA CTG CTC CAT CAG GAA CAA GGG AGA AAA TTC

6375 Lys Phe Arg Glu Glu Glu Leu Leu His Gin Glu Gin Gly Arg Lys Phe 1275 1280 1285 CTC GAG GAG GAA CAG CGG CTG CGC GAG GAA CGG GAG AGA AAA TTC CTT

6423 Leu Glu Glu Glu Gin Arg Leu Arg Glu Glu Arg Glu Arg Lys Phe Leu 1290 1295 1300 AAG GAG GAA CAG CAG CTG CGC CTC GAG GAG CGC GAG CAA CTG CGT CAG

6471 Lys Glu Glu Gin Gin Leu Arg Leu Glu Glu Arg Glu Gin Leu Arg Gin 1305 1310 1315 1320 GAC CGC GAC AGA AAA TTC CGC GAG GAG GAA CAG CAG CTG AGC CGC CAA

6519 Asp Arg Asp Arg Lys Phe Arg Glu Glu Glu Gin Gin Leu Ser Arg Gin 1325 1330 1335 . GAG CGT GAC AGA AAA TTC CGT GAA GAG GAA CAG CAG GTG CGC CGC CAG

6567 Glu Arg Asp Arg Lys Phe Arg Glu Glu Glu Gin Gin Val Arg Arg Gin 1340 1345 1350 GAA CGA GAG AGA AAA TTC CTG GAG GAG GAA CAG CAG CTG CGC CAG GAG

6615 Glu Arg Glu Arg Lys Phe Leu Glu Glu Glu Gin Gin Leu Arg Gin Glu 1355 1360 1365 CGT CAC AGA AAA TTC CGC GAA GAG GAA CAG CTG CTC CAG GAA AGG GAA

6663 Arg His Arg Lys Phe Arg Glu Glu Glu Gin Leu Leu Gin Glu Arg Glu 1370 1375 1380 GAA CAG CAG CTG CAC CGC CAA GAG CGT GAC AGA AAA TTC CTG GAG GAG

671 1 Glu Gin Gin Leu His Arg Gin Glu Arg Asp Arg Lys Phe Leu Glu Glu 1385 1390 1395 1400 GAA CAA CAG CTG CGC CGC CAA GAG CGT GAC AGA AAA TTC CGC GAA CAG

6759 Glu Gin Gin Leu Arg Arg Gin Glu Arg Asp Arg Lys Phe Arg Glu Gin 1405 1410 1415 GAA CTG CGC AGT CAG GAA CCA GAG AGA AAA TTC CTC GAG GAG GAA CAG

6807 Glu Leu Arg Ser Gin Glu Pro Glu Arg Lys Phe Leu Glu Glu Glu Gin 1420 1425 1430 CAG CTG CAC CGC CAG CAA CGG CAG AGA AAA TTC CTC CAG GAG GAA CAG

6855 Gin Leu His Arg Gin Gin Arg Gin Arg Lys Phe Leu Gin Glu Glu Gin 1435 1440 1445 CAG CTG CGC CGC CAG GAG CGC GGG CAA CAG CGG CGT CAG GAC CGT GAC

6903 Gin Leu Arg Arg Gin Glu Arg Gly Gin Gin Arg Arg Gin Asp Arg Asp 1450 1455 1460 AGA AAA TTC CGC GAG GAG GAA CAG CTG CGC CAG GAG AGG GAG GAA CAG

6951 Arg Lys Phe Arg Glu Glu Glu Gin Leu Arg Gin Glu Arg Glu Glu Gin 1465 1470 1475 1480 CAG CTG AGC CGC CAA GAG CGT GAC AGA AAA TTC CGT TTA GAG GAA CAG

6999 Gin Leu Ser Arg Gin Glu Arg Asp Arg Lys Phe Arg Leu Glu Glu Gin 1485 1490 1495 AAA GTG CGC CGC CAG GAA CAA GAG AGA AAA TTC ATG GAG GAC GAA CAG

7047 Lys Val Arg Arg Gin Glu Gin Glu Arg Lys Phe Met Glu Asp Glu Gin 1500 1505 1510 CAG CTG CGC CGC CAG GAG GGC CAA CAA CAG CTG CGC CAG GAG GAC AGA

7095 Gin Leu Arg Arg Gin Glu Gly Gin Gin Gin Leu Arg Gin Glu Asp Arg 1515 1520 1525 AAA TTC CGC GAA GAC GAA CAG CTG CTC CAG GAA AGG GAA GAA CAG CAG

7143 Lys Phe Arg Glu Asp Glu Gin Leu Leu Gin Glu Arg Glu Glu Gin Gin 1530 1535 1540 CTG CAC CGC CAA GAG CGT GAC AGA AAA TTC CTC GAG GAG GAA CCG CAG

7191 Leu His Arg Gin Glu Arg Asp Arg Lys Phe Leu Glu Glu Glu Pro Gin 1545 1550 1555 1560 CTG CGC CGC CAG GAG CGC GAA CAA CAG CTG CGT CAC GAC CGC GAC AGA

7239 Leu Arg Arg Gin Glu Arg Glu Gin Gin Leu Arg His Asp Arg Asp Arg 1565 1570 1575 AAA TTC CGT GAA GAG GAA CAG CTG CTC CAG GAA GGG GAG GAA CAG CAG

7287 Lys Phe Arg Glu Glu Glu Gin Leu Leu Gin Glu Gly Glu Glu Gin Gin 1580 1585 1590 CTG CGC CGC CAA GAG CGT GAC AGA AAA TTC CGC GAA GAG GAA CAG CAG

7335 Leu Arg Arg Gin Glu Arg Asp Arg Lys Phe Arg Glu Glu Glu Gin Gin 1595 1600 1605 CTC CGC CGT CAG GAA CGA GAG AGA AAA TTC CTC CAG GAG GAA CAG CAG

7383 Leu Arg Arg Gin Glu Arg Glu Arg Lys Phe Leu Gin Glu Glu Gin Gin 1610 1615 1620 CTG CGC CGC CAG GAA CTG GAG AGA AAA TTC CGT GAG GAG GAA CAG CTG

7431 Leu Arg Arg Gin Glu Leu Glu Arg Lys Phe Arg Glu Glu Glu Gin Leu 1625 1630 1635 1640 CGC CAA GAA ACG GAG CAA GAG CAG CTG CGC CGC CAA GAA CGC TAC AGA

7479 Arg Gin Glu Thr Glu Gin Glu Gin Leu Arg Arg Gin Glu Arg Tyr Arg 1645 1650 1655 AAA ATC CTA GAG GAA GAG CAG CTC CGT CCG GAA AGG GAA GAA CAG CAG

7527 Lys He Leu Glu Glu Glu Gin Leu Arg Pro Glu Arg Glu Glu Gin Gin 1660 1665 1670 CTG CGC CGC CAG GAG CGC GAC AGA AAA TTC CGC GAG GAG GAA CAG CTC

7575 Leu Arg Arg Gin Glu Arg Asp Arg Lys Phe Arg Glu Glu Glu Gin Leu 1675 1680 1685 CGC CAG GGA AGG GAG GAA CAG CAG CTG CGC AGC CAA GAG TCT GAC AGA

7623 Arg Gin Gly Arg Glu Glu Gin Gin Leu Arg Ser Gin Glu Ser Asp Arg 1690 1695 1700 AAA TTC CGC GAG GAG GAA CAG CTA CGC CAG GAG AGG GAA GAA CAG CAG

7671 Lys Phe Arg Glu Glu Glu Gin Leu Arg Gin Glu Arg Glu Glu Gin Gin 1705 1710 1715 1720 CTG CGC CCC CAA CAG CGT GAC GGA AAG TAT CGC TGG GAA GAA GAG CAG

7719 Leu Arg Pro Gin Gin Arg Asp Gly Lys Tyr Arg Tφ Glu Glu Glu Gin 1725 1730 1735 CTC CAA CTT GAG GAA CAA GAG CAG AGG CTG CGG CAG GAG CGA GAC CGG

7767 Leu Gin Leu Glu Glu Gin Glu Gin Arg Leu Arg Gin Glu Arg Asp Arg 1740 1745 1750 CAG TAC CGG GCG GAG GAG CAG TTT GCC ACG CAG GAG AAG AGT CGT CGT

7815 Gin Tyr Arg Ala Glu Glu Gin Phe Ala Thr Gin Glu Lys Ser Arg Arg 1755 1760 1765 GAG GAA CAA GAA CTA TGG CAA GAA GAG GAG CAG AAA CGT CGC CAG GAA

7863 Glu Glu Gin Glu Leu Tφ Gin Glu Glu Glu Gin Lys Arg Arg Gin Glu 1770 1775 1780 CGG GAA AGG AAA TTA CGG GAA GAA CAC ATC CGC CGC CAG CAG AAG GAG

791 1 Arg Glu Arg Lys Leu Arg Glu Glu His He Arg Arg Gin Gin Lys Glu 1785 1790 1795 1800 GAA CAG AGG CAC CGC CAA GTC GGG GAG ATA CAA TCC CAA GAA GGG AAG

7959 Glu Gin Arg His Arg Gin Val Gly Glu He Gin Ser Gin Glu Gly Lys 1805 1810 1815 GGC CAT GGG CGG CTT CTG GAG CCC GGC ACT CAT CAG TTT GCC AGT GTC

8007 Gly His Gly Arg Leu Leu Glu Pro Gly Thr His Gin Phe Ala Ser Val 1820 1825 1830 CCA GTG CGC TCC AGC CCT CTC TAT GAG TAC ATC CAA GAG CAG AGA TCT

8055 Pro Val Arg Ser Ser Pro Leu Tyr Glu Tyr He Gin Glu Gin Arg Ser 1835 1840 1845 CAA TAC CGC CCT TAAGTGATGT TGCCAATATC TTGACACCTG CCAAAGCTTC

8107 Gin Tyr Arg Pro 1850 CAGCACGGGA AAATGAGAAA CACTGGGTAC . CAAGTGATAA CTCAGATGTT

TCTGGTTGTG 8167

GGAAAACTCT CTGATATTAG AATGTCTTTT CTTCCAAAAT CTTAAACTAC GCTCATTTTA 8227

CGCACTTTGT ACTTCTGCTT TTTATTCTTC CTCAAGTAGT TCTTTACTGC AAGATGTCTT 8287

TCTTTTGCTC TTTGATGCAG ATGTGGTGTG CATTTAAAAA AAATATAAAT CATTTAATTT 8347

GTTTAAGAAA TTTTGTTTGA GGAACATGTT CATTTATTGC TTTCAGAAGT

AACAAGAGTA 8407 ATAGGATGAT TTGAGATTCT AAACAATGGG TCGGTTTGTT TAATGACTGA

CCCATCTTGT 8467

GGAAAGTGCA GATACTTTTA ATGTTCAAGT TGCTATTTCT TCTTGAACCT

AAATTGATCA 8527

TTGCCTCCAA ACAGCATTTC ATCCTTTTGT GGCATAGTTA GCACAAATTC CAGGTAACTA 8587

AATTTTTATA ACCCTTGAAT AGTGCAGGGG GAGTGACCTC TGCATAAAAA CTTCCTGTAA 8647

AATCAGCCCA TTACTGGAAG AAATATCTGT TAAGAATAGG TTTAGCTTTG

AAGATTTAGA 8707 ATTTAAATTA GATTTTTTTT AAACTCAACT CCACTTAAAC ACATAATCTC

ATGAAGAAAT 8767

AATGAGGTAT TTAGAATTTA AATGAGTTCA AATTTTAAAA CTGTGTCTGT

TGTAGTCTAT 8827

AGTGTTCATT CTACTTCCCC AAGTTTTGAT GAGTTTCAGA ATATTATGAA CCTTTGTTAA 8887

TTTTAGCTTG TTAGAAGGAA GCTGCTCAGA ATCCCATAAA CATCTGTCTT ACTCTAGGGC 8947

CAATAAGAGA TCACATAGAG CATGTTGGGG GTGTAAAAGG GAAAAATGTG

TGAACATAGG 9007

GGCAAATTTC TAGAGGCCCT TTGACAAGAC CCATTTGCCC ACAATCATTT GAGGCCTATT 9067

GATAATACCT TAGATATATT CTTGTTGAAA TAATTGGACT GTGAAAAATT

AATAATAAAT 9127 GTTTGGCAAG TAACTACTTT TGTCTGTTTT AACTCTGCGT CAATCATAAC

AAGATCTCAT 9187

TGTCTGGAAA CTAACACAAG TTCCCAATCA CATAAGGGCA TTTTGTTACT TATCTATGTC 9247

CAAATACGAA AAAAGAGGGG AGAGAATTCT TTG IT ITT CC CCAACCTTTT TTTTTTTTTT 9307 _{ττττττττττ TTTTGCAGTT} AGGCTGAACT CTATTTCCAT CCCCACACTG AGATTGCCTT 9367

CCAGAGTGTT TTTGTTCTTG ACCCACAGCT TTCTATGCCA TTCTTGCAGC

GACTCACTGG 9427 TCATGACAAA TACTGGTGCT CCCAATATTT GTTAATATTT CCTTTAGAGA

ATGCAGCAGC 9487

TTCTTCGTCT CTGATGTCTG ATGAGCCAAT GATAGAAAAT GGCCTGAAAC

TTCAGATCCT 9547

CGAG 9551

(2) INFORMATION FOR SEQ ID NO:94:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1899 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:

Met Ser Pro Leu Leu Arg Ser He Cys Asp He Thr Glu He Phe Asn

1 5 10 15

Gin Tyr Val Ser His Asp Cys Asp Gly Ala Ala Leu Thr Lys Lys Asp 20 25 30

Leu Lys Asn Leu Leu Glu Arg Glu Phe Gly Ala Val Leu Arg Arg Pro

35 40 45

His Asp Pro Lys Thr Val Asp Leu He Leu Glu Leu Leu Asp Arg Asp 50 55 60

Ser Asn Gly Arg Val Asp Phe Asn Glu Phe Leu Leu Phe He Phe Lys 65 70 75 80 Val Ala Gin Ala Cys Tyr Tyr Ala Leu Gly Gin Ala Thr Gly Leu Asp

85 90 95

Glu Glu Lys Arg Ala Arg Cys Asp Gly Lys Glu Ser Leu Leu Gin Asp 100 105 110

Arg Arg Thr Glu Glu Asp Gin Arg Arg Phe Glu Pro Arg Asp Arg Gin 1 15 120 125

Leu Glu Glu Glu Pro Gly Gin Arg Arg Arg Gin Lys Arg Gin Glu Gin 130 135 140

Glu Arg Glu Leu Ala Glu Gly Glu Glu Gin Ser Glu Lys Gin Glu Arg 145 150 155 160 Leu Glu Gin Arg Asp Arg Gin Arg Arg Asp Glu Glu Leu Tφ Arg Gin

165 170 175

Arg Gin Glu Tφ Gin Glu Arg Glu Glu Arg Arg Ala Glu Glu Glu Gin 180 185 190

Leu Gin Ser Cys Lys Gly His Glu Thr Glu Glu Phe Pro Asp Glu Glu 195 200 205

Gin Leu Arg Arg Arg Glu Leu Leu Glu Leu Arg Arg Lys Gly Arg Glu 210 215 220

Glu Lys Gin Gin Gin Arg Arg Glu Arg Gin Asp Arg Val Phe Gin Glu

225 230 235 240 Glu Glu Glu Lys Glu Tφ Arg Lys Arg Glu Thr Val Leu Arg Lys Glu

245 250 255

Glu Glu Lys Leu Gin Glu Glu Glu Pro Gin Arg Gin Arg Glu Leu Gin 260 265 270

Glu Glu Glu Glu Gin Leu Arg Lys Leu Glu Arg Gin Glu Leu Arg Arg

275 280 285

Glu Arg Gin Glu Glu Glu Gin Gin Gin Gin Arg Leu Arg Arg Glu Gin 290 295 300

Gln Leu Arg Arg Lys Gin Glu Glu Glu Arg Arg Glu Gin Gin Glu Glu 305 310 315 320

Arg Arg Glu Gin Gin Glu Arg Arg Glu Gin Gin Glu Glu Arg Arg Glu 325 330 335

Gin Gin Leu Arg Arg Glu Gin Glu Glu Arg Arg Glu Gin Gin Leu Arg 340 345 350 Arg Glu Gin Glu Glu Glu Arg Arg Glu Gin Gin Leu Arg Arg Glu Gin

355 360 365

Glu Glu Glu Arg Arg Glu Gin Gin Leu Arg Arg Glu Gin Glu Glu Glu 370 375 380

Arg Arg Glu Gin Gin Leu Arg Arg Glu Gin Gin Leu Arg Arg Glu Gin 385 390 395 400

Gin Leu Arg Arg Glu Gin Gin Leu Arg Arg Glu Gin Gin Leu Arg Arg 405 410 415

Glu Gin Gin Leu Arg Arg Glu Gin Gin Leu Arg Arg Glu Gin Gin Leu 420 425 430 Arg Arg Glu Gin Gin Leu Arg Arg Glu Gin Gin Leu Arg Arg Glu Gin

435 440 445

Glu Glu Glu Arg His Glu Gin Lys His Glu Gin Glu Arg Arg Glu Gin 450 455 460

Arg Leu Lys Arg Glu Gin Glu Glu Arg Arg Asp Tφ Leu Lys Arg Glu 465 470 475 480

Glu Glu Thr Glu Arg His Glu Gin Glu Arg Arg Lys Gin Gin Leu Lys 485 490 495

Arg Asp Gin Glu Glu Glu Arg Arg Glu Arg Tφ Leu Lys Leu Glu Glu 500 505 510 Glu Glu Arg Arg Glu Gin Gin Glu Arg Arg Glu Gin Gin Leu Arg Arg

515 520 525

Glu Gin Glu Glu Arg Arg Glu Gin Arg Leu Lys Arg Gin Glu Glu Glu

530 535 540

Glu Arg Leu Gin Gin Arg Leu Arg Ser Glu Gin Gin Leu Arg Arg Glu

545 550 555 560

Gin Glu Glu Arg Leu Glu Gin Leu Leu Lys Arg Glu Glu Glu Lys Arg 565 570 575

Leu Glu Gin Glu Arg Arg Glu Gin Arg Leu Lys Arg Glu Gin Glu Glu 580 585 590

Arg Arg Asp Gin Leu Leu Lys Arg Glu Glu Glu Arg Arg Gin Gin Arg 595 600 605

Leu Lys Arg Glu Gin Glu Glu Arg Leu Glu Gin Arg Leu Lys Arg Glu 610 615 620 Glu Val Glu Arg Leu Glu Gin Glu Glu Arg Arg Asp Glu Arg Leu Lys

625 630 635 640

Arg Glu Glu Pro Glu Glu Glu Arg Arg His Giu Leu Leu Lys Ser Glu 645 650 655

Glu Gin Glu Glu Arg Arg His Glu Gin Leu Arg Arg Glu Gin Gin Glu 660 665 670

Arg Arg Glu Gin Arg Leu Lys Arg Glu Glu Glu Glu Glu Arg Leu Glu 675 680 685

Gin Arg Leu Lys Arg Glu His Glu Glu Glu Arg Arg Glu Gin Glu Leu 690 695 700 Ala Glu Glu Glu Gin Glu Gin Ala Arg Glu Arg He Lys Ser Arg He

705 710 715 720

Pro Lys Tφ Gin Tφ Gin Leu Glu Ser Glu Ala Asp Ala Arg Gin Ser 725 730 735

Lys Val Leu Leu Glu Ala Pro Gin Ala Gly Arg Ala Glu Ala Pro Gin 740 745 750

Glu Gin Glu Glu Lys Arg Arg Arg Glu Ser Glu Leu Gin Tφ Gin Glu 755 760 765

Glu Glu Arg Ala His Arg Gin Gin Gin Glu Glu Glu Gin Arg Arg Asp

770 775 780 Phe Thr Tφ Gin Tφ Gin Ala Glu Glu Lys Ser Glu Arg Gly Arg Gin

785 790 795 800

Arg Leu Ser Ala Arg Pro Pro Leu Arg Glu Gin Arg Glu Arg Gin Leu 805 810 815

Arg Ala Glu Glu Arg Gin Gin Arg Glu Gin Arg Phe Leu Pro Glu Glu 820 825 830

Glu Glu Lys Glu Gin Arg Gly Arg Gin Arg Arg Glu Arg Glu Lys Glu 835 840 845

Leu Gin Phe Leu Glu Glu Glu Glu Gin Leu Gin Arg Arg Glu Arg Ala 850 855 860

Gin Gin Leu Gin Glu Glu Glu Asp Gly Leu Gin Glu Asp Gin Glu Arg 865 870 875 880

Arg Arg Gin Glu Gin Arg Arg Asp Gin Lys Tφ Arg Tφ Gin Leu Glu 885 890 895 Glu Glu Arg Lys Arg Arg Arg His Thr Leu Tyr Ala Lys Pro Ala Leu

900 905 910

Gin Glu Gin Leu Arg Lys Glu Gin Gin Leu Leu Gin Glu Glu Glu Glu 915 920 925

Glu Leu Gin Arg Glu Glu Arg Glu Lys Arg Arg Arg Gin Glu Gin Glu 930 935 940

Arg Gin Tyr Arg Glu Glu Glu Gin Leu Gin Gin Glu Glu Glu Gin Leu 945 950 955 960

Leu Arg Glu Glu Arg Glu Lys Arg Arg Arg Gin Glu Arg Glu Arg Gin 965 970 975 Tyr Arg Lys Asp Lys Lys Leu Gin Gin Lys Glu Glu Gin Leu Leu Gly

980 985 990

Glu Glu Pro Glu Lys Arg Arg Arg Gin Glu Arg Glu Lys Lys Tyr Arg 995 1000 1005

Glu Glu Glu Glu Leu Gin Gin Glu Glu Glu Gin Leu Leu Arg Glu Glu 1010 1015 1020

Arg Glu Lys Arg Arg Arg Gin Glu Tφ Glu Arg Gin Tyr Arg Lys Lys 1025 1030 1035 1040

Asp Glu Leu Gin Gin Glu Glu Glu Gin Leu Leu Arg Glu Glu Arg Glu 1045 1050 1055 Lys Arg Arg Leu Gin Glu Arg Glu Arg Gin Tyr Arg Glu Glu Glu Glu

1060 1065 1070

Leu Gin Gin Glu Glu Glu Gin Leu Leu Gly Glu Glu Arg Glu Thr Arg 1075 1080 1085

Arg Arg Gin Glu Leu Glu Arg Gin Tyr Arg Lys Glu Glu Glu Leu Gin 1090 1095 1100

Gin Glu Glu Glu Gin Leu Leu Arg Glu Glu Pro Glu Lys Arg Arg Arg 1105 1110 1115 1120

Gln Glu Arg Glu Arg Gin Cys Arg Glu Glu Glu Glu Leu Gin Gin Glu 1 125 1130 1 135

Glu Glu Gin Leu Leu Arg Glu Glu Arg Glu Lys Arg Arg Arg Gin Glu 1140 1145 1150

Leu Glu Arg Gin Tyr Arg Glu Glu Glu Glu Leu Gin Arg Gin Lys Arg 1155 1160 1165 Lys Gin Arg Tyr Arg Asp Glu Asp Gin Arg Ser Asp Leu Lys Tφ Gin

1170 1175 1180

Tφ Glu Pro Glu Lys Glu Asn Ala Val Arg Asp Asn Lys Val Tyr Cys 1185 1190 1195 1200

Lys Gly Arg Glu Asn Glu Gin Phe Arg Gin Leu Glu Asp Ser Gin Val 1205 1210 1215

Arg Asp Arg Gin Ser Gin Gin Asp Leu Gin His Leu Leu Gly Glu Gin 1220 1225 1230

Gin Glu Arg Asp Arg Glu Gin Glu Arg Arg Arg Tφ Gin Gin Ala Asn 1235 1240 1245 Arg His Phe Pro Glu Glu Glu Gin Leu Glu Arg Glu Glu Gin Lys Glu

1250 1255 1260

Ala Lys Arg Arg Asp Arg Lys Ser Gin Glu Glu Lys Gin Leu Leu Arg 1265 1270 1275 1280

Glu Glu Arg Glu Glu Lys Arg Arg Arg Gin Glu Thr Asp Arg Lys Phe 1285 1290 1295

Arg Glu Glu Glu Gin Leu Leu Gin Glu Arg Glu Glu Gin Pro Leu Leu 1300 1305 1310

Arg Gin Glu Arg Asp Arg Lys Phe Arg Glu Glu Glu Leu Leu His Gin 1315 1320 1325 Glu Gin Gly Arg Lys Phe Leu Glu Glu Glu Gin Arg Leu Arg Glu Glu

1330 1335 1340

Arg Glu Arg Lys Phe Leu Lys Glu Glu Gin Gin Leu Arg Leu Glu Glu 1345 1350 1355 1360

Arg Glu Gin Leu Arg Gin Asp Arg Asp Arg Lys Phe Arg Glu Glu Glu 1365 1370 1375

Gin Gin Leu Ser Arg Gin Glu Arg Asp Arg Lys Phe Arg Glu Glu Glu 1380 1385 1390

Gln Gin Val Arg Arg Gin Glu Arg Glu Arg Lys Phe Leu Glu Glu Glu 1395 1400 1405

Gin Gin Leu Arg Gin Glu Arg His Arg Lys Phe Arg Glu Glu Glu Gin 1410 1415 1420

Leu Leu Gin Glu Arg Glu Glu Gin Gin Leu His Arg Gin Glu Arg Asp 1425 1430 1435 1440 Arg Lys Phe Leu Glu Glu Glu Gin Gin Leu Arg Arg Gin Glu Arg Asp

1445 1450 1455

Arg Lys Phe Arg Glu Gin Glu Leu Arg Ser Gin Glu Pro Glu Arg Lys 1460 1465 1470

Phe Leu Glu Glu Glu Gin Gin Leu His Arg Gin Gin Arg Gin Arg Lys 1475 1480 1485

Phe Leu Gin Glu Glu Gin Gin Leu Arg Arg Gin Glu Arg Gly Gin Gin 1490 1495 1500

Arg Arg Gin Asp Arg Asp Arg Lys Phe Arg Glu Glu Glu Gin Leu Arg 1505 1510 1515 1520 Gin Glu Arg Glu Glu Gin Gin Leu Ser Arg Gin Glu Arg Asp Arg Lys

1525 1530 1535

Phe Arg Leu Glu Glu Gin Lys Val Arg Arg Gin Glu Gin Glu Arg Lys 1540 1545 1550

Phe Met Glu Asp Glu Gin Gin Leu Arg Arg Gin Glu Gly Gin Gin Gin 1555 1560 1565

Leu Arg Gin Glu Asp Arg Lys Phe Arg Glu Asp Glu Gin Leu Leu Gin 1570 1575 1580

Glu Arg Glu Glu Gin Gin Leu His Arg Gin Glu Arg Asp Arg Lys Phe 1585 1590 1595 1600 Leu Glu Glu Glu Pro Gin Leu Arg Arg Gin Glu Arg Glu Gin Gin Leu

1605 1610 1615

Arg His Asp Arg Asp Arg Lys Phe Arg Glu Glu Glu Gin Leu Leu Gin 1620 1625 1630

Glu Gly Glu Glu Gin Gin Leu Arg Arg Gin Glu Arg Asp Arg Lys Phe 1635 1640 1645

Arg Glu Glu Glu Gin Gin Leu Arg Arg Gin Glu Arg Glu Arg Lys Phe 1650 1655 1660

Leu Gin Glu Glu Gin Gin Leu Arg Arg Gin Glu Leu Glu Arg Lys Phe 1665 1670 1675 1680

Arg Glu Glu Glu Gin Leu Arg Gin Glu Thr Glu Gin Glu Gin Leu Arg 1685 1690 1695

Arg Gin Glu Arg Tyr Arg Lys He Leu Glu Glu Glu Gin Leu Arg Pro 1700 1705 1710 Glu Arg Glu Glu Gin Gin Leu Arg Arg Gin Glu Arg Asp Arg Lys Phe

1715 1720 1725

Arg Glu Glu Glu Gin Leu Arg Gin Gly Arg Glu Glu Gin Gin Leu Arg 1730 1735 1740

Ser Gin Glu Ser Asp Arg Lys Phe Arg Glu Glu Glu Gin Leu Arg Gin 1745 1750 1755 1760

Glu Arg Glu Glu Gin Gin Leu Arg Pro Gin Gin Arg Asp Gly Lys Tyr 1765 1770 1775

Arg Tφ Glu Glu Glu Gin Leu Gin Leu Glu Glu Gin Glu Gin Arg Leu 1780 1785 1790 Arg Gin Glu Arg Asp Arg Gin Tyr Arg Ala Glu Glu Gin Phe Ala Thr

1795 1800 1805

Gin Glu Lys Ser Arg Arg Glu Glu Gin Glu Leu Tφ Gin Glu Glu Glu 1810 1815 1820

Gin Lys Arg Arg Gin Glu Arg Glu Arg Lys Leu Arg Glu Glu His He 1825 1830 1835 1840

Arg Arg Gin Gin Lys Glu Glu Gin Arg His Arg Gin Val Gly Glu He 1845 1850 1855

Gin Ser Gin Glu Gly Lys Gly His Gly Arg Leu Leu Glu Pro Gly Thr 1860 1865 1870 His Gin Phe Ala Ser Val Pro Val Arg Ser Ser Pro Leu Tyr Glu Tyr

1875 1880 1885

He Gin Glu Gin Arg Ser Gin Tyr Arg Pro * 1890 1895

(2) INFORM ATION FOR SEQ ID NO:95: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:

Ser Pro Leu Leu Arg Ser He Cys Asp He Thr Glu He Phe Asn Gin 1 5 10 15

Tyr Val Ser His Asp Cys Asp Gly Ala Ala Leu Thr Lys Lys Asp Leu 20 25 30

Lys Asn Leu Leu Glu Arg Glu Phe Gly Ala Val Leu Arg 35 40 45

(2) INFORMATION FOR SEQ ID NO:96:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:96:

Ser Thr Leu Leu Val Phe He Phe Ala He He Asn Leu Phe Asn Glu 1 5 10 15 Tyr Ser Lys Lys Asp Lys Asn Thr Asp Thr Leu Ser Lys Lys Glu Leu

20 25 30

Lys Glu Leu Leu Glu Lys Glu Phe Arg Gin He Leu Lys 35 40 45

(2) INFORMATION FOR SEQ ID NO:97:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:

Ser Glu Leu Glu Lys Ala Met Val Ala Leu He Asp Val Phe His Gin 1 5 10 15

Tyr Ser Gly Arg Glu Gly Asp Lys His Lys Leu Lys Lys Ser Glu Leu 20 25 30

Lys Glu Leu He Asn Asn Glu Leu Ser His Phe Leu Glu

35 40 45 (2) INFORMATION FOR SEQ ID NO:98:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:

Thr Glu Leu Glu Thr Ala Met Gly Met He He Asp Val Phe Ser Arg 1 5 10 15

Tyr Ser Gly Ser Glu Gly Ser Thr Gin Thr Leu Thr Lys Gly Glu Leu 20 25 30 Lys Val Leu Met Glu Lys Glu Leu Pro Gly Phe Leu Gin

35 40 45

(2) INFORMATION FOR SEQ ID NO:99: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:

Ser Gin Met Glu His Ala Met Glu Thr Met Met Phe Thr Phe His Lys 1 5 10 15

Phe Ala Gly Asp Lys Gly Tyr Leu Thr Lys Arg Asp Leu Arg Val Leu 20 25 30

Met Glu Lys Glu Phe Pro Gly Phe Leu Glu

35 40

(2) INFORMATION FOR SEQ ID NO: 100: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100:

Cys Pro Leu Asp Gin Ala He Gly Leu Leu Val Ala He Phe His Lys 1 5 10 15

Tyr Ser Gly Arg Glu Gly Asp Lys His Thr Leu Ser Lys Lys Glu Leu 20 25 30

Lys Glu Leu He Gin Lys Glu Leu Thr Ser He Gly 35 40

(2) INFORMATION FOR SEQ ID NO: 101:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:101 :

Thr Glu Leu Glu Lys Ala Leu Asn Ser He He Asp Val Tyr His Lys 1 5 10 15 Tyr Ser Leu He Lys Gly Asn Phe His Ala Val Tyr Arg Asp Asp Leu

20 25 30

Lys Lys Leu Leu Glu Thr Glu Cys Pro Gin Tyr He Arg

35 40 45

(2) INFORMATION FOR SEQ ID NO: 102:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102:

Arg Pro His Asp Pro Lys Thr Val Asp Leu He Leu Glu Leu Leu Asp 1 5 10 15

Arg Asp Ser Asn Gly Arg Val Asp Phe Asn Glu Phe Leu Leu Phe He 20 25 30

Phe Phe Val Ala Gin Ala Cys Tyr Tyr Ala Leu Gly Gin

35 40 45 (2) INFORMATION FOR SEQ ID NO: 103:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:103: Asn Pro Asp Asp Pro Asp Met Val Asp Val Phe Met Asp His Leu Asp

1 5 10 15

He Asp His Asn Lys Lys He Asp Phe Thr Glu Phe Leu Leu Met Val 20 25 30

Phe Lys Leu Ala Gin Ala Tyr Tyr Glu Ser Thr Arg Lys

35 40 45

(2) INFORMATION FOR SEQ ID NO: 104:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO.104:

Glu He Lys Glu Gin Glu Val Val Asp Lys Val Met Glu Thr Leu Asp 1 5 10 15

Asn Asp Gly Asp Gly Glu Cys Asp Phe Gin Glu Phe Met Ala Phe Val 20 25 30 Ala Met Val Thr Thr Ala Cys His Glu Phe Phe Glu His

35 40 45

(2) INFORMATION FOR SEQ ID NO: 105: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105:

Ser Gly Lys Asp Lys Asp Ala Val Asp Lys Leu Leu Lys Asp Leu Asp 1 5 10 15 Ala Asn Gly Asp Ala Gin Val Asp Phe Ser Glu Phe He Val Phe Val

20 25 30

Ala Ala He Thr Ser Ala Cys His Lys Tyr Phe Glu Lys 35 40 45

(2) INFORMATION FOR SEQ ID NO: 106:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO: 106:

Asn Gin Lys Asp Pro Leu Ala Val Asp Lys He Met Lys Asp Leu Asp 1 5 10 15

Gin Cys Arg Asp Gly Lys Val Gly Phe Gin Ser Phe Phe Ser Leu He 20 25 30

Ala Gly Leu Thr He Ala Cys Asn Asp Tyr Phe Val Val

35 40 45 (2) INFORMATION FOR SEQ ID NO: 107:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107: Lys Leu Gin Asp Ala Glu He Ala Arg Leu Met He Phe Asp Leu Asp

1 5 10 15

Arg Asn Lys Asp Gly Glu Val Asn Phe Gin Glu Tyr Val Thr Phe Leu 20 25 30

Gly Ala Leu Ala Leu He Tyr Asn Glu Ala Leu Lys Gly

35 40 45

(2) INFORMATION FOR SEQ ID NO :108:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108:

Lys Lys Gly Ala Asp Val Tφ Phe Lys Glu Leu Asp He Asn Thr Asp 1 5 10 15

Gly Ala Val Asn Phe Gin Glu Phe Leu He Leu Val He Lys Met Gly

20 25 30

Val Ala Ala His Lys Lys Ser His Glu

35 40

(2) INFORMATION FOR SEQ ID NO: 109: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2620 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 42..2120

(xi) SEQUENCE DESCRIPTION: SEQ ID NO.109: CCTTTAGAGG AGCCTGAGAA GAGGCAGAGG AAGGGCGAAA C ATG GCT GCT CTA

53

Met Ala Ala Leu 1 GGA GTC CAG AGT ATC AAC TGG CAG AAG GCC TTC AAC CGA CAA GCG CAT

101 Gly Val Gin Ser He Asn Tφ Gin Lys Ala Phe Asn Arg Gin Ala His 5 10 15 20 CAC ACA GAC AAG TTC TCC AGC CAG GAG CTC ATC TTG CGG AGA GGC CAA

149 His Thr Asp Lys Phe Ser Ser Gin Glu Leu He Leu Arg Arg Gly Gin 25 30 35 AAC TTC CAG GTC TTA ATG ATC ATG AAC AAA GGC CTT GGC TCT AAC GAA

197 Asn Phe Gin Val Leu Met He Met Asn Lys Gly Leu Gly Ser Asn Glu 40 45 50 AGA CTG GAG TTC ATT GAC ACC ACA GGG CCT TAC CCC TCA GAG TCG GCC

245 Arg Leu Glu Phe He Asp Thr Thr Gly Pro Tyr Pro Ser Glu Ser Ala 55 60 65 ATG ACG AAG GCT GTG TTT CCA CTC TCC AAT GGC AGT AGT GGT GGC TGG

293 Met Thr Lys Ala Val Phe Pro Leu Ser Asn Gly Ser Ser Gly Gly Tφ 70 75 80 AGT GCG GTG CTT CAG GCC AGC AAT GGC AAT ACT CTG ACT ATC AGC ATC

341 Ser Ala Val Leu Gin Ala Ser Asn Gly Asn Thr Leu Thr He Ser He 85 90 95 100 TCC AGT CCT GCC AGC GCA CCC ATA GGA CGG TAC ACA ATG GCC CTC CAG

389 Ser Ser Pro Ala Ser Ala Pro He Gly Arg Tyr Thr Met Ala Leu Gin 105 1 10 1 15 ATC TTC TCC CAG GGC GGC ATC TCC TCT GTG AAA CTT GGG ACG TTC ATA

437 He Phe Ser Gin Gly Gly He Ser Ser Val Lys Leu Gly Thr Phe He

120 125 130 CTG CTT TTT AAC CCC TGG CTG AAT GTG GAT AGC GTC TTT ATG GGT AAC

485 Leu Leu Phe Asn Pro Tφ Leu Asn Val Asp Ser Val Phe Met Gly Asn 135 140 145 CAT GCT GAG AGA GAA GAG TAT GTT CAG GAA GAT GCC GGC ATC ATC TTT

533 His Ala Glu Arg Glu Glu Tyr Val Gin Glu Asp Ala Gly He He Phe 150 155 160 GTG GGA AGC ACA AAC CGA ATT GGC ATG ATT GGC TGG AAC TTT GGA CAG

581 Val Gly Ser Thr Asn Arg He Gly Met He Gly Tφ Asn Phe Gly Gin 165 170 175 180 TTT GAA GAA GAC ATT CTC AGC ATC TGC CTC TCA ATC TTG GAT AGG AGT

629 Phe Glu Glu Asp He Leu Ser He Cys Leu Ser He Leu Asp Arg Ser 185 190 195 CTG AAT TTC CGC CGT GAC GCT GCT ACT GAT GTG GCC AGC AGA AAT GAC

677 Leu Asn Phe Arg Arg Asp Ala Ala Thr Asp Val Ala Ser Arg Asn Asp 200 205 210 CCC AAA TAC GTT GGC CGG GTG CTG AGT GCC ATG ATC AAT AGC AAT GAT

725 Pro Lys Tyr Val Gly Arg Val Leu Ser Ala Met He Asn Ser Asn Asp

215 220 225 GAC AAT GGT GTG CTTGCT GGG AAT TGG AGC GGC ACT TAC ACC GGT GGC

773 Asp Asn Gly Val Leu Ala Gly Asn Tφ Ser Gly Thr Tyr Thr Gly Gly 230 235 240 CGG GAC CCA AGG AGC TGG GAC GGC AGC GTG GAG ATC CTC AAA AAT TGG

821 Arg Asp Pro Arg Ser Tφ Asp Gly Ser Val Glu He Leu Lys Asn Tφ 245 250 255 260 AAA AAA TCT GGC TTC AGC CCA GTC CGA TAT GGC CAG TGC TGG GTC TTT

869 Lys Lys Ser Gly Phe Ser Pro Val Arg Tyr Gly Gin Cys Tφ Val Phe 265 270 275 GCT GGG ACC CTC AAC ACA GCG CTG CGG TCT TTG GGG ATT CCT TCC CGG

917 Ala Gly Thr Leu Asn Thr Ala Leu Arg Ser Leu Gly He Pro Ser Arg 280 285 290 GTG ATC ACC AAC TTC AAC TCA GCT CAT GAC ACA GAC CGA AAT CTC AGT

965 Val He Thr Asn Phe Asn Ser Ala His Asp Thr Asp Arg Asn Leu Ser 295 300 305 GTG GAT GTG TAC TAC GAC CCC ATG GGA AAC CCC CTG GAC AAG GGT AGT

1013 Val Asp Val Tyr Tyr Asp Pro Met Gly Asn Pro Leu Asp Lys Gly Ser 310 315 320 GAT AGC GTA TGG AAT TTC CAT GTC TGG AAT GAA GGC TGG TTT GTG AGG

1061 Asp Ser Val Tφ Asn Phe His Val Tφ Asn Glu Gly Tφ Phe Val Arg

325 330 335 340 TCT GAC CTG GGC CCC CCG TAC GGT GGA TGG CAG GTG TTG GAT GCT ACC

1 109 Ser Asp Leu Gly Pro Pro Tyr Gly Gly Tφ Gin Val Leu Asp Ala Thr 345 350 355 CCG CAG GAA AGA AGC CAA GGG GTG TTC CAG TGC GGC CCC GCT TCG GTC

1157 Pro Gin Glu Arg Ser Gin Gly Val Phe Gin Cys Gly Pro Ala Ser Val 360 365 370 ATT GGT GTT CGA GAG GGT GAT GTG CAG CTG AAC TTC GAC ATG CCC TTT

1205 He Gly Val Arg Glu Gly Asp Val Gin Leu Asn Phe Asp Met Pro Phe

375 380 385 ATC TTC GCG GAG GTT AAT GCC GAC CGC ATC ACC TGG CTG TAC GAC AAC

1253 He Phe Ala Glu Val Asn Ala Asp Arg He Thr Tφ Leu Tyr Asp Asn 390 395 400 ACC ACT GGC AAA CAG TGG AAG AAT TCC GTG AAC AGT CAC ACC ATT GGC

1301 Thr Thr Gly Lys Gin Tφ Lys Asn Ser Val Asn Ser His Thr He Gly 405 410 415 420 AGG TAC ATC AGC ACC AAG GCG GTG GGC AGC AAT GCT CGC ATG GAC GTC

1349 Arg Tyr He Ser Thr Lys Ala Val Gly Ser Asn Ala Arg Met Asp Val 425 430 435 ACG GAC AAG TAC AAG TAC CCA GAA GGC TCT GAC CAG GAA AGA CAA GTG

1397 Thr Asp Lys Tyr Lys Tyr Pro Glu Gly Ser Asp Gin Glu Arg Gin Val

440 445 450 TTC CAA AAG GCT TTG GGG AAA CTT AAA CCC AAC ACG CCA TTT GCC GCG

1445 Phe Gin Lys Ala Leu Gly Lys Leu Lys Pro Asn Thr Pro Phe Ala Ala 455 460 465 ACG TCT TCG ATG GGT TTG GAA ACA GAG GAA CAG GAG CCC AGC ATC ATC

1493 Thr Ser Ser Met Gly Leu Glu Thr Glu Glu Gin Glu Pro Ser He He

470 475 480 GGG AAG CTG AAG GTC GCT GGC ATG CTG GCA GTA GGC AAA GAA GTC AAC

1541 Gly Lys Leu Lys Val Ala Gly Met Leu Ala Val Gly Lys Glu Val Asn 485 490 495 500 CTG GTC CTA CTG CTC AAA AAC CTG AGC AGG GAT ACG AAG ACA GTG ACA

1589 Leu Val Leu Leu Leu Lys Asn Leu Ser Arg Asp Thr Lys Thr Val Thr 505 510 515 GTG AAC ATG ACA GCC TGG ACC ATC ATC TAC AAC GGC ACG CTT GTA CAT

1637 Val Asn Met Thr Ala Tφ Thr He He Tyr Asn Gly Thr Leu Val His

520 525 530 GAA GTG TGG AAG GAC TCT GCC ACA ATG TCC CTG GAC CCT GAG GAA GAG

1685 Glu Val Tφ Lys Asp Ser Ala Thr Met Ser Leu Asp Pro Glu Glu Glu

535 540 545 GCA GAA CAT CCC ATA AAG ATC TCG TAC GCT CAG TAT GAG AGG TAC CTG

1733 Ala Glu His Pro He Lys He Ser Tyr Ala Gin Tyr Glu Arg Tyr Leu 550 555 560 AAG TCA GAC AAC ATG ATC CGG ATC ACA GCG GTG TGC AAG GTC CCA GAT

1781 Lys Ser Asp Asn Met He Arg He Thr Ala Val Cys Lys Val Pro Asp 565 570 575 580 GAG TCT GAG GTG GTG GTG GAG CGG GAC ATC ATC CTG GAC AAC CCC ACC

1829 Glu Ser Glu Val Val Val Glu Arg Asp He He Leu Asp Asn Pro Thr 585 590 595 TTG ACC CTG GAG GTG CTG AAC GAG GCT CGT GTG CGG AAG CCT GTG AAC

1877 Leu Thr Leu Glu Val Leu Asn Glu Ala Arg Val Arg Lys Pro Val Asn 600 605 610 GTG CAG ATG CTC TTC TCC AAT CCA CTG GAT GAG CCG GTG AGG GAC TGC

1925 Val Gin Met Leu Phe Ser Asn Pro Leu Asp Glu Pro Val Arg Asp Cys 615 620 625 GTG CTG ATG GTG GAG GGA AGC GGC CTG CTG TTG GGT AAC CTG AAG ATC

1973 Val Leu Met Val Glu Gly Ser Gly Leu Leu Leu Gly Asn Leu Lys He 630 635 640 GAC GTG CCG ACC CTA GGG CCC AAG GAG CGG TCC CGG GTC CGT TTT GAT

2021 Asp Val Pro Thr Leu Gly Pro Lys Glu Arg Ser Arg Val Arg Phe Asp 645 650 655 660 ATC CTG CCC TCC CGG AGT GGC ACC AAG CAA CTG CTC GCC GAC TTC TCC

2069 He Leu Pro Ser Arg Ser Gly Thr Lys Gin Leu Leu Ala Asp Phe Ser 665 670 675 TGC AAC AAG TTC CCT GCA ATC AAG GCC ATG TTG TCC ATC GAC GTA GCC

2117 Cys Asn Lys Phe Pro Ala He Lys Ala Met Leu Ser He Asp Val Ala 680 685 690 GAA TGAAGGGCGC TGGTGGCCTC CCGTACAAAC TTGGACAACA CGGAGCAGGG

2170 Glu

AGAGCTCACC ATGGAATGAA CCCCCCGCCC ATGCTGTCCG GCCTGGGAAA

CCCTCTCCAT 2230

CTCCCAAGGC TGCCAGACAT GGACTCCGGG CTCCAGCACA TCCCCCTCTC

CTCTCCCCCA 2290

GGTTGGGGCT GGGTCCACCC TGTCCTATGA CTTGATCACT TTTGCACATT

CCCTGGCCGT 2350

TTCTCCCCAG AGCTGCCTGC TCTGTGAGCC CCACAGCCCT GCTCATTCCT CACGCCCTTC 2410

AATGCTGCAG GATGGACTGG CCCCTGACCC AGGGACTCTC CAAACGGGAT ACAGGAGAGA 2470 AGCTGGTCTA GACTGTTTGC TGATCCCCAA CCTGCACGGG GCATTCCTGC

TTCTCTCTCA 2530

GGCCACCACA GAGGGCAGGG GATGGTTAGT CACCTGCCCC AGCACTCACA CCCTAACTCA 2590

AAATAAATGT TAAATAAGTG CGATCACACA 2620

(2) INFORMATION FOR SEQ ID NO:l 10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2297 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 55..2133

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:110: ACACCATCTC TGTCATTCCC AGAGGAGCCC CAGGAAAGGC AGAAGAAGCT GACC

ATG 57

Met 1 AGTGCTTTA CAG ATC CAA AAC GTC AAC TGG CAG GTG CCTATG AAT CGA

105 Ser Ala Leu Gin He Gin Asn Val Asn Tφ Gin Val Pro Met Asn Arg 5 10 15 AGG GCG CAT CAC ACA GAC AAG TTC TCC AGC CAG GAT TCT ATT GTG CGG

153 Arg Ala His His Thr Asp Lys Phe Ser Ser Gin Asp Ser He Val Arg 20 25 30 AGA GGA CAG CCC TGG GAG ATA ATA TTA GTC TGC AAC CGA AGT CTT GAG

201 Arg Gly Gin Pro Tφ Glu He He Leu Val Cys Asn Arg Ser Leu Glu

35 40 45 TCT GGA GAA GAT CTG AAT TTC ATT GTT TCC ACA GGT CCC CAA CCC TCT

249 Ser Gly Glu Asp Leu Asn Phe He Val Ser Thr Gly Pro Gin Pro Ser 50 55 60 65 GAG TCA GCC AGG ACA AAG GCT GTG TTT TCC ATC TCT GGG AGA AGC ACG

297 Glu Ser Ala Arg Thr Lys Ala Val Phe Ser He Ser Gly Arg Ser Thr 70 75 80 GGT GGC TGG AAT GCA GCG CTC AAA GCC AAC AGT GGC AAT AAT CTG GCC

345 Gly Gly Tφ Asn Ala Ala Leu Lys Ala Asn Ser Gly Asn Asn Leu Ala 85 90 95 ATT GCT ATT GCC AGT CCT GTC AGT GCT CCC ATC GGA TTG TAC ACA CTG

393 He Ala He Ala Ser Pro Val Ser Ala Pro He Gly Leu Tyr Thr Leu 100 105 1 10 AGT GTT GAG ATC TCC TCC AGG GGC AGG GCC TCC TCT CTG AAA CTT GGC

441 Ser Val Glu He Ser Ser Arg Gly Arg Ala Ser Ser Leu Lys Leu Gly 115 120 125 ACG TTT ATA ATG CTC TTC AAC CCG TGG TTG CAA GCG GAT GAT GTC TTT

489 Thr Phe He Met Leu Phe Asn Pro Tφ Leu Gin Ala Asp Asp Val Phe 130 135 140 145 ATG AGT AAC CAC GCC GAA AGA CAA GAG TAT GTT GAA GAA GAT TCT GGC

537 Met Ser Asn His Ala Glu Arg Gin Glu Tyr Val Glu Glu Asp Ser Gly 150 155 160 ATC ATC TAT GTG GGC AGC ACA AAT CGA ATT GGC ATG GTT GGC TGG AAC

585 He He Tyr Val Gly Ser Thr Asn Arg He Gly Met Val Gly Tφ Asn 165 170 175 TTT GGA CAG TTT GAA GAA GAC ATT CTG AAC ATC AGC CTC TCC ATT TTG

633 Phe Gly Gin Phe Glu Glu Asp He Leu Asn He Ser Leu Ser He Leu 180 185 190 GAT AGG AGT CTG AAT TTC CGT CGT GAC CCT GTG ACT GAT GTG GCT CGC

681 Asp Arg Ser Leu Asn Phe Arg Arg Asp Pro Val Thr Asp Val Ala Arg 195 200 205 AGA AAT GAC CCC AAA TAT GTG TGC CGG GTG CTG AGT GCC ATG ATT AAT

729 Arg Asn Asp Pro Lys Tyr Val Cys Arg Val Leu Ser Ala Met He Asn

210 215 220 225 GGC AAT GAT GAC AAC GGT GTG ATT TCT GGG AAC TGG AGT GGT AAT TAC

777 Gly Asn Asp Asp Asn Gly Val He Ser Gly Asn Tφ Ser Gly Asn Tyr 230 235 240 ACC GGT GGT GTG GAC CCA AGG ACC TGG AAT GGT AGT GTG GAG ATC CTC

825 Thr Gly Gly Val Asp Pro Arg Thr Tφ Asn Gly Ser Val Glu He Leu

245 250 255 AAG AAC TGG AAA AAA TCT GGC TTC AGG CCA GTC CAA TTT GGC CAG TGC

873 Lys Asn Tφ Lys Lys Ser Gly Phe Arg Pro Val Gin Phe Gly Gin Cys 260 265 270 TGG GTC TTT GCT GGA ACC CTC AAC ACA GTG CTG CGG TGC TTG GGG GTT

921 Tφ Val Phe Ala Gly Thr Leu Asn Thr Val Leu Arg Cys Leu Gly Val 275 280 285 CGC TCT CGG GTG ATC ACC AAC TTC AAC TCG GCT CAC GAC ACA GAT CGA

969 Arg Ser Arg Val He Thr Asn Phe Asn Ser Ala His Asp Thr Asp Arg 290 295 300 305 AAC CTC AGT GTG GAT GTG TAC TAC GAT GCC ATG GGA AAT CCC CTG GAG

1017 Asn Leu Ser Val Asp Val Tyr Tyr Asp Ala Met Gly Asn Pro Leu Glu 310 315 320 AAA GGA AGT GAT AGC GTG TGG AAT TTT CAC GTC TGG AAT GAA GGC TGG

1065 Lys Gly Ser Asp Ser Val Tφ Asn Phe His Val Tφ Asn Glu Gly Tφ

325 330 335 TTC GTG CGG ACT GAC CTA GGC CCC ACA TAC AAT GGA TGG CAG GTG CTG

1 1 13 Phe Val Arg Thr Asp Leu Gly Pro Thr Tyr Asn Gly Tφ Gin Val Leu 340 345 350 GAT GCC ACC CCC CAG GAG AGA AGC CAA GGC GTA TTC CAG TGC GGT CCA

1 161 Asp Ala Thr Pro Gin Glu Arg Ser Gin Gly Val Phe Gin Cys Gly Pro 355 360 365 GCT TCC GTT AAT GCA ATC AAA GCC GGT GAT GTG GAC CGG AAT TTT GAC

1209 Ala Ser Val Asn Ala He Lys Ala Gly Asp Val Asp Arg Asn Phe Asp 370 375 380 385 ATG ATC TTC ATC TTC GCG GAG GTT AAT GCA GAT CGC ATC ACT TGG ATC

1257 Met He Phe He Phe Ala Glu Val Asn Ala Asp Arg He Thr Tφ He 390 395 400 TAT AAT AAT AGA AAT AAC ACC CAG AAG CAG AAT TCT GTG GAC ACT CAC

1305 Tyr Asn Asn Arg Asn Asn Thr Gin Lys Gin Asn Ser Val Asp Thr His 405 410 415 TCC ATT GGC AAA TAC ATC AGC ACC AAG GCA GTA GGC AGC AAC TCT CGC

1353 Ser He Gly Lys Tyr He Ser Thr Lys Ala Val Gly Ser Asn Ser Arg 420 425 430 ATG GAT GTC ACA GAC AAG TAC AAG TAT CCA GAA GGT TCC AGT GAG GAA

1401 Met Asp Val Thr Asp Lys Tyr Lys Tyr Pro Glu Gly Ser Ser Glu Glu

435 440 445 AGA CAA GTG CAC CAA AAG GCT TTG GAC AAA CTC AAA CCT AAC GCA TCT

1449 Arg Gin Val His Gin Lys Ala Leu Asp Lys Leu Lys Pro Asn Ala Ser 450 455 460 465 TTT GGC GCA ACA TCT TCG AGG AAT CCA GAA GGG GAA GAC AAG GAG CCC

1497 Phe Gly Ala Thr Ser Ser Arg Asn Pro Glu Gly Glu Asp Lys Glu Pro

470 475 480 AGC ATT TCT GGG AAG TTC AAG GTC ACG GGC ATA CTG GCA GTA GGC AAA

1545 Ser He Ser Gly Lys Phe Lys Val Thr Gly He Leu Ala Val Gly Lys 485 490 495 GAA GTC AGT CTG TCC CTG ATG CTC AAA AAC ATG ACT AAT GAC AGG AAG

1593 Glu Val Ser Leu Ser Leu Met Leu Lys Asn Met Thr Asn Asp Arg Lys 500 505 510 ACA GTG ACG ATG AAC ATG ACA GCC TGG ACC ATC GTC TAC AAT GGT ACC

1641 Thr Val Thr Met Asn Met Thr Ala Tφ Thr He Val Tyr Asn Gly Thr 515 520 525 CTT GTC CAC GAA GTG TGG AAG GAC TCA GCC ACA ATA TCC TTG GAT CCT

1689 Leu Val His Glu Val Tφ Lys Asp Ser Ala Thr He Ser Leu Asp Pro

530 535 540 545 GAA GAA GAA ATA CAG TAT CCT GTG AAG ATC GCA TAC TCT CAG TAT GAG

1737 Glu Glu Glu He Gin Tyr Pro Val Lys He Ala Tyr Ser Gin Tyr Glu 550 555 560 AGA TAC CTG AAG GCA GAC AAC ATG ATC CGG ATC TCA GCC GTT TGC AAG

1785 Arg Tyr Leu Lys Ala Asp Asn Met He Arg He Ser Ala Val Cys Lys

565 570 575 GTG CCC GAT GAG GCT GAG GTG GTG GTG GAA TGG GAT GTC ATC CTG GAT

1833 Val Pro Asp Glu Ala Glu Val Val Val Glu Tφ Asp Val He Leu Asp 580 585 590 AAT CCT GCT TTG ACC CTG GAG GTG CTG GAA CAG GCT CAT GTG CGG AAG

1881 Asn Pro Ala Leu Thr Leu Glu Val Leu Glu Gin Ala His Val Arg Lys 595 600 605 CCC GTG AAC GTG CAG ATG ATT TTC TCC AAC CCC CTG GAC CAG CCG GTG

1929 Pro Val Asn Val Gin Met He Phe Ser Asn Pro Leu Asp Gin Pro Val 610 615 620 625 AGG AAC TGC GTG CTG CTG GTG GAG GGC AGC GGC TGC TCG GTG GCA GCC

1977 Arg Asn Cys Val Leu Leu Val Glu Gly Ser Gly Cys Ser Val Ala Ala 630 635 640 TCA AGA TTG ATG TGC CAT CCC TGC GTC CCC AAG GAG AAG TCC CGC ATC

2025 Ser Arg Leu Met Cys His Pro Cys Val Pro Lys Glu Lys Ser Arg He 645 650 655 CGA TTT GAG ATT TTC CCC ACT CGG AGT GGC ACC AAG CAA CTG CTC GCT

2073 Arg Phe Glu He Phe Pro Thr Arg Ser Gly Thr Lys Gin Leu Leu Ala 660 665 670 GAC TTT TCC TGC AAT AAA TTC CCT ACT ATC AAG GCC ATG CTG CCC ATT

2121

Asp Phe Ser Cys Asn Lys Phe Pro Thr He Lys Ala Met Leu Pro He 675 680 685 GAT GTC TCT GAG TGACCGACCC AGCAGCACTC CCACAGACGT CGGTGACACA 2173 Asp Val Ser Glu 690 GACCAGACAG CGCTCTCCTG TGGAGTGAAA CTGTTGCCTA TGTTGTCCAG CCTGAGAAGC 2233

CCTCCATGTC CCCAAGGCTG CCAGACATGG ACTTCTAGCA AGTCCCCCAA CCCCCCATTC 2293

AACC 2297

(2) INFORMATION FOR SEQ ID NO:l l l :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 693 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l l l : Met Ser Ala Leu Gin He Gin Asn Val Asn Tφ Gin Val Pro Met Asn

1 5 10 15

Arg Arg Ala His His Thr Asp Lys Phe Ser Ser Gin Asp Ser He Val 20 25 30

Arg Arg Gly Gin Pro Tφ Glu He He Leu Val Cys Asn Arg Ser Leu

35 40 45

Glu Ser Gly Glu Asp Leu Asn Phe He Val Ser Thr Gly Pro Gin Pro

50 55 60

Ser Glu Ser Ala Arg Thr Lys Ala Val Phe Ser He Ser Gly Arg Ser 65 70 75 80

Thr Gly Gly Tφ Asn Ala Ala Leu Lys Ala Asn Ser Gly Asn Asn Leu 85 90 95

Ala He Ala He Ala Ser Pro Val Ser Ala Pro He Gly Leu Tyr Thr 100 105 110 Leu Ser Val Glu He Ser Ser Arg Gly Arg Ala Ser Ser Leu Lys Leu

115 120 125

Gly Thr Phe He Met Leu Phe Asn Pro Tφ Leu Gin Ala Asp Asp Val 130 135 140

Phe Met Ser Asn His Ala Glu Arg Gin Glu Tyr Val Glu Glu Asp Ser 145 150 155 160

Gly He He Tyr Val Gly Ser Thr Asn Arg He Gly Met Val Gly Tφ 165 170 175

Asn Phe Gly Gin Phe Glu Glu Asp He Leu Asn He Ser Leu Ser He 180 185 190 Leu Asp Arg Ser Leu Asn Phe Arg Arg Asp Pro Val Thr Asp Val Ala

195 200 205

Arg Arg Asn Asp Pro Lys Tyr Val Cys Arg Val Leu Ser Ala Met He 210 215 220

Asn Gly Asn Asp Asp Asn Gly Val He Ser Gly Asn Tφ Ser Gly Asn

225 230 235 240

Tyr Thr Gly Gly Val Asp Pro Arg Thr Tφ Asn Gly Ser Val Glu He 245 250 255

Leu Lys Asn Tφ Lys Lys Ser Gly Phe Arg Pro Val Gin Phe Gly Gin 260 265 270 Cys Tφ Val Phe Ala Gly Thr Leu Asn Thr Val Leu Arg Cys Leu Gly

275 280 285

Val Arg Ser Arg Val He Thr Asn Phe Asn Ser Ala His Asp Thr Asp 290 295 300

Arg Asn Leu Ser Val Asp Val Tyr Tyr Asp Ala Met Gly Asn Pro Leu 305 310 315 320

Glu Lys Gly Ser Asp Ser Val Tφ Asn Phe His Val Tφ Asn Glu Gly 325 330 335

Tφ Phe Val Arg Thr Asp Leu Gly Pro Thr Tyr Asn Gly Tφ Gin Val 340 345 350

Leu Asp Ala Thr Pro Gin Glu Arg Ser Gin Gly Val Phe Gin Cys Gly 355 360 365

Pro Ala Ser Val Asn Ala He Lys Ala Gly Asp Val Asp Arg Asn Phe 370 375 380 Asp Met He Phe He Phe Ala Glu Val Asn Ala Asp Arg He Thr Tφ

385 390 395 400

He Tyr Asn Asn Arg Asn Asn Thr Gin Lys Gin Asn Ser Val Asp Thr 405 410 415

His Ser He Gly Lys Tyr He Ser Thr Lys Ala Val Gly Ser Asn Ser 420 425 430

Arg Met Asp Val Thr Asp Lys Tyr Lys Tyr Pro Glu Gly Ser Ser Glu

435 440 445

Glu Arg Gin Val His Gin Lys Ala Leu Asp Lys Leu Lys Pro Asn Ala 450 455 460 Ser Phe Gly Ala Thr Ser Ser Arg Asn Pro Glu Gly Glu Asp Lys Glu

465 470 475 480

Pro Ser He Ser Gly Lys Phe Lys Val Thr Gly He Leu Ala Val Gly 485 490 495

Lys Glu Val Ser Leu Ser Leu Met Leu Lys Asn Met Thr Asn Asp Arg 500 505 510

Lys Thr Val Thr Met Asn Met Thr Ala Tφ Thr He Val Tyr Asn Gly 515 520 525

Thr Leu Val His Glu Val Tφ Lys Asp Ser Ala Thr He Ser Leu Asp 530 535 540 Pro Glu Glu Glu He Gin Tyr Pro Val Lys He Ala Tyr Ser Gin Tyr

545 550 555 560

Glu Arg Tyr Leu Lys Ala Asp Asn Met He Arg He Ser Ala Val Cys

565 570 575

Lys Val Pro Asp Glu Ala Glu Val Val Val Glu Tφ Asp Val He Leu 580 585 590

Asp Asn Pro Ala Leu Thr Leu Glu Val Leu Glu Gin Ala His Val Arg 595 600 605

Lys Pro Val Asn Val Gin Met He Phe Ser Asn Pro Leu Asp Gin Pro 610 615 620

Val Arg Asn Cys Val Leu Leu Val Glu Gly Ser Gly Cys Ser Val Ala 625 630 635 640

Ala Ser Arg Leu Met Cys His Pro Cys Val Pro Lys Glu Lys Ser Arg 645 650 655 He Arg Phe Glu He Phe Pro Thr Arg Ser Gly Thr Lys Gin Leu Leu

660 665 670

Ala Asp Phe Ser Cys Asn Lys Phe Pro Thr He Lys Ala Met Leu Pro 675 680 685

He Asp Val Ser Glu 690

(2) INFORMATION FOR SEQ ID NO: 112:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 693 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112: Met Ala Ala Leu Gly Val Gin Ser He Asn Tφ Gin Lys Ala Phe Asn

1 5 10 15

Arg Gin Ala His His Thr Asp Lys Phe Ser Ser Gin Glu Leu He Leu

20 25 30

Arg Arg Gly Gin Asn Phe Gin Val Leu Met He Met Asn Lys Gly Leu

35 40 45

Gly Ser Asn Glu Arg Leu Glu Phe He Asp Thr Thr Gly Pro Tyr Pro

50 55 60

Ser Glu Ser Ala Met Thr Lys Ala Val Phe Pro Leu Ser Asn Gly Ser 65 70 75 80 Ser Gly Gly Tφ Ser Ala Val Leu Gin Ala Ser Asn Gly Asn Thr Leu

85 90 95

Thr He Ser He Ser Ser Pro Ala Ser Ala Pro He Gly Arg Tyr Thr 100 105 110

Met Ala Leu Gin He Phe Ser Gin Gly Gly He Ser Ser Val Lys Leu 1 15 120 125

Gly Thr Phe He Leu Leu Phe Asn Pro Tφ Leu Asn Val Asp Ser Val 130 135 140

Phe Met Gly Asn His Ala Glu Arg Glu Glu Tyr Val Gin Glu Asp Ala 145 150 155 160 Gly He He Phe Val Gly Ser Thr Asn Arg He Gly Met He Gly Tφ

165 170 175

Asn Phe Gly Gin Phe Glu Glu Asp He Leu Ser He Cys Leu Ser He 180 185 190

Leu Asp Arg Ser Leu Asn Phe Arg Arg Asp Ala Ala Thr Asp Val Ala 195 200 205

Ser Arg Asn Asp Pro Lys Tyr Val Gly Arg Val Leu Ser Ala Met He 210 215 220

Asn Ser Asn Asp Asp Asn Gly Val Leu Ala Gly Asn Tφ Ser Gly Thr 225 230 235 240 Tyr Thr Gly Gly Arg Asp Pro Arg Ser Tφ Asp Gly Ser Val Glu He

245 250 255

Leu Lys Asn Tφ Lys Lys Ser Gly Phe Ser Pro Val Arg Tyr Gly Gin 260 265 270

Cys Tφ Val Phe Ala Gly Thr Leu Asn Thr Ala Leu Arg Ser Leu Gly

275 280 285

He Pro Ser Arg Val He Thr Asn Phe Asn Ser Ala His Asp Thr Asp 290 295 300

Arg Asn Leu Ser Val Asp Val Tyr Tyr Asp Pro Met Gly Asn Pro Leu 305 310 315 320 Asp Lys Gly Ser Asp Ser Val Tφ Asn Phe His Val Tφ Asn Glu Gly

325 330 335

Tφ Phe Val Arg Ser Asp Leu Gly Pro Pro Tyr Gly Gly Tφ Gin Val 340 345 350

Leu Asp Ala Thr Pro Gin Glu Arg Ser Gin Gly Val Phe Gin Cys Gly 355 360 365

Pro Ala Ser Val He Gly Val Arg Glu Gly Asp Val Gin Leu Asn Phe 370 375 380

Asp Met Pro Phe He Phe Ala Glu Val Asn Ala Asp Arg He Thr Tφ 385 390 395 400

Leu Tyr Asp Asn Thr Thr Gly Lys Gin Tφ Lys Asn Ser Val Asn Ser 405 410 415

His Thr He Gly Arg Tyr He Ser Thr Lys Ala Val Gly Ser Asn Ala 420 425 430 Arg Met Asp Val Thr Asp Lys Tyr Lys Tyr Pro Glu Gly Ser Asp Gin

435 440 445

Glu Arg Gin Val Phe Gin Lys Ala Leu Gly Lys Leu Lys Pro Asn Thr 450 455 460

Pro Phe Ala Ala Thr Ser Ser Met Gly Leu Glu Thr Glu Glu Gin Glu 465 470 475 480

Pro Ser He He Gly Lys Leu Lys Val Ala Gly Met Leu Ala Val Gly 485 490 495

Lys Glu Val Asn Leu Val Leu Leu Leu Lys Asn Leu Ser Arg Asp Thr 500 505 510 Lys Thr Val Thr Val Asn Met Thr Ala Tφ Thr He He Tyr Asn Gly

515 520 525

Thr Leu Val His Glu Val Tφ Lys Asp Ser Ala Thr Met Ser Leu Asp 530 535 540

Pro Glu Glu Glu Ala Glu His Pro He Lys He Ser Tyr Ala Gin Tyr 545 550 555 560

Glu Arg Tyr Leu Lys Ser Asp Asn Met He Arg He Thr Ala Val Cys 565 570 575

Lys Val Pro Asp Glu Ser Glu Val Val Val Glu Arg Asp He He Leu 580 585 590 Asp Asn Pro Thr Leu Thr Leu Glu Val Leu Asn Glu Ala Arg Val Arg

595 600 605

Lys Pro Val Asn Val Gin Met Leu Phe Ser Asn Pro Leu Asp Glu Pro 610 615 620

Val Arg Asp Cys Val Leu Met Val Glu Gly Ser Gly Leu Leu Leu Gly 625 630 635 640

Asn Leu Lys He Asp Val Pro Thr Leu Gly Pro Lys Glu Arg Ser Arg 645 650 655

Val Arg Phe Asp He Leu Pro Ser Arg Ser Gly Thr Lys Gin Leu Leu 660 665 670

Ala Asp Phe Ser Cys Asn Lys Phe Pro Ala He Lys Ala Met Leu Ser 675 680 685

He Asp Val Ala Glu 690 (2) INFORMATION FOR SEQ ID NO: 113 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 51 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:113: Thr Ala Ala Ala His Gly Ser Lys Pro Asn Val Tyr Ala Asn Arg Gly

1 5 10 15

Ser Ala Glu Asp Val Ala Met Gin Val Glu Ala Gin Asp Ala Val Met 20 25 30

Gly Gin Asp Leu Met Val Ser Val Met Leu He Asn His Ser Ser Ser 35 40 45

Arg Arg Thr 50

(2) INFORMATION FOR SEQ ID NO: 114:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 52 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO: 114:

Arg Ala Asn His Leu Asn Lys Leu Ala Glu Lys Glu Glu Thr Gin Glu 1 5 10 15

Met Ala Thr Gly Val Ala Met Arg He Arg Val Gly Gin Ser Met Asn 20 25 30

Met Gly Ser Asp Phe Asp Val Phe Ala His He Thr Asn Asn Thr Ala

35 40 45 Glu Glu Tyr Val

50

(2) INFORMATION FOR SEQ ID NO: 115: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 69 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:115:

Lys Ala Leu Gly Lys Leu Lys Pro Asn Thr Pro Phe Ala Ala Thr Ser 1 5 10 15

Ser Met Gly Leu Glu Thr Glu Glu Gin Glu Pro Ser He Ser Gly Lys

20 25 30

Leu Lys Val Ala Gly Met Leu Ala Val Gly Lys Glu Val Asn Leu Val

35 40 45

Leu Leu Leu Lys Asn Leu Ser Arg Asp Thr Lys Thr Val Thr Val Asn 50 55 60

Met Thr Ala Tφ Thr 65

(2) INFORMATION FOR SEQ ID NO: 116:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 53 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l 16:

Thr Ala Leu Met Tyr Gly Ala Lys Lys Pro Leu Asn Thr Glu Gly Val

1 5 10 15 Met Lys Ser Arg Ser Asn Val Asp Met Asp Phe Glu Val Glu Asn Ala

20 25 30

Val Leu Gly Lys Asp Phe Lys Leu Ser He Thr Glu Arg Asn Asn Ser

35 40 45

His Asn Arg Tyr Thr 50

(2) INFORMATION FOR SEQ ID NO: 117:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 57 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 117:

Arg Val Glu Lys Glu Lys Met Glu Arg Glu Lys Asp Asn Gly He Arg 1 5 10 15

Pro Pro Ser Leu Glu Thr Ala Ser Pro Leu Tyr Leu Leu Leu Lys Ala 20 25 30 Pro Ser Ser Leu Pro Leu Arg Gly Asp Ala Gin He Ser Val Thr Leu

35 40 45

Val Asn His Ser Glu Gin Glu Lys Ala

50 55