FIELD OF THE INVENTION

The invention relates to cathepsin 02 proteins, nucleic acids, and antibodies.

BACKGROUND OF THE INVENΗON

The cathepsins belong to the papain superfamily of cysteine proteases. Cysteine or thiol proteases contain a cysteine residue, as well as a histidine and an asparagine, at the active site responsible for proteolysis. This superfamily also has a glutamine at the oxy-anion hole.

Recent work has implicated cysteine proteases in binding to DNA with putative transcription factor activity (Xu et al., J. Biol. Chem. 269(33):21177-21183 (1994)), and as a long term immunosuppressor (Hamajima et al., Parasite Immunology 16:261 (1994)).

To date, a number of cathepsins have been identified and sequenced from a number of animals. For example, cathepsin S has been cloned from rat (Petanceska et al., J. Biol. Chem. 267:26038-20643 (1992)), bovine (Wiederanders et al., FEBS Lett. 286:189-192 (1991)) and humans (Wideranders et al., J. Biol.

Chem. 267:13708-13713 (1992); and Shi et al., J. Biol. Chem. 267:7258-7262 ( 1992)). Cathepsin L has been cloned from humans, rat, mouse and chicken (Gal et al. Biochem. J., 253:303-306 (1988); Ishidoh et al., FEBS Lett. 223:69-73 (1987); Joseph et al., J. Clin. Invest. 81:1621-1629 (1988); Ritonja et al., FEBS Lett. 283:329-331 (1991)). Cathepsin H has been cloned from human and rat

(Fuchs et al., Biol. Chem. Hoppe-Seyler 369-375 (1988); Fuchs et al., Nucleic Acid Res. 17:9471 (1989); Whittier et al., Nucleic Acid Res. 15:2515-2535 (1987)). Cathepsin B has been cloned from human and mouse (Ferrara et al., FEBS Lett. 273:195-199 (1990); Chan et al., Proc. Natl. Acad. Sci. USA 83:7721- 7725 (1986)).

A cysteine protease from rabbit osteoclasts was recently cloned, and is structurally related to cathepsins L and S. Tezuka et al., J. Biol. Chem. 269(2): 1106 (1994).

Cathepsins are naturally found in a wide variety of tissues. For example, cathepsin L is found in tissues including heart, brain, placenta, lung, skeletal muscle, kidney, liver, testis and pancreas. Cathepsin S is found in lung, liver, spleen and skeletal muscle.

Cathepsins have been implicated in a number of disease conditions. For example, enzymes similar to cathepsins B and L are released from tumors and may be involved in tumor metastasis. Cathepsin L is present in diseased human synovia! fluid and transformed tissues. Similarly, the release of cathepsin B and other lysosomal proteases from polymorphonuclear granulocytes and macrophages is observed in trauma and inflammation. Cathepsins have been implicated in arthritis. In addition, cathepsins are found in abnormally high amounts in several tumor cell lines.

Cysteine proteases have also been implicated in bone remodeling. Bone remodeling is a process coupling bone formation and bone resorption, and is part

of bone growth. Bone resorption includes demineralization and degradation of extracellular matrix proteins (Delaisse et al., Biochem. J. 279:167-174 (1991)). Type I collagen constitutes ninety-five percent of the organic matrix (Krane et al., in Scientific American Medicine (Rubensttein, E., and Federman, D.D., eds) Vol. 3, 15 Rheumatism, XI Bone Formation and Resorption, pp. 1 -26, Scientific

American, Inc. New York. In addition to the interstitial collagenase, the lysosomal cysteine proteases cathepsins B and L are thought to be involved in osteoclastic bone resorption (Delaisse et al., 1991, supra). Both enzymes are present in the lysosomes as well as in the acidified extracellular resorption lacuna of the osteoclast (Goto et al, Histochemistry 99, 411-414(1993)) and both proteases display the in vitro ability to degrade collagen Type I at acidic pH (Maciewicz et al, Collagen Rel. Res. 7, 295-304 (1987), Delaisse et al, (1991), supra). Cysteine protease inhibitors, such as E-64 and leupeptin, have been shown to prevent osteoclastic bone resorption (Delaisse et al., Bone 8, 305-313 (1987), Everts et al, Calcif. Tissue Int. 43, 172-178 (1988)). Cathepsin L is considered to be one of the main proteases involved in collagen degradation in bone (Maciewiecz et al., Biochem. J. 256, 433-440 (1988); Kakegawa et al, FEBS Lett. 321, 247-250 (1993)).

The solid state of bone material is due to the low solubility of hydroxyapatite and other calcium-phosphate bone salts at physiological pH, but bone may break down at acidic pH.

Osteoclasts are multinucleate cells that play key roles in bone resorption. Attached to the bone surface, osteoclasts produce an acidic microenvironment in a tightly defined junction between the specialized osteoclast border membrane and the bone matrix, thus allowing the localized solubilization of bone matrix.

This in turn facilitates the proteolysis of demineralized bone collagen.

It is thought that the collagenolytic action of cysteine proteases is exerted preferentially in the most acidic part of the bone resorption lacuna close to the ruffled border at a pH around 3.5 or 4.5, whereas the Zn-containing collagenases are more active in the neutral environment at the interface between the demineralized and mineralized matrix (Delaisse et al., supra, (1991)). Besides cathepsins L and B, a variety of cathepsin L- and B-like activities may participate in collagenolytic bone degradation. Page et a Biochim. Biophys. Acta 1116, 57-66 (1992) isolated multiple forms of cathepsin B from osteoclastomas. These have an acidic pH optimum and the ability to degrade soluble and insoluble Type I collagen. Delaisse et al, 1991, supra, identified a 70 kDa thiol-dependent protease in bone tissue which is also capable of degrading Type I collagen.

Cysteine protease inhibitors have been shown to inhibit osteoclastic bone resorption by inhibiting degradation of collagen fibers. Cathepsins B, L, N and S can degrade type-I collagen at acidic pH. Three cathepsin-type proteases have been isolated from mouse calvaria; putative cathepsins B and L, and a cathepsin

L-like protease (Delaisse et al., Biochem. J. 279:167 (1991). However, it is still unclear as to what cysteine proteases are actually produced by osteoclasts.

Recently, a cDNA encoding a novel human cysteine protease was cloned independently by several groups (Shi et al, FEBS Lett. 357, 129-134 (1995), Inaoka et al, Biochem. Biophys. Res. Commun. 206, 89-96 (1995); Brδmme and Okamoto, Biol. Chem. Hoppe-Seyler 376, 379-384 (1995)) and named cathepsin O, cathepsin K, and cathepsin 02, respectively.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide for a new class of recombinant cathepsins, cathepsin O2, and variants thereof, and to produce useful quantities of these cathepsin 02 proteins using recombinant DNA techniques.

It is a further object of the invention to provide recombinant nucleic acids encoding cathepsin 02 proteins, and expression vectors and host cells containing the nucleic acid encoding the cathepsin O2 protein.

An addition object of the invention is to provide poly- and monoclonal antibodies for the detection of the presence of cathepsin O2 and diagnosis of conditions associated to cathepsin 02.

A further object of the invention is to provide methods for producing the cathepsin 02 proteins.

In accordance with the foregoing objects, the present invention provides recombinant cathepsin 02 proteins, and isolated or recombinant nucleic acids which encode the cathepsin 02 proteins of the present invention. Also provided are expression vectors which comprise DNA encoding a cathepsin 02 protein operably linked to transcriptional and translational regulatory DNA, and host cells which contain the expression vectors.

Additional aspect of the present invention provides methods for producing cathepsin 02 proteins which comprise culturing a host cell transformed with an expression vector and causing expression of the nucleic acid encoding the cathepsin O2 protein to produce a recombinant cathepsin 02 protein.

A further aspect of the present invention provides poly- and monoclonal antibodies to cathepsin 02 proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and IB depict the nucleotide sequence (SEQ ID NO:l) and deduced amino acid sequence (SEQ ID NO:2) of human cathepsin 02 cDNA. The amino

acid sequence (SEQ ID NO:2) is shown in single letter code beneath the nucleotide sequence (SEQ ID NO:l). The active site residues (C25, H159 and N175; papain numbering) are indicated by boldface typing, and the potential N- glycosylation site is underlined once. Arrowheads show the putative post- translational cleavage sites between the presignal and the proregion as well as between the proregion and the mature enzyme. The cleavage between the proregion and the mature protein was confirmed by protein sequencing (double underline).

Figures 2 A and 2B depict the multiple amino acid sequence alignment of human cathepsin 02 (SEQ ID NO:2) with the human cathepsins S (SEQ ID NO:4) and

L (SEQ ID NO:5) and rabbit 0C2. (SEQ ID NO:3) * active site residues; boldface type, residue conserved in all known cysteine proteases of the papain family.

Amino acids identical in all six proteases are assigned as upper case letters in the consensus sequence, and amino acids identical in five out of six are assigned in lower case letters. Gaps are indicated by hyphens. Numbers indicate the position of the last amino acid in each line and arrowheads show the putative post-translational cleavage sites.

Figure 3 depicts the maturation of procathepsin 02 with pepsin. Aliquots of the culture supernatant containing procathepsin 02 were incubated with pepsin (0.4 mg/mL) at 40°C in lOOmM-sodium acetate buffer, pH 4.0. The incubation was stopped by adding sample buffer. The times of digestion are as indicated. Molecular mass standards (kDa) are indicated in the left margin.

Figure 4 depicts the SDS-PAGE of purified recombinant human cathepsin O2 (Coomassie Blue staining). Lane 1, crude Sβ fraction; Lane 2, after passage through n-Butyl fast Flow; 3, after passage through Mono S. Molecular mass standards are indicated in the right lane.

Figure 5 depicts the pH activity profile for recombinant human cathepsin 02. The k _α /K _m values were obtained by measuring the initial rates of Z-FR-MCA hydrolysis and by dividing by enzyme and substrate concentration.

Figure 6 depicts JK^ values for the hydrolysis of Z-X-R-MCA by cathepsins 02, S, L and B (normalized to the best substrate =1). Cathepsin 02

(Z-LR-MCA) 257,900 M 's ¹ ; cathepsin S (Z-LR-MCA) 243,000 M 's ¹ ; cathepsin L (Z-FR-MCA) 5,111 ,000 M 's ¹ ); cathepsin B (Z-FR-MCA) 460,000 M 's ' (data for cathepsins S, L and B from Brδmme et al., 1994).

Figure 7 depicts elastinolytic activity of recombinant human cathepsin O2 and pH 4.5, 5.5 and 7.0 in comparison to cathepsins S and L and pancreatic elastase.

The substrate is ³ H labelled insoluble elastin.

Figure 8 depicts northern blot analyses of the human cathepsins O2, L and S in osteoclastoma preparations. Lane 1, patient (fibrous and cellular tissue); lane 2, patient 2 (cellular tissue); lane 3, patient 2 (fibrous tissue). Nitrocellulose blots were hybridized with 32P-labelled probes of human cathepsins O2, L and S.

Figures 9A and 9B depict SDS PAGE of type I collagen (soluble calf skin collagen) after digestion with recombinant human cathepsin O2 and L and bovine trypsin. Figure 9 A: Collagenase activity: Digestion of soluble calfskin collagen at 28 ^* C and at pH 4.0, 5.0, .5, 6.0, 6.5, 7.0 by human cathepsins O2, S and L (each 50 nM) for 12 hours. The reaction was stopped by addition of 10 μM

E-64. Untreated soluble collagen was used as standard (S). Figure 9B: Gelatinase activity: Digestion of denatured soluble calf skin collagen (10 min heated at 70°C) at 28°C and at pH 4.0, 5.0, 5.5, 6.0, 6.5, 7.0 by human cathepsin O2 (0.1 nM), cathepsin L (0.2 nM) and human cathepsin S (InM). Molecular mass standards are indicated in the left lane.

Figure 10 depicts an SDS-PAGE of the purification of the propart of human cathepsin 02.

Figures 11 A, 1 IB, 11C, 1 ID, 1 IE, 1 IF, 11G, 11H, 1 II, 1 IJ, 1 IK and 1 IL depict immunohistochemical staining of human cathepsin 02 in human tissues. (A) osteoclastoma, (B) lung macrophages, (C) bronchiole, (D) endometrium, (E) stomach, (F) colon, (G) kidney, (H) placenta, (I) liver, (J) ovary, (K) adrenal, (L) testis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel cathepsin 02 proteins and nucleic acids.

The cathepsin 02 proteins of the present invention may be identified in several ways. Cathepsin 02 nucleic acids or cathepsin 02 proteins are initially identified by substantial nucleic acid and/or amino acid sequence homology to the sequences shown in Figure 1. Such homology can be based upon the overall nucleic acid or amino acid sequence.

The cathepsin 02 proteins of the present invention have limited homology to other cathepsins. For example, the mature human cathepsin 02 has roughly 59% homology to mature human cathepsin L, a 58% homology to mature human cathepsin S, a 26% homology to mature human cathepsin B, and a 47% homology to mature human cathepsin H. In addition, the propart of human cathepsin 02 has a 38% homology to the propart of human cathepsin L, a 51% homology to the propart of human cathepsin S, a 13% homology to the propart of human cathepsin B, and a 23% homology to the propart of human cathepsin H. In addition, the human cathepsin 02 protein has roughly 90% homology to a rabbit osteoclast protein.

As used herein, a protein is a "cathepsin 02 protein" if the overall homology of the protein sequence to the amino acid sequence shown in Figure 1 is preferably greater than about 90%, more preferably greater than about 95% and most preferably greater than 98%. This homology will be determined using standard techniques known in the art, such as the Best Fit sequence program described by Oeveτeux etal., Nucl. Acid Res. 72:387-395 (1984). The alignment may include the introduction of gaps in the sequences to be aligned. In addition, for sequences which contain either more or fewer amino acids than the protein shown in Figure 1, it is understood that the percentage of homology will be determined based on the number of homologous amino acids in relation to the total number of amino acids. Thus, for example, homology of sequences shorter than that shown in Figure 1, as discussed below, will be determined using the number of amino acids in the shorter sequence.

In a preferred embodiment, the cathepsin 02 proteins of the present invention are human cathepsin 02 proteins.

Cathepsin 02 proteins of the present invention may be shorter than the amino acid sequence shown in Figure 1. As shown in Example 2, the human cathepsin 02 protein may undergo post-translational processing similar to that seen for cathepsins B and S, and papain (Brδmme et al., J. Biol. Chem. 268:4832-4838 (1993); Vernat et al., J. Biol. Chem. 266:21451-21457 (1991); and Rowan et al., J. Biol. Chem. 267:15993-15999 (1992)). The cathepsin 02 protein is made as a preproprotein, with a traditional presequence, a prosequence or "propart", and the mature sequence. These are depicted in Figure 1, with the sequence of human cathepsin 02, including the pre, pro and mature coding sequences, shown in Figure 1. The presequence comprises the first 15 amino acids of the sequence shown in Figure 1, the propart spans from amino acid 16 to amino acid 114 (98 amino acids), and the mature protein spans from position 115 to 329 (215 amino acids). The prosequence, or propart, is hypothesized to serve as an inhibitor of

the enzyme until the enzyme is activated, most probably as a result of a change in pH. The proteolytic processing of the propart is autoproteolytic for papain (Vernet et al., supra), cathepsin S and cathepsin L. The definition of cathepsin 02 includes preprocathepsin 02, procathepsin 02, mature cathepsin 02, and the propart, separate from the mature cathepsin 02.

In a preferred embodiment, also included within the definition of cathepsin O2 proteins are portions or fragments of the sequence shown in Figure 1. In one embodiment, the fragments range from about 40 to about 200 amino acids. Preferably, the fragments are not identical to the rabbit osteoclast protein of Tezuka et al., supra, and at least about 95 - 98% homologous to the human cathepsin O2 protein. In a preferred embodiment, when the cathepsin 02 protein is to be used to generate antibodies, for example for diagnostic purposes, the cathepsin 02 protein must share at least one epitope or determinant with either the propart or the mature protein shown in Figure 1. By "epitope" or "deterrninant" herein is meant a portion of a protein which will generate and bind an antibody. Thus, in most instances, antibodies made to a smaller cathepsin 02 protein will be able to bind to the full length protein. In a preferred embodiment, the antibodies are generated to a unique epitope; that is, the antibodies exhibit little or no cross reactivity to other proteins such as other cathepsin proteins, or to cathepsins from other organisms.

In the case of the nucleic acid, the overall homology of the nucleic acid sequence is commensurate with amino acid homology but takes into account the degeneracy in the genetic code and codon bias of different organisms. Accordingly, the nucleic acid sequence homology may be either lower or higher than that of the protein sequence. Thus the homology of the nucleic acid sequence as compared to the nucleic acid sequence of Figure 1 is preferably greater than 65%, more preferably greater than about 75% and most preferably greater than 85%. In some embodiments the homology will be as high as about 95 to 98 or 99%.

In one embodiment, the nucleic acid homology is determined through hybridization studies. Thus, for example, nucleic acids which hybridize under high stringency to the nucleic acid sequences shown in Figure 1 are considered cathepsin 02 genes. High stringency conditions are generally 0.1 XSSC at 37 - 65°C.

In another embodiment, less stringent hybridization conditions are used; for example, reduced stringency conditions are generally 2XSSC and 0.1 %SDS.

The cathepsin 02 proteins and nucleic acids of the present invention are preferably recombinant. As used herein, "nucleic acid" may refer to either DNA or RNA, or molecules which contain both deoxy- and ribonucleotides. The nucleic acids include genomic DNA, cDNA and oligonucleotides including sense and anti-sense nucleic acids. Specifically included within the definition of nucleic acid are anti-sense nucleic acids. An anti-sense nucleic acid will hybridize to the corresponding non-coding strand of the nucleic acid sequence shown in Figure 1 , but may contain ribonucleotides as well as deoxyribonucleotides. Generally, anti-sense nucleic acids function to prevent expression of mRNA, such that a cathepsin 02 protein is not made. The nucleic acid may be double stranded, single stranded, or contain portions of both double stranded or single stranded sequence.

By the term "recombinant nucleic acid" herein is meant nucleic acid, originally formed in vitro by the manipulation of nucleic acid by endonucleases, in a form not normally found in nature. Thus an isolated cathepsin 02 protein gene, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e. using the in vivo cellular machinery of the host cell rather than in vitro

manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention.

Similarly, a "recombinant protein" is a protein made using recombinant techniques, i.e. through the expression of a recombinant nucleic acid as depicted above. A recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics. For example, the protein may be isolated away from some or all of the proteins and compounds with which it is normally associated in its wild type host. Thus, for example, cathepsin 02 proteins which are substantially or partially purified, or are present in the absence of cells, are considered recombinant. The definition includes the production of a cathepsin 02 protein from one organism in a different organism or host cell. Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of a inducible promoter or high expression promoter, such that the protein is made at increased concentration levels. Alternatively, the protein may be in a form not normally found in nature, as in the addition of an epitope tag or amino acid substitutions, insertions and deletions.

Also included with the definition of cathepsin 02 protein are cathepsin 02 proteins from other organisms, which are cloned and expressed as outlined below.

In the case of anti-sense nucleic acids, an anti-sense nucleic acid is defined as one which will hybridize to all or part of the corresponding non-coding sequence shown in Figure 1. Generally, the hybridization conditions used for the determination of anti-sense hybridization will be high stringency conditions, such as 0.1XSSC at 65°C.

Once the cathepsin 02 protein nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombined to form the entire cathepsin 02

protein nucleic acid. Once isolated from its natural source, e.g., contained within a plasmid or other vector or excised therefrom as a linear nucleic acid segment, the recombinant cathepsin 02 protein nucleic acid can be further used as a probe to identify and isolate other cathepsin 02 protein nucleic acids. It can also be used as a "precursor" nucleic acid to make modified or variant cathepsin O2 protein nucleic acids and proteins.

Using the nucleic acids of the present invention which encode cathepsin 02 protein, a variety of expression vectors are made. The expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome. Generally, these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the cathepsin 02 protein. "Operably linked" in this context means that the transcriptional and translational regulatory DNA is positioned relative to the coding sequence of the cathepsin 02 protein in such a manner that transcription is initiated. Generally, this will mean that the promoter and transcriptional initiation or start sequences are positioned 5' to the cathepsin 02 protein coding region. The transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the cathepsin 02 protein; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus will be used to express the cathepsin 02 protein in Bacillus.

Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.

In general, the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. In a preferred embodiment, the regulatory sequences include a promoter and transcriptional start and stop sequences.

Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also i known in the art, and are useful in the present invention.

In addition, the expression vector may comprise additional elements. For example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification. Furthermore, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct. The integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.

In addition, in a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.

The cathepsin 02 proteins of the present invention are produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a cathepsin 02 protein, under the appropriate conditions to induce or cause expression of the cathepsin 02 protein. The conditions appropriate for cathepsin 02 protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation. For example, the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction. In addition, in some embodiments, the timing of the harvest is

important. For example, the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.

Appropriate host cells include yeast, bacteria, archebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Drosophila melaneaster cells, Saccharomvces cerevisiae and other yeasts, E. coli. Bacillus subtilis. SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, HeLa cells, and immortalized mammalian myeloid and lymphoid cell lines.

In a preferred embodiment, cathepsin 02 proteins are expressed in bacterial systems. Bacterial expression systems are well known in the art.

A suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of the coding sequence of cathepsin 02 protein into mRNA. A bacterial promoter has a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan. Promoters from bacteriophage may also be used and are known in the art. In addition, synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid of the trp and lac promoter sequences. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.

In addition to a functioning promoter sequence, an efficient ribosome binding site is desirable. In E. coli, the ribosome binding site is called the Shine-Delgarno

(SD) sequence and includes an initiation codon and a sequence 3-9 nucleotides in length located 3 - 11 nucleotides upstream of the initiation codon.

The expression vector may also include a signal peptide sequence that provides for secretion of the cathepsin 02 protein in bacteria. The signal sequence typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell, as is well known in the art. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria).

The bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed. Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways.

These components are assembled into expression vectors. Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans, among others.

The bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others.

In one embodiment, cathepsin 02 proteins are produced in insect cells.

Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art. Briefly, baculovirus is a very large DNA virus which produces its coat protein at very

high levels. Due to the size of the baculoviral genome, exogenous genes must be placed in the viral genome by recombination. Accordingly, the components of the expression system include: a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the cathepsin 02 protein; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene into the baculovirus genome); and appropriate insect host cells and growth media.

Mammalian expression systems are also known in the art and are used in one embodiment. A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence for cathepsin 02 protein into mRNA. A promoter will have a transcription initiating region, which is usually place proximal to the 5' end of the coding sequence, and a TATA box, using a located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct

RNA polymerase II to begin RNA synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element, typically located within 100 to 200 base pairs upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. O2f particular use as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adeno virus major late promoter, and herpes simplex virus promoter.

Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3' terminus of the mature mRNA is formed by site-specific post-translational

cleavage and polyadenylation. Examples of transcription terminator and polyadenlytion signals include those derived form SV40.

The methods of introducing exogenous nucleic acid into mammalian hosts, as well as other hosts, is well known in the art, and will vary with the host cell used. Techniques include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

In a preferred embodiment, cathepsin 02 protein is produced in yeast cells. Yeast expression systems are well known in the art, and include expression vectors for Saccharomvces cerevisiae. Candida albicans and C. maltosa. Hansenula polvmorpha. Kluweromvces fraeilis and K. lactis. Pichia guillerimondii and I\ pastoris. Schizosaccharomvces pombe. and Yarrowia lipolvtica. Preferred promoter sequences for expression in yeast include the inducible GAL 1,10 promoter, the promoters from alcohol dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase, hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and the acid phosphatase gene. Yeast selectable markers include ADE2, HJ.S4,

LEU2, TRP1, and ALG7, which confers resistance to tunicamycin; the G418 resistance gene, which confers resistance to G418; and the CUPl gene, which allows yeast to grow in the presence of copper ions.

A recombinant cathepsin 02 protein may be expressed intracellularly or secreted. The cathepsin O2 protein may also be made as a fusion protein, using techniques well known in the art. Thus, for example, if the desired epitope is small, the cathepsin 02 protein may be fused to a carrier protein to form an immunogen.

Alternatively, the cathepsin 02 protein may be made as a fusion protein to increase expression, or for other reasons.

Also included within the definition of cathepsin 02 proteins of the present invention are amino acid sequence variants. These variants fall into one or more of three classes: substitutional, insertional or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the cathepsin 02 protein, using cassette mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above. However, variant cathepsin 02 protein fragments having up to about 100- 150 residues may be prepared by in vitro synthesis using established techniques. Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the cathepsin 02 protein amino acid sequence. The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below.

While the site or region for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed cathepsin 02 protein variants screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, Ml 3 primer mutagenesis. Screening of the mutants is done using assays of cathepsin 02 protein activities; for example, purified or partially purified cathepsin 02 may be used in kinetic assays such as those depicted in the examples, to determine the effect of the amino acid substitutions, insertions or deletions. Alternatively, mutated cathepsin 02 genes are placed in cathepsin 02 deletion strains and tested for cathepsin 02 activity, as disclosed herein. The creation of deletion strains, given a gene sequence, is known in the art.

Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to 30 residues, although in some cases deletions may be much larger, as for example when the prosequence or the mature part of the cathepsin 02 protein is deleted. In addition, as outlined above, it is possible to use much smaller fragments of the cathepsin 02 protein to generate antibodies.

Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to riiinimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances.

When small alterations in the characteristics of the cathepsin 02 protein are desired, substitutions are generally made in accordance with the following chart:

Chart l

Original Residue Exemplary Substitutions

Ala Ser

Arg Lys

Asn Gin, His

Asp Glu

Cys Ser

Gin Asn

Glu Asp

Gly Pro

His Asn, Gin

He Leu, Val

Leu He, Val

Lys Arg, G n, Glu

Met Leu, He

Phe Met, Leu, Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp, Phe

Val He, Leu

Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those shown in Chart I. For example, substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the polypeptide' s properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residue having

a bulky side chain, e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine.

The variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analogue, although variants also are selected to modify the characteristics of the polypeptide as needed. Alternatively, the variant may be designed such that the biological activity of the cathepsin 02 protein is altered. For example, the proteolytic activity of the cathepsin 02 protein may be altered, through the substitution of the amino acids of the catalytic triad. The catalytic triad, consisting of a cysteine at position 25, a histidine at position 162 and an asparagine at position 182, may be individually or simultaneously altered to decrease or eliminate proteolytic activity. This may be done to decrease the toxicity of administered cathepsin 02. Similarly, the cleavage site between the prosequence and the mature sequence may be altered, for example to eliminate proteolytic processing.

In a preferred embodiment, the cathepsin 02 protein is purified or isolated after expression. Cathepsin 02 proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing. For example, the cathepsin 02 protein may be purified using a standard anti-cathepsin 02 antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, R., Protein Purification, Springer- Verlag, NY (1982). The degree of purification necessary will vary depending on the use of the cathepsin 02 protein. In some instances no purification will be necessary.

In some embodiments, the cathepsin 02 enzyme is expressed as a proenzyme. As depicted in the examples, the proenzyme may be treated with exogenous protease to convert the enzyme to the mature, active form, as is known in the art. Suitable exogenous proteases include, but are not limited to, pepsin and cathepsin D.

Once expressed and purified if necessary, the cathepsin 02 proteins are useful in a number of applications.

For example, as shown in Example 5, the cathepsin 02 proteins of the present invention have collagenase activity. Thus, the cathepsin 02 proteins may be used as a collagenase, both in vitro and in vivo. For example, cathepsin 02 may be used to treat analytical samples which contain interfering or problematic levels of collagen.

Similarly, cathepsin 02 proteins may be used to degrade excess collagen within the body. There are a variety of conditions associated with excess collagen. For example, one treatment of spinal disk problems such as severe disk inflammation and hemiation involves the injection of collagenase or chymopapain to degrade the disk collagen (Leonardo et al., Ann. Chirm Gyneacol. 82: 141-148 (1993); Gogan et al., Spine 17:388-94 (1992); Stula, Nerochirurgia 33:169-172 (1990); andBoccaneraetal., Chir. Organi. Mov.75:25-32 (1990)). Alternatively, the treatment of adhesions, such as pelvic adhesions, post surgical adhesions, pulmonary adhesions, abdominal adhesions and the like may be treated or dissolved with cathepsin O2. Similarly, scars and keloids may be treated with cathepsin 02 to remove or decrease the excessive amounts of collagen present. In addition, endometriosis is another significant clinical problem involving the deposit of excess amounts of collagen and other substances within the uterus and surrounding tissue; certain forms of endometriosis may also be treated with the cathepsin 02 of the present invention.

In an alternative embodiment, cathepsin 02 may be used to dissolve the matrices around tumors. Generally, tumor pH is lower than physiological pH, and, as outlined in the Examples, cathepsin 02 is active at acidic pH. Therefore, cathepsin 02 is suited to dissolve the collagen-based matrix generally surrounding a tumor.

In one embodiment, the cathepsin 02 proteins of the present invention may also be administered to treat pycnody sostosis, an osteopetrosislike bone disorder. This disorder appears to be caused by insufficient activity of osteoclastic cysteine- proteinases. In some embodiments, gene therapy may be used to administer the cathepsin 02.

In addition, since cathepsin 02 is functional at acidic pH, cathepsin 02 can be administered in conjunction with bone demineralization compounds, such as acids, to degrade bone tissue. Thus, aberrant or excess bone growths may be treated.

The cathepsin 02 proteins of the present invention are also useful to screen for cathepsin 02 protease inhibitors and for cysteine protease inhibitors. Cysteine protease inhibitors have a variety of uses, as will be appreciated in the art, including purification of cysteine proteases via coupling to affinity chromatography columns, and inhibition of cysteine proteases, similar to known cysteine protease inhibitors. In addition, cysteine protease inhibitors may have therapeutic uses, since a wide variety of physiological disorders are associated with increased levels of cysteine proteases, including arthritis, inflammation, osteoporosis, muscular dystrophy, tumor invasion, multiple myeloma and glomerulonephritis, as is known in the art.

In a preferred embodiment, the propart of cathepsin 02 may be used as a specific inhibitor of cathepsin 02. Thus, for example, the propart may be separately expressed, that is, without the mature sequence, and used as a highly specific

tight-binding inhibitor of cathepsin 02, as is shown in Example 3. Thus, the propart may be added therapeutically to samples or tissues which contain excess cathepsin 02; for example, in the treatment of bone disorders or tumors, as outlined below.

In one embodiment, the propart of cathepsin 02 is labeled, and used to diagnose, quantify or identify the presence of cathepsin 02 within a sample or tissue.

Additionally, the cathepsin 02 proteins may be used to generate polyclonal and monoclonal antibodies to cathepsin 02 proteins, which are useful as described below. Similarly, the cathepsin 02 proteins can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to purify cathepsin 02 antibodies.

In a preferred embodiment, monoclonal antibodies are generated to the cathepsin 02 protein, using techniques well known in the art. As outlined above, the antibodies may be generated to the full length cathepsin 02 protein, or a portion of the cathepsin 02 protein.

In a preferred embodiment, the antibodies are generated to epitopes unique to the human cathepsin 02 protein; that is, the antibodies show little or no cross- reactivity to antibodies generated to cathepsin 02 proteins from other organisms, such as cathepsins from rabbits or rats.

These antibodies find use in a number of applications. In a preferred embodiment, the antibodies are used to diagnose the presence of cathepsin 02 in a sample or patient. For example, an excess of cathepsin 02 protein, such as may exist in osteoclast related disorders and bone diseases, as well as tumors, may be diagnosed using these antibodies.

Similarly, high levels of cathepsin 02 are associated with certain ovarian or cervical carcinomas, as evidenced by high levels of cathepsin 02 in HeLa cells. Thus, these types of tumors may be detected or diagnosed using the antibodies of the present invention.

The detection of cathepsin 02 will be done using techniques well known in the art; for example, samples such as blood or tissue samples may be obtained from a patient and tested for reactivity with labelled cathepsin 02 antibodies, for example using standard techniques such as RIA and ELISA.

In one embodiment, the antibodies may be directly or indirectly labelled. By "labelled" herein is meant a compound that has at least one element, isotope or chemical compound attached to enable the detection of the compound. In general, labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens; and c) colored or fluorescent dyes. The labels may be incorporated into the compound at any position. Thus, for example, the cathepsin 02 protein antibody may be labelled for detection, or a secondary antibody to the cathepsin 02 protein antibody may be created and labelled.

In one embodiment, the antibodies generated to the cathepsin 02 proteins of the present invention are used to purify or separate cathepsin 02 proteins from a sample. Thus for example, antibodies generated to cathepsin 02 proteins may be coupled, using standard technology, to affinity chromatography columns. These columns can be used to pull out the cathepsin 02 protein from tissue samples.

Recent work has suggested that cysteine proteases may be used as DNA binding transcription factors (Xu et al., supra). In some embodiments, the cathepsin 02 proteins of the present invention may be used as transcription factors.

The parasite Paragonimus westermani was recently shown to express an immunosuppressor with homology to cysteine proteases (Hamajima et al., supra). In fact, the homology to the cathepsin 02 proteins of the present invention is roughly 40%. Thus, in one embodiment, the cathepsin 02 proteins may be useful as immunosuppressors.

In a preferred embodiment, when the cathepsin 02 proteins are to be administered to a human, the cathepsin 02 proteins are human cathepsin 02 proteins. This is therapeutically desirable in order to ensure that undesirable immune reactions to the administered cathepsin 02 are minimized.

The administration of the cathepsin 02 protein of the present invention can be done in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.

The pharmaceutical compositions of the present invention comprise a cathepsin 02 protein in a form suitable for administration to a patient. The pharmaceutical compositions may include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol. Additives are well known in the art, and are used in a variety of formulations.

The pharmaceutical compositions of the present invention are generally administered at therapeutically effective dosages, as can be routinely determined by those in the art.

It is believed that the human cathepsin 02 protein of the invention has characteristics which render the human protein more acceptable than cathepsin

02 proteins from other species for therapeutic purposes. In particular, the antigenicity of cathepsin 02 proteins from other species in humans makes these proteins less acceptable as therapeutic compositions; i.e. cathepsins from other species may elicit undesirable immunological responses in humans.

The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. The references cited herein are incorporated by reference.

EXAMPLES

Example 1

Cloning of Human Cathepsin 02

Unless otherwise specified, all general recombinant DNA techniques followed the methods described in Sambrook et al. (Molecular Cloning, A Laboratory

Manual, Cold Spring Harbor Press, 1989).

Two degenerate PCR primers were designed based on the published sequence of a rabbit osteoclastin gene (Tezuko et al. 1994):

5'-GGA-TAC-GTT-ACN-CCN-GT-3' (SEQ ID NO:8) 5'-GC-CAT-GAG-G/ATA-NCC-3' (SEQ ID NO:9)

These primers were used for screening a human spleen Quick Clone cDNA preparation (Clontech). An amplified 450 base pair fragment was isolated and purified and used as a cDNA probe for screening a human spleen cDNA library ( gtlO from Clontech). 600,000 clones were screened on 20 filters using a technique in which the plaques reform directly on the filter (Woo, Methods

Enzymol. 68:389-395 (1979)). This allows an amplification of the signal from positive plaques allowing for shorter exposure times, thus decreasing background and the visualization of false positives. The filters were washed at moderate stringency conditions: once with 2 x SSC, 0.1%SDS at room temperature for 10 min and once with 2 x SSC, 0.1% SDS at 68 ^* C for 20 min.

Phages from two positive plaques were isolated and cloned into the EcoRI site of pBluescript SK+ vector (Stratagene).

One positive clone was completely sequenced on an ABI sequencer model 373 A; the sequence (SEQ ID NO: 1 ) is shown in Figure 1. Sequence alignments of the protein of human cathepsin 02 (SEQ ID NO:2), human cathepsin S (SEQ ID

NO:4) and human cathepsin L (SEQ ID NO: 5) are shown in Figure 2.

Example 2 Expression of human Cathepsin O2

The human cathepsin 02 cDNA was cloned into the polyhedrin gene of the baculovirus transfer vectors using standard methods. The cDNA encoding the complete open reading frame of the prepro enzyme was inserted into the BgHI and BamHl site of the pVL1392 transfer vector (PharMingen). Recombinant baculoviruses were generated by homologous recombination following co- transfection of the baculovirus transfer vector and linearized AcNPV genomic DNA (PharMingen) into Sβ cells. Following end point dilution human cathepsin

02 expression is measured in a fluorimetric substrate assay, outlined below.

Pure virus (_4cNPVCO2) was obtained by plaque purification. Sf9 cells were grown in Sf900II media (Gibco BRL, Grand Island, NY) to a density of 2 x 10 ⁶ cells ml and infected at a moi of 1. Total cell number and cellular as well as

secreted activity of cathepsin 02 were monitored every 24 h. After 3.5 days the cells were harvested.

The majority of immunoreactive material of about 43kDa was found within the infected cells. In contrast to the single product of 43kDa in the culture medium an additional slight band of 44 kDa was detected in the cellular extract. The higher molecular weight band putatively represents unprocessed preprocathepsin 02 whereas the 43kDa protein putatively is proenzyme. No activity was observed immediately after lysis of the cells nor during autoactivating conditions at 40°C between pH 4.0 and 4.5 in the presence of dithiothreitol using the synthetic substrate Z-FR-MCA at pH 7.5. The increase of an E-64 inhibitable activity under autoactivating conditions and measured at pH 5.5 was assigned to an endogenous Sf9 cysteine protease (unpublished results). No processing of the cathepsin 02 precursor was observed with human cathepsin B incubated at pH' s 4.0 and 5.5 for 2 hours at 37 ^* C (data not shown).

Activation, purification and N-terminal sequencing of recombinant human cathepsin 02:

The intracellular cathepsin 02 was produced within the SF9 cells as an inactive precursor. The enzyme was activated in the cell lysate under reducing and acidic conditions as follows. The Sf9 cells were harvested from the production media by centrifugation at 2,000 x g and were lysed in a Dounce homogenizer. The cell lysate containing the inactive cathepsin 02 precursor was brought up to 100 ml with 100 mM-sodium acetate buffer, pH 3.75 containing 0.5 % triton X-100, 5 mM-dithiothreitol and 2.5 mM-Na^DTA and the pH was adjusted to 4.0. The conversion of the proform into the active enzyme was accomplished by treatment with pepsin. After addition of porcine pepsin (Sigma, St. Louis, MO) at a final concentration of 0.4mg/mL the activation mixture was incubated in a shaker for 90 min at 40 ^* C at 200 rpm. The activation was monitored using

Z-FR-MCA (10 μM) as a fluorogenic substrate measured in 100 mM Tris/HCl buffer, pH 7.5.

The precursor of cathepsin 02 was efficiently transformed into mature active enzyme by treatment with pepsin at pH 4.0. The digest of crude cellular extract or of concentrated culture media supernatant resulted in a time-dependent disappearance of precursor and generation of mature enzyme (29 kD) via an intermediate of 36 kD (Fig. 3). In parallel with this process an increase of E-64 inhibitable activity measured at pH 7.5 was observed.

No activation of the precursor was observed by addition of purified active cathepsin O2 at pH 4.5 (data not shown) indicating that neither a cis nor trans autoactivation of cathepsin O2 within the lysosomes is likely. This contrasts related cysteine proteases such as papain and cathepsin S which exhibit a potential autocatalytic activation pathway (Vernet et al., J. Biol. Chem., 265:1661-1666 (1990), Brδmme et al., J. Biol. Chem. 268:4832-4838 (1993)). A natural activating enzyme of cathepsin O2 within the osteoclast could be the aspartyl protease cathepsin D which is present in osteoclastic lysosomes but secreted at low levels into the resorption lacuna (Goto et al, 1993).

The activated lysate was adjusted to pH 7.0 with 2M Tris base, clarified by centrifugation at 10,000 x g and the supernatant was adjusted to 2.5 M ammonium sulfate at pH 5.5. After centrifugation at 16,000 x g the cleared supernatant was concentrated to 50 mL by ultrafiltration (YM10 Amicon). After additional centrifugation at 10,000 x g, the cleared supernatant was loaded onto butyl Sepharose 4 Fast Flow (Pharmacia, Sweden) and the column was washed with an ammonium sulfate gradient (2.5 M to 0 M in 25 mM acetate buffer, pH 5.5). The activity was eluted at 0 M ammonium sulfate. The pooled and concentrated fractions were than applied to an FPLC Mono S column (Pharmacia, Sweden) and eluted with a linear NaCl gradient (0-2 M) in 20 mM sodium acetate, pH

5.5. Electrophoretically homogeneous cathepsin 02 was eluted at 1.4M NaCl.

The average yield of a IL Sf9 cell culture (appr. 2xl0 ⁹ cells) was approximately 1 mg purified enzyme (Table 1).

Table 1 Purification of recombinant human cathepsin 02*

Assay Total Total Specific Purification Yield protein activity activity factor % mg μMol/min μMol/mg/ min

Crude ^b 800 1,753 2.2 1 100

2.5 M 276 1,412 5.1 2.3 81 (NH ₄ ) ₂ SO ₄ soluble fraction

Butyl 2.9 1,143 394 179 65 Sepharose 4

MonoS 1.1 512 465 211 29

* from 1 L Sf9 culture ^b after activation with pepsin

The purified enzyme was a single chain enzyme and exhibited an apparent molecular weight of 29 kDa in a 4-20 % Tris/Glycine SDS gel under reducing conditions. Treatment with endoglycosidases H and F as well as N-glycosidase F did not result in a shift in the molecular weight which implies that the protease is not gly cosy lated (data not shown). Human cathepsin O2 has two potential glycosylation sites in its mature sequence. However, both sites have either a proline residue consecutive to the asparagine or to the threonine, so that their

use is unlikely. Cathepsin 02 contains furthermore one putative glycosylation site in the propart close to the processing site between the mature enzyme and the propart. Again, no shift in molecular weight of the proenzyme was observed after overnight treatment with endoglycosidases H and F as well as N-glycosidase F.

NH ₂ -terminal sequencing was carried out by automated Edman degradation. N-terminal sequencing of the mature protease revealed the natural processing site for cysteine proteases of the papain family with a proline adjacent to the N-terminal alanine ( TH ₂ -APDSVDYRICKGYVTPVKN) (SEQ ID NO: 10). In contrast, autocatalytically activated cysteine proteases frequently have at their processing site an N-terminal extension of 3 to 6 amino acids from the propart (Brδmme et al. 1993). The calculated molecular mass of mature cathepsin 02 would be 23,495 which seems to be the actual weight of the enzyme. Trypsin (24 kDa) displayed the same apparent molecular weight of 29 kDa when tested under analogous conditions.

Recombinant human cathepsin S was expressed using the baculovirus expression system and purified as described elsewhere (Brδmme and McGrath, unpublished results). Recombinant human cathepsin L was kindly provided by Dr. Mort, Shriner's Hospital for Crippled Children, Montreal, Quebec). All cathepsins used were electrophoretical.y homogeneous and their molarities were determined by active-site titration with E-64 as described by Barrett and Kirschke (1981).

Fluorimetric enzvme assay

Human cathepsin 02 was assayed with a fluorogenic substrate Z-FR-MCA (MCA, methyl coumarylamide) in 100 mM sodium acetate buffer, containing 2.5 mM dithioerythreitol and 2.5 mM EDTA. Initial rates of hydrolysis of the

MCA-substrate are monitored in 1-cm cuvettes at 25°C at an excitation

wavelength at 380 nm and an emission wavelength at 450 nm. The concentration of Z-FR-MCA is 5 μM under standard conditions.

The kinetic constants V^ and K,,, were obtained by non-linear regression analysis using the program Enzfitter (Leatherbarrow, Enzfitter, Elsevier Biosoft, Cambridge, United Kingdom (1987).

The inhibition of cathepsin 02 was assayed at a constant substrate (5 μM Z-FR-MCA) and enzyme concentration (InM) in the presence of different inhibitor concentrations in the substrate assay buffer. Cathepsin 02 was preincubated with the inhibitors for 10 min and the reaction was started with substrate. The residual activity was monitored and percent inhibition was calculated from the uninhibited rate.

Example 3 Cloning and Expression of the propart of cathepsin O2

The propart of human cathepsin 02 was amplified by PCR using standard techniques using the following primers:

5'-CTGGATCCCTGTACCCTGAGGAGATACTG-3' (SEQIDNO:l1) 5'-CTA AGC TTC TAT CTA CCT TCC CATTCT GGGATA-3' (SEQ ID NO:12)

The proregion was expressed in the pTrcHis vector (Invitrogen Corp., San Diego, CA), which contains a series of six histidine residues that function as a metal binding domain in the translated protein. This metal binding domain was used to purify the propart of cathepsin 02 over Invitrogen' s ProBond Resin included in their Xpress system Protein Expression kit. A gel of the purified propart is shown in Figure 10.

The purified propart inhibited the parent enzyme with a K, value of 0.1 nM.

Example 4 Antibodies to human Cathepsin 02 and Immunohistochemistry

Polyclonal antibodies were made in New Zealand white rabbits to the proenzyme of human cathepsin 02. The cDNA encoding the proenzyme was amplified by

PCR from a preparation of its preproenzyme sequence using Pfu DNA polymerase (Promega). The primers used were made to the 5 'end of the proenzyme with an Nhel site and to the 3 'end with a BamHI site. Human cathepsin 02 was cloned and expressed in E.coli (BL21(DE3)) in the pETl lc vector from Novagen. Expression was induced with 0.4 mM IPTG at OD600 = 0.6 and cells were harvested 2 hours after induction. After collection, the expressed proteins were run on Novex 12% Tris-Glycine SDS gels which were Coomassie stained and destained. The proenzyme band of cathepsin 02 which was confirmed by N-terminal sequencing was cut out. The protein was electroeluted from the gel slices and concentrated on a Centriconl 0 which was pretreated with 1 x elution buffer. The antigen was brought up to 1 ml in lxPBS and used for immunization (EL Labs, Soquel, CA).

The antibodies were purified from the whole serum with acetone powder made to an induced culture of BL21(DE3) and by affinity binding to and elution from the antigen on nitrocellulose. The purified antibodies were specific for human procathepsin 02, the propart, and for the mature enzyme, and do not exhibit cross-reactivity with human cathepsins S, L and B in Western Blot analysis at a 1:2000 dilution.

Formalin fixed and paraffin-embedded human tissue sections (Biogenex, San Ramon, CA) were prepared as described previously (Cattoretti et al., 1992) and were stained with control rabbit IgG or affinity purified anticathepsin 02

antibodies using the StrAviGen detection system (Biogenex). Section were counterstained with Mayer's hematoxylin.

Immunostaining of an osteoclastoma revealed an intense specific staining of multinucleated osteoclasts whereas stromal cells displayed no reaction (Fig. 11). Intense immunohistochemical staining of osteoclasts in prenatal human bones was also observed (data not shown). In lung, cathepsin 02 was detected at two sites; first in lung alveolar macrophages (Fig. 11) and second in bronchiolar epithelial cells. Cathepsin 02 was found also in epithelial cells of gastric glands in stomach, of intestinal glands in colon, of proximal and distal tubuli in kidney and in the epithelium of the uterine glands in the endometrium. Furthermore,

Kupfer cells in liver as well as developing sperm cells in testis exhibit a strong staining against cathepsin 02. A more uniform staining was observed in the cortex of the adrenal, in ovary and placenta (Fig. 11).

Similarly, polyclonal antibodies against the electrophoretically homogenous propart of human cathepsin 02 are produced in New Zealand white rabbits, and monoclonal antibodies to the propart, procathepsin 02 and mature cathepsin 02 by standard techniques.

Example 5 Characterization of human cathepsin 02

The following experiments were done with the partially purified human cathepsin

02 of example 2.

pH activity profile and pH-stability of recombinant human cathepsin 02 The pH-stability of cathepsin 02 was determined by incubation of the active protease at different pH values in presence of 5 mM dithioerythreitol and 5 mM

EDTA at 25 °C. The residual activity was measured in time intervals using the above described fluorimetric substrate assay.

Initial rates of substrate hydrolysis were monitored as described above. The pH activity profile of human cathepsin 02 was obtained at 1 μM substrate (Z-FR-MCA) concentration ([S] «K _tn where the initial rate v ₀ is directly proportional to the k^/K,,, value). The following buffers were used for the pH activity profile: 100 mM sodium citrate (pH 2.8-5.6) and 100 mM sodium phosphate (pH 5.8-8.0). All buffers contained 1 mM EDTA and 0.4 M NaCl to minimize the variation in ionic strength. A three protonation model (Khouri et al., Biochem. 30:8929-8936 (1991)) was used for least square regression analysis of the pH activity data. The data were fitted to the following equation.

(KJK _o * - <k _β /KJ ([HT/K ₁ + 1 + K ₂ /[H ⁺ ])

The pH stability of cathepsins O2, S and L was studied at three different pH values. Recombinant human cathepsins O2, S and L were incubated at 37°C in 100 mM sodium acetate buffer, pH 5.5, in 100 mM potassium phosphate buffer, pH 6.5 and in 100 mM Tris/HCl, pH 7.5 containing 5 mM dithiothreitol and 2.5 mM EDTA. Incubating for 0.5, 1, 2 and 4 hours, the activity remaining was determined using 5 μM Z-FR-MCA for cathepsin O2 (100 mM potassium phosphate buffer, pH 6.5) and cathepsin L (100 mM sodium acetate buffer, pH 5.5) and 5 μM Z-WR-MCA for cathepsin S (100 mM potassium phosphate buffer, pH 6.5).

Profiles of pH activity are sensitive measures of enzymatic functional and structural integrity. A comparison of pH profiles from different but related proteases reveals differences in intrinsic activity and stability of these proteases. Human cathepsin O2 displays a bell-shaped pH profile with flanking pK values of 4.0 and 8.13 (Table 2; Fig. 5).

Table 2 pK values of pH activity profile of recombinant human cathepsin 02 in comparison with pK values described for cathepsins S and L and papain

Protease PKΓ pK, pK ₂ pH optimum* human 3.43±0.05 4.00±0.02 8.1310.01 6.1 cathepsin 02 human 4.49±0.03 7.8210.03 6.1 cathepsin S ^b human 3.33±0.14 4.22±0.28 6.9510.09 5.6 cathepsin L ^b papain ⁰ 3.58±0.29 4.5410.29 8.4510.02 6.5

^• calculated from (pK, + pK ₂ )/2 ^b from Brδmme et al., 1993, supra ^c from Khouri et al., 1991, supra

The pH optimum of Human cathepsin O2 was between 6.0 and 6.5 and comparable to that observed for cathepsin S (Brδmme et al., supra, 1993). The width of the pH profile, which mirrors the stability of the ion-pair (Menard et al., Biochem. 30:5531-5538 (1991)), is 4.15 for cathepsin O2 but only 3.35 for cathepsin S (Brδmme et al., 1993, supra). This parameter for human cathepsin O2 is more similar to that observed for the very stable papain which displays a profile width of 3.91 (Khouri et al., supra, 1991).

Human cathepsin O2 was more stable than cathepsin L at slightly acidic to neutral pH values but less stable than cathepsin S (Table 3).

Table 3 pH stability at 37°C of recombinant human cathepsin 02 in comparison with recombinant human cathepsins S and L

Protease Incubation Residual activity (%) time pH 5.5 pH 6.5 pH 7.5 hr cathepsin 02 0.5 91 85 11 1 88 49 0

2 70 22 0

4 52 15 0 cathepsin S 0.5 100 100 91 1 95 100 72

2 92 94 61

4 83 71 60 cathepsin L 0.5 87 12 0 1 78 3 0

2 71 0 0

4 51 0 0

Approximately 50 % of the cathepsin O2 activity remained after 1 hour at 37°C and pH 6.5 whereas essentially no cathepsin L activity could be observed under these conditions.

However, it must be considered that the pH stability was determined without substrate protection which usually increases the pH stability. In the ³ H elastin degradation assay with cathepsin O2 an increase of solubilized ³ H fragments was still observed after 2 hours at pH 7.0.

Inhibitor profile of recombinant human cathepsin O2

The efficacy of protease class specific inhibitors to inhibit cathepsin O2 was determined by adding the inhibitor to the purified enzyme in a fluorimetric enzyme assay (described above).

Human cathepsin 02 displays a typical inhibitor profile of a cysteine protease. It is inhibited by cysteine protease inhibitors and by inhibitors of both cysteine and serine proteases (Table 4). At concentrations above 0.1 μM, peptide aldehydes, diazomethanes, E-64 and chicken cystatin completely inhibit enzyme activity. On the other hand, specific serine and aspartic protease inhibitors did not affect enzyme activity. No effect of EDTA at a concentration of 4mM was observed on the activity of cathepsin 02. At higher concentrations (>5mM) a partial non-specific inhibition was observed.

Table 4

Inhibitor profile of recombinant human cathepsin 02

inhibitor [inhibitor] % inhibition serine protease inhibitors PMSF 1 mM 0

Befablock 0.2 mM 0

DCl 0.1 mM 0 serine/cysteine protease inhibitors leupeptin 0.05 μM 85 chymostatin 0.05 μM 64 calpeptin 0.1 μM 100 aspartate protease pepstatin 0.1 μM 0 inhibitor metallo-protease inhibitor EDTA 4 mM 0 cysteine protease inhibitor iodo acetate 50 μM 60

Z-FF-CHN ₂ 0.1 μM 90

Z-FA-CHN ₂ 0.1 μM 100

E-64 0.1 μM 100 chicken cysteine 0.1 μM 100

Cathepsin O2 activity is only inhibited by cysteine protease specific inhibitors.

Substrate Specificity of recombinant human cathepsin 02

The substrate specificity towards synthetic substrates was determined using the above described substrate assay.

The S ₂ P ₂ specificity of human cathepsin 02 was characterized using synthetic substrates of the type Z-X-R-MCA with X equal to F, L, V or R. The S ₂ subsite pocket of cysteine proteases is structurally well defined and determines the primary specificity of this protease class. For example, cathepsin B contains a glutamate (E245) residue at the bottom of the S ₂ subsite pocket which favours the binding of basic residues like arginine. This glutamate residue is replaced by neutral residues in all other known human cathepsins resulting in a very low hydrolysis rate of the Z-R-R-MCA substrate. Cathepsin 02 contains a leucine residue in position 205 which makes Z-R-R-MCA a very poor substrate (Fig. 6). The specificity of cathepsin 02 towards P ₂ residues resembles that of cathepsin S. Both enzymes prefer a leucine over a phenylalanine in this position while cathepsin L is characterized by an inverse specificity (Table 5, Fig. 6).

Valine in position P ₂ is relatively well accepted by cathepsin 02, whereas the presence of this beta-branched residue in P ₂ results in a poor substrate for cathepsins L, S and B.

Table 5

Kinetic parameters for the Z-X-R-MCA catalyzed hydrolysis by recombinant human cathepsin 02

Substrate ys- ¹ ) κ-(μ ) k _ca K M- ¹ )

Z-FR-MCA 0.9010.20 7.513.4 120,000

Z-LR-MCA 0.9810.39 3.810.8 257,900

Z-VR-MCA 1.0610.16 13.115.6 80,900

Z-RR-MCA 0.000510.0002 2314 22

Z-WR-MCA 0.0110.004 18.511.5 540

Z-LLR-MCA 0.0210.008 0.410.1 50,000

For the calculation of the kinetic parameters k^ and K,,, the initial rates were obtained typically at 9-11 different substrate concentrations, and the results are fitted to equation (1). The enzyme concentration is determined by active site titration with E-64 (Kinder et al., Biochem. J. 201:367-372 (1982)).

kcat x E0 x [S] v = equation (1)

(Km + [S])

The catalytic efficiency (%, K^) of cathepsin O2 towards dipeptide substrates was comparable to that of cathepsins S and B, but was approximately one order of magnitude lower than that of cathepsin L. Interestingly, the K,., values for cathepsin O2 were comparable to those determined for cathepsin L. The ¥^ value reflects to some extent the affinity of the substrates for the protease. This trend is even more obvious for the tripeptide substrate, Z-LLR-MCA, which displays a K,,, value as low as 4 x 10 ^"7 M (Table 5). However, in contrast to cathepsins S and L, the k^ values are almost two orders of magnitude lower for cathepsin O2, which may reflect non-productive binding.

Activities of recombinant human cathepsin 02 towards extracellular matrix proteins

[ ³ H] elastin was prepared as described (Banda et al, Methods Enzymol 144, 288-305 (1987)) and had a specific activity of 113,000 cpm/mg protein. Elastin (2mg) was incubated in 1 ml buffer containing 2.5 mM dithiothreitol, 2.5 mM

EDTA and 0.05 % Triton X-100 for the cathepsin O2, S and L assays. Aliquots were withdrawn after 10, 20, 30, 50, 90, 120 and 180 min, centrifuged for 1 min at 14,000 x g and counted in a 24- well plate containing scintillation fluid with Liquid Scintillation counter (1450 Microbeta Plus, Wallac/Pharmacia). Concentrations of human cathepsins O2, S and L and bovine elastase in the elastin degradation assay were 65 nM, 28 nM, 80 nM and 80 nM, respectively. To determine the pH effect on protease activity the digests were carried out at pH 4.5 and 5.5 (100 mM sodium acetate, 2.5 mM each dithiothreitol and EDTA, 0.05% Triton X-100), and at pH 7.0 (100 mM Tris/HCl, 2.5 mM each of dithiothreitol and EDTA, 0.05% Triton X-100). Pancreatic bovine elastase

(Boehringer, Mannheim, IN) was assayed under the same conditions except that neither dithiothreitol nor EDTA was added to the incubation mixture.

Maximal activity was observed at pH 5.5. Cathepsin O2 has between pH 4.5 to 7.0 an elastinolytic activity which is 1.7 to 3.5 times higher than that of cathepsin S. Its elastinolytic activity at the pH optimum of cathepsin L (pH 5.5) and at neutral pH was almost 9-times and 2.4-times higher when compared to cathepsin L and pancreatic elastase, respectively (Fig. 7). The values determined for cathepsin L and S are in good accordance with published data (Kirschke et al., in: Proteolysis and Protein Turnover (Bond, J. S. and Barrett, A.J., eds.) pp 33-37, Portland Press, London and Chapel Hill (1993), Kirschke and Wiederanders,

Methods Enzymol 244, 500-511 (1994)).

Soluble calfskin Type I collagen was diluted to 0.4 mg/ml into lOOmM-sodium acetate buffer, pH 4.5, 5.0, 5.5, in lOOmM-potassium phosphate buffer, pH 6.0,

6.5 and lOOmM-Tris/HCl, pH 7.0 containing 2mM-dithiotreitol/2mM-EDTA. Human cathepsins 02, S and L and bovine trypsin (Sigma) were incubated at concentrations of 100 nM enzyme concentration for 10 hours at 28°C. To measure the gelatinase activity of cathepsins 02 and S, Type I collagen was heated for 10 min at 70°C prior to incubation with the proteases. In the presence of InM proteases the reaction mix was incubated for 30 min at 28°C. The samples were subjected to SDS polyacrylamide electrophoresis using 4-20 % Tris-glycine gels (Novex, San Diego, CA).

Cathepsin O2 extensively degraded Type I collagen between pH 5.0 and 6.0 at 28°C whereas the degradation at pH 4.5 and pH 7.0 is much less pronounced

(Fig. 9a). The primary cleavage seemed to occur in the telopeptide region since the alpha monomers released from the beta and gamma components were slightly smaller. Additionally cleavage may also occur within the alpha monomers. It is yet unclear whether the cleavage occurrs in the intact helical region or in unraveled alpha monomers. Major fragments of Type I collagen observed after cathepsin O2 action had the size of 70-80 kDa. Cathepsin L also cleaved in the telopeptide region, but essentially no small molecular weight fragments were detected. The effective pH range for the collagenolytic activity of cathepsin L is more acidic when compared with that observed for cathepsin O2 (between pH 4.0 and 5.5). Cathepsin S seemed to reveal only a very weak collagenolytic activity. In contrast, tissue collagenases cleave the alpha monomers into 3/4 and 1/4 fragments (Gross and Nagai, Proc. Natl. Acad Sci. U.S.A. 54, 1197-1204 (1965)). No degradation of Type I collagen was observed with trypsin at equal enzyme concentration compared to cathepsin O2 showing that the integrity of the triple helix of the collagen used was not impaired (data not shown).

In addition to its collagenase activity cathepsin 02 displayed a powerful gelatinase activity. At 0.1 nM concentration of the enzyme, denatured collagen was totally degraded within 30 min within a pH range of 5.0 to 7.0.

In contrast, cathepsin L displayed its gelatinase activity only in the pH range between 4.5-5.5 (Fig.6 b). Cathepsin S was active between pH 4.0 and 7.0, but displayed a significant weaker activity than the cathepsins 02 and L.

Relative elastinolytic activities of cathepsins compared with the bovine pancreatic elastase

Protease pH 4.5 pH 5.5 pH 7.0 mg min/μmol mg/min/μmol mg/min/μmol enzyme enzyme enzyme cathepsin 02 245 286 170 cathepsin L 18 32 0 cathepsin S 146 102 55 pancreatic 8 18 79 elastase

Tissue distribution of human cathepsin 02 on the message level

The tissue distribution of the message level of human cathepsins 02, L and S was determined by Northern blotting using cDNA probes of the appropriate human enzymes. The probes were approximately 450 base pairs long and stretched over the region coding for the residues between the active site residues cysteine-25 (according to the papain numbering) and asparagine-175. Figure 8 shows Northern blots for human cathepsin 02. As shown in Figure 8, message levels in human osteoclastoma preparations exhibit a manyfold higher level of expression of cathepsin 02 than cathepsin L.

The tissue distribution of human cathepsin 02 mRNA showed some similarities to cathepsin L, however, its tissue concentration seemed significantly lower in most of the organs (heart, placenta, lung, pancreas and kidney). On the other hand human cathepsin 02 displayed remarkable differences in its distribution

in human tissues and cell lines when compared with the human cathepsins L and S. Cathepsin 02 showed high levels of transcription in ovary, small intestine and colon but no message in liver, which is rich for cathepsin L. It was also found in HeLa cells.

Tissue and cell line distribution (Northern Blotting)

Tissue HCATO HCATL HCATS heart XX XXXX - brain - X - placenta XX XXXX XX lung XX XXX XXX liver - XXXX XX skeletal muscle XX XX - kidney X XXXX - pancreas X XX - spleen X - X thymus X X - prostate X X - testis X XX - ovary XXX X - small intestine XX - - colon XXX X - leukocytes - - XXX promyelocyt. leukemia - - X HL-60

HeLa S3 XX X X lymphoblasϋeukemia - XX X MOLT-4

Burkitt's lymphoma Raji - - X colect. adenocarcinoma - X - lung carcinoma A549 - XXXX X melanoma G361 - xxxxx -

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Khepri Pharmaceuticals, Inc. (ii) TITLE OF INVENTION: CATHEPSIN 02 PROTEASE (iii) NUMBER OF SEQUENCES: 12

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Flehr, Hohbach, Test, Albritton & Herbert

(B) STREET: Four Embarcadero Center, Suite 3400

(C) CITY: San Francisco

(D) STATE: California

(E) COUNTRY: United States

(F) ZIP: 94111-4187

(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 #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT/US95

(B) FILING DATE: 26-OCT-1995

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US UNKNOWN

(B) FILING DATE: 02-OCT-1995

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/330,121

(B) FILING DATE: 27-OCT-1994

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Silva, Robin M.

(B) REGISTRATION NUMBER: 38,304

(C) REFERENCE/DOCKET NUMBER: FP-60261-l-PC/DJB/RMS

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (415) 781-1989

(B) TELEFAX: (415) 398-3249

(C) TELEX: 910 277299

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1482 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 142..1128

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GCGCACTCAC AGTCGCAACC TTTCCCCTTC CTGACTTCCC GCTGACTTCC GCAATCCCGA 60

TGGAATAAAT CTAGCACCCC TGATGGTGTG CCCACACTTT GCTGCCGAAA CGAAGCCAGA 120

CAACAGATTT CCATCAGCAG C ATG TGG GGG CTC AAG GTT CTG CTG CTA CCT 171

Met Trp Gly Leu Lys Val Leu Leu Leu Pro 1 5 10

GTG GTG AGC TTT GCT CTG TAC CCT GAG GAG ATA CTG GAC ACC CAC TGG 219 Val Val Ser Phe Ala Leu Tyr Pro Glu Glu He Leu Asp Thr His Trp 15 20 25

GAG CTA TGG AAG AAG ACC CAC AGG AAG CAA TAT AAC AAC AAG GTG GAT 267 Glu Leu Trp Lys Lys Thr His Arg Lys Gin Tyr Asn Asn Lys Val Asp 30 35 40

GAA ATC TCT CGG CGT TTA ATT TGG GAA AAA AAC CTG AAG TAT ATT TCC 315 Glu He Ser Arg Arg Leu He Trp Glu Lys Asn Leu Lys Tyr He Ser 45 50 55

ATC CAT AAC CTT GAG GCT TCT CTT GGT GTC CAT ACA TAT GAA CTG GCT 363 He His Asn Leu Glu Ala Ser Leu Gly Val His Thr Tyr Glu Leu Ala 60 65 70

ATG AAC CAC CTG GGG GAC ATG ACC AGT GAA GAG GTG GTT CAG AAG ATG 411 Met Asn His Leu Gly Asp Met Thr Ser Glu Glu Val Val Gin Lys Met 75 80 85 90

ACT GGA CTC AAA GTA CCC CTG TCT CAT TCC CGC AGT AAT GAC ACC CTT 459 Thr Gly Leu Lys Val Pro Leu Ser His Ser Arg Ser Asn Asp Thr Leu 95 100 105

TAT ATC CCA GAA TGG GAA GGT AGA GCC CCA GAC TCT GTC GAC TAT CGA 507 Tyr He Pro Glu Trp Glu Gly Arg Ala Pro Asp Ser Val Asp Tyr Arg 110 115 120

AAG AAA GGA TAT GTT ACT CCT GTC AAA AAT CAG GGT CAG TGT GGT TCC 555 Lys Lys Gly Tyr Val Thr Pro Val Lys Asn Gin Gly Gin Cys Gly Ser 125 130 135

TGT TGG GCT TTT AGC TCT GTG GGT GCC CTG GAG GGC CAA CTC AAG AAG 603 Cys Trp Ala Phe Ser Ser Val Gly Ala Leu Glu Gly Gin Leu Lys Lys 140 145 150

AAA ACT GGC AAA CTC TTA AAT CTG AGT CCC CAG AAC CTA GTG GAT TGT 651 Lys Thr Gly Lys Leu Leu Asn Leu Ser Pro Gin Asn Leu Val Asp Cys 155 160 165 170

GTG TCT GAG AAT GAT GGC TGT GGA GGG GGC TAC ATG ACC AAT GCC TTC 699 Val Ser Glu Asn Asp Gly Cys Gly Gly Gly Tyr Met Thr Asn Ala Phe 175 180 185

CAA TAT GTG CAG AAG AAC ^GG GGT ATT GAC TCT GAA GAT GCC TAC CCA 747 Gin Tyr Val Gin Lys Asn Arg Gly He Asp Ser Glu Asp Ala Tyr Pro 190 195 200

TAT GTG GGA CAG GAA GAG AGT TGT ATG TAC AAC CCA ACA GGC AAG GCA 795 Tyr Val Gly Gin Glu Glu Ser Cys Met Tyr Asn Pro Thr Gly Lys Ala 205 210 215

GCT AAA TGC AGA GGG TAC AGA GAG ATC CCC GAG GGG AAT GAG AAA GCC 843 Ala Lys Cys Arg Gly Tyr Arg Glu He Pro Glu Gly Asn Glu Lys Ala 220 225 230

CTG AAG AGG GCA GTG GCC CGA GTG GGA CCT GTC TCT GTG GCC ATT GAT 891 Leu Lys Arg Ala Val Ala Arg Val Gly Pro Val Ser Val Ala He Asp 235 240 245 250

GCA AGC CTG ACC TCC TTC CAG TTT TAC AGC AAA GGT GTG TAT TAT GAT 939 Ala Ser Leu Thr Ser Phe Gin Phe Tyr Ser Lys Gly Val Tyr Tyr Asp 255 260 265

GAA AGC TGC AAT AGC GAT AAT CTG AAC CAT GCG GTT TTG GCA GTG GGA 987 Glu Ser Cys Asn Ser Asp Asn Leu Asn His Ala Val Leu Ala Val Gly 270 275 280

TAT GGA ATC CAG AAG GGA AAC AAG CAC TGG ATA ATT AAA AAC AGC TGG 1035 Tyr Gly He Gin Lys Gly Asn Lys His Trp He He Lys Asn Ser Trp 285 290 295

GGA GAA AAC TGG GGA AAC AAA GGA TAT ATC CTC ATG GCT CGA AAT AAG 1083 Gly Glu Asn Trp Gly Asn Lys Gly Tyr He Leu Met Ala Arg Asn Lys 300 305 310

AAC AAC GCC TGT GGC ATT GCC AAC CTG GCC AGC TTC CCC AAG ATG 1128

Asn Asn Ala Cys Gly He Ala Asn Leu Ala Ser Phe Pro Lys Met 315 320 325

TGACTCCAGC CAGCCAAATC CATCCTGCTC TTCCATTTCT TCCACGATGG TGCAGTGTAA 1188

CGATGCACTT TGGAAGGGAG TTGGTGTGCT ATTTTTGAAG CAGATGTGGT GATACTGAGA 1248

TTGTCTGTTC AGTTTCCCCA TTTGTTTGTG CTTCAAATGA TCCTTCCTAC TTTGCTTCTC 1308

TCCACCCATG ACCTTTTTCA CTGTGGCCAT CAGGACTTTC CCTGACAGCT GTGTACTCTT 1368

AGGCTAAGAG ATGTGACTAC AGCCTGCCCC TGACTGTGTT GTCCCAGGGC TGATGCTGTA 1428

CAGGTACAGG CTGGAGATTT TCACATAGGT TAGATTCTCA TTCACGGGAC CCGG 1482

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 329 amino acids

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

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Trp Gly Leu Lys Val Leu Leu Leu Pro Val Val Ser Phe Ala Leu 1 5 10 15

Tyr Pro Glu Glu He Leu Asp Thr His Trp Glu Leu Trp Lys Lys Thr

20 25 -- — - 30

His Arg Lys Gin Tyr Asn Asn Lys Val Asp Glu He Ser Arg Arg Leu 35 40 45

He Trp Glu Lys Asn Leu Lys Tyr He Ser He His Asn Leu Glu Ala 50 55 60

Ser Leu Gly Val His Thr Tyr Glu Leu Ala Met Asn His Leu Gly Asp 65 70 75 80

Met Thr Ser Glu Glu Val Val Gin Lys Met Thr Gly Leu Lys Val Pro 85 90 95

Leu Ser His Ser Arg Ser Asn Asp Thr Leu Tyr He Pro Glu Trp Glu 100 105 110

Gly Arg Ala Pro Asp Ser Val Asp Tyr Arg Lys Lys Gly Tyr Val Thr 115 120 125

Pro Val Lys Asn Gin Gly Gin Cys Gly Ser Cys Trp Ala Phe Ser Ser 130 135 140

Val Gly Ala Leu Glu Gly Gin Leu Lys Lys Lys Thr Gly Lys Leu Leu 145 150 155 160

Asn Leu Ser Pro Gin Asn Leu Val Asp Cys Val Ser Glu Asn Asp Gly 165 170 175

Cys Gly Gly Gly Tyr Met Thr Asn Ala Phe Gin Tyr Val Gin Lys Asn 180 185 190

Arg Gly He Asp Ser Glu Asp Ala Tyr Pro Tyr Val Gly Gin Glu Glu 195 200 205

Ser Cys Met Tyr Asn Pro Thr Gly Lys Ala Ala Lys Cys Arg Gly Tyr 210 215 220

Arg Glu He Pro Glu Gly Asn Glu Lys Ala Leu Lys Arg Ala Val Ala 225 230 235 240

Arg Val Gly Pro Val Ser Val Ala He Asp Ala Ser Leu Thr Ser Phe 245 250 255

Gin Phe Tyr Ser Lys Gly Val Tyr Tyr Asp Glu Ser Cys Asn Ser Asp 260 265 270

Asn Leu Asn His Ala Val Leu Ala Val Gly Tyr Gly He Gin Lys Gly 275 280 285

Asn Lys His Trp He He Lys Asn Ser Trp Gly Glu Asn Trp Gly Asn 290 295 300

Lys Gly Tyr He Leu Met Ala Arg Asn Lys Asn Asn Ala Cys Gly He 305 310 315 320

Ala Asn Leu Ala Ser Phe Pro Lys Met 325

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 329 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Met Trp Gly Leu Lys Val Leu Leu Leu Pro Val Val Ser Phe Ala Leu 1 5 10 15

His Pro Glu Glu He Leu Asp Thr Gin Trp Glu Leu Trp Lys Lys Thr 20 25 30

Tyr Ser Lys Gin Tyr Asn Ser Lys Val Asp Glu He Ser Arg Arg Leu 35 40 45

He Trp Glu Lys Asn Leu Lys His He Ser He His Asn Leu Glu Ala 50 55 60

Ser Leu Gly Val His Thr Tyr Glu Leu Ala Met Asn His Leu Gly Asp 65 70 75 80

Met Thr Ser Glu Glu Val Val Gin Lys Met Thr Gly Leu Lys Val Pro 85 90 95

Pro Ser Arg Ser His Ser Asn Asp Thr Leu Tyr He Pro Asp Trp Glu 100 105 110

Gly Arg Thr Pro Asp Ser He Asp Tyr Arg Lys Lys Gly Tyr Val Thr 115 120 125

Pro Val Lys Asn Gin Gly Gin Cys Gly Ser Cys Trp Ala Phe Ser Ser 130 135 140

Val Gly Ala Leu Glu Gly Gin Leu Lys Lys Lys Thr Gly Lys Leu Leu 145 150 155 160

Asn Leu Ser Pro Gin Asn Leu Val Asp Cys Val Ser Glu Asn Tyr Gly 165 170 175

Cys Gly Gly Gly Tyr Met Thr Asn Ala Phe Gin Tyr Val Gin Arg Asn 180 185 190

Arg Gly He Asp Ser Glu Asp Ala Tyr Pro Tyr Val Gly Gin Asp Glu 195 200 205

Ser Cys Met Tyr Asn Pro Thr Gly Lys Ala Ala Lys Cys Arg Gly Tyr 210 215 220

Arg Glu He Pro Glu Gly Asn Glu Lys Ala Leu Lys Arg Ala Val Ala 225 230 235 240

Arg Val Gly Pro Val Ser Val Ala He Asp Ala Ser Leu Thr Ser Phe 245 250 255

Gin Phe Tyr Ser Lys Gly Val Tyr Tyr Asp Glu Asn Cys Ser Ser Asp 260 265 270

Asn Val Asn His Ala Val Leu Ala Val Gly Tyr Gly He Gin Lys Gly 275 280 285

Asn Lys His Trp He He Lys Asn Ser Trp Gly Glu Ser Trp Gly Asn 290 295 300

Lys Gly Tyr He Leu Met Ala Arg Asn Lys Asn Asn Ala Cys Gly He 305 310 315 320

Ala Asn Leu Ala Ser Phe Pro Lys Met 325

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 331 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

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

Met Lys Arg Leu Val Cys Val Leu Leu Val Cys Ser Ser Ala Val Ala 1 5 10 15

Gin Leu His Lys Asp Pro Thr Leu Asp His His Trp His Leu Trp Lys 20 25 30

Lys Thr Tyr Gly Lys Gin Tyr Lys Glu Lys Asn Glu Glu Ala Val Arg 35 40 45

Arg Leu He Trp Glu Lys Asn Leu Lys Phe Val Met Leu His Asn Leu 50 55 60

Glu His Ser Met Gly Met His Ser Tyr Asp Leu Gly Met Asn His Leu 65 70 75 80

Gly Asp Met Thr Ser Glu Glu Val Met Ser Leu Met Ser Ser Leu Arg 85 90 95

Val Pro Ser Gin Trp Gin Arg Asn He Thr Tyr Lys Ser Asn Pro Asn 100 105 110

Arg He Leu Pro Asp Ser Val Asp Trp Arg Glu Lys Gly Cys Val Thr 115 120 125

Glu Val Lys Tyr Gin Gly Ser Cys Gly Ala Cys Trp Ala Phe Ser Ala 130 135 140

Val Gly Ala Leu Glu Ala Gin Leu Lys Leu Lys Thr Gly Lys Leu Val 145 150 155 160

Ser Leu Ser Ala Gin Asn Leu Val Asp Cys Ser Thr Glu Lys Tyr Gly 165 170 175

Asn Lys Gly Cys Asn Gly Gly Phe Met Thr Thr Ala Phe Gin Tyr He 180 185 190

He Asp Asn Lys Gly He Asp Ser Asp Ala Ser Tyr Pro Tyr Lys Ala 195 200 205

Met Asp Gin Lys Cys Gin Tyr Asp Ser Lys Tyr Arg Ala Ala Thr Cys 210 215 220

Ser Lys Tyr Thr Glu Leu Pro Tyr Gly Arg Glu Val Asp Leu Lys Glu 225 230 235 240

Ala Val Ala Asn Lys Gly Pro Val Ser Val Gly Val Asp Ala Arg His 245 250 255

Pro Ser Phe Phe Leu Tyr Arg Ser Gly Val Tyr Tyr Glu Pro Ser Cys 260 265 270

Thr Gin Asn Val Asn His Gly Val Leu Val Val Gly Tyr Gly Asp Leu 275 280 285

Asn Gly Lys Glu Tyr Trp Leu Val Lys Asn Ser Trp Gly His Asn Phe 290 295 300

Gly Glu Glu Gly Tyr He Arg Met Ala Arg Asn Lys Gly Asn His Cys 305 310 315 320

Gly He Ala Ser Phe Pro Ser Tyr Pro Glu He 325 330

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 333 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

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

Met Asn Pro Thr Leu He Leu Ala Ala Phe Cys Leu Gly He Ala Ser 1 5 10 15

Ala Thr Leu Thr Phe Asp His Ser Leu Glu Ala Gin Trp Thr Lys Trp 20 25 30

Lys Ala Met His Asn Arg Leu Tyr Gly Met Asn Glu Glu Gly Trp Arg 35 40 45

Arg Ala Val Trp Glu Lys Asn Met Lys Met He Glu Leu His Asn Gin 50 55 60

Glu Tyr Arg Glu Gly Lys His Ser Phe Thr Met Ala Met Asn Ala Phe 65 70 75 80

Gly Asp Met Thr Ser Glu Glu Phe Arg Gin Val Met Asn Gly Phe Gin 85 90 95

Asn Arg Lys Pro Arg Lys Gly Lys Val Phe Gin Glu Pro Leu Phe Tyr 100 105 110

Glu Ala Pro Arg Ser Val Asp Trp Arg Glu Lys Gly Tyr Val Thr Pro 115 120 125

Val Lys Asn Gin Gly Gin Cys Gly Ser Cys Trp Ala Phe Ser Ala Thr 130 135 140

Gly Ala Leu Glu Gly Gin Met Phe Arg Lys Thr Gly Arg Leu He Ser 145 150 155 160

Leu Ser Glu Gin Asn Leu Val Asp Cys Ser Gly Pro Gin Gly Asn Glu 165 170 175

Gly Cys Asn Gly Gly Leu Met Asp Tyr Ala Phe Gin Tyr Val Gin Asp 180 185 190

Asn Gly Gly Leu Asp Ser Glu Glu Ser Tyr Pro Tyr Glu Ala Thr Glu 195 200 205

Glu Ser Cys Lys Tyr Asn Pro Lys Tyr Ser Val Ala Asn Asp Thr Gly 210 215 220

Phe Val Asp He Pro Lys Gin Glu Lys Ala Leu Met Lys Ala Val Ala 225 230 235 240

Thr Val Gly Pro He Ser Val Ala He Asp Ala Gly His Glu Ser Phe 245 250 255

Leu Phe Tyr Lys Glu Gly He Tyr Phe Glu Pro Asp Cys Ser Ser Glu 260 265 270

Asp Met Asp His Gly Val Leu Val Val Gly Tyr Gly Phe Glu Ser Thr 275 280 285

55

Glu Ser Asp Asn Asn Lys Tyr Trp Leu Val Lys Asn Ser Trp Gly Glu 290 295 300

Glu Trp Gly Met Gly Gly Tyr Val Lys Met Ala Lys Asp Arg Arg Asn 305 310 315 320

His Cys Gly He Ala Ser Ala Ala Ser Tyr Pro Thr Val 325 330

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 335 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

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

Met Trp Ala Thr Leu Pro Leu Leu Cys Ala Gly Ala Trp Leu Leu Cys 1 5 10 15

Val Pro Val Cys Gly Ala Ala Glu Leu Cys Val Asn Ser Leu Glu Lys 20 25 30

Phe His Phe Lys Ser Trp Met Ser Lys His Arg Lys Thr Tyr Ser Thr 35 40 45

Glu Glu Tyr His His Arg Leu Gin Thr Phe Ala Ser Asn Trp Arg Lys 50 55 60

He Asn Ala His Asn Asn Gly Asn His Thr Phe Lys Met Ala Leu Asn 65 70 75 80

Gin Phe Ser Asp Met Ser Phe Ala Glu He Lys His Lys Tyr Leu Trp 85 90 95

Ser Glu Pro Gin Asn Cys Ser Ala Thr Lys Ser Asn Tyr Leu Arg Gly 100 105 110

Thr Gly Pro Tyr Pro Pro Ser Val Asp Trp Arg Lys Lys Gly Asn Phe 115 120 125

Val Ser Pro Val Lys Asn Gin Gly Ala Cys Gly Ser Cys Trp Thr Phe 130 135 140

Ser Thr Thr Gly Ala Leu Glu Ser Ala He Ala He Ala Thr Gly Lys 145 150 155 160

Met Leu Ser Leu Ala Glu Gin Gin Leu Val Asp Cys Ala Gin Asp Phe 165 170 175

Asn Asn Tyr Gly Cys Gin Gly Gly Leu Pro Ser Gin Ala Phe Glu Tyr 180 185 190

He Leu Tyr Asn Lys Gly He Met Gly Glu Asp Thr Tyr Pro Tyr Gin 195 200 205

Gly Lys Asp Gly Tyr Cys Lys Phe Gin Pro Gly Lys Ala He Gly Phe 210 215 220

Val Lys Asp Val Ala Asn He Thr He Tyr Asp Glu Glu Ala Met Val 225 230 235 240

Glu Ala Val Ala Leu Tyr Asn Pro Val Ser Phe Ala Phe Glu Val Thr 245 250 255

Gin Asp Phe Met Met Tyr Arg Thr Gly He Tyr Ser Ser Thr Ser Cys 260 265 270

His Lys Thr Pro Asp Lys Val Asn His Ala Val Leu Ala Val Gly Tyr 275 280 285

Gly Glu Lys Asn Gly He Pro Tyr Trp He Val Lys Asn Ser Trp Gly 290 295 300

Pro Gin Trp Gly Met Asn Gly Tyr Phe Leu He Glu Arg Gly Lys Asn 305 310 315 320

Met Cys Gly Leu Ala Ala Cys Ala Ser Tyr Pro He Pro Leu Val 325 330 335

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 339 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

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

Met Trp Gin Leu Trp Ala Ser Leu Cys Cys Leu Leu Val Leu Ala Asn 1 5 10 15

Ala Arg Ser Arg Pro Ser Phe His Pro Val Ser Asp Glu Leu Val Asn 20 25 30

Tyr Val Asn Lys Arg Asn Thr Thr Trp Gin Ala Gly His Asn Phe Tyr 35 40 45

Asn Val Asp Met Ser Tyr Leu Lys Arg Leu Cys Gly Thr Phe Leu Gly 50 55 60

Gly Pro Lys Pro Pro Gin Arg Val Met Phe Thr Glu Asp Leu Lys Leu 65 70 75 80

Pro Ala Ser Phe Asp Ala Arg Glu Gin Trp Pro Gin Cys Pro Thr He 85 90 95

Lys Glu He Arg Asp Gin Gly Ser Cys Gly Ser Cys Trp Ala Phe Gly 100 105 110

Ala Val Glu Ala He Ser Asp Arg He Cys He His Thr Asn Ala His 115 120 125

Val Ser Val Glu Val Ser Ala Glu Asp Leu Leu Thr Cys Cys Gly Ser 130 135 140

Met Cys Gly Asp Gly Cys Asn Gly Gly Tyr Pro Ala Glu Ala Trp Asn 145 150 155 160

Phe Trp Thr Arg Lys Gly Leu Val Ser Gly Gly Leu Tyr Glu Ser His 165 170 175

Val Gly Cys Arg Pro Tyr Ser He Pro Pro Cys Glu His His Val Asn 180 185 190

Gly Ser Arg Pro Pro Cys Thr Gly Glu Gly Asp Thr Pro Lys Cys Ser 195 200 205

Lys He Cys Glu Pro Gly Tyr Ser Pro Thr Tyr Lys Gin Asp Lys His 210 215 220

Tyr Gly Tyr Asn Ser Tyr Ser Val Ser Asn Ser Glu Lys Asp He Met 225 230 235 240

Ala Glu He Tyr Lys Asn Gly Pro Val Glu Gly Ala Phe Ser Val Tyr 245 250 255

Ser Asp Phe Leu Leu Tyr Lys Ser Gly Val Tyr Gin His Val Thr Gly 260 265 270

Glu Met Met Gly Gly His Ala He Arg He Leu Gly Trp Gly Val Glu 275 280 285

Asn Gly Thr Pro Tyr Trp Leu Val Ala Asn Ser Trp Asn Thr Asp Trp 290 295 300

Gly Asp Asn Gly Phe Phe Lys He Leu Gly Gly Gin Asp His Cys Gly 305 310 315 320

He Glu Ser Glu Val Val Ala Gly He Pro Arg Thr Asp Gin Tyr Trp 325 330 335

Glu Lys He

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GGATACGTTA CNCCNGT 17

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GCCATGAGRT ANCC 14

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

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

Ala Pro Asp Ser Val Asp Tyr Arg Lys Lys Gly Tyr Val Thr Pro Val 1 5 10 15

Lys Asn

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CTGGATCCCT GTACCCTGAG GAGATACTG 29

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CTAAGCTTCT ATCTACCTTC CCATTCTGGG ATA 33