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Title:
CLONING, EXPRESSION AND USES OF A NOVEL SECRETED PROTEIN, F-SPONDIN
Document Type and Number:
WIPO Patent Application WO/1993/020196
Kind Code:
A1
Abstract:
This invention provides an isolated vertebrate nucleic acid molecule encoding F-spondin. This invention also provides a probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding an F-spondin. This invention further provides a method of attaching nerve cells to a matrix comprising contacting the matrix with nerve cell and purified F-spondin at a concentration effective to effect attachment of the cells to the matrix. This invention also provides a method of stimulating growth of a nerve cell comprising contacting the nerve cell with purified F-spondin at a concentration effective to stimulate growth of the nerve cell. This invention provides a method of regenerating nerve cells in a subject comprising administering to the subject purified F-spondin at a concentration effective to regenerate nerve cells in the subject. Finally, this invention provides a pharmaceutical composition for stimulating nerve cell growth comprising a pharmaceutically acceptable carrier and purified F-spondin at a concentration effective to stimulate nerve cell growth.

Inventors:
JESSELL THOMAS M (US)
KLAR AVIHU (US)
Application Number:
PCT/US1993/003164
Publication Date:
October 14, 1993
Filing Date:
April 02, 1993
Export Citation:
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Assignee:
UNIV COLUMBIA (US)
JESSELL THOMAS M (US)
KLAR AVIHU (US)
International Classes:
C12N15/09; C07K14/46; C07K14/465; C07K14/47; C07K14/48; C12N1/21; C12N5/10; C12P21/02; A61K38/00; (IPC1-7): C12N15/00; C12N15/12; C07K13/00
Foreign References:
Other References:
KLAR A., BALDASSARE M., JESSELL T. M.: "F-SPONDIN: A GENE EXPRESSED AT HIGH LEVELS IN THE FLOOR PLATE ENCODES A SECRETED PROTEIN THAT PROMOTES NEURAL CELL ADHESION AND NEURITE EXTENSION.", CELL, CELL PRESS, US, vol. 69., 3 April 1992 (1992-04-03), US, pages 95 - 110., XP002042409, ISSN: 0092-8674, DOI: 10.1016/0092-8674(92)90121-R
Proceedings of the National Academy of Sciences USA, Volume 78, No. 4, issued April 1981, C.E. HENDERSON et al., "Neurite Outgrowth from Embryonic Chicken Spinal Neurons is Promoted by Media Conditioned by Muscle Cells", pages 2925-2929, see entire document.
The Journal of Biological Chemistry, Volume 266, No. 36, issued 25 December 1991, M.D. KOSFELD et al., "Cell Attachment Activity of the Carboxyl-Terminal Domain of Human Thrombospondin Expressed in Escherichia coli", pages 24257-24259, see entire document.
Developmental Biology, Volume 150, No. 2, issued April 1992, D.J. OSTERHOUT et al., "Thrombospondin Promotes Process Outgrowth in Neurons from the Peripheral and Central Nervous Systems", pages 256-265, see entire document.
Science, Volume 250, issued 16 November 1990, M. PLACZEK et al., "Mesodermal Control of Neural Cell Identity: Floor Plate Induction by the Notochord", pages 985-988, see entire document.
The Journal of Cell Biology, Volume 105, issued August 1987, W.A. FRAZIER, "Thrombospondin: A Molecular Adhesive Glycoprotein of Platelets and Nucleated Cells", pages 625-632, see entire document.
Neuron, Volume 7, issued August 1991, K.S. O'SHEA et al., "Thrombospondin and a 140 kd Fragment Promote Adhesion and Neurite Outgrowth from Embryonic Central and Peripheral Neurons and from PC12 Cells", pages 231-237, see entire document.
See also references of EP 0670895A4
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Claims:
What is claimed is:
1. An isolated vertebrate nucleic acid molecule encoding Fspondin.
2. An isolated vertebrate nucleic acid molecule of claim 1, wherein the nucleic acid is DNA.
3. An isolated vertebrate nucleic acid molecule of claim 2, wherein the DNA is cDNA.
4. An isolated vertebrate nucleic acid molecule of claim 1, wherein the nucleic acid is RNA.
5. An isolated human nucleic acid molecule of claim 1.
6. An isolated rat nucleic acid molecule of claim 1.
7. An isolated chicken nucleic acid molecule of claim 1.
8. An isolated Xenopus nucleic acid molecule of claim 1.
9. A nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of the nucleic acid molecule of claim 1.
10. A nucleic acid probe of claim 9 wherein the nucleic acid is DNA.
11. A nucleic acid probe of claim 9 wherein the nucleic acid is RNA.
12. An isolated nucleic acid molecule of claim l operatively linked to a . promoter of RNA transcription.
13. A vector which comprises the isolated nucleic acid molecule of claim 1.
14. A vector of claim 13, wherein the isolated nucleic acid molecule is linked to a plasmid.
15. A host vector system for the production of a polypeptide having the biological activity of F spondin which comprises the vector of claim 13 in a suitable host.
16. A host vector system of claim 15, wherein the suitable host is a bacterial cell, insect cell, or animal cell.
17. A method of producing a polypeptide having the biological activity of Fspondin which comprising growing the host vector system of claim 15 under conditions permitting production of the polypeptide and recovering the polypeptide so produced.
18. Purified, vertebrate Fspondin polypeptide.
19. A polypeptide encoded by the isolated vertebrate nucleic acid molecule of claim 1.
20. A method of attaching nerve cells to a matrix comprising contacting the matrix with nerve cell and purified Fspondin at a concentration effective to effect attachment of the cells to the matrix.
21. A method of stimulating growth of a nerve cell comprising contacting the nerve cell with purified Fspondin at a concentration effective to stimulate growth of the nerve cell.
22. A method of regenerating nerve cells in a subject comprising administering to the subject purified F spondin at a concentration effective to regenerate nerve cells in the subject.
23. A pharmaceutical composition for stimulating nerve cell growth comprising a pharmaceutically acceptable carrier and purified Fspondin at a concentration effective to stimulate nerve cell growth.
Description:
CLONING. EXPRESSION AND USES OF A NOVEL SECRETED PROTEIN. F-SPONDIN

Background of the invention

Throughout this application various references are referred to within parenthesis. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the sequence listing and the claims.

The early development of the vertebrate nervous system is controlled by local cell interactions that determine the identity of specific neural cell types and the pathways of growing axons. One of the first cell types to differentiate within the embryonic nervous system is the floor plate, a small group of epithelial cells located at the ventral midline of the neural tube (Schoenwolf and Smith, 1990) . The differentiation of the floor plate is induced by local, possibly contact-dependent signals from the notochord (Figure 1) (van Straaten et al., 1988; Placzek et al. , 1990c; Hatta et al., 1991). Signals that derive from the floor plate have been implicated in the control of cell identity in the neural tube and in the guidance of axons (Figure 1) (Jessell and Dodd, 1991) .

Evidence that the floor plate is a source of polarizing signals that control cell identity and pattern in the neural tube has come from experiments in chick embryos in which floor plate cells grafted next to the neural tube of host embryos give rise to additional ectopic motor

neurons and to other ventral neuronal types defined by cell specific antigenic markers (Yamada et al., 1991; Placzek et al. , 1991) . Inversely, preventing floor plate differentiation by removing the notochord leads to the formation of a spinal cord that is devoid of motor neurons and other ventral neurons. These grafting experiments suggest that the floor plate has a central role in establishing the identity and pattern of neuronal cell types present in the ventral spinal cord. The floor plate also has limb polarizing activity when grated into the chick wing bud, possibly through the release of orphogenically active retinoids (Wagner et al., 1990).

After the identity of spinal cord neurons has been established, the floor plate appears to provide both long-range and local guidance cues that promote the growth of axons to and across the ventral midline of the spinal cord. First, the floor plate secretes a diffusible che oattractant which can orient the growth of axons of commissural neuron in vitro (Figure 1) (Tessier- Lavigne et al., 1988; Placzek et al., 1990a; Tessier- Lavigne and Placzek, 1991) and may account for the homing of these axons to the floor plate in vitro (Weber, 1938; Placzek et al., 1990b; Bovolenta and Dodd, 1991; Yaginuma and Oppenheim, 1991) . Second, the floor plate may contribute to the change in trajectory of commissural axons from the transverse to the longitudinal plane that occurs immediately after crossing the ventral midline

(Figure 1) (Holley and Silver, 1987; Dodd et al., 1988; Bovolenta and Dodd, 1990) . In support of this proposal, genetic mutations in mice and zebrafish that result in the absence of the floor plate during embryonic development lead to errors in the pathfinding of commissural axons at the midline of the spinal cord

(Bovolenta and Dodd, 1991; Bernhardt and Kuwada, 1990).

Third, the floor plate may promote the fasciculation of commissural axons that occurs after they cross the midline of the spinal cord (Holley and Silver, 1987) by regulating the expression of glycoproteins of the immunoglobulin superfamily (Dodd et al., 1988; Schachner et al., 1990; Furley et al., 1990). The specialized role of the floor plate in vertebrate neural development has parallels in invertebrate organisms in that cells at the midline of the embryonic drosophila and C. elegans central nervous systems have been implicated in neural patterning and axon guidance (Klambt et al., 1991; Nambu et al., 1991; Hedgecock and Hall, 1990).

To identify molecules that may mediate the diverse functions of the floor plate during early neural development, subtractive hybridization techniques have been used to isolate cDNA clones expressed selectively by the floor plate. The characterization of cDNA clones encoding a novel secreted protein, F-spondin, that is expressed at high levels by the rate floor plate during embryonic development is described here. The predicted amino acid sequence of F-spondin reveals that the protein contains domains similar to those present in the thro bospondin and other proteins implicated in cell adhesion and neurite outgrowth. In vitro assays show that F-spondin promotes neural cell adhesion and neurite outgrowth suggesting that the secretion of this protein by the loor plate contributes to the growth and guidance of axons in the developing CNS.

g "P -f-.rγ of the invention

This invention provides isolated vertebrate nucleic acid molecule encoding F-spondin. The isolated nucleic acid may be cDNA or SNA. The isolated vertebrate nucleic acid may be derived from human, rat, chicken or Xenopus.

This invention also provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a F-spondin. The nucleic acid probe may be DNA or SNA.

This invention provides the method to obtain F-spondin nucleic acid molecule. In an embodiment, a rat F-spondin gene is isolated by substractive hybridization. In another embodiment, a chicken F-spondin gene is isolated by screening a chicken cDNA library using a rat F-spondin probe. In a further embodiment, a Xenopus F-spondin is also isolated.

This invention further provides a host vector system for the production of a polypeptide having the biological activity of F-spondin. The isolated vertebrate F-spondin nucleic acid molecule is linked to a promoter of SNA transcription and then to a plasmid. The suitable host is a bacterial cell, insect cell, or animal cell, depending on the type of promoter and plasmid used. This invention also provides a method of producing a polypeptide having the biological activity of F-spondin, which comprises growing the selected host vector system under suitable conditions permitting production of the polypeptide and recovering the polypeptide so produced.

This invention further provides purified vertebrate F-

spondin. Such purified F-spondin will be useful for adhesion and outgrowth of axon. This invention provides a method of attaching nerve cells to a matrix comprising contacting the matrix with nerve cell and purified F- spondin at a concentration effective to effect attachment of the cells to the matrix. This invention further provides a method of stimulating growth of a nerve cell comprising contacting the nerve cell with purified F- spondin at a concentration effective to stimulate growth of the nerve cell. This invention provides a method of regenerating nerve cells in a subject comprising administering to the subject purified F-spondin at a concentration effective to regenerate nerve cells in the subject. Finally, this invention provides a pharmaceutical composition for stimulating nerve cell growth comprising a pharmaceutically acceptable carrier and purified F-spondin at a concentration effective to stimulate nerve cell growth.

Brief Description of Figures

Figure 1. Diagram showing the induction and proposed functions of the floor plate during early spinal cord development. For details see text.

Figure 2. Schematic diagram of the subtractive hybridization protocol used to identify floor plate specific cDNA clones.For details see text.

Figure 3. Expression of F-spondin mBNA. Total cellular

SNA or poly (A) + RNA was isolated from different tissues and separated on 1% agarose- formaldehyde gels and blotted to nylon membranes. The blot was analyzed with cDNA probes derived from the F-spondin 3' noncoding region labelled by random priming.

A. Preferential expression of F-spondin mRNA in E13 (embryonic day 13) floor plate compared with E13 dorsal spinal cord at adult spleen. Two transcripts of 4.5 and 4.7 kb are detected in floor plate RNA.

B. NCAM, Neural Cell Adhesion Molecule, mRNA is expressed at approximately equivalent levels in E13 floor plate and dorsal spinal cord and P0 (postnatal; day 0) brain.

C. F-spondin mRNA is detected in blots of total RNA adult kidney and brain but not in adult liver or sciatic nerve.

Figure 4. Restriction map of the F-spondin cDNA. The arrow indicates the direction of translation.

The restriction sites are indicated above the cDNA.

Figure 5. cDNA and predicted amino acid sequence of F- spondin.

A. Nucleotide and amino acid sequence of rat F-spondin determined from cDNA clones. The numbering of amino acids starts at the first methionine. Underline NH 2 terminal residues indicates the putative signal sequence. Potential sites of N-linked glycosylation are indicated by double lines.

B. Analysis of the hydrophobicity of the predicted F-spondin amino acid sequence. The plot was generated using the parameters given in Kyte and Doolittle (1982) . The NH 2 terminus of the protein is to the left. Negative values indicate hydrophobic residues.

Figure 6. Alignment of the carboxy terminal domain F- spondin and homology to thrombospondin type one repeats in other proteins.

A. Schematic representation of the domain structure of F-spondin. The black box represents the signal sequence. The hatched box represents the thrombospondin type 1 repeats (TSRs) .

B. Alignment of the six repeats motifs in F- spondin which occupy residues 440-807 of the protein. The position of the first and last amino acids of each repeat is shown on the left. Numbers over each repeat refer to the position of residues.

Positions in which there are four or more identical residues are enclosed in boxes.

C. Comparison of the conserved F-spondin motif with the conserved TSRs found in thrombospondin I, thrombospondin II, region II of the plasmodial circumsporooite (cs) proteins thrombospondin-related anonymous protein (TRAP) , properdin and in the N-and C- terminal regions . of the complement proteins C6, C7, C8a, C8b and C9. The number at the right of the figure indicates the number of TSR domains that contain VTCG sequence as a proportion of the total number of TSR domains.

Figure 7. Localization of F-spondin mRNA in the developing spinal cord.

A. Autoradiographic localizationof F-spondin mRNA in the hindbrain of a day 10 rat embryo by in situ hybridization using an antisense RNA probe. Intense hybridization is detected at the ventral midline of the neural tube and possible also in the axial mesoderm underlying the neural tube.

B. Localization of whole mount in situ F- spondin mRNA by Ell (embryonic day 11) rat embryos hybridizationhistochemistryusing digoxigenin-labelled antisense probe. Hybridization is detected in the floor plate of the midbrain, hindbrain and spinal cord (arrow heads) .

C. Bright field micrograph showing localization of F-spondin mRNA in E12

(embryonic day 12) rat spinal cord. The floor plate is intensely labelled.

D. Dark field micrograph of a similar section showing a low level of hybridization is in the ventral horn in addition to intense labelling in the floor plate.

_ Hybridization is also detected in the b ventral root.

E. Dark field micrograph showing the floor plate and the ventral ventricular zone of E13 spinal cord express high levels of F- 0 spondin mRNA.

F. Bright field micrograph of E16 (embryonic day 16) spinal cord showing that F-spondin mRNA levels are still high in the floor plate and the ventral ventricular zone. 5 G. Dark field micrograph showing that by E16, significant hybridization is also detected in ventral and intermediate regions of the spinal cord.

H. Dark field micrographs showing a uniform distribution of F-spondin mRNA. Scale bar: A=100 μm; B=350 μm; C=80 μm; E=100 μm; F=170 μm; G=170 μm; H=120μm.

Figure 8. F-Spondin myc is secreted by cos cells and associated with the cell surface.

A. Position of insertion of an oligonucleotide encoding for a 10 amino acid region of the c-myc oncogene ligated into unique Ncol site or Spel sites within the F-spondin cDNA. . . .

B. Immunoprecipitation of conditioned media obtained by exposure of Oh to cos cells transfected with pFPδmyS, pFP5myN and to

mock transfected cells. Both constructs generated a single protein band at 116 kDa.

C. Phase contrast micrograph showing a small group of transfected cos cells. . D. Immunofluorescence micrograph showing the localization of F-spondin myc on the cell surface. Immunoreactivity is detectable at much higher levels at cell-cell rather than at cell-substrate contacts.

Scale bar in C, D = 20μm

Figure 9. F-spondin myc promotes the extension of neurites from DRG neurons jLn vitro. F-spondin myc protein obtained from transfected cos cells supernatants was affinity purified and analyzed by SDS-PAGE(8-25%) and silver staining. (A)

Two stained bands are observed, which may re lect differences in the glycosylation of F- spondin. Neural cells isolated from E14 rat dorsal root ganglia were plated on F-spondin (B) or on cos cell-conditioned media (C) or BSA

(not shown) substrates for 14h and then fixed and labelled with MAb 3A10 and visualized by indirect immunofluorescence. (D) The length of the longest neurite of each 3A10-positive neurons was measured (or recorded as 0 mm if no neurite was seen) . The percentage of neurons

(ordinate) with neurites longer than a given length in μm (abscissa) is plotted. Similar results were obtained in 5 experiments. Only non-fasciculated neurites were included in the plots shown in D. Scale bar in B and C = 100 μm.

Figure 10. F-spondin promotes the adhesion of dorsal spinal cord cells. A single cell suspension of E13 dorsal spinal cord cells (10 6 cells/35mm disk) was plated on, F- spondin myc (A, B) , on BSA (C) and on F- spondin myc substrate in the presence of heparin (1 mg/ml) (D) , for lh. Cells were then washed in PBS, fixed and counted.

E. Box plot showing dose-dependent adhesion of E13 dorsal spinal cord cells to different amounts of F- spondin myc substrate. Each box represents cell counts from 10 different fields. Similar results were obtained in 3 separate experiments.

F. Box plot showing inhibition of the adhesion of E13 dorsal spinal cord cells to F-spondin myc in the presence of different concentrations of heparin and chondroitin sulfate. The inhibition at all concentrations of chondroitin sulfate and heparin is significant (p<0.001; Ttest) . Scale bar in A, C, D = 200 μm, B = 50 μm

Box plot: The box enclosed 50% of the population with the median marked as a bold line and the mean as a dot. The range of the data is indicated by the extent of the lines. Each plot represents 10 determinations form one of three similar experiments.

Detailed Description of the Invention

This invention provides isolated vertebrate nucleic acid molecules which encode F-spondin. As used herein, the term F-spondin encompasses any amino acid sequence, polypeptide or protein having the biological activities

_ provided by the F-spondin. b

In one embodiment of this invention, the isolated nucleic acid molecules described hereinabove are DNA. In other embodiments of this invention, the isolated nucleic acid 0 molecules described hereinabove are cDNA, or RNA. In the preferred embodiment of this invention, the isolated nucleic molecules are cDNAs as shown in sequence ID numbers (9, 11 and 13)

5 This invention also encompasses DNAs and cDNAs which encode amino acid sequences which differ from those of F- spondin, but which should not produce phenotypic changes. Alternatively, this invention also encompasses DNAs and cDNAs which hybridize to the DNA and cDNA of the subject invention. Hybridization methods are well known to those of skill in the art.

The DNA molecules of the subject invention also include DNA molecules coding for polypeptide analogs, fragments or derivatives of antigenic polypeptides which differ from naturally-occurring forms in terms of the identity or location of one or more amino acid residues (deletion analogs containing less than all of the residues specified for the protein, substitution analogs wherein one or more residues specified are replaced by other residues and addition analogs wherein one or more amino acid residues is added to a terminal or medial portion of the polypeptides) and which share some or all properties

of naturally-occurring forms. These sequences include: the incorporation of codons "preferred" for expression by selected non-mammalian host; the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate

DNA sequences that facilitate construction of readily expressed vectors.

The DNA molecule described and claimed herein are useful for the information which they provide concerning the amino acid sequence of the polypeptide and as products for the large scale synthesis of the polypeptide by a variety of recombinant techniques. The molecule is useful for generating new cloning and expression vectors, transformed and transfected procaryotic and eucaryotic host cells, and new and useful methods for cultured growth of such host cells capable of expression of the polypeptide and related products.

Moreover, the isolated nucleic acid molecules are useful for the development of probes to study the neurodevelopment.

F-spondin may be produced by a variety of vertebrates. In an embodiment, a rat F-spondin nucleic acid is isolated. A restriction map of the cDNA of rat F-spondin is shown in Figure 4. The Xhol-Dral fragment of rat F-spondin was excised from the F-spondin cDNA. The Xhol site was blunt-ended with T4 DNA polymerase, and Bgl2 linkers (12 mers) was ligated. The fragment was subcloned into BamHl site of pBluescript SK (Strategene) . The 5" of the gene is located near the T3 promoter. The resulting plasmid, pFP5/KS, encoding the rat F-spondin was deposited on March 19, 1992 with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852,

U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganism for the Purposes of Patent Procedure. Plasmid, pFP5/KS was accorded ATCC accession number 75215.

In another embodiment, a chicken F-spondin cDNA was isolated (Seq. ID No. 11) . The translation initiates at nucleotide position 136. In a further embodiment, a partial Xenopus F-spondin was isolated (Seq. ID No. 13) .

Throughout this application, references to specific nucleotides are to nucleotides present on the coding strand of the nucleic acid. The following standard abbreviations are used throughout the specification to indicate specific nucleotides:

C=cytosine A=adenosine

T=thymidine G=guanosine

For the purpose of illustration only, applicants used a substractive hybridization techniques to isolate and characterized F-spondin cDNA clones in rats. Similar substractive hybridization techniques are applicable to isolate and characterize the F-spondin genes in different vertebrates.

Alternatively, the F-spondin genes may be isolated using the probe generated from the rat F-spondin gene. The chicken and Xenopus homologous F-spondin genes have recently been cloned by applicants. These genes are extremely conserved and share 90% homology at the amino acid level and about 70% homology at the nucleic acid level. The chicken gene was isolated by low stringency screening of embryonic spinal cord cDNA library whereas the Xenopus F-spondin gene was isolated by low stringency

screening of the whole embryo cDNA library, both using probes from the coding region of rat F-spondin.

For the human F-spondin gene, it is conceivable that the degree of homology between rat and human would be even greater since both rat and humans are mammals. Human embryonic brain cDNA library, available from Clontech, and human genomic library may be used for such screening.

Duplicated filters of human libraries may be screened with radiolabelled probe derived from the rat F-spondin.

The probe may be encompassing the coding region, since the homology of F-spondin across species is through the whole coding region. , The filters containing the human libraries will be hybridized with the probes at low stringency (Sambrook et al. 1989) and positive clone will be further analyzed by DNA sequencing techniques which are well known to an ordinary skilled artisan.

This invention provides a nucleic probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a F-spondin, for example, with a coding sequencing included within the sequence shown in Figure 5 and Sequence ID number 9. As used herein, the phrase "specifically hybridizing" means the ability of a nucleic acid molecule to recognize a nucleic acid sequence complementary to its own and to form double-helical segments through hydrogen bonding between complementary base pairs. Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary greatly in length and may be labeled with a detectable label, such as a radioisotope or fluorescent dye, to facilitate detection of the probe. DNA probe molecules may be produced by insertion of a DNA

molecule which encodes F-spondin into suitable vectors, such as piasmids or bacteriophages, followed by transforming into suitable bacterial host cells, replication in the transformed bacterial host cells and harvesting of the DNA probes, using methods well known in the art. Alternatively, probes may be generated chemically from DNA synthesizers.

The probes are useful for 'in situ' hybridization to locate tissues which express this gene, or for other hybridization assays for the presence of this gene or its mRNA in various biological tissues.

Vectors which comprise the isolated nucleic acid molecule described hereinabove also are provided. Suitable vectors comprise, but are not limited to, a plasmid or a virus. These vectors may be transformed into a suitable host cell to form a host cell vector system for the production of a polypeptide having the biological activity of F-spondin.

This invention further provides an isolated DNA or cDNA molecule described hereinabove wherein the host cell is selected from the group consisting of bacterial cells (such as E.coli) , yeast cells, fungal cells, insect cells and animal cells. Suitable animal cells include, but are not limited to Vero cells, HeLa cells, Cos cells, CVl cells and various primary mammalian cells.

This invention provides a method to identify and purify expressed F-spondin proteins. A myσ-epitope was first introduced into the F-spondin protein. This F-spondin carrying yc-spondin may linked to an expression vector. Such vector may be used to transfect cell and the distribution of F-spondin in the cell can be detected by

reacting myc antibodies known to be reactive to the introduced myc-epitope with the transfected cells which is expressing the F-spondin carrying myc-epitope. Taking advantage of this myc-epitope, F-spondin may be purified by an antibody affinity column which binds with this myc- epitope.

In one embodiment, myc-epitope is introduced in the Ncol site of the rat F-spondin. After that the smal (125),

Dra (2731) fragment of the rat F-spondin was isolated.

Bgl2 linkers were added, and the fragment was subcloned into BamH 1 site of pcDNA neo (InVitrogene) . The 5'end of the gene is located near the T7 RNA promoter. The resulting plasmid, pcFPδ.myn, was deposited on March 19,

1992 with the American Type Culture Collection (ATCC) ,

12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. under the provisions of the Budapest Treaty for the

International Recognition of the Deposit of Microorganism for the Purposes of Patent Procedure. Plasmid, pcFPS.myn was accorded ATCC designation number 75216.

The above uses of the myc-epitope for identification and purification of F-spondin should not be considered limiting only to the myc-epitope. Other epitopes with specific antibodies against them which are well known to an ordinary skilled in the art could be similarly used.

Also provided by this invention are F-spondin complete protein sequences (seq. ID Nos. 10, 12). In an embodiment a complete rat F-spondin protein sequence is disclosed (Seq. ID No. 10) . In aother embodiment a complete chicken F-spondin protein sequence is provided (Seq. ID No. 12) . In a further embodiment a partial Xenopus F-spondin protein sequence is also provided (Seq. ID No. 14) .

Further provided by this invention is a purified, F- spondin polypeptide. As used herein, the term "purified F-spondin" shall mean isolated naturally-occurring F- spondin or protein (purified from nature or manufactured such that the primary, secondary and tertiary conformation, and posttranslational modifications are identical to naturally-occurring material) as well as non-naturally occurring polypeptides having a primary structural conformation (i.e. continuous sequence of amino acid residues) . Such polypeptides include derivatives and analogs.

Such F-spondin will be useful for adhesion and outgrowth of axon. Therefore, this invention also provides a method of attaching nerve cells to a matrix comprising contacting the matrix with nerve cell and purified F- spondin at a concentration effective to effect attachment of the cells to the matrix.

Methods to determine such a concentration are well-known in the art. The effect concentration of F-spondin may be determined by using different concentrations of purified F-spondin to the matrix and the nerve cell. The concentration in which attachment of the matrix and the nerve cell is observed is the effective concentration.

This invention further provides a method of stimulating growth of a nerve cell comprising contacting the nerve cell with puri ied F-spondin at a concentration effective to stimulate growth of the nerve cell.

This invention also provides a method of regenerating nerve cells in a subject comprising administering to the subject purified F-spondin at a concentration effective to regenerate nerve cells in the subject.

Finally, this invention provides a pharmaceutical composition for stimulating nerve cell growth comprising a pharmaceutically acceptable carrier and purified F- spondin at a concentration effective to stimulate nerve cell growth.

For the purposes of this invention "pharmaceutically acceptable carriers" means any of the standard pharmaceutical vehicles. Examples of suitable vehicles are well known in the art and may include, but not limited to, any of the standard pharmaceutical vehicles such as a phosphate buffered saline solutions, phosphate buffered saline containing Polysorb 80, water, emulsions such as oil/water emulsion, and various type of wetting agents.

This invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.

Experimental Details Experimental Procedures Library Construction and Screening

Directional cDNA libraries were constructed in Lambda ZAP® II (Stratagene®) from embryonic day (E) 13 floor plate and dorsal spinal cord poly (A) + -selected RNA. The 5' ends of the cDNA inserts were located downstream of the T3 RNA polymerase promotor, and the 3' ends downstream of the T7 RNA polymerase promotor. DNA was prepared from the library using the plate lysate method (Sambrook et al., 1989). The DNA was linearised with Xhol and RNA was transcribed with T3 RNA polymerase (Stratagene®) . RNA from the dorsal spinal cord library was transcribed in the presence of UTP-biotin (Clontec) diluted 1:10 with UTP. First-strand cDNA was transcribed from the T3 floor plate RNA using an oligo dT Xhol linker (Stratagene®) .

Solution hybridization of first strand floor plate cDNA and the dorsal T3 biotinylated RNA was performed as described by Sive and St. John (1988) . Approximately 1 μg of cDNA was hybridized with a 30-fold molar excess of RNA. The nucleic acids were dissolved in 10 μl of hybridization buffer containing 50 mM HEPES (pH 7.6), 0.2% SDS, 2 mM EDTA, 500 mM NaCl, and incubated at 68 β C. Under these conditions, CoT values greater than 100 were obtained. The hybridization mixture was diluted to 60 μl with hybridization buffer without SDS, and 10 μg streptavidin was added. The cDNA/biotin RNA hybrids were removed by phenol-chloroform extraction. The remaining single strand cDNA was isolated and hybridized with a 300 fold excess of biotinylated RNA as described above. About 10% of the starting cDNA was recovered in the first hybridization and about 15-20% from the second

hybridization.

The subtracted cDNAs were subjected to 20 cycles of a PCR reaction using oligo dT Xhol linker primer and SK primers (Stratagene®) . The products of the PCR reaction were cut with EcoRI and Xhol, the primers and the flanking sequences were removed with sephacryl S-300 spin columns (Pharmacia®) . The inserts were cloned into Lambda ZAP II arms.

Duplicate filters of the subtracted floor plate library were screened with radiolabelled first strand cDNA derived from floor plate and dorsal spinal cord. 100 ng of mRNA was incubated in 20 μl of 50 mM Tris pH 8.3, 10 mM MgCl 2 , 150 mM KCl, 1.0 mM dGTP, 1.0 mM dTTP, 100 μCj.[32p]dATP (3000 Ci/mmol) , 100 μCi[ 32 P]dCTP (3000 Cimmol) , 100 mg/ml oligo dT, 10 mM DTT, 10 U of RNasin (Promega) , 20 U of MulV reverse transcriptase (BRL) , for 30 min in 37 β c. 4xl0 3 recombinant phage were plated and screened. Hybridization and washes were performed at high stringency (Sambrook et al., 1989). The floor plate cDNA probe hybridized selectively with 24 phages. Cross hybridization analysis revealed that these corresponded to three difference cDNAs designated FP2, FP5 and FP24. The pattern of expression in the spinal cord was determined by in situ hybridization. FP2 and FP5 are expressed selectively in the floor plate while FP24 is expressed in the floor plate, roofplate and in the ventricular zone of the spinal cord. The degree of enrichment as determined by screening the floor plate enriched library and floor plate library with FP2, FP5 and FP35, which is expressed selectively in the floor plate (McKanna & Cohen, 1989) is about 50-fold. RNA Transfer Analysis

Total RNA was prepared from various tissues using the RNA Azol method (Biotex Laboratories) and then enriched for poly (A) + containing transcripts by passage over an oligo (dT) cellulose matrix. RNA transfer was performed as described by Thomas (1980) . Probes were labelled by random priming (Feinberg and Vogelstein, 1984) and 5 hybridized under standard conditions.

DNA Sequencing and Analysis cDNA inserts were excised directly as Bluescript plasmids (Stratagene®) . The nucleotide sequence of the inserts ■ jQ were determined by the dideoxy chain-termination method (Sanger et al. , 1977) using both double-stranded and single-stranded DNA as template for T7 DNA polymerase (Sequenase, United States Biochemicals) . The nucleotide sequence of the entire coding region was determined by

15 sequencing both strands. Sequences were assembled on an Apple Maclintosh computer using MacVector (IBI) program.

In Situ Hybridization

In situ hybridization was preformed as described 0 previously (Wilkinson et al., 1987) using a T3 or T7 RNA polymerase-derived [ 35 S]UTP-labelled single stranded antisense RNA probe which encompasses a region of the 3" untranslated region of F-spondin (nt 3359-4029) , or the TSRs (nt 1545-2626) . Exposure times range from four to 5 fourteen days. Sense probes were used as controls.

For whole mount in situ hybridization, Ell rat embryos were fixed in 0.1 M MOPS, 2 mM EGTA, 1 mM Mg S04, 3.7% formaldehyde for 2 hours. i situ hybridization was preformed essentially as described by Harland (1991) , 0 with a few modifications: anti-digoxygenin antibody (Boehringer Mannheim) , was preabsorbed to E14 rat acetone powder (1%) (Harlow and Lane, 1988) before addition to

5

the hybridization mixture. The chromogenic reaction was carried out for l-2h.

DNA Constructs

The myc epitope was introduced as follows: Two partially complementary oligonucleotides with the sequence: 5'- CTAGCGAGCAGAAGCTGATCTCCGAGGAGGACCTCA-3' (Seq. ID No. 1) and 5'-CTAGTGAGGTCCTCCTCGGAGATCAGCTTCTGCTCG-3' (Seq. ID No. 2) were annealed to obtain a double-stranded DNA fragment coding for the c-myc proto-oncogene epitope EQKLISEEDL (Seq. ID No. 3) flanked by a Spel site, the fragment was cloned into a unique Spel site (nt 1365) in F-spondin. The same epitope was also introduced into a Ncol site (nt 1575) using the oligonucleotides: 5'- CATGGGAGCAGAAGCTGATCTCCGAGGAGGACCTCG-3' (Seq. ID No. 4) and 5'-CATGCGAGGTCCTCCTCGGAGATCAGCTTCTGCTCC-3' (Seq. ID No. 5) . The tagged F-spondin DNA was subcloned into the expression vector pMT21 (provided by Genetics Institute) , or pcDNA-I (InVitrogen) .

cos Cells Transfection

Cos cells were transfected by the DEAE-Dextran method as follows: 80% confluent overnight cultures were transfected with 5 μg DNA, per 100 mm dish, in 250 μg/ml DEAE Dextran (Pharmacia®), 100 mM Tris pH 7.3, in DMEM. After 6h cells were waished and incubated in DMEM 10% calf serum, O.lmM choloroquine (Sigma) for 2.5h, followed by incubation in DMEM 10% calf serum overnight. For isolation of F-spondin the medium was changed to OPTI-MEM (BRL) , and the cells were incubated for 48h.

Metabolic Labeling of Cos Cells and Tτn * mtιnoprecipitation Transfected cos cells were preincubated in methionine- free DMEM (BRL®-GIBCO) . After lh at 37 β C, 250μCi/ml[ 35 S] methionine (NEN) was added, and the cells were incubated

for an additional 3h. The medium was collected and incubated with anti-myc antibody (MAb 9E10) for lh. The immune complex was precipitated with fixed Staphylococcus aureus (BRL®) for lh. Pellets were washed three times with PBS, before resuspension in lx sample buffer. 35 S- labelled immunoprecipitated proteins were visualized after electrophoresis on 10% SDS-polyacrylamide gels.

χ * m-mιmnr_γ- oc ** hemistry

F-spondin tagged with the c-myc epitope was detected with MAb 9E10 (Evan et al., 1985). Fluoresceinated isotype- specific second antibody (Boehringer® Mannheim; goat antimouse IgG) was used at a dilution of 1:100. For Immunofluorescence labelling (Dodd and Jessell, 1985) , cultures were washed once at 22°C with L15 and then incubated with primary antibody for 30 min at 22°C. Cultures were then washed twice in L15-l% normal goat serum (NGS) and incubated with secondary FITC conjugated isotype-specific antibody diluted in L15-l% NGS for 30 min at 22°C. Cultures were washed twice and fixed in 4% paraformaldehyde in 0.2 M phosphate buffer (PB) for 20 min, rinsed in 0.12M PB and coverslipped in 0.05% paraphenylenediamme (Sigma) in 0.2 M sodium carbonate (pH 9.0); glycerol (1:1). Cultures were viewed on a Zeiss Axioplan microscope under epifluorescence optics.

Cell Culture

Spinal cords were dissected from embryonic day (E) 13 rats and placed into L15 medium at 4°C. The dorsal region of the spinal cord were dissected and incubated with 0.05 trypsin (Gibco) for 20 min in a Ca 2+ /Mg 2+ -free modified essential medium (S-MEM) (Gibco) supplemented with 8 mg ml" 1 glucose. The tissue was then washed with S-MEM and triturated to give a single cell suspension. Spinal cord cells were plated in 35 mm tissue culture

dishes on appropriate substrates and grown in Ham's E12 medium (Gibco) supplemented with N3 additive (F12-N3) (Romijin et al., 1982) at a density of 10 6 cells/dish in a 5% C02 humidified incubator at 37°C. Dorsal root ganglia were dissected from E14 rats and treated as described above. Cells were incubated with 0.1 trypsin, and plated with F12-N3 supplemented with 100 ng NGF at a density of 4xl0 /dish.

Neurite Outgrowth Assays

5xl0 10 cos cells were transfected with pFP5myN and conditioned medium was collected. F-spondin myc , was affinity purified on a monoclonal anti-myc (9E10) affinity column. Affinity purified F-spondin myc (20 μl/ml) was absorbed onto nitrocellulose (Lemmon et al., 1989) . For controls, parental cos cell conditioned medium was purified on the same column and used as a substrate on nitrocellulose. The nitrocellulose was then blocked with bovine serum albumin (10 mg/ml) which provided a further control for background neurite outgrowth. E14 dorsal root ganglion (DRG) neurons were plated on immobilized protein substrates at a density of 2-lOxlO 4 cells/35 mm tissue culture dish (Nunc, 35 mm diameter) and grown for 14h. Cultures were then fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and stained using MAb 3A10 (Furley et al., 1990; available from Developmental Studies Hybridoma Bank) , which recognizes a neuronal filament-associated protein and serves as a marker for fine neurites. Neuronal cell bodies and neurites were visualized by indirect immunofluorescence on a Zeiss Axioplan microscope. Neurite lengths were measured as the distance from the edge of the soma (sharply defined by 3A10 fluorescence) to the tip of its longest neurite. Neurite lengths were only measured if the entire length to the neurite could

be unambiguously identified. About 25 neurites were measurable within each protein-coated area (3-4 mm 2 ) .

Adhesion Assay

Dissociated E13 dorsal spinal cord cells were plated on immobilized protein substrate at a density of 10 6 cells/35 mm tissue culture dish (Nunc, 35 mm diameter) . After one hour the cultures were washed twice with PBS and fixed in 4% parafor aldehyde. Cells were counted on a Zeiss Axioplan microscope at 400x magnification. Ten independent counts were taken from each experiment. The floor plate is a transient neural cell group implicated in the control of cell pattern and axonal growth in the developing vertebrate nervous system.

Experimental Results Identification and Sequence of a Floor Plate-Enriched cDNA Clone

Cellular assays have revealed that the floor plate has several specializing signalling functions during the embryonic development of the spinal cord. Floor plate- derived signals are likely to be encoded by proteins whose mRNAs are restricted to or are highly enriched in the floor plate. In order to identify such molecules subtractive hybridization techniques have been used to isolate cDNA clones that are expressed by the floor plate but not by the dorsal spinal cord in embryonic day (E) 13 rat embryos (see Figure 2 and Experimental Procedures) . One cDNA clone identified in this screen, designated FP5, contained a 0.5 kb insert which hybridized to two major transcripts of 4.5 and 4.7 kb in poly (A) + -selected RNA derived from E13 rat floor plate (Figure 3A) . Very faint hybridization to the same two transcripts was detected in RNA derived from E13 dorsal spinal cord (Figure 3A) and post-natal day (P) 0 brain (Figure 3C) , whereas no

hybridization was detected to RNA derived from adult liver and spleen (Figures 3A, C) . The specificity of expression of FP5 transcripts within E13 rat spinal cord was confirmed by in situ hybridization histochemistry which showed that FP5 mRNA is expressed at very high levels in the floor plate but is undetectable in the dorsal region of E13 rat spinal cord (see below) . These studies indicate that FP5 transcripts are highly enriched in the floor plate.

Screening of an E13 rat floor plate cDNA library with the 0.5 kb cDNA insert from the FP5 clone identified several additional cDNA clones of which clone FP5-9 contained a 4 kb insert. The FP5-9 cDNA contains a single long open reading frame that starts with a methionine codon at nucleotide 226 associated with a conventional translation initiation sequence (Kozak, 1984) and ends with a TGA stop codon at nucleotide 2646 (Fig. 5A) . No in-frame methionine codons were found upstream of the putative translation initiation site and sequences 5' of the initiation site contain stop codons in all three reading frames. Sequencing of several other independently isolated FP5 cDNA subclones spanning the entire coding region did not reveal any differences in the nucleotide sequence of the open reading frame.

Translation of the open reading frame FP5-9 predicts a protein 807 amino acids with a molecular mass of 90,766 daltons, and N-terminal hydrophobic leader sequence (Figure 5A; Seq. ID No. 9) with a consensus signal peptide cleavage site (von Heijne, 1985) . No other long stretches of hydrophobic residues were observed (Figure 5B) suggesting that the protein does not possess a transmembrane spanning domain. The amino terminal domain of FP5-9 contains a region of clustered basic residues

(residues 138-142) which could represent a site for proteolytic processing by mammalian subtilisin-like cleavage enzymes (Steiner, 1991) . In addition, the predicted protein contains three N-linked glycosylation sites (Figure 5A) . Collectively, these features suggest that the FP5-9 cDNA encodes a secreted protein.

The Protein Encoded bv the FP5-9 cDNA has Structural

Features of Cell and Substrate Adhesion Molecules

Analysis of the predicted amino acid sequence of the FP5-

9 encoded protein reveals that it is separable into two major domains (Figure 6A) . The NH 2 -terminal domain of

440 residues contains 10 cysteine residues and exhibits no sequence homology to other proteins in the Genbank database. The COOH terminal of the protein extends from residues 441-807 and contains six repeats of a domain 55-

59 amino acids in length which can be aligned on the basis of conserved cysteine, tryptophan and arginine residues (Figures 6B, C) .

Similar domains are present in a small number of proteins (Patthy, 1988; Smith et al., 1991). In particular, the adhesive glycoprotein encoded by the thrombospondin I and II genes (Lawler and Hynes, 1986; Bornstein et al. , 1991) each possess 3 of these domains which have been designated thrombospondin type 1 repeats (TSRs) (Lawler and Hynes, 1986) (Figure 6C) . Two TSRs are found in protein C6-C9 of the alternative complement cascade, one at the NH 2 -terminal and one at the COOH-terminal of each protein (Haefliger et al., 1989; Smith et al., 1991). Moreover, the complement-binding protein properdin contains 6 TSRs which comprise 80% of the protein (Goundis and Reid, 1988) . In addition to these vertebrate proteins, the central core of the TSR is similar to region II of malarial circumsporozoite (CS)

and other plasmodial proteins (Figure 6C) (Rich et al., 1990; Robson et al., 1988) which appear to mediate the binding of malarial sporozoites to host cells in the early stages of parasitic infection (Dame et al., 1984). Finally, two TSRs are present in the C.elegans gene Unc- 5, which appears to regulate axonal pathfinding in a subset of neurons (Hedgecock et al., 1990; Culotti et al., 1991). The organization of cysteine and tryptophan residues in the TSRs of the FP5-9 encoded protein is not similar to that of the NH 2 -terminal TSRs of the C6-C9 complement proteins (Figure 6B) . However, the core region of the TSRs in FP5-9 (residues 14-19) is most similar to that of thrombospondin, properdin and the malarial CS proteins (Figure 6B) . We have named the FP5- 9 gene F-spondin to reflect its high level of expression in the floor plate (see below) and the presence of the TSRs.

The TSRs in thrombospondin promote the adhesion of a variety of different cell types (Prater et al., 1991). Similarly, the TSR core region of the plasmodium vivax CS protein promotes the attachment of human hematopoietic cell lines in vitro (Rich et al., 1990). The amino acid sequence VTCG which is contained within this common motif appears to be critical to the cell adhesive properties of the CS proteins. A VTCG sequence (Seq. ID No. 6) is also present in the two TSRs of thrombospondin that promote cell adhesion (Prater et al., 1991). Strikingly, there is a VTCG in the fourth TSR of F-spondin and the second and third TSRs of F-spondin contain sequences (VSCG, Seq. ID No. 7; ATCG, Seq. ID No. 8) that vary by a single conservative substitution (Figure 6B) . These observations raise the possibility that the TSRs in F- spondin mediate cell adhesion. A search of the Genbank database for other proteins implicated in cell adhesion

arid recognition that contain a VTCG sequence identified V-CAMl (Hession et al., 1991) and the VLA4 integrin subunit (Takada et al. , 1989).

Analysis of the predicted amino acid sequence of F- spondin reveals several other structural features that may contribute to the functional properties of the protein. The charged region that is interposed between the fifth and sixth TSRs contains the sequence LRE that has been shown to function as a neuronal cell attachment site in the extracellular matrix glycoprotein S-laminin (Hunter et al. , 1989a, b) . The first, third, fifth and sixth TSR's of F-spondin contain clusters of basic residues that have been implicated in the binding of proteins to heparin and other sulfated glycosaminoglycans (Cardin and Weintraub, 1989) . The first, fourth and fifth TSRs of F-spondin also contain a WSXWS sequence (Figure 6B) which is present in the variant fibronectin type III repeats found in the receptors for several growth and differentiation factors, including ciliary neurotrophic factor (CNTF) , leukemia inhibitory factor (LIF) and the interleukins (ILs) 2-7 (Bazan, 1990; Davis, et al., 1991; Patthy, 1990). The function of the WSXWS motif is unclear although mutation at this site in the IL2 receptor blocks transmembrane signalling (Miyazaki et al., 1991).

Expression Pattern of F-Spondin mRNA

Northern blot analyses of E13 embryos indicate that F- spondin is expressed at much higher levels in the floor plate than in the dorsal spinal cord. More detailed information on the distribution of F-spondin was provided by localizing its mRNA in developing rat embryos by n situ hybridization. F-spondin mRNA was first detected at E10.5 in cells located at the ventral midline of the

neural tube at the level of the prospective midbrain, hindbrain and spinal cord (Figure 7A) . At this stage, cells, at the ventral midline of the neural tube have acquired floor plate-derived chemoattractant activity (Placzek, et al., 1990c) although no antigenic markers of floor plate differentiation can be detected. The expression of F-spondin mRNA therefore provides an early molecular marker of floor plate differentiation.

The expression of F-spondin mRNA is maintained at high levels in E11-E12 floor plate (Figure 7B) whereas other regions of the spinal cord and hindbrain exhibit undetectable levels of hybridization at this stage. By E12-E13 low levels of mRNA are detected in the ventral horn although there is still no detectable mRNA in the dorsal horn (Figures 7C, D) . In addition, the ventral ventricular zone immediately above the floor plate begins to express high levels of F-spondin mRNA (Figure 7) whereas hybridization to cells in the ventricular zone in the dorsal half of the spinal cord is not detectable (Figure 7E) . Thus, expression of F-spondin mRNA reveals a molecular difference between ventricular zone cells in the dorsal and ventral spinal cord. Recent studies have suggested that the ventral ventricular zone is the site of origin of oligodendrocyte and astrocyte precursors that subsequently migrate laterally and dorsally to populate the remainder of the spinal cord (Miller, 1991) . F-spondin mRNA levels remain high in the floor plate and ventral ventricular zone at E16 and by this stage significant hybridization is also detected in cells in the ventral and intermediate regions of the spinal cord (Figures 7F, G) . By P0, the levels of F-spondin mRNA in the floor plate have decreased and there is an increase in hybridization to other cells in the spinal cord, resulting in an uniform expression of F-spondin mRNA

(Figure 7H) . F-spondin mRNA is also preferentially expressed in the floor plate of ° the E11-E16 hindbrain and midbrain and becomes more widely expressed in the brain at later embryonic stages (not shown) .

In addition to the expression of F-spondin in the

_ embryonic CNS, from E11-E12 onwards hybridization is also b detected in association with sensory and motor nerve branches that project into the periphery (Figure 7D) . The association with peripheral nerve branches suggests that F-spondin mRNA is expressed in Schwann cells. The expression of F-spondin mRNA in association with 0 peripheral nerves persists till E16, but appears to decrease at later stages, and by P0, little or no hybridization is detected in peripheral nerve (Figure 3C) . These results provide evidence that over the period of initial outgrowth of central and peripheral axons, F- spondin mRNA is expressed predominantly by the floor plate with lower levels of expression in cells of the peripheral nerves, probably Schwann cells.

F-spondin mRNA is also expressed outside the nervous system. In particular, mesodermal cells underlying the ventral midline of the spinal cord express low levels of F-spondin mRNA from Ell (Figure 7D) . In addition, embryonic and PO kidney (Figure 3C) , lung and condensing cartilage (not shown) expresses F-spondin mRNA. Expression of mRNA in the CNS, lung and kidney persists post-natally and in the adult (not shown) .

Secretion and Cell Surface Association of F-Spondin To determine the cellular localization of the F-spondin protein when expressed in mammalian cells, two epitope- tagged derivatives, F-spondin myc were generated, each of which contain a 10 amino acid insert derived from the

human c-myc proto-oncogene that can be detected by MAb 9E10 (Evan et al., 1985) (Figui-. 8A) . The cDNAs encoding F-spondin myc were cloned into a mammalian expression vector and transfected into cos cells. To examine whether F-spondin myc is present in medium conditioned by transfected cells, cos cells were labelled with 35 S- methionine for 3-4h and the released proteins were immunprecipitated with MAb 9E10. Immunoprecipitates from cos cells transfected with two different F-spondin myc constructs revealed a single major band of -116 kDa that was absent from mock-transfected cells (Figure 8B) . Immunoprecipitation of proteins extracted from the cos cells indicated that the amount of F-spondin recovered from the medium was similar to that associated with the cells (not shown) . Thus cos cells release a significant fraction of synthesized F-spondin myc . Other myc epitope- tagged proteins, for example the drosophila wingless protein, are synthesized by cos cells but are not detected in the medium (K. Basler, Personal communication) suggesting that the presence of F- spondin myc in the medium does not result from leakage from damaged cells. Thus, under these in vitro conditions F- spondin myc is secreted from cells. The apparent molecular weight of F-spondin determined by SDS-PAGE (-116kDa) is significantly greater than that predicted from the amino acid sequence (-90kDa) . This difference in molecular weight may derive, in part, from glycoslyation of the core protein.

The cellular localization of F-spondin myc in transfected cos cells was also determined by immunocytochemistry. High levels of immunoreactivity were associated with the cell surface (Figures 8C, D) with both F-spondin myc constructs (Figure 8A) . No immunoreactivity was detected on the surface of untransfected cos cells (not shown) .

The absence of a membra e spanning region and the presence of multiple heparin attachment sites in F- spondin suggests that the cell surface association of F- spondin myc involves the binding of the secreted protein to. the cell surface or extracellular matrix. In support of this, F-spondin myc present in the medium removed from transfected cos cells was found to bind to the surface of untransfected cos cells in vitro (not shown) .

F-Spondin Promotes Neural Cell Adhesion and Neurite Outgrowth in vitro The structural features of F-spondin combined with its secretion and association with the cell surface raise the possibility that F-spondin can promote the adhesion of neural cells and the outgrowth of axons. Since F-spondin is expressed at highest levels in the floor plate, the effect of F-spondin on the adhesion and outgrowth of dorsal spinal cord cells to include the population of commissural neurons that project to and across the floor plate was examined. In addition, the expression of F- spondin mRNA in peripheral nerve suggested that the dorsal root ganglion (DRG) neurons might adhere to and extend neurites on F-spondin.

The F-spondin myc protein was purified on a MAb 9E10 affinity column from medium exposed to transfected cos cells (Figure 9A) and immobilized onto a nitrocellulose substrate (Lem on et al., 1989). The ability of F- spondin myc to promote the outgrowth of E14 DRG neurons was compared with that of MAb 9E10 affinity-purified proteins secreted from untransfected cos cells and BSA. Outgrowth of DRG neurons on EHS laminin was used as a positive control. Over 80% of DRG neurons extended neurites on F- spondin (Figures 9B, D) and the length of DRG neurites that extended on F-spondin was similar to that on laminin

(not shown) and significantly greater than that on parental cos cell proteins and on BSA (Figures 9C, D) . Similar results were obtained with both versions of F- spondin myc (not shown) . In addition, the number of DRG neurons that adhered to a substrate of F-spondin myc after 18h was about 3 fold greater than that to BSA and parental cos cell proteins, and similar to that on laminin (not shown) . These observations provide evidence that F-spondin can promote the adhesion of DRG neurons and the extension of neurites in vitro. The expression of F-spondin by peripheral nerve cells i vivo occurs before many sensory neurons have extended peripheral projections and could therefore contribute to the growth of developing sensory axons in the peripheral nervous system.

The ability of F-spondin myc to promote the adhesion and outgrowth of dorsal spinal cord cells was also examined. We found that dorsal spinal cord cells adhered well to F- spondin myc . Within 60 min (Figures 10A, E) the number of cells adhering to F-spondin was 10-15 fold greater than that to MAb 9E10 affinity-purified proteins secreted from untransfected cos cells or to BSA (Figures 10C, E) . The majority (>60%) of the adherent cells are neurons as determined by detection of the polysialic acid side chain of NCAM with MAb 5A5 (not shown; see Dodd et al., 1988; Karagogeos et al., 1991). Moreover, many adherent spinal cord neurons extended short neurites during this time period (Figure 10B) . To examine further whether F- spondin promotes the outgrowth of spinal cord neurites the neurite length of adherent spinal cord neurons after 18h in vitro was determined. The length of spinal cord neurites on F-spondin myc had increased by 18 hours; however neurites outgrowth on purified cos cell proteins and on BSA has also increased significantly and was not

detectably different from that on F-spondin myc . Thus it remains unclear whether F-spondin promotes extensive neurite outgrowth as well as the adhesion of spinal cord neurons.

The adhesion of a variety of cell lines to TSRs or to peptide derived from these repeats has been shown to be inhibited by glycosaminoglycans and other sulfated glycoconjugates (Roberts, 1988; Bernfield and Sanderson, 1990; Prater et al., 1991). Moreover, heparin sulfate proteoglycans have been suggested to function as cell surface receptors for thrombospondin (ftolt et al., 1984; Sun et al., 1989; Bernfield and Sanderson, 1990). It is possible therefore that the interactions of neural cells with F-spondin may be inhibitable by addition of soluble glycosaminoglycans. It was found that adhesion of dorsal spinal cord neurons to F-spondin was markedly inhibited by heparin, dextran sulfate (not shown) and to a lesser extent by chondroitin sulfate (Figures 10D, F) . To control for non-specific inhibition of the interactions of spinal cord cells with all adhesive substrates, the spinal cord neurons adhere well to fibronectin was determined and it was found that their adhesion is not significantly affected by concentrations of heparin that block adhesion to F-spondin (not shown) . Heparin also reduced to near background levels the adhesion of DRG neurons to F-spondin (not shown) . It was not possible to determine whether the outgrowth of neurites from DRG neurons is also blocked by addition of glycosaminoglycans because heparin caused the detachment of virtually all neurons from the F-spondin substrate, even when added to DRG neurons that had been permitted to settle on F- spondin for 2-3h.

Experimental discussion

Floor plate cells are located at the ventral midline of the developing nervous system and have been implicated in the control of neural cell identity and in the guidance of developing axons (Jessell and Dodd, 1991) . In order to identify genes that might contribute to the functions of the floor plate, subtractive hybridization techniques have been used to isolate cDNA clones encoding a novel protein, F-spondin. F-spondin mRNA expressed at high levels by the developing floor plate and at low or undetectable levels in other regions of the embryonic spinal cord over the period that axons first extend. The predicted structure of F-spondin together with its biochemical properties indicates that it is a secreted glycoprotein with homology to other proteins that mediate cell adhesion and neurite outgrowth. F-spondin promotes the adhesion and outgrowth of axons from embryonic neurons in vitro. suggesting that it may contribute to the growth and guidance of commissural axons at the ventral midline of the spinal cord and of sensory axons in the periphery.

Localization of F-Spondin

Several lines of evidence suggest that the F-spondin protein may be associated with the extracellular matrix. First, F-spondin has several clusters of basic residues that function as glycosaminoglycan binding domains in other secreted proteins. Second, F-spondin is associated with the surface of cos cell transfectants. Third, the complement binding protein properdin which consists almost entirely of 6 TSRs has been shown to bind sulfated glycoconjugates (Holt et al., 1990).

The restricted distribution of F-spondin mRNA in the embryonic nervous system contrasts with that of other secreted glycoproteins which promote neural cell adhesion

and neurite outgrowth. For example, the expression of F- spondin mRNA is more restricted than that of thrombospondin I (O'Shea and Dixit, 1988; O'Shea et al., 1990) and of tenascin/cytotactin (Wehrle and Chiqet, 1990) which appears to be widely expressed in the embryonic central nervous system. Similarly, laminin and fibronectin are expressed in many regions of the developing peripheral nervous system (Sanes et al., 1990) . One glycoprotein which has a restricted distribution during nervous system development is S- laminin, an isoform of the laminin B chain (Hunter et al., 1989a) .

The TSRs of F-Spondin may be Responsible for Neural Cell Adhesion and Axon Extension

The domains of F-spondin that mediate neural cell adhesion and neurite extension have not been mapped although several indirect lines of evidence suggest that the TSRs may be involved. First, proteolytic fragments of thrombospondin which contain the TSRs promote the adhesion of melanoma cells and antibodies directed against the TSRs domain block cell adhesion (Prater et al. , 1991) . Second, both native thrombospondin and a 140 kDa proteolytic fragment which includes the TSR domains promote the outgrowth of neurites from central and peripheral neurons in vitro (Osterhout and Higgins, 1990; Osterhout et al. , 1992; Neugebauer et al., 1991; O'Shea et al., 1991). In addition, antibodies directed against the TSR domains block neurite outgrowth on thrombospondin (Osterhout and Higgins, 1990; Osterhout et al. , 1992). Third, the plasmodial CS proteins, which contain the core domain of the TSRs also promote the adhesion of a wide variety of mammalian cells (Rich et al., 1990).

The adhesive properties of the CS proteins have been

mapped to the VTCG sequence (Rich et al., 1990). In addition,, the two peptides derived from the TSRs in thrombospondin that are potent attachment factors for melanoma cells also contain the VTCG sequence whereas the peptide derived from the third TSR which does not contain this sequence is not adhesive (Prater et al., 1991).

Thus, the presence of a VTCG in the fourth TSR of F- spondin suggests that this domain could be involved in the adhesive properties of F-spondin. Nevertheless, other domains within F-spondin may be involved in neural cell adhesion or neurite outgrowth. For example, the region interposed between the fifth and sixth TSP-1 repeats of F-spondin contains an LRE sequence that mediates the neuronal attachment properties of S-laminin (Hunter et al., 1989b).

The ability of neural cells to adhere to and extend neurites on F-spondin suggests that there are neural receptors for this protein. The inhibition by heparin of the adhesion of dorsal spinal cord cells and DRG neurons to F-spondin suggests that proteoglycans may constitute neuronal F-spondin receptors or may regulate receptor function.

The conservation of TSRs in F-spondin and thrombospondin also raises the possibility that receptors for the TSR domains of thrombospondin may interact with the related domains of F-spondin. There is evidence that the TSRs of thrombospondin can interact with 3 distinct classes of cellular receptors (Frazier, 1991) . First, thrombospondin and a VTCG-containing peptide from the TSR core region can bind to an 88 kDa membrane glycoprotein, GPIV, or CD36, which is present on many cell types (Asch et al., 1990, 1991). Second, thrombospondin can bind to sulfated glycoconjugates including the heparin sulfate

proteoglycan syndecan (Roberts, 1988; Sun et al. , 1989; Holt et al., 1989; Bernfied and Sanderson, 1990). In addition, the adhesion of cells to VTCG-containing peptides derived from the TSR domains of thrombospondin and plasmodial CS proteins can be inhibited by heparin and other glycosaminoglycans (Holt et al., 1990; Prater et al., 1991; Rich et al., 1991). Third, antibodies against integrins block neurite outgrowth on thrombospondin (Neugebauer et al., 1991). Since antibodies to the TSR domains of thrombospondin block the outgrowth of neurites on thrombospondin (Osterhout and Higgins, 1990; Osterhout et al., 1992) it is possible that sequences within the TSRs interact with neuronal integrins.

Possible Functions of F-Spondin in Neural Development The most prominent expression of F-spondin in the embryonic nervous system is in the floor plate, an epithelial cell group that has been implicated in several aspects of spinal cord development. Midline neural plate cells that give rise to the floor plate undergo marked cell shape changes during the closure of the neural tube. Thus, one possible function of F-spondin could be to mediate adhesive interactions between floor plate cells that maintain the integrity of the floor plate during the formation of the embryonic spinal cord. The expression of F-spondin mRNA in floor plate cells is highest at the time that the floor plate has been suggested to have roles in the chemotropic (Tessier-Lavigne et al., 1988; Placzek et al. , 1990a) and contact (Dodd et al., 1988) guidance of commissural axons. It is found that recombinant F-spondin yc secreted from cos cells does not mimic the ability of the floor plate derived chemoattractant to promote the outgrowth of commissural axons from dorsal spinal cord explants (Klar, Placzek,

Tessier-Lavigne, Dodd and Jessell, unpublished observations) . This suggests that F-spondin may not be involved in the long-range guidance of commissural axons to the floor plate, at least through chemotropism.

F-spondin could be involved in the contact-dependent guidance of commissural axons once they reach the ventral midline of the spinal cord under the influence of chemotropic guidance cues. The growth cones of commissural neurons cross the midline by growing between the basal surface of floor plate cells and the underlying basal lamina (Kuwada et al., 1990; Yaginuma et al., 1991) . F-spondin secreted by the floor plate may accumulate at high levels in association with the basal surface of floor plate cells or with the underlying basal lamina thus generating a difference in adhesive properties of the floor plate and the lateral neuroepithelium. The growth cones of commissural neurons may adhere preferentially to F-spondin, prompting them to change trajectory at the boundary of the floor plate and lateral neuroepithelium. It is also possible that F- spondin has a more active signalling role which induces changes in the properties of commissural growth cones that permits them to respond to other midline guidance cues. Several proteins are expressed selectively on the surface of floor plate cells at this stage of spinal cord development (Dodd and Jessell, 1988; Chuang and Lagenaur, 1990) and could provide cues that contribute to the guidance of commissural axons at the midline.

F-spondin mRNA is also expressed by cells in the peripheral nerve, presumably Schwann cells, from Ell to E16 over the period that motor and sensory axons project to their peripheral targets. Non-neuronal cells in peripheral nerve are known to secrete a variety of

extracellular matrix glycoprotein, including laminin and fibronectin that can promote the growth of developing axons. Antibody inhibition studies have provided evidence for the existence of additional molecules that mediate neuronal outgrowth on peripheral nerve substrates (Tuttle et al. , 1989) . The ability of recombinant F- spondin to promote the outgrowth of embryonic sensory neurons in vitro suggests that the protein may be released by non-neuronal cells in the peripheral nerve and could contribute to the initial outgrowth of sensory axons in vivo.

Taken together, the present studies identify F-spondin as a novel secreted protein with potential roles in neural cell adhesion and neurite outgrowth in vivo. The development of antibodies that recognize native F-spondin will be important in determining the localization of the protein within the nervous system and in assessing its function in more detail.

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SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Jessell, Thomas M Klar, Avihu

(ii) TITLE OF INVENTION: CLONING, EXPRESSION AND USES OF A NOVEL SECRETED PROTEIN, F-SPONDIN

(iii) NUMBER OF SEQUENCES: 20

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(A) ADDRESSEE: Cooper & Dunham

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(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: White, John P

(B) REGISTRATION NUMBER: 28,678

(C) REFERENCE/DOCKET NUMBER: 40028 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (212) 977-9550

(B) TELEFAX: (212) 664-0525

(C) TELEX: 422523 COOP UI

(2) INFORMATION FOR SEQ ID NO:l:

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(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

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

CTAGCGAGCA GAAGCTGATC TCCGAGGAGG ACCTCA 36

(2) INFORMATION FOR SEQ ID NO:2:

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 36 base pairs

(B ) TYPE : nucleic acid

(C) STRANDEDNESS : single

(D ) TOPOLOGY : linear

( ii ) MOLECULE TYPE : cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CTAGTGAGGT CCTCCTCGGA GATCAGCTTC TGCTCG (2) INFORMATION FOR SEQ ID NO:3:

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(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Glu Gin Lys Leu lie Ser Glu Glu Asp Leu 1 5 10

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CATGGGAGCA GAAGCTGATC TCCGAGGAGG ACCTCG (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CATGCGAGGT CCTCCTCGGA GATCAGCTTC TGCTCC

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Val Thr Cys Gly

1

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS: 0 (A) LENGTH: 4 amino acids

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(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Val Ser Cys Gly

1

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids Q (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 5 Ala Thr Cys Gly

1

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(A) LENGTH: 4029 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single 0 (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

5

( ix) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 226..2647

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

CCCTCCCTCT TCGCGCTCCT TCGCCACCGC CCGCCCCTCA GCTCCGCTGC TCGGCTCCGC

TCAGAGCAGC GCAGCTCCGC AGCCAAAGCG AGGCGGGCTC GGGCTCCCCA CCGCCAGTGC 1 CACCCGGGCT CCTCCAGCTT TCGCCTCTGC AGCTCCCGTC ACTTGGAGTA AAAGTGTCCT 1

GACAGGGGTC TGCAACATCA GCAGAAAGTT GGGAGGTCCT CGAGA ATG AGG CTA 2

Met Arg Leu

1

TCT CCC GCG CCC CTG AGG CTT AGC CGG GGT CCG GCG CTG CTG GCC CTG 2 Ser Pro Ala Pro Leu Arg Leu Ser Arg Gly Pro Ala Leu Leu Ala Leu 5 10 15

GCG CTG CCC CTG GCC GCA GCG CTC GCT TTC TCG GAT GAG ACC CTG GAC 3

Ala Leu Pro Leu Ala Ala Ala Leu Ala Phe Ser Asp Glu Thr Leu Asp

20 25 30 35

AAA GTG GCC AAG TCG GAG GGC TAC TGC AGC CGC ATC TTG CGC GCC CAG 3 Lys Val Ala Lys Ser Glu Gly Tyr Cys Ser Arg lie Leu Arg Ala Gin 40 45 50 GGC ACA CGG CGT GAG GGA TAC ACA GAG TTC AGC CTC CGC GTG GAA GGC 4 Gly Thr Arg Arg Glu Gly Tyr Thr Glu Phe Ser Leu Arg Val Glu Gly 55 60 65

GAC CCT GAC TTC TAT AAG CCA GGA AGC AGC TAC CGA GTG ACA CTC TCG 4 Asp Pro Asp Phe Tyr Lys Pro Gly Ser Ser Tyr Arg Val Thr Leu Ser 70 75 80

GCT GCC CCT CCC TCC TAC TTC AGA GGC TTC ACG TTA ATT GCT CTC AAA 5 Ala Ala Pro Pro Ser Tyr Phe Arg Gly Phe Thr Leu lie Ala Leu Lys 85 90 95

GAG AAC CGC GAA GGC GAT AAG GAA GAA GAC CAC GCG GGC ACC TTC CAG 5 Glu Asn Arg Glu Gly Asp Lys Glu Glu Asp His Ala Gly Thr Phe Gin 100 105 110 115

ATC ATA GAT GAA GAA GAA ACC CAG TTT ATG AGT AAC TGT CCT GTG GCA 6 He He Asp Glu Glu Glu Thr Gin Phe Met Ser Asn Cys Pro Val Ala 120 ' 125 130

GTC ACT GAA AGC ACC CCT CGG AGG AGG ACA CGG ATC CAG GTG TTT TGG 6 Val Thr Glu Ser Thr Pro Arg Arg Arg Thr Arg He Gin Val Phe Trp 135 140 145

ATA GCG CCA CCC ACA GGG ACA GGC TGT GTG ATT CTG AAG GCC AGC ATT 7 He Ala Pro Pro Thr Gly Thr Gly Cyβ Val He Leu Lys Ala Ser He 150 155 160

GTA CAG AAA CGC ATT ATC TAT TTT CAA GAC GAG GGC TCC CTG ACC AAG 7 Val Gin Lys Arg He He Tyr Phe Gin Asp Glu Gly Ser Leu Thr Lys 165 170 175

AAG CTG TGT GAA CAG GAT CCC ACA CTT GAT GGA GTG ACG GAC AGA CCG 8

Lys Leu Cys Glu Gin Asp Pro Thr Leu Asp Gly Val Thr Asp Arg Pro 180 185 190 195

ATC TTA GAC TGC TGC GCC TGC GGA ACT GCC AAG TAC AGA CTC ACG TTT 858 He Leu Asp Cys Cys Ala Cys Gly Thr Ala Lys Tyr Arg Leu Thr Phe . 200 205 210

TAT GGG AAC TGG TCG GAG AAG ACT CAT CCA AAG GAT TAC CCT CGT CGG 906 Tyr Gly Asn Trp Ser Glu Lys Thr His Pro Lys Asp Tyr Pro Arg Arg 215 220 225 c GCT AAT CAC TGG TCT GCC ATC ATT GGC GGA TCC CAC TCC AAG AAC TAC 954 Ala Asn His Trp Ser Ala He He Gly Gly Ser His Ser Lys Asn Tyr 230 235 240

GTG CTG TGG GAG TAC GGA GGG TAT GCC AGT GAA GGG GTC AAG CAA GTT 1002 Val Leu Trp Glu Tyr Gly Gly Tyr Ala Ser Glu Gly Val Lys Gin Val 245 250 255

GCT GAA. CTT GGC TCA CCA GTA AAA ATG GAG GAA GAA ATT CGA CAA CAG 1050 ιn Ala Glu Leu Gly Ser Pro Val Lys Met Glu Glu Glu He Arg Gin Gin ,u 260 265 270 275

AGT GAT GAA GTC CTC ACT GTC ATC AAA GCC AAA GCC CAG TGG CCA TCC 1098 Ser Asp Glu Val Leu Thr Val He Lys Ala Lys Ala Gin Trp Pro Ser 280 285 290

TGG CAG CCT GTC AAT GTG AGA GCA GCA CCC TCA GCC GAA TTC TCA GTG 1146 Trp Gin Pro Val Asn Val Arg Ala Ala Pro Ser Ala Glu Phe Ser Val

15 295 300 305

GAC AGG ACA CGC CAC TTG ATG TCC TTC CTA ACC ATG ATG GGC CCC AGT 1194 Asp Arg Thr Arg His Leu Met Ser Phe Leu Thr Met Met Gly Pro Ser 310 315 320

CCT GAC TGG AAC GTG GGC CTA TCT GCA GAG GAT CTG TGC ACC AAG GAG 1242 Pro Asp Trp Asn Val Gly Leu Ser Ala Glu Asp Leu Cys Thr Lys Glu 325 330 335 0 TGT GGC TGG GTC CAG AAA GTG GTG CAG GAC CTA ATT CCC TGG GAT GCT 1290 Cyβ Gly Trp Val Gin Lys Val Val Gin Asp Leu He Pro Trp Asp Ala 340 345 350 355

GGC ACG GAC AGC GGG GTG ACC TAC GAG TCA CCA AAC AAG CCC ACA ATT 1338 Gly Thr Asp Ser Gly Val Thr Tyr Glu Ser Pro Asn Lys Pro Thr He 360 365 370 5 CCT CAG GAA AAA ATC CGA CCC CTG ACT AGT CTG GAC CAT CCT CAG AGT 1386 Pro Gin Glu Lye He Arg Pro Leu Thr Ser Leu Asp His Pro Gin Ser 375 380 385

CCT TTC TAT GAC CCG GAA GGT GGG TCC ATC ACA CAA GTG GCC AGA GTC 1434 Pro Phe Tyr Asp Pro Glu Gly Gly Ser He Thr Gin Val Ala Arg Val 390 395 400

GTC ATC GAG AGA ATT GCC CGG AAG GGA GAA CAA TGC AAC ATT GTA CCT 1482 Val He Glu Arg He Ala Arg Lys Gly Glu Gin Cyβ Asn He Val Pro 0 405 410 415

GAC AAT GTG GAT GAT ATT GTA GCC GAC CTG GCT CCA GAA GAG AAA GAT 1530 Asp Asn Val Asp Asp He Val Ala Asp Leu Ala Pro Glu Glu Lye Asp 420 425 430 435

5

GAA GAT GAC ACC CCT GAA ACC TGC ATC TAC TCC AAC TGG TCC CCA TGG 15 Glu Aβp Aβp Thr Pro Glu Thr Cys He Tyr Ser Asn Trp Ser Pro Trp 440 445 450

TCG GCC TGC AGC TCT TCC ACT TGT GAA AAG GGT AAG AGG ATG CGG CAA 16 Ser Ala Cyβ Ser Ser Ser Thr Cys Glu Lys Gly Lys Arg Met Arg Gin 455 460 465

CGC ATG CTG AAG GCA CAG CTG GAC CTC AGT GTC CCC TGT CCT GAC ACC 16 Arg Met Leu Lys Ala Gin Leu Asp Leu Ser Val Pro Cys Pro Aβp Thr 470 475 480

CAG GAC TTC CAG CCC TGC ATG GGC CCC GGC TGC AGC GAT GAA GAT GGC 17 Gin Aβp Phe Gin Pro Cyβ Met Gly Pro Gly Cyβ Ser Aβp Glu Aβp Gly 485 490 495

TCC ACC TGT ACC ATG TCG GAG TGG ATC ACC TGG TCA CCC TGC AGT GTC 17 Ser Thr Cys Thr Met Ser Glu Trp He Thr Trp Ser Pro Cys Ser Val 500 505 510 515

TCG TGT GGC ATG GGT ATG AGG TCC CGG GAG AGG TAC GTG AAG CAG TTC 18 Ser Cyβ Gly Met Gly Met Arg Ser Arg Glu Arg Tyr Val Lye Gin Phe 520 525 530

CCG GAA GAC GGC TCG GTG TGC ATG CTG CCC ACG GAA GAG ACA GAG AAG 18 Pro Glu Asp Gly Ser Val Cys Met Leu Pro Thr Glu Glu Thr Glu Lys 535 540 545

TGC ACG GTC AAC GAG GAG TGC TCT CCT AGC AGC TGC CTG GTG ACT GAG 19 Cys Thr Val Asn Glu Glu Cyβ Ser Pro Ser Ser Cyβ Leu Val Thr Glu 550 555 560

TGG GGT GAG TGG GAT GAC TGC AGC GCC ACC TGT GGA ATG GGC ATG AAG 19 Trp Gly Glu Trp Aβp Aβp Cyβ Ser Ala Thr Cyβ Gly Met Gly Met Lye 565 570 575

AAG CGG CAC CGC ATG GTC AAG ATG AGC CCC GCG GAC GGC TCC ATG TGC 20 Lye Arg Hie Arg Met Val Lys Met Ser Pro Ala Asp Gly Ser Met Cyβ 580 585 590 595

AAG GCG GAG ACT TCG CAG GCG GAG AAA TGC ATG ATG CCT GAG TGC CAT 20 Lye Ala Glu Thr Ser Gin Ala Glu Lye Cyβ Met Met Pro Glu Cyβ Hie 600 605 610

ACC ATC CCG TGC TTG CTG TCT CCT TGG TCC GAG TGG AGC GAC TGT AGC 21 Thr He Pro Cys Leu Leu Ser Pro Trp Ser Glu Trp Ser Aβp Cyβ Ser 615 620 625

GTG ACC TGT GGG AAG GGC ATG CGG ACG CGC CAG CGG ATG CTC AAG TCT 21 Val Thr Cys Gly Lys Gly Met Arg Thr Arg Gin Arg Met Leu Lye Ser 630 635 640

CTG GCA GAG CTG GGG GAC TGT AAT GAG GAT CTG GAG CAG GCG GAG AAG 22 Leu Ala Glu Leu Gly Aβp Cyβ Asn Glu Aβp Leu Glu Gin Ala Glu Lye 645 650 655

TGT ATG CTG CCA GAG TGC CCC ATT GAC TGC GAA CTC AGT GAG TGG TCC 22 Cyβ Met Leu Pro Glu Cyβ Pro He Aβp Cyβ Glu Leu Ser Glu Trp Ser 660 665 670 675

CAG TGG TCT GAA TGT AAC AAG TCC TGT GGG AAA GGT CAC ATG ATT CGA 22 Gin Trp Ser Glu Cyβ Asn Lys Ser Cys Gly Lys Gly Hie Met He Arg

680 685 690

ACC CGG ACA ATC CAA ATG GAA CCT CAG TTT GGA GGT GCA CCC TGC CCA 23 Thr Arg Thr He Gin Met Glu Pro Gin Phe Gly Gly Ala Pro Cyβ Pro 695 700 705

GAG ACT GTG CAA CGC AAG AAG TGC CGT GCC CGG AAA TGC CTT CGC AGC 23 Glu Thr Val Gin Arg Lye Lye Cyβ Arg Ala Arg Lye Cye Leu Arg Ser 710 715 720

CCA TCG ATC CAG AAG CTG CGC TGG AGG GAG GCC CGA GAG AGC AGG AGG 24

- Pro Ser He Gin Lye Leu Arg Trp Arg Glu Ala Arg Glu Ser Arg Arg

- 725 730 735

AGT GAG CAG CTG AGG GAA GAG TCA GAT GGA GAG CAG TTC CCA GGC TGT 249 Ser Glu Gin Leu Arg Glu Glu Ser Aβp Gly Glu Gin Phe Pro Gly Cyβ 740 745 750 755

CGG ATG CGC CCG TGG ACA GCC TGG TCA GAG TGC ACC AAA CTG TGC GGA 253 Arg Met Arg Pro Trp Thr Ala Trp Ser Glu Cyβ Thr Lye Leu Cyβ Gly 0 760 765 770

GGT GGG ATC CAA GAA CGC TAC ATG ACT GTG AAG AAG AGG TTC AAA AGC 258 Gly Gly He Gin Glu Arg Tyr Met Thr Val Lye Lye Arg Phe Lye Ser 775 780 785

TCC CAG TTT ACC AGC TGC AAA GAC AAG AAG GAG ATC AGA GCG TGC AAC 263 Ser Gin Phe Thr Ser Cyβ Lye Aβp Lye Lye Glu He Arg Ala Cyβ Aβn 790 795 800

GTG CAC CCT TGT T AGTAGGGGTT CAACTCCCCA GGGCTGCATT CCAGATTCTA 26 Val Hie Pro Cye 805

GTCACCAATG GTTGGGTGGT GTATTTGCTT GTTTAAGATG ATTTAAATTG TGTCCACATG 27

TTTTCATTTT TACCGGTGTG GTTTGCCCAA TAGTCTTATG GAGGCCGAGG GACATCTTGT 28

CTGAATACTT CTTGGTGAGT ACAGGCCAAG CGGGGCATCT TGTCCCCAGG CGCCATCTTC 286

CTGCACTGAG TTGAGTAGTG TTGGTTCACC TTGGTACTAA ACTGAATCGT GTCCCTCTGG 292

AGCATCCCCT GGTCAAGCAG GGTGGAGACT TTGGCCATCC ACAAGGAGAA GCAACCAGGA 29

TGCAGCATGC GGGAGACACA GCCATTAATT GCAAAGGACA GATCCTCCTC TCTCACCTTT 30

GGCCTGCTCA CTCTTACAGA AACCTGTTTG TCCGCCTCCT TTTTTATTTA GCACAACTCC 31

AGGCATCTTG GTAAGTCTCC AGGGTCATGG GTTCTTCGGT GCCCTGAAGG AGAAGCCCTG 3

AGGTGAGGTG GCATTTGTTA CAAACCTCCC AATACTGCTT TACTGGCATC ACAAGGTCAG 32

CAGGTGATGA TGGCTACTTC ATTTCATTGT GAGCCGTGAT TTCCGTTGAG TTTTGATTGT 32

TGGTGCCATA AATGTCCTAG GATGCTGGAC GGACACATCA GCCTTGTCAG CAGATCCTTC 33

TTTGAGCCAA TGTAGACAGT AAGCTGGGCA CTGGTTCCAA AGCCAACTTA AAATCTTCCT 34

ACACATATCC AGACCTTTTT TTAGGTTGCC CAAACTTCCT TAGAATAAAG CATTTTAGCT 34

CTGAGAACTA CTTGATAAGT CTGCCAGGAA GCCCCCAAGT CAATTCTTCA ACAAAAATAC 35

TATCTTCCCT ACTTAATTTT TTTTAAGTCA TGATATTTTA TAGTTAGAGG AGAGAGAGAC 3

AATCTATTCC CATGACTAAG ACACAAACCT ACAAGAAAGG GTTACTCAGT CAAGCCTGTG 3

CCTGACTTCT GGACCAGGCC CCTGATTTTC ATGGATAGTC CAAAGGAAGG CCAGGGGTTC 3

CCACTGACTC CAAGCCATCA GCAGCACCCA AACCCAGGAG CAACAAATAT TCAGAGAAAG 3

AGGATGTTTA TCTCAGCTAT GAGCTCATTG GCAGGTTGTA CTCATGCATC TGTTAAAAGC 3

ACCACCACAT CCTTTTGCAA GTCTGTTTAT TACCGCTTCA TCCAAATACA TTTTGTGGTC 3 AAGATCGACA CAGTGCTATG AATACAGTAC TTTAAGGTCT GCATTAAACA CATCAGAATA 3

TTTCCTGCCA CATCTATGTA CAACCCCTGA ATATGTATTT TTCCTTAACA CAAGAGAGCC 4

TGTTCAATTA AAAAAAAAAA AA 4

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 807 amino acids

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

(ii) MOLECULE TYPE: protein

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

Met Arg Leu Ser Pro Ala Pro Leu Arg Leu Ser Arg Gly Pro Ala Leu 1 5 10 15

Leu Ala Leu Ala Leu Pro Leu Ala Ala Ala Leu Ala Phe Ser Aβp Glu 20 25 30

Thr Leu Asp Lys Val Ala Lys Ser Glu Gly Tyr Cyβ Ser Arg He Leu 35 40 45

Arg Ala Gin Gly Thr Arg Arg Glu Gly Tyr Thr Glu Phe Ser Leu Arg 50 55 60

Val Glu Gly Aβp Pro Aβp Phe Tyr Lye Pro Gly Ser Ser Tyr Arg Val 65 70 75 80

Thr Leu Ser Ala Ala Pro Pro Ser Tyr Phe Arg Gly Phe Thr Leu He 85 90 95

Ala Leu Lye Glu Asn Arg Glu Gly Aβp Lye Glu Glu Aβp Hie Ala Gly 100 105 110

Thr Phe Gin He He Aβp Glu Glu Glu Thr Gin Phe Met Ser Aβn Cye 115 120 125

Pro Val Ala Val Thr Glu Ser Thr Pro Arg Arg Arg Thr Arg He Gin 130 135 140

Val Phe Trp He Ala Pro Pro Thr Gly Thr Gly Cye Val He Leu Lye 145 150 155 160

Ala Ser He Val Gin Lye Arg He He Tyr Phe Gin Aβp Glu Gly Ser 165 170 175

Leu Thr Lys Lys Leu Cys Glu Gin Asp Pro Thr Leu Asp Gly Val Thr 180 185 190

Asp Arg Pro He Leu Asp Cys Cye Ala Cyβ Gly Thr Ala Lye Tyr Arg 195 200 205

Leu Thr Phe Tyr Gly Aen Trp Ser Glu Lys Thr Hie Pro Lye Aβp Tyr 210 215 220

Pro Arg Arg Ala Aen His Trp Ser Ala He He Gly Gly Ser His Ser 225 230 235 240 Lys Asn Tyr Val Leu Trp Glu Tyr Gly Gly Tyr Ala Ser Glu Gly Val

245 250 255

Lys Gin Val Ala Glu Leu Gly Ser Pro Val Lye Met Glu Glu Glu He 260 265 270

Arg Gin Gin Ser Asp Glu Val Leu Thr Val He Lye Ala Lys Ala Gin 275 280 285

Trp Pro Ser Trp Gin Pro Val Asn Val Arg Ala Ala Pro Ser Ala Glu 290 295 300

Phe Ser Val Asp Arg Thr Arg His Leu Met Ser Phe Leu Thr Met Met 305 310 315 320

Gly Pro Ser Pro Aβp Trp Asn Val Gly Leu Ser Ala Glu Asp Leu Cyβ 325 330 335

Thr Lye Glu Cyβ Gly Trp Val Gin Lye Val Val Gin Aβp Leu He Pro 340 345 350

Trp Aβp Ala Gly Thr Aβp Ser Gly Val Thr Tyr Glu Ser Pro Aen Lye 355 360 365

Pro Thr He Pro Gin Glu Lye He Arg Pro Leu Thr Ser Leu Aβp Hie 370 375 380

Pro Gin Ser Pro Phe Tyr Aβp Pro Glu Gly Gly Ser He Thr Gin Val 385 390 395 400

Ala Arg Val Val He Glu Arg lie Ala Arg Lye Gly Glu Gin Cyβ Aen 405 410 415

He Val Pro Aβp Aen Val Aβp Aβp He Val Ala Aβp Leu Ala Pro Glu 420 425 430

Glu Lye Aβp Glu Asp Aβp Thr Pro Glu Thr Cyβ He Tyr Ser Aen Trp 435 440 445

Ser Pro Trp Ser Ala Cyβ Ser Ser Ser Thr Cyβ Glu Lye Gly Lye Arg 450 455 460

Met Arg Gin Arg Met Leu Lye Ala Gin Leu Aβp Leu Ser Val Pro Cyβ 465 470 475 480

Pro Aβp Thr Gin Aep Phe Gin Pro Cye Met Gly Pro Gly Cyβ Ser Aβp 485 490 495

Glu Aβp Gly Ser Thr Cye Thr Met Ser Glu Trp He Thr Trp Ser Pro 500 505 510

Cyβ Ser Val Ser Cyβ Gly Met Gly Met Arg Ser Arg Glu Arg Tyr Val

515 520 525

Lye Gin Phe Pro Glu Aep Gly Ser Val Cyβ Met Leu Pro Thr Glu Glu 530 535 540

Thr Glu Lys Cys Thr Val Asn Glu Glu Cys Ser Pro Ser Ser Cys Leu 545 550 555 560

Val Thr Glu Trp Gly Glu Trp Asp Asp Cys Ser Ala Thr Cys Gly Met 565 570 575 Gly Met Lys Lye Arg His Arg Met Val Lye Met Ser Pro Ala Aβp Gly

580 585 590

Ser Met Cyβ Lye Ala Glu Thr Ser Gin Ala Glu Lye Cyβ Met Met Pro 595 600 605

Glu Cyβ His Thr He Pro Cys Leu Leu Ser Pro Trp Ser Glu Trp Ser 610 615 620

Aβp Cyβ Ser Val Thr Cys Gly Lye Gly Met Arg Thr Arg Gin Arg Met 625 630 635 640

Leu Lye Ser Leu Ala Glu Leu Gly Aβp Cyβ Aen Glu Aep Leu Glu Gin 645 650 655

Ala Glu Lys Cyβ Met Leu Pro Glu Cyβ Pro He Aβp Cyβ Glu Leu Ser 66G 665 670

Glu Trp Ser Gin Trp Ser Glu Cyβ Aen Lye Ser Cyβ Gly Lye Gly Hie 675 680 685

Met He Arg Thr Arg Thr He Gin Met Glu Pro Gin Phe Gly Gly Ala 690 695 700

Pro Cyβ Pro Glu Thr Val Gin Arg Lye Lye Cyβ Arg Ala Arg Lye Cyβ 705 710 715 720

Leu Arg Ser Pro Ser He Gin Lye Leu Arg Trp Arg Glu Ala Arg Glu 725 730 735

Ser Arg Arg Ser Glu Gin Leu Arg Glu Glu Ser Aβp Gly Glu Gin Phe 740 745 750

Pro Gly Cyβ Arg Met Arg Pro Trp Thr Ala Trp Ser Glu Cyβ Thr Lye 755 760 765

Leu Cye Gly Gly Gly He Gin Glu Arg Tyr Met Thr Val Lye Lye Arg 770 775 780

Phe Lye Ser Ser Gin Phe Thr Ser Cyβ Lye Aβp Lye Lye Glu He Arg 785 790 795 800

Ala Cyβ Aen Val Hie Pro Cyβ 805

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3226 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 136..2543

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

GTGTCCCTCC CTCCTCCCTC CCTCCCTCTC TCCCTCCCTC CCCGCCTGCC CCCTCCCCGC 6

CTCTCCCCTC CCCTCTCCCG CGCCGCAGCC TCCCCCGGGC CGCCCGGCGC TGCCCGAGCT 12

GTGCGGGCGC CGAGG ATG GCA GCG CGG CTG CGG CCC CTG GCC CTG CGG CTG 17 Met Ala Ala Arg Leu Arg Pro Leu Ala Leu Arg Leu 1 5 10

CTG GCG CGC ACC TTC CCC TTG GTG GCG AGG GGC TTC TCC GAC GAG ACC 21 Leu Ala Arg Thr Phe Pro Leu Val Ala Arg Gly Phe Ser Aβp Glu Thr 15 20 25

CTG GAG AAA GCC GCC AAA TCC GAG GGC TAC TGC AGC CGG ATC CTG CGA 26 Leu Glu Lye Ala Ala Lye Ser Glu Gly Tyr Cyβ Ser Arg He Leu Arg 30 35 40

GCC CAA GGC ACC AGG AGG GAA GGG TAC AAT GAA TTT AGC CTG AGG GTG 31 Ala Gin Gly Thr Arg Arg Glu Gly Tyr Aen Glu Phe Ser Leu Arg Val 45 50 55 60

GAG GGC GAT CCG GAA TTC TAC AAG CCT GGG AAC AGT TAC CGC GTG ACG 36 Glu Gly Aβp Pro Glu Phe Tyr Lye Pro Gly Aen Ser Tyr Arg Val Thr 65 70 75

CTT TCT GCT GCC ACT CCT GCG TAC TTT CGA GGA TTC ACA TTG ATT GCT 41 Leu Ser Ala Ala Thr Pro Ala Tyr Phe Arg Gly Phe Thr Leu He Ala

80 85 90

CTG AAG GAA GGA AAA GAA GGT GAT AAA GAG GAA GAC CAT GCG GGA ACT 45

Leu Lye Glu Gly Lye Glu Gly Aβp Lye Glu Glu Aβp Hie Ala Gly Thr 95 100 105

TTT CAG ATC ATA GAT GAA GAA GAG ACG CAG TTC ATG AGC AAT TGT CCC 50 Phe Gin He He Aβp Glu Glu Glu Thr Gin Phe Met Ser Aen Cyβ Pro 110 115 120

GTC GCG GTT ACT GAG AGC ACA CCT AGA AGG AGG ACA CGC ATC CAG GTC 55 Val Ala Val Thr Glu Ser Thr Pro Arg Arg Arg Thr Arg He Gin Val 125 130 135 140

TTC TGG ACA GCT CCT CCT ACT GGT ACG GGC TGT GTC ATT CTG AAA GCC 60 Phe Trp Thr Ala Pro Pro Thr Gly Thr Gly Cyβ Val He Leu Lye Ala 145 150 155

AGT ATT GTG CAG AAG CGC ATT ATT TAT TTT CAG GAC GAG GGT TCT CTC 65 Ser He Val Gin Lye Arg He He Tyr Phe Gin Aep Glu Gly Ser Leu 160 165 170

ACC AAA AGA ATC TGT GAA CAA GAT TCA GCC TCT GAA GGT GTG ACT GAC 69

Thr Lye Arg Ha Cye Glu Gin Aβp Ser Ala Ser Glu Gly Val Thr Aβp 175 180 185

AAA CCA ACA TTA GAT TGC TGT GCC TGT GGA ACT GCC AAA TAC AGG CTA Lye Pro Thr Leu Aβp Cyβ Cyβ Ala Cyβ Gly Thr Ala Lye Tyr Arg Leu 190 195 200

ACG TTT TAT GGA AAT TGG TCG GAA AAA ACA CAT CCC AAA GAC TTT CCT Thr Phe Tyr Gly Aen Trp Ser Glu Lye Thr Hie Pro Lye Aβp Phe Pro 205 210 215 220

5 CGG CGC ACC AAC CAT TGG TCT GCG ATC ATT GGT AGC TCT CAC TCA AAG Arg Arg Thr Asn Hie Trp Ser Ala He He Gly Ser Ser Hie Ser Lye 225 230 235

AAC TAC ATC CTT TGG GAG TAT GGA GGG TAT GCT AGT GAA GGT GTC AAG Aen Tyr He Leu Trp Glu Tyr Gly Gly Tyr Ala Ser Glu Gly Val Lye 240 245 250

CAG GTT GCA GAG CTG GGA TCC CCA GTC AAG ATG GAA GAA GAA ATT CGA 0 Gin Val Ala Glu Leu Gly Ser Pro Val Lye Met Glu Glu Glu He Arg 255 260 265

CAA CAA AGT GAT GAG GTT TTA ACA GTC ATC AAG GCA AAA GCA CAG TGG Gin Gin Ser Aβp Glu Val Leu Thr Val He Lye Ala Lye Ala Gin Trp 270 275 280

CCT GCC TGG CAG CCT CTG AAT GTG AGA GCT GCT CCC TCT GCT GAG TTT 1 Pro Ala Trp Gin Pro Leu Aen Val Arg Ala Ala Pro Ser Ala Glu Phe 15 285 290 295 300

TCT GTT GAT CGC CAC CGG CAC CTG ATG TCC TTC CTC ACC ATG CTG GGG 1 Ser Val Aβp Arg Hie Arg Hie Leu Met Ser Phe Leu Thr Met Leu Gly 305 310 315

CCC AGT CCC GAC TGG AAT GTG GGC CTG TCT GCT GAG GAC CTC TGC ACC 11 Pro Ser Pro Aβp Trp Aen Val Gly Leu Ser Ala Glu Aβp Leu Cye Thr 320 325 330

20

AAG GAC TGT GGC TGG GTT CAG AAA GTC GTG CAG GAT TTA ATC CCC TGG 11

Lye Aβp Cyβ Gly Trp Val Gin Lye Val Val Gin Aep Leu He Pro Trp

335 340 345

GAT GCC GGC ACA GAC AGT GGC GTC ACC TAT GAG TCA CCC AAC AAA CCT 12 Aβp Ala Gly Thr Aep Ser Gly Val Thr Tyr Glu Ser Pro Aen Lye Pro 350 355 360

25 ACA GTT CCT CAA GAG AAG ATT AGA CCA CTT ACA AGC TTA GAT CAC CCT 12 Thr Val Pro Gin Glu Lye He Arg Pro Leu Thr Ser Leu Aβp Hie Pro 365 370 375 380

CAG AGT CCA TTT TAT GAT CCA GAA GGA GGA TCT ATC AAG CTT GTA GCC 13 Gin Ser Pro Phe Tyr Aep Pro Glu Gly Gly Ser He Lye Leu Val Ala 385 390 395

AGA GTC GTG CTT GAA AGA ATT GCA CGC AAG GGG GAG CAG TGC AAC TTC 13 on Arg Val Val Leu Glu Arg He Ala Arg Lye Gly Glu Gin Cyβ Aen Phe ~- 400 405 410

GTA CCT GAT AAC ATA GAT GAT ATT GTG GCA GAC CTA GCA CCA GAA GAA 1 Val Pro Aβp Aen He Aβp Aep He Val Ala Aβp Leu Ala Pro Glu Glu 415 420 425

35

AAλ GAA GAA GAT GAT ACC CCT GAG ACC TGC ATA TAT TCA AAC TGG TCC 1467 Lye Glu Glu Asp Asp Thr Pro Glu Thr Cys He Tyr Ser Aen Trp Ser 430 435 440

CCC TGG TCA GCC TGC AGC TCC TCT ACC TGT GAG AAG GGC AAG AGG ATG 1515 Pro Trp Ser Ala Cyβ Ser Ser Ser Thr Cyβ Glu Lye Gly Lye Arg Met 445 450 455 460

AGG CAG AGA ATG CTT AAA GCT CAG CTG GAC CTC AGT GTG CCC TGT CCT 1563 Arg Gin Arg Met Leu Lye Ala Gin Leu Aβp Leu Ser Val Pro Cyβ Pro 465 , 470 475

5 GAT ACC CAA GAT TTT CAG CCA TGC ATG GGT CCA GGC TGC AGT GAT GAA 1611 Aβp Thr Gin Aβp Phe Gin Pro Cyβ Met Gly Pro Gly Cye Ser Aep Glu 480 485 490

GAT GGT TCA ACT TGC ATG ATG TCT GAC TGG ATT ACA TGG TCC CCC TGT 1659 Aβp Gly Ser Thr Cye Met Met Ser Aβp Trp He Thr Trp Ser Pro Cyβ 495 500 505 ln AGT GTT TCC TGT GGA ATG GGA ACG CGA TCT AGA GAG AGA TAT GTA AAG 1707 1U Ser Val Ser Cys Gly Met Gly Thr Arg Ser Arg Glu Arg Tyr Val Lye '510 515 520

CAA TTC CCC GAA GAT GGC TCT ATG TGC AAA GTG CCT ACT GAA GAA ACT 1755 Gin Phe Pro Glu Aβp Gly Ser Met Cyβ Lye Val Pro Thr Glu Glu Thr 525 530 535 540

GAG AAA TGT ATT GTA AAT GAG GAA TGC TCC CCT AGC AGC TGC CTT GTC 1803 _._ Glu Lye Cyβ He Val Aen Glu Glu Cye Ser Pro Ser Ser Cyβ Leu Val lb 545 550 555

ACC GAA TGG GGA GAG TGG GAT GAA TGC AGT GCT AGC TGT GGC ACA GGA 1851 Thr Glu Trp Gly Glu Trp Aβp Glu Cyβ Ser Ala Ser Cyβ Gly Thr Gly 560 565 570

ATG AAA AGG CGA CAC AGA ATG ATC AAG ATG ACT CCT GCT GAT GGA TCT 1899 Met Lye Arg Arg Hie Arg Met He Lye Met Thr Pro Ala Aβp Gly Ser 20 575 580 585

ATG TGC AAG GCA GAA ACT ACA GAG GCA GAG AAA TGC ATG ATG CCC GAA 1947 Met Cyβ Lye Ala Glu Thr Thr Glu Ala Glu Lye Cyβ Met Met Pro Glu 590 595 600

TGC CAT ACT ATT CCC TGC CTT CTA TCC CCA TGG TCT GAA TGG AGC GAC 1995 Cyβ Hie Thr He Pro Cye Leu Leu Ser Pro Trp Ser Glu Trp Ser Aβp 605 610 615 620 5 TGC AGC GTG ACA TGT GGG AAG GGA ATG CGA ACC CGG CAA AGG ATG CTG 2043 Cye Ser Val Thr Cyβ Gly Lye Gly Met Arg Thr Arg Gin Arg Met Leu 625 630 635

AAA TCT GCA GCT GAG CTT GGA GAC TGC AAT GAG GAA CTG GAG CAA GCA 2091 Lye Ser Ala Ala Glu Leu Gly Aβp Cyβ Aen Glu Glu Leu Glu Gin Ala 640 645 650

GAG AAA TGC ATG CTA CCT GAA TGC CCC ATT GAC TGT GAG CTA ACG GAG 2139 0 Glu Lye Cyβ Met Leu Pro Glu Cyβ Pro He Aβp Cyβ Glu Leu Thr Glu 655 660 665

TGG TCC CAG TGG TCC GAG TGC AAT ACC TCC TGT GGG AAG GGC CAC ATG 2187 Trp Ser Gin Trp Ser Glu Cyβ Aen Thr Ser Cyβ Gly Lye Gly Hie Met

5

670 675 680

ATC AGA ACA AGA ATG ATC AAA ATA GAA CCA CAG TTT GGA GGA ACA GCA 22 He Arg Thr Arg Met He Lye He Glu Pro Gin Phe Gly Gly Thr Ala 685 690 695 700

TGC CCA GAA ACT GTC CAA CGT ACT AAA TGT CGA GTA AGG AAA TGC CTG 22 Cye Pro Glu Thr Val Gin Arg Thr Lye Cyβ Arg Val Arg Lye Cyβ Leu 705 710 715

AGA GGC CCA GGT ATG GAA AAG AGG CGT TGG AAG GAG GCC CGG GAG AAA 23 Arg Gly Pro Gly Met Glu Lye Arg Arg Trp Lye Glu Ala Arg Glu Lye 720 725 730

AGA AGA AGT GAA CAA GCA AAA AAA AAT ATT GAT AAT GAG CAA TAT CCA 23 Arg Arg Ser Glu Gin Ala Lys Lye Aen He Aβp Aen Glu Gin Tyr Pro 735 740 745

GTT TGT AGG CTG AAA CCA TGG ACT GCT TGG ACA GAA TGT TCT ACA CTC 24 Val Cye Arg Leu Lys Pro Trp Thr Ala Trp Thr Glu Cys Ser Thr Leu 750 755 760

TGT GGA GGT GGA ATT CAG GAG CGC TAC ATG ATG GTA AAG AAG AGG TCC 24 Cys Gly Gly Gly He Gin Glu Arg Tyr Met Met Val Lys Lye Arg Ser 765 770 775 780

AAA AGC ACT CAG TTT ACT AGC TGC AAA GAC AAλ AAG GAG CTA AGA GCA 25 Lye Ser Thr Gin Phe Thr Ser Cyβ Lye Aep Lye Lye Glu Leu Arg λla 785 790 795

TGT AAC GTT CAT CCT TGT TA GGAAAACACA AGGCTTCCAA GTGATGCACT 25

Cys Aen Val Hie Pro Cye 800

CTGAGCTATA AGGAAAGTCA λCCTTGGTTT GGTTTTTλλλ ACλλλCλλλλ GTλTλAAGTG 26

TATATTAGTT TTCλTTTTTG CλGTGTGGTT TGCTTTTλGT CTTGCTGGTG CλλGλλATAT 26

ATTTTATAAA TATTTCCTCC GATTAATCTA GGTAAACTTT GATGCTCCAG CTAGCCCTTA 27

CTGCATAAλλ ATAGTAGGTC ATTGTGAGTC ATTTAACTGA AGTACAGACA TATCTGTGGA 28

CATGGAATλG CCATATAGAλ ATACTλCTTG TAAAGACATG GGλTGCATGC ATATTAACAT 28

AACTAATTTG AλGTGACATG TTTCATATGT GGGAGGATTT CTCTCTTGAT TTGATTTAAA 29

AATCCAAAGC AGTGCCTλTG TGATTATACA ACTλTGCCAλ GGAGλλλTTT CλGTAATGCT 29

GGTTCAATAA TλTTAAAGGT GCATGTTTAT CTTTTTACAλ TATTGGGTTλ AGCGATλGTT 30

GAAATAλTTλ CCCTACATλC TTTTGTTCAC ATGGλTGCTG CGTTCCλTGC λλλλTCλTCT 31

TTGTTTCTCλ AATλGCλλCT TACTTAAATA ATCTGTGCAG CTCAATλGTG λTGTCλGCCC 31

ATAACACAGT CACAλCλCAC AAAGACATGT GGCTATCACA GTλCCTGTCA CTG 3

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 802 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Met Ala Ala Arg Leu Arg Pro Leu Ala Leu Arg Leu Leu Ala Arg Thr 1 5 10 15

Phe Pro Leu Val Ala Arg Gly Phe Ser Aβp Glu Thr Leu Glu Lye Ala 20 25 30

Ala Lye Ser Glu Gly Tyr Cyβ Ser Arg He Leu Arg Ala Gin Gly Thr

35 40 45 v

Arg Arg Glu Gly Tyr Aen Glu Phe Ser Leu Arg Val Glu Gly Aβp Pro 50 55 60

Glu Phe Tyr Lye Pro Gly Aen Ser Tyr Arg Val Thr Leu Ser Ala Ala 65 70 75 80

Thr Pro Ala Tyr Phe Arg Gly Phe Thr Leu He Ala Leu Lye Glu Gly 85 90 95

Lye Glu Gly Aβp Lye Glu Glu Aep Hie Ala Gly Thr Phe Gin He He 100 105 110

Aβp Glu Glu Glu Thr Gin Phe Met Ser Aen Cyβ Pro Val Ala Val Thr 115 120 125

Glu Ser Thr Pro Arg Arg Arg Thr Arg He Gin Val Phe Trp Thr Ala 130 135 140

Pro Pro Thr Gly Thr Gly Cyβ Val He Leu Lye Ala Ser He Val Gin 145 150 155 160

Lye Arg He He Tyr Phe Gin Aβp Glu Gly Ser Leu Thr Lye Arg He 165 170 175

Cyβ Glu Gin Aβp Ser Ala Ser Glu Gly Val Thr λβp Lye Pro Thr Leu 180 185 190

Aβp Cyβ Cyβ Ala Cyβ Gly Thr Ala Lye Tyr λrg Leu Thr Phe Tyr Gly 195 200 205

Aen Trp Ser Glu Lye Thr Hie Pro Lye Aβp Phe Pro λrg Arg Thr Aen 210 215 220

His Trp Ser Ala He He Gly Ser Ser Hie Ser Lye Aen Tyr He Leu 225 230 235 240

Trp Glu Tyr Gly Gly Tyr Ala Ser Glu Gly Val Lye Gin Val Ala Glu 245 250 255

Leu Gly Ser Pro Val Lye Met Glu Glu Glu He Arg Gin Gin Ser λβp 260 265 270

Glu Val Leu Thr Val He Lye λla Lye λla Gin Trp Pro λla Trp Gin 275 280 285

Pro Leu Asn Val λrg λla λla Pro Ser Ala Glu Phe Ser Val Asp Arg 290 295 300

Hie λrg Hie Leu Met Ser Phe Leu Thr Met Leu Gly Pro Ser Pro λβp 305 310 315 320

Trp λβn Val Gly Leu Ser λla Glu λβp Leu Cyβ Thr Lye λβp Cyβ Gly 325 330 335

Trp Val Gin Lye Val Val Gin λβp Leu He Pro Trp λβp λla Gly Thr 340 345 350 λep Ser Gly Val Thr Tyr Glu Ser Pro λen Lye Pro Thr Val Pro Gin 355 360 365 ς

- Glu Lye He λrg Pro Leu Thr Ser Leu Aep Hie Pro Gin Ser Pro Phe

370 375 380

Tyr Aβp Pro Glu Gly Gly Ser He Lye Leu Val λla λrg Val Val Leu 385 390 395 400

Glu λrg He Ala Arg Lye Gly Glu Gin Cyβ Aen Phe Val Pro Aβp λβn 405 410 415 0 He λβp Asp He Val λla λβp Leu λla Pro Glu Glu Lye Glu Glu λβp 420 425 430 λβp Thr Pro Glu Thr Cyβ He Tyr Ser λβn Trp Ser Pro Trp Ser λla 435 440 445

Cyβ Ser Ser Ser Thr Cyβ Glu Lye Gly Lye λrg Met λrg Gin λrg Met 450 455 460

Leu Lye λla Gin Leu λβp Leu Ser Val Pro Cyβ Pro λβp Thr Gin λβp 465 470 475 480

Phe Gin Pro Cyβ Met Gly Pro Gly Cyβ Ser λβp Glu λβp Gly Ser Thr 485 490 495

Cyβ Met Met Ser λβp Trp He Thr Trp Ser Pro Cyβ Ser Val Ser Cyβ 500 505 510

Gly Met Gly Thr λrg Ser λrg Glu λrg Tyr Val Lye Gin Phe Pro Glu 515 520 525 λβp Gly Ser Met Cyβ Lye Val Pro Thr Glu Glu Thr Glu Lye Cyβ He 530 535 540

Val λen Glu Glu Cyβ Ser Pro Ser Ser Cyβ Leu Val Thr Glu Trp Gly 545 550 555 560

Glu Trp λβp Glu Cye Ser λla Ser Cyβ Gly Thr Gly Met Lye λrg λrg 565 570 575

Hie λrg Met He Lye Met Thr Pro λla Asp Gly Ser Met Cyβ Lye λla 580 585 590

Glu Thr Thr Glu λla Glu Lye Cyβ Met Met Pro Glu Cyβ Hie Thr He 595 600 605

Pro Cyβ Leu Leu Ser Pro Trp Ser Glu Trp Ser Aβp Cyβ Ser Val Thr 610 615 620

Cyβ Gly Lye Gly Met Arg Thr Arg Gin Arg Met Leu Lye Ser Ala Ala 625 630 635 640

Glu Leu Gly Asp Cys Asn Glu Glu Leu Glu Gin Ala Glu Lye Cyβ Met 645 650 655

Leu Pro Glu Cyβ Pro He Aep Cys Glu Leu Thr Glu Trp Ser Gin Trp 660 665 670

Ser Glu Cys Asn Thr Ser Cys Gly Lys Gly His Met He Arg Thr Arg 675 680 685

Met He Lys He Glu Pro Gin Phe Gly Gly Thr Ala Cyβ Pro Glu Thr 690 695 700

- Val Gin λrg Thr Lye Cye λrg Val λrg Lye Cyβ Leu λrg Gly Pro Gly 705 710 715 720

Met Glu Lye λrg Arg Trp Lys Glu Ala λrg Glu Lys λrg λrg Ser Glu 725 730 735

Gin λla Lys Lys λsn He λsp λsn Glu Gin Tyr Pro Val Cys λrg Leu 740 745 750

- Lys Pro Trp Thr λla Trp Thr Glu Cys Ser Thr Leu Cyβ Gly Gly Gly

755 760 765

He Gin Glu λrg Tyr Met Met Val Lye Lye λrg Ser Lye Ser Thr Gin 770 775 780

Phe Thr Ser Cye Lye λβp Lye Lye Glu Leu λrg λla Cye λβn Val Hie 785 790 795 800

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1816 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEλTURE:

(λ) NAME/KEY: CDS (B) LOCATION: 2..1705

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

T TCλ GGT Gλλ TλT GTT CTT TGG AGT λTG AGA Cλλ GCC AGT GAT GGT 46

Ser Gly Glu Tyr Val Leu Trp Ser Met Arg Gin Ala Ser λβp Gly

1 5 10 15

GTC λλλ Cλλ GTλ GCT GλG TTG GGT TCT CCλ GTC λλλ λTG Gλλ Gλλ Gλλ 94 Val Lye Gin Val λla Glu Leu Gly Ser Pro Val Lye Met Glu Glu Glu 20 25 30 λTT CGλ CλG λλG GGλ GλT Gλλ GTT CTλ ACA GTλ λTC λλλ GCC AAA GCT 142 He Arg Gin Lye Gly Aβp Glu Val Leu Thr Val He Lye λla Lye λla 35 40 45

CλG TGG CCG GCC TGG CλG CCC CTC λλT GTG λGG GCC GCC CCT TCλ GCT 1 Gin Trp Pro λla Trp Gin Pro Leu λsn Val λrg λla λla Pro Ser λla 50 55 60

GλG TTC TCT GTG GλC λGλ λGC CGT CλC CTG λTG TCλ TTT CTG GCC λTG 2 Glu Phe Ser Val Asp Arg Ser Arg His Leu Met Ser Phe Leu Ala Met 65 70 75

ATG GGT CCT AGC CCA GAC TGG AAT GTA GGA CTC λCC TCC GλG GλT CTC 2 Met Gly Pro Ser Pro λβp Trp λsn Val Gly Leu Thr Ser Glu λsp Leu 80 85 90 95

TGT λCC λλλ GλG TGT GGC TGG GTT CλG AAG GTG GTC CAG GAT TTG ATT 3 Cyβ Thr Lye Glu Cyβ Gly Trp Val Gin Lye Val Val Gin Aβp Leu He 100 105 110

CCλ TGG GλT GCλ GGC λCT GλC λGT GGG GTλ λCC TλC GλG TCT CCλ AAC 3 Pro Trp Aep Ala Gly Thr Asp Ser Gly Val Thr Tyr Glu Ser Pro Aen 115 120 125 0 λλG CCC λCC λTT CCC CAG GAT AAA ATC CGA CCT CTG ACλ λGT CTG GλT 4 Lye Pro Thr He Pro Gin λsp Lys He λrg Pro Leu Thr Ser Leu λsp 130 135 140

CλC CCλ CAA AGC CCT TCT λTG λCC λGλ GGT GGG CCλ λTC λTλ CCT ATA 4 His Pro Gin Ser Pro Ser Met Thr Arg Gly Gly Pro He He Pro He 145 150 155

GCT CGA GTT GTG ATT GAA λGG λTT GCC λGG λλG GGλ Gλλ CλG TGC λλT 5 ic λla λrg Val Val He Glu λrg He λla λrg Lye Gly Glu Gin Cyβ λβn 160 165 170 175 λTT λTλ CCC GλC AAC GTG GAT GAC ATλ GTλ GCλ GλT CTG GTλ ACG Gλλ 5 He He Pro λβp λβn Val λβp λβp He Val λla λβp Leu Val Thr Glu 180 185 190

GλG λλλ GλC GAA GλT GλT λCC CCG GλG λCC TGC λTλ TAT TCC AAC TGG 6 Glu Lye Aep Glu λβp λβp Thr Pro Glu Thr Cyβ He Tyr Ser λβn Trp 20 195 200 205

TCC CCC TGG TCG GCC TGC AGC TCG GCC ACC TGC GAC AAG GGC λλG CGG 6 Ser Pro Trp Ser λla Cyβ Ser Ser λla Thr Cyβ Asp Lys Gly Lye Arg 210 215 220

ATG AGλ CAG CGC ATG TTA AAG GCT CAG TTA GλT CTC λGT GTT CCC TGC 7 Met λrg G n λrg Met Leu Lye λla Gin Leu λβp Leu Ser Val Pro Cyβ 225 230 235

25

CCλ GλC λCT CλG GλC TTT Gλλ CCC TGC λTG GGG CCC GGC TGC λGC GλT 7

Pro λβp Thr Gin λβp Phe Glu Pro Cyβ Met Gly Pro Gly Cyβ Ser λβp

240 245 250 255

GλC Gλλ GCC TCT λCC TGC λTG λTG TCλ Gλλ TGG λTC λCC TGG TCG CCG 8 λβp Glu λla Ser Thr Cyβ Met Met Ser Glu Trp He Thr Trp Ser Pro 260 265 270 n TGC λGC GCC TCC TGC GGG λTG GGλ λTT GAG GTC AGG GAG AGλ TλC GTC 8 J Cyβ Ser λla Ser Cyβ Gly Met Gly He Glu Val λrg Glu λrg Tyr Val

275 280 285 λλG CλG TTC CCλ Gλλ GλC GGT TCC TTG TGT AAA GTC CCA ACG GAA Gλλ 9 Lye Gin Phe Pro Glu λβp Gly Ser Leu Cyβ Lye Val Pro Thr Glu Glu

35

290 295 300

ACT GAG λλλ TGC λTT GTC λλT GλG GλG TGT GλG CCλ λGC λGC TGT λTλ 9 Thr Glu Lye Cyβ He Val λen Glu Glu Cyβ Glu Pro Ser Ser Cyβ He 305 310 315

GTC ACG GAA TGG GCA GAG TGG GAG GAG TGC AGC GCT ACA TGC CGG ATG 10 Val Thr Glu Trp Ala Glu Trp Glu Glu Cyβ Ser Ala Thr Cyβ Arg Met 320 325 330 335

GGT ATG AAG λλG CGG CλC λGG λTG λTλ AAG λTG ACT CCA GCG GAT GGA 10 Gly Met Lye Lye λrg Hie λrg Met He Lye Met Thr Pro λla λβp Gly

340 345 350

TCT λTG TGC AAλ GCC GλC λCλ λCλ GλG GTT GλG AAA TGC ATG ATG CCC 11 Ser Met Cyβ Lye Ala λβp Thr Thr Glu Val Glu Lye Cyβ Met Met Pro 355 360 365

GAλ TGT CAT ACC ATC CCG TGC GTG TTG TCC CCT TGG TCT Gλλ TGG λGT 11 Glu Cyβ Hie Thr He Pro Cyβ Val Leu Ser Pro Trp Ser Glu Trp Ser 370 375 ' 380

GλT TGC λGC GTT λCC TGT GGC AAA GGC ACC AGλ λCC λGλ CλG λGλ λTG 11 λsp Cys Ser Val Thr Cye Gly Lye Gly Thr λrg Thr λrg Gin λrg Met 385 390 395

TTG λλG TCC CCG TCT Gλλ CTT GGλ GλT TGC λλT GλG Gλλ CTG Gλλ CTG 12 Leu Lye Ser Pro Ser Glu Leu Gly λβp Cyβ λβn Glu Glu Leu Glu Leu 400 405 410 415 λλλ CAA GTG GAλ λλG TGC λTG CTT CCT Gλλ TGC CCT λTλ λGC TGT Gλλ 12 Lys Gin Val Glu Lye Cyβ Met Leu Pro Glu Cye Pro He Ser Cyβ Glu 420 425 430

TTG ACA GAG TGG TCT TAC TGG TCT GAG TGT AAC A A TGC TCG GGC AAG 13 Leu Thr Glu Trp Ser Tyr Trp Ser Glu Cyβ Aen Lye Cyβ Ser Gly Lye 435 440 445 GGT CAC ATG ATT CGT ACC CGA ATG ATC ACA ATG Gλλ CCλ CλG TTT GGλ 13 Gly Hie Met He λrg Thr λrg Met He Thr Met Glu Pro Gin Phe Gly 450 455 460

GGλ GCC GTC TGT CCG Gλλ λCC GTG Cλλ CGC AAA λλλ TGC CGλ TTλ CGT 14 Gly λla Val Cyβ Pro Glu Thr Val Gin Arg Lye Lye Cyβ λrg Leu λrg 465 470 475 λλλ TGT Cλλ AAA AGT TCC GGG AAT GAG CGλ λGG CλT TTλ λλG GλT GCC 14 Lye Cyβ Gin Lye Ser Ser Gly λβn Glu λrg λrg Hie Leu Lye λβp λla 480 485 490 495

CGλ GλG λλG λGλ λGG λGT Gλλ λλλ λTλ λλG Gλλ GλT TCλ GλT GGλ Gλλ 15 λrg Glu Lye λrg λrg Ser Glu Lye He Lye Glu λβp Ser λep Gly Glu 500 505 510

CλG TλC CCT GTλ TGT λλλ λTG λ A CCA TGG λCT GCλ TGG λCG Gλλ TGT 15 Gin Tyr Pro Val Cyβ Lye Met Lye Pro Trp Thr λla Trp Thr Glu Cyβ 515 520 525 λCC λ λ TTC TGC GGT GGC GGG ATλ CAλ GAG CGG TTC ATG λCT GTG AAG 16 Thr Lye Phe Cyβ Gly Gly Gly He Gin Glu Arg Phe Met Thr Val Lye 530 535 540

λλG λGλ TTC λλλ λGT TCT CλG TTC λCC λGC TGC λλG GλC λAG AλG GλG 1 Lye λrg Phe Lye Ser Ser Gin Phe Thr Ser Cyβ Lye λβp Lye Lye Glu 545 550 555

ATC CGG GCT TGC AλT GTC CλT CCλ TGT TλACCTGCCT GλAAλGλGGG 1

He λrg λla Cyβ λβn Val His Pro Cys 560 565

ATTGACACTλ CλλTCGCλλC AGAAGTCAAT CTTTATTλGλ TλTTTTTTλT CλTλGAATλT 1 λTλCλTGTGC TTTCλTTTTG CλTGTλCTTT T 18

(2) INFORMλTION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(λ) LENGTH: 568 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ϋ) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Ser Gly Glu Tyr Val Leu Trp Ser Met λrg Gin λla Ser λsp Gly Val

1 5 10 15

Lys Gin Val λla Glu Leu Gly Ser Pro Val Lye Met Glu Glu Glu He 20 25 30 λrg Gin Lye Gly λβp Glu Val Leu Thr Val He Lye λla Lye λla Gin 35 40 45

Trp Pro λla Trp Gin Pro Leu λβn Val λrg λla λla Pro Ser λla Glu 50 55 60

Phe Ser Val λβp λrg Ser λrg Hie Leu Met Ser Phe Leu λla Met Met 65 70 75 80

Gly Pro Ser Pro λβp Trp λβn Val Gly Leu Thr Ser Glu λβp Leu Cyβ 85 90 95

Thr Lye Glu Cyβ Gly Trp Val Gin Lye Val Val Gin λβp Leu He Pro 100 105 110

Trp λβp λla Gly Thr Asp Ser Gly Val Thr Tyr Glu Ser Pro Asn Lye 115 120 125

Pro Thr He Pro Gin Aep Lye He Arg Pro Leu Thr Ser Leu Aβp Hie 130 135 140

Pro G n Ser Pro Ser Met Thr Arg Gly Gly Pro He He Pro He λla 145 150 155 160 λrg Val Val He Glu λrg He Ala Arg Lye Gly Glu Gin Cyβ Aen He 165 170 175

He Pro Aβp Aen Val λβp Asp He Val Ala Asp Leu Val Thr Glu Glu 180 185 190

Lye Aβp Glu Aβp Aβp Thr Pro Glu Thr Cyβ He Tyr Ser Asn Trp Ser 195 200 205

Pro Trp Ser Ala Cyβ Ser Ser Ala Thr Cyβ λβp Lye Gly Lye λrg Met 210 215 220 λrg Gin Arg Met Leu Lye Ala Gin Leu Aep Leu Ser Val Pro Cyβ Pro 225 230 235 240

Aβp Thr Gin λep Phe Glu Pro Cys Met Gly Pro Gly Cys Ser λβp λβp 245 250 255

Glu λla Ser Thr Cyβ Met Met Ser Glu Trp He Thr Trp Ser Pro Cye 260 265 270

Ser λla Ser Cyβ Gly Met Gly He Glu Val λrg Glu Arg Tyr Val Lye 275 280 285

Gin Phe Pro Glu Aβp Gly Ser Leu Cye Lye Val Pro Thr Glu Glu Thr 290 295 300

Glu Lye Cyβ He Val λβn Glu Glu Cyβ Glu Pro Ser Ser Cyβ He Val 305 310 315 320

Thr Glu Trp λla Glu Trp Glu Glu Cyβ Ser λla Thr Cyβ λrg Met Gly 325 330 335

Met Lye Lye λrg Hie λrg Met He Lye Met Thr Pro λla λβp Gly Ser 340 345 350

Met Cyβ Lye λla λβp Thr Thr Glu Val Glu Lye Cyβ Met Met Pro Glu 355 360 365

Cyβ Hie Thr He Pro Cyβ Val Leu Ser Pro Trp Ser Glu Trp Ser λβp 370 375 380

Cyβ Ser Val Thr Cyβ Gly Lye Gly Thr λrg Thr λrg Gin λrg Met Leu 385 390 395 400

Lye Ser Pro Ser Glu Leu Gly λβp Cyβ λβn Glu Glu Leu Glu Leu Lye 405 410 415

Gin Val Glu Lye Cyβ Met Leu Pro Glu Cyβ Pro He Ser Cyβ Glu Leu 420 425 430

Thr Glu Trp Ser Tyr Trp ,Ser Glu Cyβ λen Lye Cyβ Ser Gly Lye Gly 435 440 445

Hie Met He Arg Thr λrg Met He Thr Met Glu Pro Gin Phe Gly Gly 450 455 460 λla Val Cyβ Pro Glu Thr Val Gin λrg Lye Lye Cyβ λrg Leu λrg Lye 465 470 475 480

Cye Gin Lys Ser Ser Gly λβn Glu λrg λrg Hie Leu Lye λβp λla λrg 485 490 495

Glu Lye λrg λrg Ser Glu Lye He Lye Glu λβp Ser λβp Gly Glu Gin 500 505 510

Tyr Pro Val Cyβ Lye Met Lye Pro Trp Thr λla Trp Thr Glu Cyβ Thr 515 520 525

Lye Phe Cyβ Gly Gly Gly He Gin Glu λrg Phe Met Thr Val Lye Lye 530 535 540

λrg Phe Lye Ser Ser Gin Phe Thr Ser Cys Lys λsp Lys Lys Glu He 545 550 555 560 λrg λla Cyβ λβn Val Hie Pro Cys 565

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(λ) LENGTH: 59 amino acids

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

0 (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

«n Glu Thr Cye He Tyr Ser λβn Trp Ser Pro Trp Ser λla Cyβ Ser Ser ιυ 1 5 10 15

Ser Thr Cyβ Glu Lye Gly Lye λrg Met λrg Gin λrg Met Leu Lye λla 20 25 30

Gin Leu λβp Leu Ser Val Pro Cyβ Pro λβp Thr Gin λβp Phe Gin Pro 35 40 45

15 Cyβ Met Gly Pro Gly Cyβ Ser λβp Glu λsp Gly 50 55

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 56 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single 0 (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Ser Thr Cyβ Thr Met Ser Glu Trp He Thr Trp Ser Pro Cye Ser Val 5 1 5 ' 10 15

Ser Cyβ Gly Met Gly Met Arg Ser Arg Glu λrg Tyr Val Lye Gin Phe 20 25 30

Pro λβp Gly Ser Val Cyβ Met Leu Pro Thr Glu Glu Thr Glu Lye Cye 35 40 45

Thr Val λβn Glu Glu Cyβ Ser Pro 50 55 0

(2) INFORMλTION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(λ) LENGTH: 56 amino acids

5

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Ser Ser Cyβ Leu Val Thr Glu Trp Gly Glu Trp Aβp Aβp Cyβ Ser λla 1 5 10 15

Thr Cye Gly Met Gly Met Lye Lye Arg Hie Arg Met Val Lye Met Ser 20 25 30

Pro Ala Aep Gly Ser Met Cyβ Lye Ala Glu Thr Ser Gin Ala Glu Lys 35 40 45

Cys Met Met Pro Glu Cys His Thr 50 55

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 53 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

He Pro Cyβ Leu Leu Ser Pro Trp Glu Trp Ser Asp Cyβ Ser Val Thr 1 5 10 15

Cyβ Gly Lye Gly Met Arg Thr Arg Gin λrg Met Leu Lye Ser Leu λla 20 25 30

Glu Leu Gly λβp Cyβ λβn Glu λβp Leu Glu Gin λla Glu Lye Cye Met 35 40 45

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 56 amino acidβ

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

He Asp Cyβ Glu Leu Ser Glu Trp Ser Gin Trp Ser Glu Cyβ Aen Lys

1 5 10 15

Ser Cyβ Gly Lye Gly Hie Met He Arg Thr Arg Thr He Gin Met Glu 20 25 30

Pro Gin Phe Gly Gly Ala Pro Cys Pro Glu Thr Val Gin λrg Lys Lys .35 40 45

Cys λrg λla λrg Lys Cys Leu Arg 50 55

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 55 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear 0 (ϋ) MOLECULE TYPE: protein

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

Pro Gin Cys Arg Met λrg Pro Trp Thr λla Trp Ser Glu Cys Thr Lys 1 5 10 15 ( - Leu Cyβ Gly Gly Gly He Gin Glu λrg Tyr Met Thr Val Lye Lye λrg

'- 20 25 30

Phe Lye Ser Ser Gin Phe Thr Ser Cyβ Lye λβp Lye Lye Glu He λrg 35 40 45 λla Cyβ λβn Val Hie Pro Cyβ 50 55 0

5

0

5