AU713198B2 - Cloning, expression and uses of a novel secreted protein, F-spondin - Google Patents

Description

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AUSTRALIA

PATENTS ACT 1990 DIVISIONAL APPLICATION NAME OF APPLICANT(S): The Trustees of Columbia University in the City of New York ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys 1 Little Collins Street Melbourne, 3000.

INVENTION TITLE: Cloning, expression and uses of a novel secreted protein, F-spondin The following statement is a full description of this invention, including the best method of performing it known to us: Q:\oPER\EJH\39455-DL44 13t2/97 P:\OPER\MRO\1883748.SPE 28/9/99 -1- 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.

10 Throughout this specification and the claims which follow, unless the context requires .otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

As used herein, the term "derived from" shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source.

The early development of the vertebrate nervous system is controlled by local cell interactions 20 that determine the identity of specific neural cell types and the pathways of growing axons.

S* 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 Ii~.

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 morphogenically 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 chemoattractant which can orient the growth of axons of commissural neuron in vitro (Figure 1) (Tessier- Lavigne et al., 1988; Placzek et al., 1990a; Tessier- 20 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, Sgenetic 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; Purley 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. Th? 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 thrombospondin 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 floor plate contributes to the growth and guidance of axons in the developing CNS.

4ii-;r-~rr Summarv of the invention This invention provides isolated vertebrate nucleic acid molecule encoding F-spondin. The isolated nucleic acid may be cDNA or RNA. 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 nucleotides capable of specifically hybridizing with a 1 sequence included within the sequence of a nucleic acid molecule encoding a F-spondin. The nucleic acid probe may be DNA or RNA.

This invention provides the method to obtain F-spondin 1 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 20 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 RNA 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- 1 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 Fspondin 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 Fspondin 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.

*3 a. 6 Brief Description of FiGures Figure 1.

Figure 2.

Figure 3.

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

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

Expression of F-spondin mRNA. Total cellular RNA or poly RNA was isolated from different tissues and separated on 1% agaroseformaldehyde 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 PO (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 Fspondin.

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 NHZ 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 o hydrophobic residues.

Figure 6. Alignment of the carboxy terminal domain Fspondin 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- Sspondin 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.

T

8 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 Cterminal 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 localization of F-spondin mRNA in the hindbrain of a day 10 rat embryo by in situ hybridization using an 20 antisense RNA probe. Intense hybridization is detected at the ventral .1 midline of the neural tube and possible also in the axial mesoderm underlying the neural tube.

B. Localization of whole mount in situ Fspondin mRNA by Ell (embryonic day 11) rat embryos hybridization histochemistry using digoxigenin-labelled antisense probe.

.p Hybridization is detected in the floor 30 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 ventral root.

E. Dark field micrograph showing the floor plate and the ventral ventricular zone of E13 spinal cord express high levels of Fspondin 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.

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 Am; B-350 ym; C-80 mm; E-100 Am; F-170 Am; G-170 Am; H-120am.

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 NcoI site or Spel sites within the F-spondin cDNA.

B. Immunoprecipitation of conditioned media obtained by exposure of 40h to cos cells transfected with pFP5myS, pFP5myN and to 9.

9 $9 9*e* 9* .9 9 9.

$9 9* 9 9 9* 9 Figure 8.

Figure 9.

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-spondinmyc 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 204m F-spondinmye promotes the extension of neurites from DRG neurons in vitro. F-spondinye 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 reflect differences in the glycosylation of Fspondin. Neural cells isolated from E14 rat dorsal root ganglia were plated on F-spondin or on cos cell-conditioned media or BSA (not shown) substrates for 14h and then fixed and labelled with MAb 3A10 and visualized by indirect immunofluorescence. 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 gm (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 Am.

4 04 S. 4 54

S.

S. 4 *4SS Figure 10. F-spondin promotes the adhesion of dorsal spinal cord cells. A single cell suspension of E13 dorsal spinal cord cells (106 cells/35mm disk) was plated on, Fspondin m ye on BSA and on Fspondinmyc 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 Fspondinmyc substrate. Each box represents cell counts from 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-spondinyc 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 gm, B 50 Mm 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 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.

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 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 11 and 13) 1 This invention also encompasses DNAs and cDNAs which encode amino acid sequences which differ from those of Fspondin, 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 20 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 2 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 p6rtion 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.

20 F-spondin may be produced by a variety of vertebrates. In *e 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 .A mers) was ligated. The fragment was subcloned into BamH1 site of pBluescript SK (Strategene). The 5' of the gene is located near the T3 promoter. The resulting plasmid, encoding the rat F-spondin was deposited on March 19, 1992 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852,

_I

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).

1 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 20 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 Sextremely 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 Sbase pairs. Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate Sthat 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 plasmids 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.

20 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 Snot limited to Vero cells, HeLa cells, Cos cells, CV1 cells and various primary mammalian cells.

This invention provides a method to identify and purify expressed F-spondin proteins. A myc-epitope was first S introduced into the F-spondin protein. This F-spondin carrying myc-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 17 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 mycepitope.

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 of the gene is located near the T7 RNA promoter. The resulting plasmid, pcFP5.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.

S. 20 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 Sembodiment 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, Fspondin polypeptide. As used herein, the term "purified F-spondin" shall mean isolated naturally-occurring Fspondin 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 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- 1 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 20 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 purified F-spondin at a concentration effective to stimulate growth of the nerve cell.

SThis 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 Fspondin 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.

S

Experimental Details Experimental Procedures Library Construction and Screening Directional cDNA libraries were constructed in Lambda ZAP* II (Stratagene®) from embryonic day 13 floor plate and dorsal spinal cord poly (A)'-selected RNA. The 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 XhoI 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 1 from the T3 floor plate RNA using an oligo dT XhoI linker (Stratagene*).

Solution hybridization of first strand floor plate cDNA and the dorsal T3 biotinylated RNA was performed as 20 described by Sive and St. John (1988). Approximately 1 Ag of cDNA was hybridized with a 30-fold molar excess of RNA. The nucleic acids were dissolved in 10 ul of hybridization buffer containing 50 mM HEPES (pH 7.6), 0.2% SDS, 2 mM EDTA, 500 mM NaC1, and incubated at 68*C.

Under these conditions, CoT values greater than 100 were obtained. The hybridization mixture was diluted to 60 Ml with hybridization buffer without SDS, and 10 Mg streptavidin was added. The cDNA/biotin RNA hybrids were Sremoved 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 T-i-ri Xi--C hybridization.

The subtracted cDNAs were subjected to 20 cycles of a PCR reaction using oligo dT XhoI linker primer and SK primers (Stratagene®). The products of the PCR reaction were cut with EcoRI and XhoI, 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 1 were screened with radiolabelled first strand cDNA derived from floor plate and dorsal spinal cord. 100 ng of mRNA was incubated in 20 Al of 50 mM Tris pH 8.3, mM MgCl 2 150 mM KC1, 1.0 mM dGTP, 1.0 mM dTTP, 100 MCi[32p]dATP (3000 Ci/mmol), 100 pCi[3 2 P]dCTP (3000 Ci/mmol), 100 mg/ml oligo dT, 10 mM DTT, 10 U of RNasin (Promega), 20 U of MulV reverse transcriptase (BRL), for min in 37*C. 4x10 3 recombinant phage were plated and screened. Hybridization and washes were performed at high stringency (Sambrook et al., 1989). The floor plate 20 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 25 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 r enriched library and floor plate library with FP2, and FP35, which is expressed selectively in the floor plate (McKanna Cohen, 1989) is about RNA Transfer Analysis Total RNA was prepared from various tissues using the RNA Azol method (Biotex Laboratories) and then enriched for poly 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 hybridized under standard conditions.

DNA Seauencin and Analysis cDNA inserts were excised directly as Bluescript plasmids (Stratagenee). The nucleotide sequence of the inserts 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 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 20 previously (Wilkinson et al., 1987) using a T3 or T7 RNA polymerase-derived [3 5 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 25 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. In Asit hybridization was preformed essentially as described by Harland (1991), with a few modifications: anti-digoxygenin antibody (Boehringer Mannheim), was preabsorbed to E14 rat acetone powder (Harlow and Lane, 1988) before addition to the hybridization mixture. The chromoqenic reaction was carried out for l-2h.

DNA Constructs The myc epitope was introduced as follows: Two partially complementary ol igonuc leot ides with the sequence: 5'1- 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 SpeI site. the fragment was cloned into a unique SpeI site (nt 1365) in F-spondin. The same epitope was also introduced into a NcoI site (nt 1575) using the oligonucleotides: CATGGGAGCAGAAGCTGATCTCCGAGGAGGACCTCG-3' (Seq. ID No. 4) and 5' -CATGCGAGGTCCTCCTCGGAGATCAGC~rCTGCTCC-3' (Seq. ID No. The tagged F-spondin DNA was subcloned into the expression vector pMT21 (provided by Genetics Institute), or pcDNA-I (InVitrogen).

cos Cells Transfection a. *aa*a.

a a a. a. a a.

20 Cos cells were transfected follows: 80% confluent transfected with 5 jsg DNA, DEAE Dextran (Pharmacia@), After 6h cells were washed serum, 0.1mM choloroquine incubation in DNEN 10% isolation of F-spondin the (BRL), and the cells were by the DEAE-Dextran method as overnight cultures were per 100 zmdish, in 250 g/ml 100 mM Tris pH 7.3, in DMEM.

and incubated in DMD( 10% calf (Sigma) for 2.5h, followed by calf seru.m overnight. For medium was changed to OPTI-ND( incubated f or 48h.

Met&boli Labeling of Cos Cells and Immunooeciitation' Transfected cos cells were- preincubated in =ethioninefree DMEM (BRL@-GIBCO). After 1h at 370C, 25OjsCi/ul[ 3 5

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 hl. 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.

Immunocvtochemistrv F-spondin tagged with the c-myc epitope was detected with MAb 9E10 (Evan et al., 1985). Fluoresceinated isotypespecific second antibody (BoehringerO 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 220C.

1 Cultures were then washed twice in L15-1% normal goat serum (NGS) and incubated with secondary FITC conjugated isotype-specific antibody diluted in L15-1% NGS for min at 229C. Cultures were washed twice and fixed in 4% paraformaldehyde in 0.2 M phosphate buffer (PB) for min, rinsed in 0.12M PB and coverslipped in 0.05% paraphenylenediamine (Sigma) in 0.2 M sodium carbonate (pH glycerol Cultures were viewed on a Zeiss Axioplan microscope under epifluorescence optics.

*SSS

25 Cell Culture Spinal cords were dissected from embryonic day 13 rats and placed into L15 medium at 4*C. The dorsal region of the spinal cord were dissected and incubated Swith 0.05 trypsin (Gibco) for 20 min in a Ca 2 +/Mg2+-free modified essential medium (S-MEM) (Gibco) supplemented with 8 mg ml 1 glucose. The tissue was then washed with S-MEN 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 106 cells/dish in a 5% C02 humidified incubator at 370C. 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 4 /dish.

Neurite Outgrowth Assays 5x10 10 cos cells were transfected with pFP5myN and 1 conditioned medium was collected. F-spondin m yc, was affinity purified on a monoclonal anti-myc (9E10) affinity column. Affinity purified F-spondinmYc pl/ml) was absorbed onto nitrocellulose (Lemmon et al., 1989). For controls, parental cos cell conditioned 1 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 20 plated on immobilized protein substrates at a density of 20 2-10x104 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 Larker 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 30 Sedge 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 106 cells/35 mm tissue culture dish (Nunc, 35 mm diameter).

After one hour the cultures were washed twice with PBS and fixed in 4% paraformaldehyde. 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.

ExDerimental 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- 20 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 25 but not by the dorsal spinal cord in embryonic day 13 rat embryos (see Figure 2 and Experimental Procedures).

One cDNA clone identified in this screen, designated contained a 0.5 kb insert which hybridized to two major transcripts of 4.5 and 4.7 kb in poly -selected RNA 30 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 0 brain (Figure 3C), whereas no 27 hybridization was detected to RNA derived from adult liver and spleen (Figures 3A, 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 20 isolated FP5 cDNA subclones spanning the entire coding region did not reveal any differences in the nucleotide sequence of the open reading frame.

2. Translation of the open reading frame FP5-9 predicts a 25 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 by the FP5-9 cDNA has Structural Features of Cell and Substrate Adhesion Molecules Analysis of the predicted amino acid sequence of the 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 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 20 (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).

Il Moreover, the complement-binding protein properdin 30 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) 29 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 Uncwhich 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 FPS- 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 '20 protein promotes the attachment of human hematopoietic 20 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.

SID No. 7; ATCG, Seq. ID No. 8) that vary by a single conservative substitution (Figure 6B). These 30 observations raise the possibi :-ty that the TSRs in Fspondin mediate cell adhesion. A search of the Genbank database for other proteins implicated in cell adhesion and recognition that contain a VTCG sequence identified V-CAM1 (Hession et al., 1991) and the VLA4 integrin a subunit (Takada et al., 1989).

Analysis of the predicted amino acid sequence of Fspondin 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, 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 20 (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 Fspondin 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 in situ hybridization. F-spondin mRNA was first detected at E10.5 in cells located at the ventral midline of the 31 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, 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 Sa 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 EP the ventral and intermediate regions of the spinal cord (Figures 7F, By PO, 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 Ell-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 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 peripheral nerves persists till E16, but appears to decrease at later stages, and by PO, 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, Fspondin mRNA is expressed predominantly by the floor plate with lower levels of expression in cells of the peripheral nerves, probably Schwann cells.

V.

20 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 25 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).

*V Secretion and Cell Surface Association of F-Soondin 30 To determine the cellular localization of the F-spondin protein when expressed in mammalian cells, two epitopetagged derivatives, F-spondinmYc were generated, each of which contain a 10 amino acid insert derived from the

I

human c-myc proto-oncogene that can be detected by MAb 9E10 (Evan et al., 1985) (Figu:. 8A). The cDNAs encoding F-spondin my c were cloned into a mammalian expression vector and transfected into cos cells. To examine whether F-spondin m yc is present in medium conditioned by transfected cells, cos cells were labelled with 3 5

S-

methionine for 3-4h and the released proteins were immunprecipitated with MAb 9E10. Immunoprecipitates from cos cells transfected with two different F-spondinmyc 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 m YC. Other myc epitopetagged proteins, for example the drosophila wingless protein, are synthesized by cos cells but are not detected in the medium Basler, Personal communication) suggesting that the presence of Fspondinmyc in the medium does not result from leakage from 20 damaged cells. Thus, under these in vitro conditions Fspondinmye 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-spondinmyc in transfected cos cells was also determined by immunocytochemistry.

30 High levels of immunoreactivity were associated with the cell surface (Figures 8C, D) with both F-spondin m ye constructs (Figure 8A). No immunoreactivity was detected on the surface of untransfected cos cells (not shown).

The absence of a membrane spanning region and the presence of multiple heparin attachment sites in Fspondin suggests that the cell surface association of Fspondin m y c involves the binding of the secreted protein to the cell surface or extracellular matrix. In support of this, F-spondinmye 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-SDondin 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 Fspondin mRNA in peripheral nerve suggested that the dorsal root ganglion (DRG) neurons might adhere to and extend neurites on F-spondin.

*54* The F-spondin m yc protein was purified on a MAb 9E10 affinity column from medium exposed to transfected cos cells (Figure 9A) and immobilized onto a nitrocellulose substrate (Lemmon et al., 1989). The ability of FspondinmYe 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 i 3 of DRG neurons on EHS laminin was used as a positive control. Over 80% of DRG neurons extended neurites on Fspondin (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 Fspondinmyc (not shown). In addition, the number of DRG neurons that adhered to a substrate of F-spondinye 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 in 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 my to promote the adhesion and outgrowth of dorsal spinal cord cells was also examined.

We found that dorsal spinal cord cells adhered well to Fspondin myc Within 60 min (Figures 10A, E) the number of cells adhering to F-spondin was 10-15 fold greater than 20 that to MAb 9E10 affinity-purified proteins secreted from untransfected cos cells or to BSA (Figures 10C, The majority 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 Fspondin 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 my 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-spondinmyc. 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 (Holt 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, To control for non-specific inhibition of the interactions of spinal cord cells with all adhesive substrates, the 20 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 Fspondin 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 homo'ogy 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.

20 Localization of F-SDondin 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 -ne embryonic nervous system contrasts with that of other secreted glycoproteins which promote neural cell adhesion and neurite outgrowth. For example, the expression of Fspondin 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 Slaminin, an isoform of the laminin B chain (Hunter et al., 1989a).

The TSRs of F-Snondin 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 20 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 at al., 1992).

Third, the plasmodial CS proteins, which contain the core 30 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 Fspondin 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 20 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-Soondin in Neural DeveloDment 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 20 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-spondinmYc 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- 20 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 Ssurface 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 S 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 42 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 Fspondin 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 GENERAL INFORMATION: 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: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Cooper Dunham STREET: 30 Rockefeller Plaza CITY: New York STATE: New York COUNTRY: USA ZIP: 10112 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: US FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAMtE White, John P REGISTRATION NUMBER: 28,678 REFERENCE/DOCKET NUMBER: 40028

S*

20 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (212) 977-9550 TELEFAX: (212) 664-0525 S* TELEX: 422523 COOP UI INFORMATION FOR SEQ ID NO:1: S(1) SEQUENCE CHARACTERISTICS: 25 LENGTH: 36 base pairs TYPEI nucleic acid STRANDEDNESS: singl* TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CTAGCGAGCA GAAGCTGATC TCCGAGGAGG ACCTCA 36 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDZDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: eDNA

S*

S

S.

S

S**S

a S S. S *5 p5 *5e5 5 (xi) SEQUENCE DESCRIPTION: SEQ ID 11:2: CTAGTGAGGT CCTCCTCGGA GATCAGCTTC TGCTCG INFORMATION FOR SEQ ID 11:3: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID 11:3s Glu Gln Lys Lou Ile Ser Glu Glu Asp Lou 1 5 INFORMATION FOR SEQ ID 110:4: SEQUENCE CHARACTERISTICS:- LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 20 (1i) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID 110:4: CATGGGAGCA GAAGCTGATC TCCGAGGAGG ACCTCG 25 INFORMATION FOR SEQ ID 11:5: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID N110: CATGCGAGGT CCTCCTCGGA GATCAGCTTC TGCTCC INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Val Thr Cyn Gly 1 INFORMATION FOR SEQ ID NO:7: Wi SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (1i) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Val Ser Cys Gly 1 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids 20 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Ala Thr Cys Gly INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 4029 base pairs TYPE: nucleic acid STRANDRDNESS: single *1 TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 57 (Ix) FEATURE: NAME/KEY: CDS LOCATION: 226..2647 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CCCTCCCTCT TCGCGCTCCT T0GCCAC0GC CCGCCCCTCA GCTCCGCTGC ToGr4CTCCGC TCAGAGCAGC GCAGCTCCGC AGCCAAAGCG AGOGGGCTC GGGCTCCCCA CCGCCAGTGC 120 CACCCGGOCT CCTCCAGCTT TCGCCTCTGC AGCTCCCGTC ACTTGOAGTA AAAGTGTCCT 180 GACAG=GTC TGCAACATCA GCAGAAAGTT GGGAOOTCCT CGAGA ATG AGO CTA 234 Me0t Arg Lou

I

TCT CCC OCO CCC CTG AGO CTT AGC CGO GOT CCG GCG CTG CTOG CC CTG 282 Ser Pro Ala Pro Lou Ar; Lou Ser Ar; Gly Pro Ala Lou Lou Ala Lou 10 GCG CTG CCC CTG GCC OCA GCG CTC OCT TTC TCG GAT GAG ACC CTG GAC 330 Ala Lou Pro Lou Ala Ala Ala Lou Ala Phe Ser Asp Glu Thr Lou Asp 25 30 AAA GTG 0CC AAO TCG GAG 000 TAC TGC AGC CGC ATC TTG CCC 0CC CAG 378 Lys Val Ala Lys Sor Glu Gly Tyr Cys 8cr Ar; Ile Lou Ar; Ala Gin 45 000 ACA COG COT GAG GGA TAO ACA GAG TTC AGO CTC COO GTG OAA 000 426 Gly Thr Arg Arg Glu Gly Tyr Thr Glu Phe So: Lou Ar; Val Glu Oly 60 GAO COT GAO TTC TAT AAG CR GGA AGO AGC TAO CGA GTO ACA CTC TCG 474 *Asp Pro Asp Ph* Tyr Lys Pro Oly Ser Tyr Ar; Val Tb: Lou Ser *70 75 OCT 0CC CCT CCC TCC TAC TTC AGA GOC TTC ACG TTA ATT OCT CTC AAA 522 *Ala Ala Pro Pro Ser Tyr Phe Ar; Oly Phe Tb: Lou Ile Ala Lou Lys 90 GAO AAC COO GAA 000 GAT AAG OAK GAA GAO CRC CG 000 ACC TTC CR0 570 Olu Ann Ar; Olu Gly Asp Lys Glu Glu Asp His Ala Cly Thr Ph. Gin 100 105 110 115 ATO ATA OAT OAK GAA OAK ACC CAG TTT ATO AGT AAC TOT CCT OTO CR 618 Ile Ile Asp Glu Glu Glu Tb: Gin Ph. Not Ser Ann Cys Pro Val Ala 120 125 130 OTC ACT OAK AGO ACC COT COO AGO AGO ACA COG ATC CAG OTO TTT TOO 666 *.:Val Tb: Olu $or Thr Pro Ar; Ar; Ar; Tb: Arg Ile Gin Val Ph& Try, 135 140 145 ATA 0GM CM CCC-ACK 000 ACA 000 TOT GTO ATT CTO KAG 0CC AGC ATT 714 Ile Ala Pro Pro Th: Oly Tb: Gly Cys Val Ile Lou Lys Ala 8cr Ile 150 155 160 OTA CA AA CGO ATT ATC TAT TTT OAK GAC GAG 000 TCC CTC hOCC hAG 762 Val Gin Lys Ar; Ile Ile Tyr Pb. Gin Asp Glu Gly So: Lou Tb: Lye 165 170 175 hAG CTO TOT OAK CAG OAT CC ACA OTT GAT GGA OTC AG GAO AGA CCC 810 LLili;-~i~ 58 Lys Lou Cys Glu Gin Amp Pro Thr Lou Asp Gly Val Thr Asp Arg Pro 180 185 190 195 ATC TTA GAC TOC TC GCC TOC OGA ACT 0CC AAG TAC AGA CTC AGO TTT 858 Ile Lou Ap Cys Cym Ala Cys Gly Thr Ala Lys Tyr Ar; Lou Thr Ph.

200 205 210 TAT GGG AAC TOG TOO GAG AAG ACT CAT CCA AAG GAT TAC CCT COT COG 906 Tyr Gly Ann Trp Ser Glu Lys Thr His Pro Lys Amp Tyr Pro Arg Arg 215 220 225 OCT hAT CAC TOG TT GCC ATC ATT GOC GGA TCC CAC TCC AAG AAC TAC 954 Ala Mn Hi Trp So: Ala Ile Ile Oly Gly Scr Him So: Lys Ann Tyr 230 235 240 OTO CTO TOG GAG TAC OGA 000 TAT 0CC AGT OAA 000 OTC AAG CAA GTT 1002 Val Lou Trp Glu Tyr Oly Gly Tyr Ala Sor Glu Gly Val Lys Gin Val 245 250 255 OCT OAA CTT GGC TCA CCA GTA AM ATO GAG OAA OAK ATT COA CM CAG 1050 Ala Oiu Lou Gly Pro Val Lys Mot Giu Glu Oiu Ile Arg Gin Gln 260 265 270 275 AOT OAT OAA OTC CTC ACT OTC ATC MA 0CC AM 0CC CAG TOG CCA TCC 1098 Sor Asp Glu Val Lou Thr Val Ile Lys Ala Lys Ala Gin Trp Pro Ser 280 285 290 TOG CAG CCT OTC MT GTO AGA 0CA 0CA CCC TCA 0CC OAA TTC TCA OTG 1146 Trp Gin Pro Val Mn Val Arg Ala Ala Pro 8or Ala Glu Ph. Sor Val 295 300 305 GAC AGO ACK COC CAC TTG ATO TOO TTC CTA ACC ATO ATG GC CCC AGT 1194 Amp Ar; Thr Arg Him Lou Not So: Ph* Lou Thr Not Not Oly Pro So: 310 315 320 CCT GAC TOG MC oTG 000 CTA TOT CA GAG OAT CTO TOO ACC AA GAG 1242 Pro Amp Trp Ann Val Oly Lou Ser Ala Olu Asp Lou Cym Tb: Lys Olu 325 330 335 TT WGC TOO GTC CAG AM OTO OTO CAG GAC CTA ATT CCC TOG OAT OCT 1290 Cy: Gly Trp Val Gin Lys Val Val Gin Amp Lou le Pro Trp Asp Ala 340 345 350 355 GOC ACO GAO AGC 000 OW' AC TAC GAG TCA CCA MC MG CCC ACA ATT 1338 Oly Thr Amp So: Oly Val Thr Tyr Olu Sor Pro Mn Lys Pro Thr Ile 360 365 370 OCT CAG GM AM ATC C0 CCC CTO ACT AGT CTO GAO CAT CCT CAG AOT 1386 Pro Gin Olu Lye le Ar; Pro Lou Thr So: Lou Amp His Pro Gin So: 375 380 385 COT TC TAT GAC CG GM GOT 000 TCC ATC ACA CM TO 0CC AGA GTC 1434 Pro Ph Tmyr Asp Pro Clu Gly Gly So Ile Thr Gin Val Ala A Val 390 395 400 GT ATC GAG AGRAKT 0CC COO MO GOA GM OA TOO MC ATT OTA COT 1482 Val Ile Giu Arg Ile Ala Ar; Lys Oly Glu Oln Cym Mn le Val Pro 405 410 415 GAC MT OTO CAT OAT ATT 0TA 0= GAC CT OCT CCA GM GAG AR OAT 1530 Amp Mn Val Amp Amp Ii Val Ala Amp Lou Ala Pro Oiu Giu Lys Ap 420 425 430 435 if I liii Thii~ n~Ti}1i1JiTi1Fffi n I GAh GAT GAC Glu Asp Asp TCQ o=c TGC Sur coc Arg

CAG

Gin

TCC

Sur 500

TOO

Sur ccc Pro

TGC

Cys

TOG

Trp hAG Lys 580

AAO

Lys

ACC

Thr

OTG

Val

CTG

Lou Ala

ATG

Hot

GAC

Asp 485

AC

Thr

TOT

cys

GAA

o iu Aco Th:

GOT

Gly 565

CG

Ar;

GOO

Ala

ATC

Ile

AC

Thbr

OCA

Ala 645 Cys

CTG

Lou 470

TTC

Phs

TOT

Cy.

GGC

Oly

GAC

Asp

GTC

Val 550

GAG

Glu

CAC

His

GAG

Glu

CO

Pro

TOT

cys 630

GAG

Glu 59 A=C C= GAA A=C TOC ATC TAC T=C AAC TOOGC C TOG Thr Pro Giu Tb: Cys Ile Tyr Sur Amin Try, 8ur Pro Trp 440 445 450 ACC TCT TCC ACT TOT OAA AAG GOT AAG AGO ATO COO CAA 8ur 8ur 8ur Tb: Cys Glu Lys Gly Lys Arg Met Arg Gin 455 460 465 AMG GCA CMG CTG GAC CTC AGT OTC CCC TOT CCT GAC ACC Lys Ala Gin Lou Asp Lou Sur Val Pro Cys Pro Asp Th: 475 480 CAG CCC TGC ATO 0CC =C 0CC TOC MGC GAT GMA GAT C Gin Pro Cy. Net Gly Pro Gly Cys Sur Asp Glu Asp Gly 490 495 ACC ATOv TCO CAG TOG ATC ACC TOO TCA CCC TGC MGT GTC Tb: Met Sur Glu Trp, Ile Tb: Trp Sur Pro Cys Sur Val 505 510 515 ATOv GOT ATO& AGO TCC CG GAG AGO TAC GTG AMG CMG TTC Met Oly Hot Ar; Sur Ar; Olu Ar; Tyr Val Lys Gin Ph.

520 525 530 0CC TCG OTC TOC ATG CTG CC ACO GMA GAG ACA GAG AAG Gly Sc: Val Cys Not Lou Pro Tb: Glu 02.u Tb: Giu Lys 535 540 545 MAC GAG GAG TOC TCT CCT AMC MGC TOC CTG OTG ACT GMG Asn Glu Glu Cym 8cr Pro Sur 8cr Cy. Lou Val Tb: Giu 555 560 TOG GAT GAC TOC MGC GCC ACC TOT OGA ATO GCC ATO AMG Trp Asp Asp Cys 8cr Ala Tb: Cys Gly Not Gly Met Lys 570 575 OC ATGOGTC AMG ATO ACC CCC GOG GAC GCC TCC ATO TOC Ar; Met Val Lys Met Sur Pro Ala Asp Oly Sur Met Cye 585 590 595 ACT TO CM OO GAG MAA TOC ATO ATG CCT GAG TGC CAT Tb: 8cr Gin Ala Giu Lys Cys Met Met Pro Glu Cys His 600 605 610 TOC ?YG CTG TCT CCT TOO TCC GMA TOO AGC GAC TOT MGC Cys Lou Lou 8cr Pro Trp Sur Olu Trp Sur Asp Cys 8ur 615 620 625 0OO, AMG CCC ATO COG A0O COC CMG COO ATG CTC AMG TCT Gly Lys Oly Met Ar; Tb: Arg Gin Arg Met Lou Lys Sur 635 640 CTO COG, CAC TOT MAT GAG GAT CTG GAG CMG GC GAG AMG Lou Gly Asp Cys Asn Giu Asp Lou Glu Gin Ala Giu Lys 650 655 1578 1626 1674 1722 1770 1818 1866 1914 1962 2010 2058 2106 2154 2202 2250 2298 C.

C.

S C C. C C. C C

.C

TOT ATO Cys 660 CMo Gin not ToG Trp, =T CCA 04 TOC CC KTT GAC T= GM =CTC T GAG TOO T= Lou Pro Giu Cys Pro Ile Asp Cym Giu Lou Sur Glu Trp, 8cr 665 670 675 TCT GMA TOT MAC AMQ TCC TOT 00O MAA GOT CRC ATO ATT CGA Sur Giu Cys Ann Lye 8cr Cys Gly Lys Oly His Met Ile Ar; 680 685 690 ACCC= ACM ATC MAA MT GAA C= CAG WA WOT GCA =CC Tc ccA 2346 Thr Arg Thr Ile Gin Met Glu Pro Gin Ph* Gly Gly Ala Pro Cys Pro 695 700 705 GAG ACT GTG MAA COC AAG AAG TGC COT GCC CGG AAA TGC CT? cc AGC 2394 Glu Thr Val Gin Arg Lys Lys Cys Arg Ala Arg Lys Cys Lou Arg Ser 710 715 720 CCA TCO ATC CAG AAG CTG OC TOG AGO GAG GCC COA GAG AGC AGO AGO 2442 Pro 8cr Ile Gin Lys Lou Arg Try Arg Glu Ala Arq Glu 8cr Arg Arg 725 730 735 AG? GAG CAG CTO AGO GAA GAG TCA OAT GGA GAG CAG TTC CCA GGC TOT 2490 8cr Glu Gin Lou Arg Giu Giu 8cr Amp Gly Giu Gin Ph. Pro Gly Cys 740 745 750 755 OG ATGO CC COG TOG ACA GCC TOG TCA GMG TOC ACC AAA CTG TGC OGA 2538 Arg Met Arg Pro Trp Thr Ala Trp 8cr Giu Cym Thr Lys Lou Cys Gly 760 765 770 GOT 000 ATC MAA GAR CGC TAO ATO ACT 070 AAG AAG AGO TTC AAA AGC 2586 Gly Gly Ile Gin Giu Arg Tyr Met Thr Val Lys Lys Arg Ph. Lys 8cr 775 780 785 TOO CAG TTT ACC AGC TGC AAA CAC AAG AAG GAG ATC AGA OCO TOC AAC 2634 Gin Ph. Thr 8cr Cym Lys Asp Lys Lys Glu Ile Arq Ala Cys Amn 790 795 800 OTO CAC CCT TOT T AGTAQGOGG? CAACTCCOCA GCTGCATT CCAGATTCTA 2687 Val Hisi Pro Cys 805 GTCACCAATG GTTGGGTOOT GTATTTGCTT OTTTAAGATG ATTTAAATTG TGTCCACATG 2747 TTTTCATTT-v- TACOGOTOTO GTTTGCCCAA TAGTCTTATO GAGGOOOGAGG GACATCTTGT 2807 CTGAATACTT CTTGGTGAGT ACAGGCCAAG COGGOCATCT TGTCCCCAGG CGCCATCTTC 2867 *CTGCACTGAG TTGAGTAGTG TTGGOTCACC TTGGTACTAA ACTOAAT~GT OTCOCTCTGG 2927 AGCATOC? GGTCAAGCAG GGTGGAGACT TOCTCACAGAAGCAACCAGGA 2987 TGCAGCATGC OAACC CCTTAATT GCAAAGGACAGTCOT TCTCOTTT 3047 OGOCTGTCA CTCTACAGA AACCTGTTTG TTTAT? GCAACTCC 3107 *AGGCATCTTO GTAAOTCTCC AGGGTCATOG CTTCTTCGG GCCCTGAAGG AGAAGCCCTG 3167 *AGGTGAGOTO GCATTTA CAAACCTCCC AATACTGCTT TACTGOCATC ACAAOGTCAO 3227 CAGGTGAATQA TGCAfCATTTCATTOT GAGCTOAT TTTGATTOT 3287 TGGTGCCATA AATGTCCTAG GATQCTOGAC GGAOCATCA U £A CAaATCCTTC 3347 GAGOCRA TOTAGACACT AAGCTGGGCR CTOOTTC A AGCCAACTTA AAATCTTCCT 3407 ACACATATCC AGACCTTTTAGTO CAAACTTCCT TAGAATAAAG CATTTTAGCT 3467 CTGAGAACTA CTTGATAAGT CTOCCAGQAA GOCOCCAAGT CRATTCTTCA ACAAAAATAC 3527 61 TATCTTCCCT ACTTAATTTT TTTTAAGTCA TGATATTTTA TAGTTAGAGG AGAGAGAGAC 351 AATCTATTCC CATGACTAAG ACACAAACCT ACAAGAAAGG GTTACTCAGT CAAGCCTGTG 36, CCTGACTTCT GGACCPAGGCC CCTGATTTT-17C ATGGATAGTC CAAAGGAAcX CCAGGGGTTC 37( CCACTGACTC CAAGCCATCA GCAGCACCCA AACCCAGGAG CAACAAATAT TCAGAGAAAG 37f AGGATGTTTA TCTCAGCTAT GAGCTCATTG GCAGGTTGTA CTCATGCATC TGTTAAAAGC 38 ACCACCACAT CCTTTTGCAA GTCTGTTTAT TACCGCTTCA TCCAAATACA TTTTGTGGTC 388 AAGATCGACA CAGTGCTATG AATACAGTAC TTTAAGGTCT GCATTAAACA CATCAGAATA 394 TTTCCTGCCA CATCTATGTA CAACCCCTGA ATATGTATTT TTCCTTAACA CAAGAGAGCC 400 TGTTCAATTA AAAAAA AA 402 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICSi LENGTH: 807 amino acids TYPE: amino acid TOPOLOGY; linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l0: Het Arg Leu Ser Pro Ala Pro Lou Arg Lou 5cr Arg Gly Pro Ala Lou 10 is Leu Ala Lau Ala Lou Pro Lou Ala Ala Ala Lou Ala Ph. Ser Amp Glu 25 Thr Lou Asp Lys Val Ala Lys Sec Glu Gly Tyr Cys Ser Arg Ile Lou to135 40 4S .20 Arg Ala Gin Gly Thr Arg Arg Glu Gly Tyr Thr Glu Ph. Ser Lou Arg :50 55 Val Glu Gly Asp Pro Asp Ph. Tyr Lys Pro Gly Ser Ser Tyr Arg Val 70 75 to to.Thr Lou Ser Ala Ala Pro Pro Sor Tyr Ph. Arg Gly Ph. Thr Lou Ile 90 too 25 Ala Lou Lys Glu Ann Arq Glu Gly Asp Lys Glu Glu Asp His Ala Gly 100 105 110 Thr Ph. Gin Ile Ile Asp Glu Glu Glu Thr Gln Phe Not Soc Ann Cys 115..ii 120 125 Pro Val I Val Thr Glu Sor Thr Pro Arg Arq Arg Thr 4krq Ila Gin 130 135 140 Val Ph. Trp Ile Ala Pro Pro Thr Gly Thr Gly Cys Val Ile Lou Lys Al Sr IeVlGin Lys Arg Ile I1e Tyr Ph* Gin Asp Glu, Gly Sor 165 170 175 62 Lou Thr Lye Lye Leu Cys Glu Gin Asp Pro Thr Leu Asp Gly Val Thr 180 185 190 Asp Arg Pro 119 Leu Asp Cye Cys Ala Cye Gly Thr Ala Lye Tyr Arg 195 200 205 Leu Thr Phe Tyr Gly Ann Trp Ser Glu Lye Thr His Pro Lye Asp Tyr 210 215 220 Pro Arg Arg Ala Asn His Trp Ser Ala Ile Ile 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 Lye Gln Val Ala Glu Leu Gly Ser Pro Val Lye Met Glu Giu Glu Ile 260 265 270 Arg Gin Gin Ser Asp Glu Val Leu Thr Val Ile Lye Ala Lye Ala Gln 275 280 285 Trp Pro Ser Trp Gin Pro Val Aen 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 Asp Trp Aen Val Gly Leu Ser Ala Glu Asp Lou Cys 325 330 335 Thr Lye Glu Cym Gly Trp Val Gin Lye Val Val Gin Asp Leu 11e Pro 340 345 350 Trp Asp Ala Gly Thr Asp Ser Gly Val Thr Tyr Glu Ser Pro Ann Lye 355 360 365 Pro Thr Ile Pro Gin Glu Lye Ile Arg Pro Leu Thr Ser Lou Asp His 370 375 380 Pro Gin Ser Pro Phe Tyr Asp Pro Glu Gly Gly Ser Ile Thr Gin Val 385 390 395 400 Ala Arg Val Val Ile Glu Arg Ile Ala Arg Lye Gly Glu Gin Cys Aen 405 410 415 Ile Val Pro Asp Asn Val Asp Asp Ile Val Ala Asp Leu Ala Pro Glu 420 425 430 25 Glu Lys Asp Glu Asp Asp Thr Pro Glu Thr Cys Ile Tyr Ser Aen Trp 435 440 445 Ser Pro Trp Ser Ala Cys Ser Ser Ser Thr Cys Glu Lys Gly Lys Arg .450 455 460 Met Arg Gin Arg Met Lei Gin Lou Asp Lou Ser Val Pro Cys 465 470 475 480 30 Pro Asp Thr Gin Asp Phe Gin Pro Cyn Met Gly Pro Gly cys Ser Asp 485 490 495 Glu Asp Gly Ser Thr Cys Thr Met Ser Glu Trp Ile Thr Trp Ser Pro 500 505 510 Cys Lys Thr 545 Val Gly Ser Glu Asp 625 Lou Ala Glu mot Pro 705 20 Lou sor Pro Lou Phe 785 Ala scr Gin 530 Glu Thr mot met Cy.

610 Cys Lys Glu Trp Ile 690 cys Arg Arg Gly Cys 770 Lys Cys Val 515 Phe Lys Glu Lys Cys 595 His 5cr 5cr Lys Ser 675 Arg Pro 5cr Arg Cys 755 Gly Ser Aen ser Pro CYl Trp Lys 580 Lys Thr Val Lou Cys 66C Gin Thr Glu Pro 5cr 740 Arg Gly 8cr Val Cys Gly Met Glu Asp Gly 535 Thr Val Asn 550 Gly Glu Trp 565 A~rg His Arg Ala Glu Thr Il Pro Cys 615 Thr Cys Gly 630 Ala Giu Lou 645 Met Lou Pro Trp Ser Glu Arg Thr 11e 695 Thr Val Gin 710 5cr Ile Gin 725 Glu Gin Lou Met Ar; Pro Gly Ile Gin 775 Gin Pho Thr 790 His Pro Cys 805 Gly 520 5cr Glu Asp Mot Ser 600 Lou Lys Gly Glu Cya 680 Gin Arg Lys Arg Trp 760 Giu met Val Glu Asp Val 585 Gin Lou Gly Asp Cys 665 Asn met Lys Lou Glu 745 Thr Arg 63 A~r Cys Cy.

Cys 570 Lys Ala Ser Met Cys 650 Pro Lys C u Lys Ar; 730 Glu Ala Tyr Ser mot Ser 555 5cr met Glu Pro Arg 635 hAn Ser Pro Cys 715 Trp Saw Trp met Ap 795 Ar; Lou 540 Pro Ala Ser Lye Trp 620 Thr Glu ASp Cys Gin 700 Arg Arg ap Ser Thr 780 Glu 525 Pro Ser Thr Pro Cys 605 Sec Arg Asp Cye Gly 685 Ph* Ala Glu Gly Glu 765 Val Akr Thr Ser Cys Al a 590 met Glu Lou Glu 670 Lys Gly Ar; Ala Glu 750 Cys Lys Tyr Glu Cys Gly 575 Asp met Trp Ar; Glu 655 Lou Gly Gly Lys Arg 735 Gln Thr Lys Val Glu Lou 560 mot Gly Pro Sec mot 640 Gin 8cr His Ala Cys 720 Glu Ph@ Lys Ar; Ar; 800 9 99 9 9999

I.

9. 9 9* 9 99 9* 9* *99 9 r 5cr Cys Lys Lys Lys Glu Ili INORMATION FOR SEQ ID NO:11: sEQUENCE CHARACTERISTICS: LEOTH: 3226 base pairs TYPE: nucleic acid 64 STRANDEDNESSI Single (D),TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 136..2S43 (xi) SEQUENCE DEsCRZPTION: SEQ ID NO:11: GTGTCCCTC TCTC ccCTCTC :CCCGCCTGCC CCCTCCCGCc ~&sI~j~CCCCTCC GCCGCAGC TccCOCGGC CGC=G= TGCCCGAGCT GTGCGGOGCGC CGAGG ATG GCA OW COO CTG COO CCC CTG GCC CTG COG CTG Not Ala Ala Ar; Lou Arg Pro Lou Ala Lou Ar; Lou 1 5 @0 00 S

S.

OS

*5 5

S.

5 0*O@ 0* S S S

S.

*5S55* S S 0*

SSOS

S

5* Sb 0 S. S Ge 45 .5.5 0 .9 055055

S

CTO

Lou

CTO

Lou

GC

Ala

GAG

Glu

CTI

Lou

CTG

Lou

TX?

Ph.

GTC

Val 125

TTC

Phe

MGT

A=C

OG

Ala

GAG

Glu

CAA

Gin

GOC

Oly

TCT

S.:

AMG

Lys

CMG

Gin 110 Ala Too Trp

ATT

11e

AA

COC

Ar; is

A

Lys

GOC

Oly

CAT

Amp

OCT

Ala

GFAA

ciu 95

ATC

11e

OTT

Val

ACA

Thr anO Val

A

ACC

Tb: 0CC Ala

ACC

Tb: cco Pro

G=C

Ala so

OA

Oly

ATA

110

ACT

Thr

OCT

Ala Cho Gin 160

ATC

Phe 0CC Ala

AGO

Arg

GAA

Olu 65

ACT

Thr

AAA

Lys

GAT

Asp Gma clu

CCT

pro 145

MAG

Lys

TOT

Pro

AAA

Lys

AGO

Ar; so

TTC

Phe

CCT

pro

GAA

Giu

OAA

Glu

MGC

Bar 130

CCT

Pro

COC

Ar;

A

Lou

TCC

Ser 35

GAA

Giu

TAC

Tyv Ala

GOT

Oly

GAA

Olu 115 Ach Tb:

ACT

Thr

AT?

CAR

Val 20 Glu Gly

AMG

Lys

TAC

Tyr

GAT

Asp 100

GAG

Giu

CCT

Pro

GOT

Gly

AT?

GAT

TTC CCC TTG OTO OO AGG GOC TTC TOC GAC GAG ACC Ala Ar; Oly Phe 8cr Asp Giu Thr GGC TAC TGC MCC COO ATC CTG CGA Gly Tyr Cya 8cr Ar; Ile Lou Ar; TAC AAT CIM TTT AGC CTG AGO GTG Tyr Amn Giu Ph* 8cr Lou Ar; Val 55 CC? 000 MAC MGT TAC CGC OTG ACO Pro Gly Awn 8cr Tyr Arg Val Thr 70 TTT COA GOA TTC ACA TTG ATT OCT Ph@ Ar; Gly Phe Tb: Lou Ile Ala e5 MAA GMG GMA GAC CAT GCM GGA ACT Lys Glu Glu Asp His Ala Gly Tb: 105 ACO CMG TTC ATG MGC MAT TGT CCC Tb: Gin Ph* Not 8cr Asn Cys Pro 120 AGA AGO AGO ACA CGC ATC CAG GTC Ar; Ar; Ar; Tb: Ar; Ile Gin Val 135 140 ACG GOC TOT GTC ATT CTO MAA GCC Tb: oly Cys Val Ile Lou Lys Ala 150 155 TAT TrT CMG QAC GAG GOT TCT CTC Tyr Phs Gin Asp Oiu Oly 8cr Lou 165 170 TcA 0CC TCT GMA GOT GTO ACT GAC 120 171 219 267 315 363 411 459 507 603 651 699 Thr

AAA

Lys

ACO

Thr 205 cGO Arg

AAC

Asn

CAG

Gln

CAA

Gin

CCT

Pro 285

TCT

Ser

CCC

Pro

AAG

Lys

GAT

Asp Pro 190 Phe CGc Arg

TAC

Tyr

OTT

Val

CAA

Gin 270 0CC Ala

GTT

Val

AGT

Ser

GAC

Asp

GCC

Ala 350 Ar; 110 Cys Glu 175 ACA TTA GAT TGC Thr Lou Asp CYl TAT GGA AAT TG Tyr Gly Ann Trp 210 ACC AAC CAT TOG Tb: Ann His Trp 225 ATC CT? TOG GAG Ila Lou Trp Glu 240 GCA GAG CTG OGA Ala Glu Lou Gly 255 AG? GAT GAG .uT.

Ser Asp Glu Val TOG CAG CC? CTO Try Gin Pro Lou 290 Gin

TOT

Cys 195

TOG

Ser

TCT

Ser

TAT

Tyr

TCC

So:

TTA

LOU

275

AAT

Asn Asp Sor Ala Sor Oiu Oly Val Tb: Asp 180 185 0cc TOT OA ACT GCC AAA TAC AGO CTA Ala Cy8 Gly Tb: Ala Lys Tyr Ar; Lou 200 GAA AAM ACA CAT CCC MAA GAC TTT CC? Glu Lys Tb: His Pro Lys Asp Ph* Pro 215 220 GCO, ATC ATT GOT AGC TCT CAC TCA AG, Ala Ile Ile Gly So: Ser His Sor Lys 230 235 GOA 000 TAT OCT AGT GMA GOT GTC MAG Gly Gly Tyr Ala So: Glu Gly Val Lys 245 250 CCA OTC MAG ATG GAA GAM GM AT? CGA Pro Val Lys Met Glu Giu Giu Ile Ar; 260 265 ACP~ GTC ATC MAG GCA AAA GMA CAG TG Tb: Val Ile Lys Ala Lys Ala Gin Trp 280 OTO AGA OCT OCT CCC TCT OCT GAO TTT Val Arg Ala Ala Pro So: Ala Giu Phe 295 300 t OAT cC ASP A~r; CCC GAC Pro Asp 320 TOT GGC Cye Oly 335 GO= ACA Gly Tb: CA COG CA CT ATO T= MT CT A=C M A M CGG0 His Ar; His Lou Not Sor Ph* Lou Tb: got Lou Oly 305 310 315 TOO MAT GTO GOC CTG TCT OCT GAG GAC CTC TGC ACC Trp Asn Val Gly Lou Ala Glu Asp Lou Cys Tb: 325 330 TOG OTT CM MAA GTC OTO CMG GAT TA ATC CCC TG Trp Val Gin Lys Val Val Gin Asp Lou I1e Pro Trp 340 345 GAC ACT GGC OTC ACC TAT GAG TCA CCC MAC AMA CCT Asp So: Gly Val Tb: Tyr 01u Pro Asn Lys Pro 35S 360 747 795 843 891 939 987 1035 1083 1131 1179 1227 1275 1323 1371 1419 A CT? CCT CM GMAOM AT? A CC CT? AC AMC TA OAT CA CC Tb: Val Pro Gin Giu Lys Ile Arg Pro Lou Tb: So: Lou Asp His Pro 365 370 375 380 CMG MT CCA TTT TAT CAT CCA GMA GGA OGA TCT ATC MhG CT? OTA C Gin Sor Pro Phe Tyr Asp Pro 01u Gly Gly Ile Lys Lou Val Ala 385 390 395 MGA OTC OTO CT? GM MGA AT? OCA CGC MAG 000G GAG CMG TOC MC TTC Ar; Val Val Lou Giu Arg Ile Ala Arg Lys Gly Giu Gin Cys Amn Ph* 400 405 410 OTA CC? GAT MAC ATA GA? OAT AT? OTW WA GAC CTA GMA CCA GAA GAA Val Pro Asp Ann Ile Asp Asp Ile Val Ala Asp Lou Ala Pro Glu.Giu 415 420 425 66 AM GMA GAA GAT GAT ACC C= GAG A=C TGC ATA TAT MC AAC TGG TCC 1467 Lys Olu, Glu hop Asp Thr Pro Glu Th: Cys Ile Tyr 8cr Ann Trp Ser 430 435 440 CCC TOO TCA GCC TGC AGC TCC TCT ACC TOT GAG AAG GGC AAG AGO ATG 1515 Pro Trp Ser Ala Cys Scr Ser Ser Thr Cys Glu Lys Gly Lys Arq Met 445 450 455 460 AGO CAG AGA ATG CTT AMA OCT CAG CTO GAC CTC AGT OTO CCC TOT CCT 1563 Arg Gin Ar; Met Lou Lys Ala Gin Lou Asp Lou Ser Val Pro Cys Pro 465 470 475 GAT ACC CAA GAT TTT CAG CCA TOC ATO GOT CCA GOC TGC ACT GAT GAA 1611 Asp Thr Gin Asp Phe Gin Pro Cyrn Net Gly Pro Gly Cys 8cr Asp Glu 480 485 490 GAT GOT TCA ACT TGC ATG ATG TCT GAC TGG ATT ACA TOG TCC CCC TOT 1659 Asp Gly 8cr Thr Cys Met Not 8cr Asp Trp Ile Thr Trp, Ser Pro Cys 495 500 505 AG? OTT TCC TOT GGA ATO OGA ACG CGA TCT AGA GAG AGA TAT GTA hAG 1707 Ser Val Ser Cys Oly Hot Gly Tkir Arg 8cr Arg Glu Ar; Tyr Val Lys 510 515 CAA TTC CCC GAA OAT GOC TCT ATO TGC AAA OTO CC? ACT GAA GMh ACT 1755 Gin Pb. Pro Giu hop Oly 8cr Met Cys Lys Val Pro Thr Glu Glu Thr 525 530 535 540 GAG AAA TOT AT? OTA hT GAG GAA TGC TCC CC? ACC AGC TGC CT? GC 1803 Glu Lys Cys Ile Val Ann Giu Glu Cyu 8cr Pro 8cr 8cr Cys Lou Val 545 550 555 .ACC GAA TOO GGA GAG TOG OAT GAM TOC AG? OCT AGC TOT GGC ACA OGA 1851 Thr Glu Trp, Gly Giu Trp Asp Oiu Cys 8cr Ala 8cr Cys Oly Thr Gly .*560 565 570 ATO AAA AGGOCGA CAC A ATO ATC AMG ATO ACT CC? OCT GAT GOA TCT 1899 *.:Met Lys Ax; Arg His Arq Met Ile Lys Met Thr Pro Ala Asp Giy 8cr *575 580 585 *ATO TOC AMG OCA OAA AC? ACA GAG GCA GAG AAA TOC ATG ATG CCC GAA 1947 Met Cys Lys Ala Glu Tbr Tb: Olu, Ala Glu Lys Cyu Met Met Pro 01u 590 595 600 *TOC CAT ACT AT? CCC TGC CT'? CTA TCC CCA TOG TC? OAA TOO MGC GAC 1995 Cy. His Thr Ile Pro Cys Lou Lou 8cr Pro Trp 8cr Glu Try, 5cr Asp 605 610 615 620 :TOC MGC GTO WA TOT 000 hG OGA ATO CGA ACC GG CRA MG ATO CTO 2043 *Cys 8cr Val Thr Cyn Oly Lys Oly Mist Ar; Tb: Ar; Gin Ar; Not Lou 625 630 635 AM TCT GCA OCT GMG GOA GAC TOC GAG OAk CTO GAG CAA OCA 2091 Lys 8cr Ala Ala Oiu Lou Oly Asp Cys Aen Oiu Oiu Lou Glu Gin Ala 645 650 GAG AMA TOC ATG CTA CC? GMA TOC CCC ATT GAC TOT GKO CTA AG GAO 2139 Glu Lye Cys Met Lou Pro Oiu Cys Pro Ile Asp Cys Oiu Lou Thr Giu 655 660 665 TOG TC= CMG TOG TCC GAG TGC Ah? ACC TCC TOT 000 AMG GOC CAC ATO 2187 ?rp 5cr Gin Trp 8cr Glu Cys Amn Tb: 8cr Cys Oly Lye Oly Big Met 670 ATC AGA Ile Ar; C. a 675 680 ACA AOA ATG ATC AAM AA GMA CCA CAG GOA GGA ACA OCA Thr Arig Hot Ile Lys Ile Giu Pro Gin Phst Gly Gly Thr Ala 690 695 700

TOC

Cy.

AGA

Ar;

AGA

Ar; CCA GAA ACT OTC CAA COT ACT AMA TOT COA OTA AGO AAA TGC CTG Pro Oiu Thr Val Gin Ar; Th: Lys Cy. Arg Val Ar; Lys Cys Lou 705 710 715 GGC CCA GOT ATO GMA MO AGO COT TOOG MO GAG 0CC OG GAG AMA Gly Pro Gly Hot Gilu Lys Arg Ar; Trp, Lys Glu Ala Ar; Glu Lys 720 .725 730 AGA AG? GMA CAA GCA AMA AMA MT AT? OAT MAT GAG CMA TAT CCA Arg Ser Giu Gin Ala Lys Lys Asn Ile Amp Asn Giu Gin Tyr Pro 735 740 745 OTT TGT Val Cyrn 750

AGO

Ar; CTG AM CCA TOO ACT OCT TOG ACA GAA TOT TCT ACA CT Lou LyS Pro Trp Thr Ala Trp, Thr O.lu Cys Ser Thr Lou 755 760

TOT

Cys 765

AAA

Lys

TOT

GGA GOT OGA ATT CAG GAG =O TAC ATO ATO OTA MOG MO AGO T= Gly Oly Gly Ile Gin Giu Ar; Tyr Het Miet Val Lys Lys Ar; Ser 770 775 780 AGC ACT CAG TTT ACT AGC TGC AMA GAC AMA MO GAG CTA AGA GCA Ser Tb: Gin Phis Th: Ser Cys Lys Asp Lys Lys Giu Lou Ar; Ala 785 790 795 MAC OTT? CAT CCT TOT TA GGAMAACACA AGGCTTCCAA

GTGATOCACT

2235 2283 2331 2379 2427 2475 2523 2573 2633 2693, 2753 2813 2873 2933 2993 3053 3113 3173 3226 Cys Aen Val

S.

.5

S

S

S

S.

S

55..

S

5* *5 S S His Pro Cys S00 CTGAGCTATA AGGAAMGTCA ACCTTOOTTT GGTTTTTAMA

ACAMACMMA

TATATTAGTT :?ATTTTTG CAGTGTGGTT TGCTTTTAGT CTI 5

GCTGGTO

ATTTTATAMA TATTTCCTCC GATTAATCTA GGTAMACTTT GATGCTCCAG CTGCATAAMA ATAOTAGGTC ATTOTGAGTC ATTTMACTGA AGTACAGACA CATGGAATAG CCATATAGMA ATACTACTTG TAMOGACATO OGATOCATGC MACTAATTTG AAGTGACATO TTTCATATOT GAGGATTT

CTCTCTTGAT

MATCCMAGCC AGTGOCATG TGATTATACA ACTATGCCMA

GGAGAAATTT

GGTTCAATAA TATTAAAGGT GCATGTTTAT CTTTTACM ATOTA GAAATAATTA CCCTACATAC TTTTTCAC ATGGATGCTG

GGTTATOC

TTGTTTCTCA AATAGCAACT TACTTAAATA ATCTOGCAG CTCAATAGTG

OTATAMOGTG

CAAGAMATAT

CTAGCCCTTA

TATCTGTGGA

ATATTAACAT

TTGATTTAAM

CAGTAATGCT

AGCOATAGTT

MMATCATCT

ATGTCAGCCC

ATAACACACT CAAC CAAAGACATGT GGCATCACA GTAcCTGCA

CTG

IKIoRNATIoII FoR SQ iD 110:12: SZQUZNcg COARACTER!STICS? LENGTH: 802 aino acdsm Typigs amino acid 68 TOPOLOGY: linear (Li) moLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Met Ala Ala Ar; Lou Ar; Pro Lou Ala Lou Ar; Lou Lou Ala Ar; Thr 1 5 10 Ph. Pro Lou Val Ala Arg Gly Phe 5cr Amp Glu Thr Lou Glu Lys Ala 25 Ala Lys 5cr Giu Gly Tyr Cym Ser Ar; Ile Lou Ar; Ala Gin Gly Thr 4045 Ar; Arg Giu Gly Tyr Aen Giu Ph* 5cr Lou Ar; Val Glu Gly Amp Pro 55 Glu Ph. Tyr Lys Pro Gly Asn 5cr Tyr Arg Val Thr Lou 5cr Ala Ala 70 75 Thr Pro Ala Tyr Ph. Ar; Gly Ph* Thr Lou Ile Ala Lou Lys Glu Gly 90 Lys Glu Gly Asp Lys Glu Giu Asp His Ala Gly Thr Ph., Gin Ile Ile 100 105 110 Asp Giu Giu Glu Thr Gin Phe Not 5cr Asn Cym Pro Val Ala Val Thr 115 120 125 Glu 5cr Thr Pro Ar; Ar; Ar; Thr Ar; Ile Gin Val Ph* Trp Thr Ala 130 135 140 Pro Pro Thr Gly Thr Gly Cym Val Ile Lou L~ye Ala 8cr Ile Val Gin ~*145 150 155 160 Lys Ar; Ile Ii. Tyr Ph., Gin Asp Giu Gly Bar Lou Thr Lye Ar; Ile ''t165 170 175 Cym Glu Gin Asp Bar Ala 5cr Giu Gly Val Thr Asp Lys Pro Thr Lou 180 185 190 ft*otAmp Cys Cys Ala Cym Gly Thr Ala Lye Tyr Ar; Lou Thr Ph* Tyr Gly ft195 200 205 ASTI Trp 8cr Giu Lys Thr Him Pro Lys Asp Ph. Pro Ar; Ar; Thzr Aun 210 215 220 His Trp Bar Ala Ile Ile Gly 8cr Bcr His Bar Lys Asn Tyr Ile Lou 't..225 230 235 240 Trp Glu Tyr Gly Gly Tyc Ala Ser Giu Gly Val Lys Gin Val Ala Glu ~~)245 250 255 Lou Gly Bcr Pro Val Lys Met Giu Giu Giu Ile Ar; Gin Gin Bar Asp ft260 265 270 Giu Val Lou Thr Val Ile Lye Ala Lye Ala Gin Tcp, Pro Ala Trp, Gin 275 280 285 Pro Lou Asn Val Ar; Ala Ala Pro 5cr Ala Giu Phe 8cr Val Asp Ar; 290 295 300 His 305 Trp Trp Ap Tyr 385 Glu Il Asp cys Lou 465 Pri.

cys 20 Gly Ap Val 545 25 Glu His Glu Pro Arg His Lou Not Ser Phs, Lou Thr 310 Ann Val Ser Lys 370 Asp Art;

ASP

Thr Ser 450 Lys Gin met Hot Gly 530 Ann Trp Art; Thr Cyu 610 Val Gin Gly 355 Ile Pro Ile Asp Pro 435 Ser Ala Pro met Giy 515 Ser Glu Asp Net, Thr 595 Lou Gly Lys Val Art Giu Ala Ile 420 Giu Ser Gin Cym Ser 500 Thr met Giu Giu Ile Giu Lou Lou 325 Val1 Thr Pro Gly Art; 405 Val Thr Thr Lou met 485

ASP

Arg cyn Cym Cym 565 Lys Ala Ser ser Val1 Tyr Lou Giy 390 Lys Ala Gym cys Ap 470 Gly Try Sec Lys Ser 550 Ser met Glu Pro Ala Glu Asp Gin Asp Lou 345 Giu Ser Pro 360 Thr Ser Lou 375 Ser Ile Lys Gly Giu Gin Asp Lou Ala 425 Tyr Ser 440 Giu Lys Gly 455 Lou Ser Val Pro Gly Cys Ile Thr Trp 505 Art; Giu Arg 520 Val Pro Thr 535 Pro Ser Ser Ala Ser Cym Thr Pro Ala 585 Lys Cym Hot 600 Trp Ser Glu 615 69 Hot Lou 330 Ile Ann Asp Lou Gym 410 Pro Ann Lys Pro Ser 490 Ser Tyr Glu Gym Gly 570 Ap Met Trp Lou 315 Gym Pro Lys His Val 395 Ann Glu Trp Arg Gys 475 Asp Pro Val G lu

LOU

555 Thr Gly Pro Ser Gly Thr Trp Pro Pro 380 Ala Phe, Glu Sec met 460 Pro Glu Gym Lys Thr 540 Val Gly Sec Glu Asp 620 Pro Lys Asp Thr 365 Gin Ar; Val1 Lys Pro 445 Arg Ap Asp Ser Gin 525 Glu Thr Met met Gym 605 Gym Ser Asp Ala 350 Val Ser Val Pro Glu 430 TrP Gin Thr Gly Val 510 Ph* Lys Clu Lys Gym 590 His Bar Pro Gym, 335 Gly Pro Pro Val Asp 415 Giu Ser Arg Gin Ser 495 Ser Pro Gym TrP Arg 575 Lys Thr Val Asp 320 Gly Thr Gin Ph* Lou 400 Ann Asp Ala Hot Asp 480 Thr Gym Glu I le Gly 560 Art; Ala 11e Thr 9* 0 *0

M

Gym 625 Gly Lys Gly Met Art; Thr Art; Gin Art; Not Lou Lys 8cr Ala Ala 630 635 640 Giu Lou Gly Asp Cyu Ann Glu Giu Lou Giu Gin Ala Glu Lys Cys Met 645 650 655 Lou Pro Glu Cys Pro Ile Alp Cys Glu Lou Thr Glu Trp 8cr Gin Trp 660 665 670 Ser Glu Cys Ann Thr Sr Cys Gly Lys Gly His Met Ile Arg Thr Ar; 675 680 685 Hot Ile Lys Ile Glu Pro Gin Phe Gly Gly Thr Ala Cys Pro Glu Thr 690 695 700 val Gin Arg Thr Lys Cys Arg Val Ar; Lys Cys Lou Arg Gly Pro Gly 705 710 715 720 Met Giu Lys Arg Ar; Trp Lys Glu Ala Ar; Giu Lys Arg Arg Sr Glu 725 730 735 Gin Ala Lys Lys Aen Ile Asp Aen Glu Gin Tyr Pro Val Cya Ar; Lou 740 745 750 Lys Pro Trp Thr Ala Trp Thr Giu Cys Sr Thr Lou Cyn Gly Gly Gly 755 760 765 Ile Gin Glu Ar; Tyr Met Mt Val Lys Lys Arg Sr Lys Sr Thr Gln 770 775 780 Ph@ Thr 8cr Cys Lys Asp Lys Lys Giu Lou Arg Ala Cys Ann Val His 785 790 795 800 Pro Cys INrORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTRISTICS: LENGTH 1816 base pairs .i TYPZ: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPZ: cA (ii) FETURE: NANE/KEY: CDS LOCATION: 2..1705 .1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: T TCA OGT GAA TAT OTT CTT TG AG? ATG AGA CAA CC AGT GAT OT 46 8cr Oly Glu Tyr Val Lou Trp Ser Mt Arg Gin Ala Sr Asp Gly 1 5 10 GTC ARA CAA GTA OCT GAG TTG GGT TCT CCA GTC AAA ATO GAA GA GAA 94 Val Lys Gin Val Ala Olu Lou Gly 8cr Pro Val Lys Mt Olu Giu Glu 20 25 ATT CGA CAG hAG GGA GAT GAA GTT CTA ACA GTA ATC AAA CC AAA GCT 142 Ile Arg Gin Lys Gly Asp Giu Val Lou Thr Val Ile Lys Ala Lys Ala 40 71 CAGTG CG CC TGG cAG CCC CTC AK? GTG AGO GCC GCC CCT TCA GC? 190 Gin Try Pro Ala Trp Gin Pro Lou Ann Val Arg Ala Ala Pro Sex Ala so 55 GAG TTC TC?, cTO QAC AGA AGC COT CAC CTG ATO TCA TTT CTG GCC ATO 238 Giu Ph. Ser Val Asp Arg Sex Arg His Lou Hot Ser Ph. LOU Ala Hot 70 ATG GOT CCT AGC CCA GAC TOG AAT GTA GGA CTC ACC TCC GAG GAT CTC 286 Hot Gly Pro Sex Pro Asp Trp Asn Val Gly Lou Thr Ser Glu Asp Lou so 85 90 TOT ACC AAA GAG TGT GOC TOG GTT CAG AAG GTG GTC CAG GA? TTG ATT 334 Cys Thr Lys Olu Cys Gly Trp Val Gin Lys Val Val Gin Asp Lou Ile 100 105 110 CCA TGG GAT GCK GOC AC? GAC AG? GGG GTA ACC TAC GAG TCT CCA AAC 382 Pro Trp Asp Ala Gly Thr Asp Sex Gly Val Thr Tyr Glu Ser Pro Ann 115 120 125 AAG CCC ACC AT? CCC CAG GAT AAA ATC CGA CCT CTG ACA AG? CG GAT 430 Lys Pro Thr Ile Pro Gin Asp Lys Ile Ar; Pro Lou Tb: Ser Lou Asp 130 135 140 CAC CCA CAA AGC CCT TCT ATG ACC AGA GOT GGG CCA ATC ATA CC? ATA 478 His Pro Gin Ser Pro Ser Net Thr Ar; Gly Gly Pro Ile Ile Pro Ile 145 150 155 GCT CGA GTT GTO AT? OAK AGO AT? 0CC AGO AG 00K OAK CAG TGC AK? 526 Ala Arg Val Vai Ile Giu Ar; Ile Ala Ar; Lys Giy Oiu Gin Cys Ann 160 165 170 175 *ATT ATA CCC GAC AAC OTG GAT GAC ATA GTA OCK OAT CTO GTA ACG GAA 574 *Ile Ile Pro Asp Annl Val Asp Asp le Val Ala Asp Lou Val Tb: Olu 180 185 190 a..GAG AAA GAC GAA GAT GA? ACC CCG GAG ACC TGC ATA TAT TCC AAC TOG 622 :Giu Lys Asp Giu Asp Asp Tb: Pro Giu Tb: Cys Ile Tyr Ser Ann Trp 195 200 205 TCC CCC TOO TCG GCC TGC AGC TCO GCC ACC TGC GAC AKO GOC AKO COG 670 Pro Trp Ser Ala Cys Ser Ser Ala Tb: Cys Asp Lys Oly Lys Ar; 210 215 220 ATG AGA CAG CGC ATO TTA AAG OCT CAG TTA GA? CTC AG? OTT CCC TOC 718 mot Ar; Gin Arg Het Lou Lye Ala Gin Lou Asp Lou 8cr Val Pro Cys 225 230 235 .CCA GAC ACT CAG GAC TTT OAK CCC TGC ATG GGG CCC GGC TGC AGC OAT 766 Pro Asp Tb: Gin Asp Phe Giu Pro Cys Not Gly Pro Gly Cys Ser Asp 240 245 250 255 GAC GAK 0CC TCT ACC TGC ATG ATO TCA GA, TOG ATC ACC TOG TCO CCG 814 4,Asp Giu Ala Ser Tb: Cy. Not Met Ser Oiu Trp Ile Tb: Trp Ser Pro 260 265 270 TGC AGC GCC TCC TGC 000 ATG 00K ATT GAG OTC AGO GAG AGA TAC OTC 862 Cys Ser Ala Ser Cys Oly Met Oly Ile Oiu Val Ar; Giu Ar; Tyr Val 275 280 285 AKO CAG TTC CCA OA GAC GOT TCC TTN TOT AA GTC CCA AG OAK OAK 910 Lys Gin Phe Pro O.u, Asp Oly Ser Lou Cys Lys Val Pro Tb: Glu, Giu 290 295 300 ACT GAG AAA TGC AT? GTC ART GAG GAG TOT GAG CCA AGC AGC TOT ATA Thr Giu Lys Cy Ile Val Kan Giu Giu Cys Giu Pro Ser 8cr Cy. Ile 305 310 315 958 GTC ACO GMA Val Thr 320 GOT ATO Gly Met TCT ATO, Ser Met GMA TOT Giu Cys GAT TOC Ap Cys 385 TTG AG Lou Lys 400 AAA CAA Lys Gin .:TTG AM.

**Lou Thr *GGT CAC Gly His :6..,GGA GCC *Gly Aia ~:'.465 MAA TOT :Lys Cys *6 480 CGA GAG Arg Giu CAG TAC Gin Tyr ACC A Thr Lys Glu

AAG

Lys

TOC

Cy.

CAT

His 370

AGC

8cr Tcc 8cr

GTG

Val

GAG

Giu

ATO

Hot 450

GT=

Val

MA

Gin

MGO

Lys

CCT

Pro

TTC

Ph* 530 TOO GM GAG TOO GAG GAG TC MOC OC ACA TGC CO AMO Trp Aia Giu Try, Giu Giu CY8 8cr Ala Th~r Cys Ar; Met 325 330 335 MAG COO MAC AGO ATO ATA MAG ATO ACT CCA GCG GAT GGA Lye Ar; His Ar; Hot Ile Lys Hot Thr Pro Ala Asp Giy 340 345 350 MAA GC= GAC ACM ACA GAO OTT GAG AMA TGC ATO ATO CCC Lys Ala Asp Thr Tb: Giu Vai Giu Lye Cy. Met Hot Pro 355 360 1365 ACC ATC COO TOC OTO TTG TCC CCT TOO TCT GMA TOO AGT Thr Ile Pro Cys Val Lou So: Pro Try, 8cr Giu Try, 8cr 375 380 OTT ACC TOT GOC AAA GOC ACC AGA ACC AGA CAG AGA ATC Val Tbr Cy. Gly Lye Gly Thr Ar; Tb: Ar; Gln Ar; Hot 390 395 CCO TCT GMA CTT OGA. OAT TOC AAT GAG QAA CTO GMA CTO Pro 8cr Giu Lou Giy Asp Cys Ann Giu Giu. Lou Giu Lou 405 410 415 GMA MG TOC ATO CTT CCT GMA TGC CCT ATA AGC TOT GMA Giu Lye Cye Hot Lou Pro Giu Cys Pro Ile 8cr Cys Giu 420 425 430 TOO TCT TAC TOO TCT GAG TOT MC MAA TOC TCG 0CC MAG Trp 8cr Tyr Try, 8cr Oiu Cys Asn Lys Cye 8cr Giy Lye 435 440 445 ATT COT ACC COA ATO ATC ACA ATO GMR CCA CAG TTT GGA Ile Ar; Th: Ar; Hot Ile Tb: Hot Giu Pro Gin Ph* Gly 455 460 TOT COG GMA ACC OTO CMR CO MA MAA TOO OA TTA COT Cys Pro Glu Tb: Val Gin Ar; Lys Lye Cys Ar; Lou Ar; 470 475 AAA hOT TCC 000 hAT GAG CGA AGO CAT TTA MhG OAT 0CC Lye 8cr 8cr Gly hen Giu Ar; Ar; His Lou Lys Asp Ala 485 490 495 AGA AGO hOT GM MAA ATA MAG GMR OAT TCA OAT OGA CAM Ar; Arg Ser Giu Lye Ile Lye Glu Asp 8cr Asp Gly Giu 500 505 510 0TA TOT MAA ATO A CCA TOG ACT GMA TOG ACC GAR TOT Val Cye Lye Hot Lys Pro Trp Tb: Ala Try, Tb: Giu Cym 515 520 525 TOC GOT 000 000 ATA CM GAG COG TTC ATO ACT GTO hAG Cys Gly Gly Gly Ile Gin Oiu Arg Ph. Net Thr Vai Lye 535 540 1006 1054 1102 1150 1198 1246 1294 1342 1390 1438 1486 1534 1582 1630 73 AAO AGA TTC AAA hOT TCT CAG TTC ACC AGC TOC AAG GAC AAG AAG GAG 1678 Lys Arg Ph. Lys Ser Gin Ph* Thr Sor Cys Lys Asp Lys Lys Giu 550 555 ATC COG OCT TGC AAT GTC CAT CCA TOT TAACCTGCCT GAlAAGAGGG 1725 Ile Arg Ala Cy. hen Val Bin Pro Cys 560 565 ATTGACACTA CAATCGCAAC AGAAGTCAAT CTTTATTAGA TATTTTTTAT CATAGAlTAT 1785 ATACATGTGC CAI'T CATOTACTIT T 1816 INFORMATION FOR SEQ ID NOt14t SEQUENCE CHARACTERISTICS: LENGTH: 568 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

C.

C

C

Ce..

C

C

C.

CC..

C C C. CC a a C

C

CCC.

CC.*C

C

Ser Lys Arg Trp, Ph* 65 Gly Thr Try Pro Pro 145 Arg Ile Gin Gin Pro 50 Sor Pro Lys Ap Th: 130 Gin Val Val Lys Ala Val 5cr G iu Ala 115 110 8cr Val Ala Gly Trp Ap Pro Cyu 100 Gly Pro Pro Ile Oly Olu Tyr Val Olu Asp Gin Arg Asp Gly Thr Gin 5cr Glu 165 Val Lou Trp Jar Met Arg 10 Gin Ala Ser Asp Lou Glu Pro So: 70 Trp Trp Ap Ap Not 150 Arg Asp Gly Val Lou 55 Arg an Val

S.:

Lys 135 Tb: I10 Ap Se: Pro Val 25 Lou Tb: Val 40 an Val Arg His Lou Net Val Gly Lou 90 Gin Lys Val 105 Gly Val Tb: 120 Ile Arg Pro Arq Gly Gly Ala Arg Lys 170 11-w Y1t- Ala 185 Lys I10 Ala 8cr 75 Tb: Val Tyr

LOU

Pro 155 Gly Ap Net Lys Ala Phe 5cr Gin Giu Tb: 140 Ile Giu

LOU

Glu Ala Pro Lou Giu ap Ser 125 Sc: Ile Gin Val Clu Lys 8cr Ala Ap Lou 110 Pro Lou Pro Cys Tb: 190 Giu Ala Ala met Lou Ile an Ap I10 hen 175 Glu Ile Gin Oiu met s0 Cys Pro Lys His Ala 160 110 Glu Oly Val Pro Ap An 180 Lys Ap Glu 195 Ap Ap Tb: Pro Glu Tb: Cys Ile Tyr gar 200 An Trp Scr 74 Pro Trp $or Ala Cym 8cr 8cr Ala Thr Cys Asp Lys Gly Lys Ar; met 210 215 220 Ar; Gin Ar; Met Lou Lys Ala Gin Lou Asp Lou 8cr Val Pro Cys Pro 225 230 235 240 Asp Thr Gin Asp Ph. Giu Pro Cys Mot Gly Pro Gly Cys 8cr Asp Asp 245 250 255 Giu Ala Ser Thr Cys Met Met Ser Glu Trp Ile Thr Trp 5cr Pro Cys 260 265 270 Ser Ala 8cr Cy. Gly Mot Gly Ile Giu Val Ar; Giu Arg Tyr Val Lys 275 280 285 Gin Phe Pro Giu Asp Gly 8cr Lou Cym Lys Val Pro Thr Giu Giu Thr 290 295 300 Giu Lys Cys le Val Ann Giu Giu Cym Giu Pro 8cr Sec Cys Ile Vai 305 310 315 320 Thr Giu Trp Ala Giu Trp Giu Giu Cys 8cr Ala Thr Cys Ar; Met Gly 325 330 335 Mot Lys Lys Ar; His Ar; Met Ile Lys Met Thr Pro Ala Asp Gly 8cr 340 345 350 Met Cy. Lys Ala Asp Thr Thr Giu Val Glu Lys Cyu Met Met Pro Glu 355 360 365 Cys His Thr Ile Pro Cy. Val Lou 8cr Pro Trp 8cr Glu Trp, 8cr Asp 370 375 380 Cys 8cr Val Thr Cys Giy Lys Gly Thr Ar; Tkxr Ar; Gin Ar; Met Lou *385 390 395 400 Lys Ser Pro 8cr Glu Lou Gly Asp Cys Ann Giu Glu Lou Glu Lou Lys Gin Val Giu Lys Cy. Met Lou Pro Giu Cys Pro Ile 8cr Cys Giu Lou 420 425 430 Thr Giu Trp 8cr Tyr Trp 8cr Glu Cys Ann Lys Cys $or Gly Lye Gly *435 440 445 :His Met Ile Ar; Thr Ar; Met le Thr Met Giu Pro Gin Phc Giy Gly 450 455 460 Aia Val Cys Pro Giu Thr Val Gin Ar; Lys Lys Cys Ar; Lou Arq Lye 465 470 475 480 Cys Gin Lye 8cr 8cr Gly Aen Giu Ar; Ar; His Lou Lys Asp Ala Ar; 485 490 495 Glu Lys Ar; Ar; 8cr Giu Lys Ile Lye Gltv Asp 8cr Asp Gly Giu Gin 500 505 510 3' Tyr Pro Val Cys Lye Not Lye Pro Trp Thr Ala Trp Thr Glu Cys Th: 515 520 525 Lys Ph* Cys Gly Giy Giy Ile Gin Qiu Ar; Ph* Met Tb: Val Lye Lye 530 535 540 Ar; Phe Lye ser Ser Gin Phes Thr Ser Cys Lys Asp Lys Lys Gu Ile 545 550 555 560 Arg Ala Cys Ann Val His Pro Cys 565 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 59 ami.no acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID 110:15: Giu Thr Cys Ile Tyr Ser Ann Trp Scr Pro Trp Ser Ala Cys Scr Scr Thr Cys Giu Lys Gly Lys Arg Mot Ar; Gin Arg Met Lou Lys Ala 25 Gin Lou Amp Lou Ser Vai Pro Cys Pro Asp Thr Gin Asp Ph* Gin Pro 40 Cys Met Gly Pro Gly Cys Ser Asp Giu Asp Gly a. INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 56 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Ser Thr Cys Thr Met Ser Giu Trp Ile Thr Trp Ser Pro Cys Ser Val.

.25 15 10 Ser eye Gly Met Gly Not Ar; Ser Ar; Glu krg Tyr VJal Lys Gin Phe 25 Pro Asp Gly Ser Vai Cys Met Leu Pro Thr Glu Giu Thr Giu Lys Cys 40 Thr Val Ann Giu Giu Cys Ser Pro 50 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 56 amino acids 76 TYPE: amino acid (C STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID 140:17: Ser Sor Cys Leu Val Thr Glu Trp Gly Glu Trp Amp Asp Cys Ser Ala 1 5 10 Thr Cys Gly Het Gly Met Lys Lys Arg His Arg Met Val Lys Met Ser 25 Pro Ala Asp Gly Ser Met Cys Lys Ala Glu Thr Ser Gln Ala Glu Lys 40 Cys Met Met Pro Glu Cys His Thr so0S INFORMATION FOR SEQ ID 190:18: SEQUENCE CHARACTERISTICS: LENGTH: 53 amino acids TYPE: amino acid STRANDEDNESSZ single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID 190:18: Ile Pro Cys Leu Leu Ser Pro Trp Glu Trp Ser Asp Cys Ser Val Thr 5 10 Cys Gly Lys Gly Met Arq Thr Ar; Gin Arg Met Lou Lys Ser Lou Ala 25 Glu Leu Gly Asp Cys Asn Glu Asp Lou Glu Gln Ala Glu Lys Cys Met 40 Leu Pro Glu Cys Pro .5.25 INFORMATIONI FOR SEQ ID N90:19: SEQUENCE CHARACTERISTICS: LENGTH: 56 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (1i) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID 190:19: 77 Ile Asp Cys Glu Lou Ser Glu Trp, Ser Gin Trp, 5cr Glu Cys Asn Lys 1 5 10 Sor Cym Gly Lys Gly His met Ile Arg Thr Arg Thr Ile Gin Met Giu 25 Pro Gin Ph& Gly Giy Ala Pro Cys Pro Glu Thr Val Gin Ar; Lys Lys 40 Cys Arg Ala Arg Lys Cy. Lou Arg so INFORMATION FOR SEQ ID N0:20: SEQUENCE CHARACTERISTICS: LENGTH: 55 ami.no acids TYPE: amio aci.d STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Pro Gin Cys Ar; Met Arg Pro Trp Thr Ala Trp Ser Glu Cys Thr Lys 1 5 10 Lou Cys Gly Gly Gly Ile Gin Giu Ar; Tyr Met Thr Val Lys Lys Arg 20 25 Ph* Lys Ser Gin Ph. Thr Se: Cys Lys Asp Lys Lys Giu Ile Ar; 40 :.Ala Cys Aen Val His Pro Cys

Claims (16)

1. An isolated nucleic acid molecule encoding human F-spondin wherein said nucleic acid molecule comprises a nucleotide sequence that is complementary to a sequence that is capable of hybridizing under low stringency conditions to SEQ ID NO: 9 and/or SEQ ID NO: 11 and/or SEQ ID NO: 13.

2. An isolated nucleic acid molecule that encodes a human F spondin polypeptide when isolated by a process comprising contacting nucleic acid derived from a human source with a hybridisation-effective amount of a nucleic acid probe comprising at least 15 contiguous nucleotides of any one of SEQ ID NOs: 9, 11, or 13 for a time and under conditions sufficient for hybridisation to occur and then isolating the hybridised human nucleic acid.

An isolated or recombinant human F-spondin polypeptide.

4. An isolated or recombinant human F-spondin polypeptide encoded by the nucleic acid molecule of claim 1 or 2.

5. A method of attaching nerve cells to a matrix comprising contacting the matrix with nerve cell and human F-spondin at a concentration effective to effect attachment of the cells o to the matrix.

6. A method of attaching nerve cells to a matrix comprising contacting the matrix with nerve cell and human F-spondin at a concentration effective to effect attachment of the cells to the matrix, wherein said human F spondin polypeptide is encoded by the isolated nucleic acid molecule of claim 1 or 2.

7. A method of stimulating growth of a nerve cell comprising contacting the nerve cell with human F-spondin at a concentration effective to stimulate growth of the nerve cell.

8. A method of stimulating growth of a nerve cell comprising contacting the nerve cell v *I -v j P:\OPER\MRO\1883748.SPE 28/9/99 -79- with human F-spondin at a concentration effective to stimulate growth of the nerve cell, wherein said human F spondin polypeptide is encoded by the isolated nucleic acid molecule of claim 1 or 2.

9. A method of regenerating nerve cells in a subject comprising administering to the subject human F-spondin at a concentration effective to regenerate nerve cells in the subject.

A method of regenerating nerve cells in a subject comprising administering to the subject human F-spondin at a concentration effective to regenerate nerve cells in the subject, wherein said human F spondin polypeptide is encoded by the isolated nucleic acid molecule of claim 1 or 2.

11. A pharmaceutical composition for stimulating nerve cell growth comprising a pharmaceutically acceptable carrier and human F-spondin at a concentration effective to stimulate nerve cell growth. i:

12. A pharmaceutical composition for stimulating nerve cell growth comprising a pharmaceutically acceptable carrier and human F-spondin at a concentration effective to stimulate nerve cell growth, wherein said human F spondin polypeptide is encoded by the isolated nucleic acid molecule of claim 1 or 2.

13. Use of the human F spondin polypeptide according to claim 3 in the preparation of a medicament for the regeneration of a nerve cell in a subject.

14. Use of the human F spondin polypeptide according to claim 4 in the preparation of a medicament for the regeneration of a nerve cell in a subject. Use of the human F spondin polypeptide according to claim 3 in the preparation of a medicament for the stimulation of nerve cell growth in a subject.

P;\OPER\MRO\1883748.SPE 28/9/99 80

16. Use of the human F spondin polypeptide according to claim 4 in the preparation of a medicament for the stimulation of nerve cell growth in a subject. DATED this TWENTY EIGHTH day of SEPTEMBER 1999 The Trustees of Columbia University in the City of New York by their Patent Attorneys DAVIES COLLISON CAVE 0 0 0 00* S.0.0 0 00* .Z*