WO2001038491A2 - Novel semaphorin domains - Google Patents

Abstract

The invention relates to new functional domains in semaphorin polypeptides that are homologous to hanatoxin. A method of searching for other new functional domains based on toxin sequences is also disclosed.

Description

NOVEL SEMAPHORIN DOMAINS

Field of the Invention

The field of the invention is structural biology, neurology, and bioinformatics.

Background of the Invention The semaphorins are a large family of secreted and membrane-bound proteins that have multiple and diverse functions, including a contribution to axon guidance during development of the nervous system (Mark et al., Cell Tissue Res. 290:299-306, 1997). Semaphorin 3 A (Sema3A), a secreted semaphorin expressed in vertebrates, repels sensory neurons by inducing collapse of their axonal growth cones (Luo et al., Cell 75:217-227,1993; and Kolodkin et al., Cell 75:1389-1399, 1993).

Summary of the Invention The invention is based on the discovery of a new, previously unrecognized domain in semaphorins that is highly homologous to Tarantula hanatoxin, a toxin known to bind to membrane-bound Ca⁺² channels. Mutagenesis of this domain, termed the hanatoxin-like sequence (HTLS), in Sema3 A indicated that the domain is responsible for the dorsal root ganglion repulsion and growth cone collapse activities associated with semaphorins. In addition, it was discovered that the growth cone collapse activity of Sema3 A was dependent on a membrane-bound Ca⁺² channel, consistent with the recognition of the HTLS in semaphorins. Consequently, peptides containing the HTLS can be used to modulate the activity of Ca⁺² channels. Alternatively, peptides containing HTLS can be used as an antigen to generate antibodies that can then be used to modulate the activity of Ca⁺² channels by inactivating naturally-occurring channel ligands. Such peptides or antibodies can be used as drugs to treat any condition or disease that is characterized by abnormal Ca⁺² channel function.

Accordingly, the invention features a polypeptide including the degenerate sequence Xaai -Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaag-Xaa₉-Xaa i o-Xaa ^{■ •} -Xaa 1₂-Xaa ^• ₃-Xaa- ₄-Xaa i s-Xaa 16- Xaai₇-Xaai₈-Xaai₉-Xaa₂o-Xaa₂ι-Xaa₂₂, where Xaai is Lys, Arg, Thr, Ser, Ala, Glu, Gin, His, Gly or Tyr;

Xaa₂ is Thr, Ala, Val, Ser, Phe, Asp, Cys, or Gly; Xaa₃ is Thr, Cys, Arg, Gin, Met, or Lys; Xaa₄ is Ala, Thr, Nal, Tyr, Gly, Ser, Lys, Arg, His, or Glu;

Xaas is Asp, Glu, Thr, Ala, Asn, Ser, Gly, or Leu;

Xaa₇ is Cys, Nal, He, or Leu; Xaa₈ is Lys, Leu, Gly, Ala, Met, or Thr;

Xaaio is Leu or no amino acid;

Xaaπ is Gly or no amino acid;

Xaa-₂ is Cys or no amino acid; Xaaι₃ is Lys or no amino acid;

Xaa-₄ is Phe or no amino acid;

Xaai ₅ is Arg, Gin, Leu, or Thr;

Xaaiβ is Asp;

Xaaπ is Lys or Pro; Xaai ₈ is Tyr, His, or Nal;

Xaa₂o is Ala or Gly;

Xaa₂ι is Trp; and

Xaa₂₂ is Asp, Asn, Thr, Ala, Nal, His, Cys. The polypeptide is 17 to 34 (e.g., 19 to 32) amino acids in length. Examples of the sequence include any of SEQ ID ΝOs:l-14 and 16-23.

The invention also includes a polypeptide consisting of (1) a first amino acid sequence that is the degenerate sequence described above; and (2) a second amino acid sequence less than 70% (e.g., less than 60, 50, 40, or 30%) identical to each one of SEQ ID NOs:24-29. The first amino acid sequence can be selected from any one of SEQ ID NOs:l-14 and 16-23. The second amino acid sequence can be a detectable protein such as a fluorescent protein, a luminescent protein, or a chromogenic protein.

The invention also features a purified polypeptide consisting of (1) a first amino acid sequence that is the degenerate sequence; (2) a second amino acid sequence that is less than 70% (e.g., less than 60, 50, 40, or 30%) identical to each one of SEQ ID NOs:24-29, the second amino acid sequence being present C-terminal to the first amino acid sequence; and (3) a third amino acid sequence that is less than 70% (e.g., less than 60, 50, 40, or 30%) identical to each one of SEQ ID NOs:24-29, the third amino acid sequence being present N-terminal to the first amino acid sequence. The second amino acid sequence is not SEQ ID NO:39, and the third amino acid sequence is not SEQ ID NO:38. The first amino acid sequence can be any one of SEQ ID NOs: l-14 and 16-23.

The invention further includes a polypeptide consisting of (1) a first amino acid sequence consisting of Xaai-Xaa_∑-Xaa Xaa^Xaas-Xaaβ-Xaa_?-

Xaa₈-Xaa9-Xaaio-Xaaii-Xaai2-Xaai₃-Xaai₄-Xaai5-Xaai6-Xaai -Xaai₈-Xaai9-Xaa₂o-Xaa₂i-Xaa₂2, where

Xaai is Lys, Arg, Thr, Ser, Ala, Glu, Gin, His, Gly or Tyr;

Xaa₂ is Thr, Ala, Val, Ser, Phe, Asp, Cys, or Gly; Xaa₃ is Thr, Cys, Arg, Gin, Met, or Lys;

Xaa is Ala, Thr, Nal, Tyr, Gly, Ser, Lys, Arg, His, or Glu;

Xaas is Asp, Glu, Thr, Ala, Asn, Ser, Gly, or Leu;

Xaa₇ is Cys, Nal, lie, or Leu; Xaa₈ is Lys, Leu, Gly, Ala, Met, or Thr;

Xaag is Ala, Ser, Leu;

Xaaio is Leu or no amino acid;

Xaai i is Gly or no amino acid;

Xaaι₂ is Cys or no amino acid; Xaaπ is Lys or no amino acid;

Xaaι₄ is Phe or no amino acid;

Xaais is Arg, Gin, Leu, or Thr;

Xaaπ is Lys or Pro; Xaais is Tyr, His, or Val;

Xaai9 is Cys;

Xaa₂o is Ala or Gly;

Xaa₂ι is Trp; and

Xaa₂₂ is Asp, Asn, Thr, Ala, Nal, His, Cys; and (2) a second amino acid sequence. The first amino acid sequence can be selected from SEQ ID NOs:l-14 and 16-23. The second amino acid sequence can be a detectable protein such as a fluorescent, luminescent, or chromogenic protein.

A polypeptide of the invention can be substantially pure. The invention also includes an isolated nucleic acid encoding a polypeptide of the invention and a purified antibody that specifically binds to a hanatoxin-like sequence in a semaphorin polypeptide.

An "isolated nucleic acid" is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of different (i) DNA molecules, (ii) transfected cells, or (iii) cell clones: e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.

The term "substantially pure" as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological compounds, such as those in cellular material, viral material, or culture medium, with which the polypeptide was associated (e.g., in the course of production by recombinant DNA techniques or before purification from a natural biological source). The substantially pure polypeptide is at least 75% (e.g., at least 80,

85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another residue having a chemically similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). As used herein, "percent identity" of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264- 2268, 1990), modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches are performed with the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches are performed with the

XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to a reference polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov. A "detectable protein" is a protein that has an activity that is readily measurable. Luminescent and fluorescent detectable proteins (e.g., green fluorescent protein) can be detected using a photometer, phosphorimager, photographic film, or other medium or device that is affected by light. Chromogenic detectable proteins include enzymes such as β-galactosidase or horseradish peroxidase that can convert a substrate into a molecule that absorbs light at a specific wavelength.

A "toxin", as that term is used herein, is a naturally-occurring, non-mammalian polypeptide that is cytotoxic to a mammal or a mammalian cell. Toxins can be of invertebrate or vertebrate origin, and can be, for example, a snake, insect (e.g., scorpion, spider, bee), frog, mollusk, sea anemone, plant, or bacterial toxin. A "toxin domain" is a portion of a toxin that possesses the cytotoxic activity of the toxin.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The methods and materials described herein can be used to practice the present invention, although other similar or equivalent methods and material known to one skilled in the art can also be used. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples described herein are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Brief Description of the Drawings Fig. 1 is a schematic representation of the wild-type and mutant HTLS sequences within Sema3A.

Fig. 2 is a graph of protein concentration versus percent growth cone collapse in an assay for growth cone collapse activity. The boxes represent the data points for wild-type Sema3A, and the circles represent the data points for Sema3A mutant RD. Each concentration was repeated with at least 12 explants. The EC50 was 47 pM for wild-type Sema3A and 1466 pM for Sema3A mutant RD.

Figs. 3 A and 3B are Scatchard plots of wild-type Sema3A binding (Fig. 3 A) or Sema3A mutant RD binding (Fig. 3B) to PAE cells expressing neuropilin (PAE-NP-1). About 50,000 cells were treated with media containing various amounts of Sema3 A-AP or RD-AP mutant. K for Sema3A-AP was 305 pM. K_d for RD-AP was 505 pM. Error bars indicate S.E.M.

Fig. 4 is a bar graph of the relative responsiveness of DRG growth cones to Sema3A-AP in a solution containing 2 mM CoCl₂, 200 μM CdCl₂, 10 μM hanatoxin, and 1 mM l,2-bis(2- aminophenoxy)ethane-N,N,N',N^,-tetraacetic acid (BAPTA, a calcium ion chelator) or thrombin (50 u/ml); or in the same solution except without the 2 mM CoC12. The percentage of collapsed growth cones with (black bars) or without (gray bars) Sema3A-AP or with thrombin (white bars) are shown. Error bars indicate S.E.M.

Fig. 5 is a bar graph of percent growth cone collapse in response to various reagents. E13 mouse embryo DRG explant cultures were incubated with or without 2 μM BAPTA- AM and

0.08 μM calmidazolium chloride agents for 60 minutes. Control conditioned media or Sema3A- AP conditioned media were then added. The percentage of collapsed growth cones with (black bars) or without (gray bars) Sema3A-AP in the presence of these reagents is presented. Error bars indicate S.E.M. Fig. 6 is a line graph of Sema3A-AP binding to PAE-NP-1 cells. The cells were treated with media containing Sema3A-AP with or without 2 mM CoC12, or with media containing Sema3A-AP with or without 200 μM CdCl₂. Error bars indicate S.E.M. for triplicates. Detailed Description The invention relates to new peptide sequences that encompass the HTLS of semaphorins. These peptides can be produced by any means known in the art, including recombinant expression followed by isolation of the peptides (e.g., using HPLC), or by chemical synthesis.

Particularly for larger polypeptides, such as fusion proteins that contain the new peptide sequences, a nucleic acid encoding the peptide sequence can be cloned into a commercially available expression vector designed for ease of producing a desired fusion protein. For example, a nucleic acid encoding an HTLS 17 amino acids in length can be readily produced by synthesizing the plus strand encoding the 17 amino acids and the minus strand complementary to the plus strand. The ends of each strand can be designed such that, upon hybridization of the plus and minus strand, the double-stranded fragment is ready for ligation into an appropriately digested vector, such as pMCl 871 (AmershamPharmacia Biotech), to produce a plasmid that will express a fusion protein containing the 17 amino acid HTLS sequence linked to β-galactosidase. The fusion protein is produced in bacteria and isolated by affinity chromatography. This fusion protein is useful for carrying out cell binding and growth cone collapse studies, such as those described below. Generally, this procedure can be used to screen any HTLS peptide sequence for its ability to bind cells and or inhibit growth cone collapse. An HTLS peptide of the invention can serve as an immunogen to prepare antibodies by immunizing a suitable subject (e.g., rabbit, goat, mouse or other mammal) with the immunogen. An appropriate immunogenic preparation can contain, for example, recombinantly expressed HTLS peptide or a chemically synthesized HTLS peptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent Immunization of a suitable subject with an immunogenic HTLS peptide preparation induces a polyclonal anti-HTLS antibody response.

Accordingly, another aspect of the invention pertains to anti-HTLS antibodies. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as an HTLS. A molecule which specifically binds to HTLS is a molecule which binds an HTLS, but does not substantially bind other non-HTLS molecules in a sample. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')₂ fragments, which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind HTLS. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of imrnunoreacting with a particular epitope within a HTLS. A monoclonal antibody composition thus typically displays a single binding affinity for a particular HTLS with which it immunoreacts.

Polyclonal anti-HTLS antibodies can be prepared as described above by immunizing a suitable subject (e.g., a rabbit) with an HTLS immunogen. The anti-HTLS antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized HTLS peptide. If desired, the antibody molecules directed against HTLS can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography, to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti-HTLS antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al., Nature 256:495-497, 1975; Kozbor et al., Immunol Today 4:72, 1983; Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985; or trioma techniques. The technology for producing various monoclonal antibody hybridomas is well known. See, e.g., Current Protocols in Immunology, Coligan et al. (eds.), John Wiley &

Sons, Inc., New York, NY, 1994. Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a HTLS immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds an HTLS. Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-HTLS monoclonal antibody. See, e.g., Current Protocols in Immunology, supra; Galfre et al., Nature 266:550, 1977; Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York, 1980; and Lerner, Yale J. Biol. Med. 54:387-402, 1981. Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic peptide of the present invention with an immortalized mouse cell line, e.g., a myeloma cell line that is sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/0-Agl4 myeloma lines. These myeloma lines are available from the American Type Culture Collection (ATCC). Typically, HAT- sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supematants for antibodies that bind an HTLS peptide, e.g., using a standard ELISA assay.

As an alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-HTLS antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with an HTLS peptide to isolate immunoglobulin library members that bind HTLS. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP^® Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display libraries can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al., Bio/Technology 9:1370-1372, 1991; Hay et al., Hum. Antibod. Hybridomas 3:81-85, 1992; Huse et al., Science 246:1275-1281, 1989; and Griffiths et al., EMBO J. 12:725-734, 1993.

Additionally, recombinant anti-HTLS antibodies, such as chimeric and humanized monoclonal antibodies, are within the scope of the invention. Such antibodies include both human and non-human portions and can be made using standard recombinant DNA techniques. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application Nos. 184187, 171496, 173494, and 125023; PCT Publication No. WO 86/01533; U.S. Patent Nos.4,816,567 and 5,225,539; Better etal, Science 240:1041-1043, 1988; Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443, 1987; Liu et al., J. Immunol.139:3521-3526, 1987; Sun et al., Proc. Natl. Acad. Sci. USA 84:214-218, 1987; Nishimura et al., Cane. Res.47:999-1005, 1987; Wood et al., Nature 314:446-449, 1985; Shaw et al., J. Natl. Cancer Inst.80:1553-1559, 1988; Morrison, Science 229:1202-1207, 1985; Oi et al. Bio/Techniques 4:214, 1986; Jones et al., Nature 321:552-525, 1986; Nerhoeyan et al., Science 239:1534, 1988; and Beidler et al., J. Immunol.141:4053-4060, 1988.

Example Using primary sequence alignment of the semaphorin protein sequences, a domain with sequence homology to hanatoxin, a short peptide tarantula toxin that selectively blocks some voltage-gated K⁺ and Ca²⁺ channels, was discovered. The hanatoxin-like sequence (HTLS) is found in the semaphorin domain of all known chick and mammalian secreted semaphorins. An alignment of the HTLS in various semaphorins with the full length hanatoxin sequence is shown below.

Alignment of Sequences of Hanatoxin and Various Semaphorins

Hanatoxin: ECRY FGGCKTTADCCKHLGCKFRDKYCAWDFTFS (SEQ ID NO: 37) Semaphorins

Type 3

Sema3A: KACAECC A RDPYCAWD (SEQ ID NO: 16)

Sema3D: KACADCC A RDPYCAWD (SEQ ID NO: 17) mSema3B RACAECC A RDPYCAWD (SEQ ID NO: 2) hSema3B RVCTECCLA RDPYCAWD (SEQ ID NO : 3 ) hSema3C TACADCCLA RDPYCAWD (SEQ ID NO : 4 ) hSema3E SACADCCLA RDPYCAWD (SEQ ID NO: 5) hSema3F ACADCC A RDPYCAWD (SEQ ID NO : 6 )

Type 4 mSema4A ESCVDCV A RDPHCAWD (SEQ ID NO: 7) mSema4D QSCYDCI A RDPYCGWD (SEQ ID NO: 8) mSema4C RFCVDCV A RDPYCAWN (SEQ ID NO: 9) mSema4B YDCGDCLLA RDPYCAWT (SEQ ID NO: 10)

Sema4F: QSCSECILA QDPVCAWN (SEQ ID NO: 11)

Type 5

Sema5A .- RTRSTCIGA QDPYCGWD (SEQ ID NO: 18)

Sema5B : HSQGACLGA RDPYCGWD (SEQ ID NO: 19)

Type 6

SemaβB SGCMKNCIGS QDPYCGWA (SEQ ID NO: 20)

S e a 6 A GKCKKTCIAS RDPYCGWV (SEQ ID NO: 21)

SetnaδC GACQRSCLAS DPYCGWH (SEQ ID NO:22)

TVPe 7 Sema7A: GGCHGCLMS RDPYCGWD (SEQ ID NO: 23)

Invertebrate semaphorin

Sema-la: TSCSECVA QDPYCAWD (SEQ ID NO: 12)

Plexins mPlexin 1: TSCELCLGS RDPHCGWC (SEQ ID NO: 13)

PVESPR: KSCSECLTA TDPHCGWC (SEQ ID NO: 14)

Note: "h"=human; "m"=mouse

As can be seen from the above alignment, amino acids 10-31 of hanatoxin (KTTADCCKHLGCKFRDKYCAWD; SEQ ID NO: 1) align well with semaphorins, especially with types 3 and 4 semaphorins. Note that SEQ ID NO:l encompasses a substantial portion of hanatoxin, leaving out only ECRYLFGGC (SEQ ID NO:38) at the N-terminus and FTFS (SEQ ID NO:39) at the C-terminus. Each region of homology in semaphorins can be further divided into two sub-domains separated by a gap, when compared to the hanatoxin sequence. Sema3 A and Sema3D show the highest homology to hanatoxin. Other secreted semaphorins are slightly less homologous. All membrane-bound semaphorins (types 4-7) are significantly less homologous to hanatoxin, with only the C-terminus sub-domain having homology at more than one amino acid.

The full length sequences of selected semaphorins (including leader peptides, which are cleaved off prior to secretion) are shown below. _..

Human Sema3A:

MGWLTRIVCLFWGVLLTARANYQNGKNNVPRLKLSYKEMLESNNNITFNGLANSSSYH TFLLDEERSRLYVGAKDHIFSFDLVNIKDFQKIVWPNSYTRRDECKWAGKDILKECANFI KV KAYNQTHLYACGTGAFHPICTYIEIGHHPEDNTFKLENSHFENGRGKSPYDPKLLTA SLLIDGELYSGTAADFMGRDFAIFRTLGHHHPIRTEQHDSRWLNDPKFISAHLISESDNPE DDKVWFFRENAIDGEHSGKATHAWGQICK-NDFGGHRSLVNKWTTFLKARLICSVPGPN GroTHFDELQD LMNFKDPKNPVVYGVFTTSSNTFKGSAVCMYSMSDVRRVFLGPYAH RDGPNYQWVPYQGRVPYPRPGTCPSKTFGGFDSTKDLPDDVITFARSHPAMYNPVFPMN I P^v^KTDV^r QFTQIVVDRVDAEDGQYDVMFIGTDVGT\^.KVVSIPKETWYDLEEVL LEEMTVFREPTAISAMELSTKQQQLYIGSTAGVAQLPLHRCDIYGKACAECCLARDPYC AWDGSACSRYFPTAKRRTRRQDIRNGDPLTHCSDLHHDNHHGHSPEERJJYGVENSSTFL ECSPKSQRALVYWQFQRRNEERKEEIRVDDHIIRTDQGLLLRSLQQKDSGNYLCHAVEH GFIQTLLKVTLEVIDTEHLEELLHKDDDGDGSKTKEMSNSMTPSQKVWYRDFMQLrNHP NLNTMDEFCEQVWKRDRKQRRQRPGHTPGNSNKWKHLQE fKKGRNRRTHEFERAPRS V (SEQ ID NO:24) Human Sema3B:

MGRAGAAANIPGLALLWANGLGSAAPSPPRLRLSFQELQAWHGLQTFSLERTCCYQAL L\T⁾EERGRLFVGAEΝHNASLΝLDΝISKRAKKLAWPAPNEWREECΝWAGKDIGTECMΝ FVKLLHAYNRTHLLACGTGAFHPTCAFVENGHRAEEPVLRLDPGRTEDGKGKSPYDPRH RAASNLVGEELYSGVAADLMGRDFTIFRSLGQRPSLRTEPHDSRWLΝEPKFVKVFWIPES EΝPDDDKrYFFFRETAVEAAPALGRLSVSRVGQICRΝDVGGQRSLNΝKWTTFLKARLVC SVPGVEGDTHFDQLQDVFLLSSRDHRTPLLYAVFSTSSSIFQGSANCNYSMΝDVRRAFLG PFAHJ^GPMHQWVSYQGRVPYPRPGMCPSKTFGTFSSTKDFPDDVIQFARΝHPLMYΝS VLPTGGRPLFLQNGAΝYTFTQIAADRNAAADGHYDVLFIGTDVG'TVLKNISNPKGSRPS AEGLLLEELHNFEDS AANTSMQISSKRHQLYNASRSANAQIALHRCAAHGRNCTECCLA RDPYCAWDGNACTRFQPSAKRRFRRQDNRΝGDPSTLCSGDSSRPALLEHKNFGNEGSSA FLECEPRSLQARVEWTFQRAGVTAHTQVLAEERTERTARGLLLRRLRRRDSGVYLCAA VEQGFTQPLRRLSLHVLSATQAERLARAEEAAPAAPPGPKLWYRDFLQLVEPGGGGSAΝ SLRMCRPQPALQSLPLESRRKGRΝRRTHAPEPRAERGPRSATHW (SEQ ID ΝO:25)

Human Sema3C:

MAFRΗCVLVG\ΨICSICVKGSSQPQARVYLTFDELRETKTSEYFSLSHHPLDYRTLLMDE DQDRJΥVGSKDHTLSL riNMSQEALSVFWPASTIKVEECKMAGKDP FNRTHLYNCGSGAFSPNCTYLNRGRRSEDQVFMTOSKCESGKGRCSFNPNNNTNSNMIN EELFSGMYIDFMGTDAAIFRSLTKRNAVRTDQHNSKWLSEPMFVDAHVIPDGTDPNDAK VYFTFKEKLTDNNRSTKQIHSMIARICPNDTGGLRSLNNKWTTFLKAJ LNCSNTDEDGPE THFDELEDNFLLETDNPRTTLNYGffTTSSSNFKGSANCNYHLSDIQTVFNGPFAHKEGPN HQLISYQGRIPYPRPGTCPGGAFTPΝMRTTKEFPDDVVTFIRΝHPLMYΝSrYPfflKRPLrVR ΝHAPITTMKISSKKQQLYVSSΝEGVSQVSLHRCHIYGTACADCCLARDPYCAWDGHSCS RIΥPTGKJΛJ SRRQDVRHGΝPLTQCRGFM.KAYRΝAAEIVQYGVKΝΝTTFLECAPKSPQ ASIKWLLQKDKDRJΛKEVKL TERIIATSQGLLπiSVQGSDQGLYHCIATENSFKQTIAKINF K\π.DSEMVA\^\tTDKWSPWTWASSVRALPFHPKDIMGAFSHSEMQMINQYCKDTRQQH QQGDESQKMRGDYGKLKALINSRKSRNRRNQLPES (SEQ ID NO:26)

Human Sema3D:

MNANKΌERLKARSQDFHLFPALMMLSMTMLFLPVTGTLKQNTPRLKLTYKDLLLSNSCI PFLGSSEGLDFQTLLLDEERGRLLLGAKDHIFLLSLVDLNKNFKKRYWPAAKERNELCKL AGKDA TECAΝFLRVLQPY^FKTH^YVCGTGAFFIPICGYIDLG\TYKEDITFKLDTHΝLESG RLKCPFDPQQPFASVMTDEYLYSGTASDFLGKDTAFTRSLGPTHDHHYIRTDISEHYWLN GAKPIGTFFIPDTYNPDDDKRYFFFRESSQEGSTSDKΗLSRNGRNSPCRSFVLSFQRKRLKS NSRΝΝPLKIETTQID IDNGGQRSLIΝKWTTFLKΛJΛLICSIPGSDGADTYFDELQD^I LLPT RDERNP\NYGVFTTTSSIFKGSAVCVYSMADIRAVFNGPYAHKESADHRWVQYDGRIPY PRPGTCPSKTYDPLIKSTRDFPDDNISFIKRHSVMYKSΛ^YPNAGGPTFKRINNDYRLTQIN VDHNL\EDGQYDNMFLGTDIGTVLKVVSISKEKWΝMEEVVLEELQIFKHSSIILΝMELSL KQQQLYIGSRDGLVQLSLHRCDTYGKACADCCLARDPYCAWDGNACSRYAPTSKRRA RRQDNKYGDPITQCWDFFIDSISHETADEKNTFGIEFNSTFLECRPKSQQALTKWYIQRSGDE HREELKPDERΠKTEYGLLIRSLQKKDSGMYYCKAQEHTFFFLTIVKLTLNVTENEQMENTQ RAEHEEGKVKDLLAESRLRYKDYIQILSSPNFSLDQYCEQMWHREKRRQRNKGGPKWK HMQEMKKKRNRRHHRDLDELPRAVAT (SEQ ID NO:27) Mouse Sema3E:

MAPAGFFLLTLLLWGHLLELWTPGHSANPSYARLPLSHKELFELNGLQYFKAPLGFLDLH TMLLDEYQERLFVGGRDLVYSLNLERVSDGYREΓYWPSTAVKVEECIMKGKDANECAN YMVLHHYNRTHLLTCATGAFDPHCAFIRVGHHSEEPLFHLESHRSERGRGRCPFDPNSSF VSTLNGNELFAGLYSDYWGRDSAIFRSMGKLGHIRTEHDDERLLKEPKFNGSYMIPDNE DRDDNKMYIΨFTEKALEAE WAHTILHPSGRLCVNDMGGQRILNNKWSTFLKARLNCS GMΝGROT^DELEDNI^LPTRDPKΝPNTFGLFΝTTSΝRFRGHANCNYHMSSIREAFΝGP YAHKEGPEYHWSLYEGKNPYPRPRSCASKNΝGGKYGTΝQRLPDDAIRFARMHPLMYQP I NHKKPILNKTDGKYΝLRQLANDRNEAEDGQYDNLFIGTDTGINIIK\ TTYΝQETEW MEENILEELQIFKDPAPΠSMEISSKRQQLYIGSASAVAQVRFHHCDMYGSACADCCLAR DPYCAWDGISCSRYYPTGAHEKRRFRRQDVRHGNAAQQCFGQQFVGDALDRTEERLA YGFFISNSTLLECTPLSLQAKVIWFLQKGRDVRKEEΛTKTDDRNVKMDLGLLFLRNRKSDA GTYFCQT\ΕHΝFVHTVRKITLEVVEEHKVEGMFHKDHEEERHHKMPCPPLSGMSQGTK PWYKEFLQLIGYSSKFQRVEEYCEKVWCTDKKRKKLKMSPSKWKYANPQEKRLRSKAE HFRLPRHTLLS (SEQ ID NO:28)

Human Sema3F:

MLVAGLLLWASLLTGAWPSFPTQDHLPATPRVIΛLSFKELKATGTAHFFNFLLNTTDYRI LLKDEDHDIΛMYVGSKDYVLSLDLHDINREPLΠHWAASPQRIEECVLSGKDVNGECGNF VRLIQPWNRTHLYVCGTGAYNPMCTYVNRGRRAQATPWTQTQAVRGRGSRATDGALR PMPTAPRQDYTJYLEPERLESGKGKCPYDPKLDTASALINEELYAGVYΓDFMGTDAAIFR TLGKQTAMRTDQYNSRWLNDPSFIHAELIPDSAERNDDKLYFFFRERSAEAPQSPANYA RIGRICLΝDDGGHCCLVΝKWSTFLKARLVCSVPGEDGIETHFDELQDNFNQQTQDVRΝP \ Y^"A\TTSSGS\TRGSANCVYSMADIRMVFΝGPFAHKEGPΝYQWMPFSGKMPYPRPGT CPGGTFTPSMKSTKDYPDEVNNFMRSHPLMYQAVYPLQRRPLV TGA^

\TDAGDGRYEVLFLGTDRGTVQK\TVXPKDDQEMEELMLEEVEVFKDPAPVKTMΗSSK RQQLYNASAVGNTHLSLHRCQAYGAACADCCLARDPYCAWDGQACSRYTASSKRRSR RQD\TMGOTIRQCRGFΝSΝAΝKΝAVESVQYGNAGSAAFLECQPRSPQAT\Ε:WLFQRDP GDRRREIRAEDRFLRTEQGLLLRALQLSDRGLYSCTATE I IFKHVNTRNQLHNLGRDAV HAALFPPLSMSAPPPPGAGPPTPPYQELAQLLAQPENGLIHQYCQGYWRHNPPSPREAPG APRSPEPQDQKKPRΝRRHHPPDT (SEQ ID ΝO:29)

Based on the identification of a hanatoxin-like sequence in secreted semaphorins, it was hypothesized that the secreted semaphorins may produce repulsion of neuronal growth cones by an interaction with a voltage-gated K⁺ or Ca²⁺ channel. To begin testing this, certain conserved amino acids in one sub-domain of the hanatoxin-like sequence of Sema3A were mutated to alanine (Fig. 1). The two mutations generated were: RDPYCAWD to AAPYCAWD (RD mutant) and RDPYCAWD to RDPACAAA (YWD mutant). The Sema3A gene was mutated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's protocol. The oligonucleotides used to generate the YWD mutant were:

CTCGCCCGAGACCCTGC CTGTGCTGCGGCTGGTTCTGCATGT (SEQ ID NO:41) and GAACATGCAGAACC AGCCGCAGCACAGGCAGGGTCTCGGGCG (SEQ ID NO:42). The oligonucleotides used to generate the RD mutant were:

CTGAGTGTTGCCTCGCCGCAGCCCCTT ACTGTGCTTGGG (SEQ ID NO:40) and CCCAAGCACAGTAAGGGGCTGCGGCG AGGCAACACTCAG (SEQ ID NO: 15).

Plasmids encoding each of the mutants, as well as wild-type Sema3A, were transfected into COS-7 cells and tested for their capacity to repel axonal outgrowth by co-culturing them for 40 hours with an embryonic (El 3.5) mouse explant dorsal root ganglion (DRG).

Mouse El 3 DRG and Sema3A-, RD- or YWD- (pCOS H-Sema3A-myc) expressing COS-7 cells were co-cultured for 40 hours, using methodology described in Messersmith et al., Neuron 14:949-959, 1995, except that collagen was substituted with 10% growth factor-reduced Matrigel (Collaborative Biomedical Products). Paraformaldehyde-fixed co-cultures were immunostained with a neurofilament-specific antibody (1:1000 dilution; Chemicon) and AP- conjugated secondary antibody (1 :400 dilution; Boehringer-Mannheim).

DRG axons were repelled by COS-7 cells transfected with wild-type Sema3A. In contrast, COS-7 cells transfected with plasmids encoding RD or YWD mutants exhibited only very weak repellent effects on DRG axons, which grew in close proximity to the transfected COS cells.

The degree of activity retained by the RD mutant was further tested in a growth cone collapse assay, which measures the immediate effects of Sema3A on growth cone morphology. DRG explants derived from embryonic day 13 mouse embryos were cultured for 20 hours in F12/N3 medium as described in Tessier-Lavigne et al., Nature 336:775-778, 1988, except that the medium contained only 0.5% FBS, 15 mM Hepes (pH 7.5), 0.6 mg/ml hydroxypropylmethyl cellulose (5600 centripoises - Sigma), and 50 ng/ml NGF (Boehringer-Mannheim). In addition, the plates were pre-coated with growth factor-reduced Matrigel (Collaborative Biomedical Products) diluted 1:5 in F12 medium. To facilitate detection and measurement of Sema3 A, alkaline-phosphatase/Sema3 A fusion proteins were produced. To generate the H-Sema3A-AP (N-terminus) fusion protein expression vector, the human Sema3A coding sequence was inserted into the Bgl II and Xho I site of pAPtag-4 to generate a Sema3A-AP fusion (Flanagan et al., Cell 63:185-194, 1990). Then the entire Sema3A-AP sequence was excised the from the pAPtag4 vector and inserted into the Hind III and Xho I sites of pCDNA 1.1 Amp (Invitrogen). This vector also included myc-and His-epitope tags at the C-terminus. The collapse assay was performed using Sema3A-AP-containing medium, essentially as described (Luo et al., supra). The DRG were stained with 5 units/ml rhodamine-phalloidin (Molecular Probes) for 20 minutes, then washed and mounted with Vectashield^® (Vector). Pharmacological agents were added 7 minutes (time point for CoCl₂, CdCl₂, BAPTA, and hanatoxin) or 60 minutes (time point for calmidazolium chloride) before the addition of Sema3A, thrombin, or control conditioned medium. To load BAPTA-AM, the DRG explants were pre- incubated in room temperature for 30 minutes, washed once, and incubated for another 30 minutes at 37°C before the addition of Sema3A-containing or control conditioned medium. CoCl₂, CdCl₂, and thrombin were obtained from Sigma; BAPTA and BAPTA-AM were obtained from Molecular Probes; calmidazolium chloride was obtained from Calbiochem; hanatoxin, purified from Phrixotrichus spatulata venom, was generously provided by Kenton J. Swartz .

The RD mutant was 35.9-fold less potent than wild type Sema3A. The EC₅₀ was 0.047 ± 0.003 nM and 1.69 ± 0.54 nM for wild type and mutant proteins, respectively (n = 4; Fig. 2). The RD mutant did not, even at high doses, produce the level of collapse generated by the wild-type protein.

It was known that Sema3A contains two neuropilin 1 binding sites, one in the C domain and the other in the sema domain (amino acids 25-586; see He et al., Cell 90:736-751, 1997). See Fig. 1 for relative positions of the domains. A truncated sema domain (amino acids 25-526) had been shown not to exhibit neuropilin 1 binding (He et al., supra). Since the HTLS (amino acids 522-538) was in close proximity to the residues responsible for neuropilin 1 binding, it was hypothesized that mutations within the HTLS impaired binding to neuropilin 1 through local or global conformational changes.

To test this hypothesis, binding of the RD mutant to neuropilin 1 -expressing cells was compared to binding of wild-type Sema3A binding to the same cells. AP-tagged constructs, prepared as described above, were used in this assay. pCDNA 1.1 -Sema3 A-AP or RD-AP was transfected into 293T cells. The 293T cell supernatants were used for binding experiments with stably transfected PAE-neuropilin 1 and PAE parental cell lines. Cells were incubated with supernatant containing Sema3A-AP or RD-AP fusion protein. The fusion protein binding experiments were performed as described in Cheng et al., Cell 79:157-168, 1994. Sema3A and RD bound to neuropilin 1 with similar affinities, as shown in Figs. 3A and

3B. The K_d for Sema3A was calculated as 298.8 ± 40.9 pM, and the K_d for RD was calculated as 389.3 ± 97.6 pM; n = 8; p = 0.0673 in paired t-test. It was therefore concluded that the hanatoxin-like sequence is likely to play a role in the functional activity of Sema3A, independent of neuropilin 1 binding.

Since hanatoxin is a voltage-gated K⁺ or Ca²⁺ channel blocker, and mutation of the HTLS reduces Sema3A activity, the question of whether Sema3A acts through K⁺ or Ca²⁺ ion channel blockade or activation was next addressed. To test this hypothesis, it was determined whether K⁺ and Ca²⁺ ion channel blockers can mimic or inhibit the growth cone collapse activity of Sema3 A. The K⁺ channel blockers tetraethylammonium (up to 30 mM) and 4-aminopyridine (up to 10 mM) neither induced growth cone collapse by themselves, nor modified Sema3A-induced growth cone collapse. A similar lack of activity was found for a number of specific K⁺ ion channel toxins (Apamin at 130 nM, Charybdotoxin at 100 nM, α-Dendrotoxin at 100 nM, Iberiotoxin at 10 μM, Kaliotoxin at 10 μM, Margatoxin at 1 μM, Stichodactyla Toxin at 0.5 μM, and -Tityustoxin K ^t 5 μM).

The general Ca²⁺ channel blocker cobalt (CoCl₂, 2 mM) by itself also did not affect DRG growth cones (Fig. 4). However, when incubated in the presence of Sema3 A, CoCl₂ inhibited Sema3A action on DRG growth cones by 87.8% (n = 12, p < 0.0001, one-way ANOVA; Fig. 4). Another general Ca²⁺ channel blocker, cadmium (CdCl₂, 200 μM), had an effect on growth cone morphology (reduction in surface area) by itself, but when combined with Sema3A, inhibited growth cone collapse by 81.2% at concentrations of 200 μM (n = 8, p < 0.0001, one-way ANOVA; Fig. 4). Several more specific Ca²⁺ channel blockers were also tested. The activity of Sema3A was not blocked by 7 μM Nifedipine (L-type Ca channel blocker), 3 μM ω-Conotoxm GVIA (N-type Ca²⁺ channel blocker), or 0.3 μM ω-Agatoxin (P-/Q- type Ca²⁺ channel blocker). Finally, we tested the effects of hanatoxin. Hanatoxin (10 μM) reduced Sema3A activity by 23.2% (n = 12, p < 0.0086, one-way ANOVA; Fig. 4). At this dose hanatoxin had no effect on DRG growth cone collapse. To test whether extracellular Ca²⁺ blockers inhibit DRG growth cone collapse non- specifically, the effect of cobalt on another molecule capable of inducing growth cone collapse was determined. Thrombin, a G-protein dependent collapsing factor (Gomez et al., Neuron 14:1233-1246, 1995), induced collapse activity in E13.5 DRG axons. Cobalt did not block the growth cone collapse induced by thrombin, even at a dose that effectively inhibited this activity of Sema3A.

The role of Ca²⁺ on the activity of Sema3A was further characterized by reducing the extracellular concentration of Ca²⁺ with BAPTA (1 mM). The calculated Ca²⁺ concentration in the medium after BAPTA treatment was about 50 nM, similar to the resting intracellular concentrations in rodent growth cones (Kuhn et al., J. Neurosci. 18:184-194, 1998; and Ming et al., Neuron 19:1225-1235, 1997). While BAPTA itself caused only a small increase in growth cone collapse (15%) compared to control (Fig.4), it completely blocked the growth cone collapsing activity of Sema3A (Fig. 4). No consistent change in cytosolic Ca²⁺ in growth cones was detected on exposure to Sema3A, using the indicator fluo-3, although technical difficulties may have affected the results.

Intracellular Ca²⁺ manipulations also modified Sema3A activity. Preloading DRG explants with BAPTA-AM at a dose of 2 μM did not change growth cone morphology but reduced Sema3A induced growth cone collapse by 38.8% (n = 9, p < 0.0001, one-way ANOVA; Fig. 5). Ca²⁺ influx into growth cones triggered by Sema3A could activate numerous target proteins such as Ca /calmodulin, a major intracellular Ca²⁺ receptor abundant in growth cones (Wang et al., J. Neurosci. 18:4973-4984, 1998). Pretreatment of DRG explants with the calmodulin inhibitor calmidazolium chloride at 0.08 μM reduced the Sema3A-induced growth cone collapse by 33.6% (n = 12, p < 0.0001, one-way ANOVA; Fig. 5). Since higher doses of BAPTA-AM and calmidazolium chloride induced growth cone collapse, an analysis of whether a complete inhibition of Sema3A's action could be achieved by a greater intracellular calcium block could not be performed. To exclude the possibility that the blocking effects of Sema3A by heavy metals is simply due to a dependence of Sema3A binding to neuropilin 1 by a Ca²⁺-dependent protein-protein interaction, the effects of these reagents on Sema3A/neuropilin 1 binding were tested. The calculated K for Sema3A-AP binding was 298.8 ± 40.9 pM, n = 4. In the presence of CoCl₂ (2 mM) or CdC12 (200 μM) the calculated Kd for Sema3A-AP binding was 238.7 ± 139.9 pM (n = 3) and 314.3 ± 170.1 pM (n = 6), respectively. Both CoCl₂ and CdCl₂ had no significant effect on Sema3A/neuropilin 1 binding properties (p = 0.438 and 0.563 for CoCl₂ and CdCl₂, respectively; paired t-test). See Fig. 6.

Other Embodiments It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of this invention. What is claimed is:

Claims

1. A polypeptide comprising the sequence Xaaι-Xaa₂-Xaa₃-Xaa₄-Xaas-Xaa₆-Xaa₇-Xaa₈-

Xaa9-Xaaιo-Xaaιι-Xaai2-Xaaι₃-Xaaι -Xaai5-Xaai6-Xaai7-Xaaι₈-Xaai9-Xaa₂o-Xaa2i-Xaa22, wherein

Xaai is Lys, Arg, Thr, Ser, Ala, Glu, Gin, His, Gly or Tyr; Xaa₂ is Thr, Ala, Val, Ser, Phe, Asp, Cys, or Gly;

Xaa₃ is Thr, Cys, Arg, Gin, Met, or Lys;

Xaa* is Ala, Thr, Val, Tyr, Gly, Ser, Lys, Arg, His, or Glu;

Xaas is Asp, Glu, Thr, Ala, Asn, Ser, Gly, or Leu;

Xaa₇ is Cys, Val, He, or Leu;

Xaa₈ is Lys, Leu, Gly, Ala, Met, or Thr;

Xaa₉ is Ala, Ser, Leu;

Xaaio is Leu or no amino acid;

Xaan is Gly or no amino acid; Xaaπ is Cys or no amino acid;

Xaa_!3 is Lys or no amino acid;

Xaaι₄ is Phe or no amino acid;

Xaais is Arg, Gin, Leu, or Thr;

Xaaiβ is Asp; Xaaπ is Lys or Pro;

Xaaι₈ is Tyr, His, or Val;

Xaa∑o is Ala or Gly;

Xaa₂₂ is Asp, Asn, Thr, Ala, Val, His, Cys, and the polypeptide is 17 to 34 amino acids in length.

2. The polypeptide of claim 1, wherein the polypeptide is 19 to 32 amino acids in length.

3. The polypeptide of claim 1, wherein the sequence is selected from the group consisting of SEQ ID NOs:l-14 and 16-23.

4. A polypeptide consisting of a first amino acid sequence consisting of Xaaι-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaag-

Xaa9-Xaaio-X an-Xaai2-Xaai₃-Xaai₄-Xaai5-Xaai6-Xaai7-Xaai8-Xaai9-Xaa2o-Xaa2i-Xaa22, wherein Xaai is Lys, Arg, Thr, Ser, Ala, Glu, Gin, His, Gly or Tyr;

Xaa_∑ is Thr, Ala, Val, Ser, Phe, Asp, Cys, or Gly;

Xaa₃ is Thr, Cys, Arg, Gin, Met, or Lys;

Xaa is Ala, Thr, Val, Tyr, Gly, Ser, Lys, Arg, His, or Glu;

Xaas is Asp, Glu, Thr, Ala, Asn, Ser, Gly, or Leu;

Xaa is Cys, Val, He, or Leu;

Xaa₈ is Lys, Leu, Gly, Ala, Met, or Thr;

Xaa₉ is Ala, Ser, Leu;

Xaaio is Leu or no amino acid; Xaai i is Gly or no amino acid;

Xaai₂ is Cys or no amino acid;

Xaaj₃ is Lys or no amino acid;

Xaaι is Phe or no amino acid;

Xaai 5 is Arg, Gin, Leu, or Thr; Xaaiβ is Asp;

Xaaπ is Lys or Pro;

Xaaι₈ is Tyr, His, or Val;

Xaa₂o is Ala or Gly; Xaa∑i is Trp; and

Xaa_∑∑ is Asp, Asn, Thr, Ala, Val, His, Cys; and a second amino acid sequence less than 70% identical to each one of SEQ ID NOs:24-29.

5. The polypeptide of claim 4, wherein the first amino acid sequence is selected from the group consisting of SEQ ID NOs:l-14 and 16-23.

6. The polypeptide of claim 4, wherein the second amino acid sequence is less than 50% identical to each one of SEQ ID NOs:24-29.

7. The polypeptide of claim 4, wherein the second amino acid sequence is less than 30% identical to each one of SEQ ID NOs:24-29.

8. The polypeptide of claim 4, wherein the second amino acid sequence is a detectable protein selected from the group consisting of a fluorescent protein, a luminescent protein, and a chromogenic protein.

9. A purified polypeptide consisting of a first amino acid sequence consisting of Xaaι-Xaa₂-Xaa₃-Xaa₄-Xaas-Xaa₆-Xaa₇-Xaa₈- Xaa9-Xaaιo-Xaaιι-Xaai2-Xaaι₃-Xaaι -Xaai5-Xaai6-Xaaπ-Xaai8-Xaai9-Xaa2o-Xaa2i-Xaa22, wherein

Xaai is Lys, Arg, Thr, Ser, Ala, Glu, Gin, His, Gly or Tyr;

Xaa_∑ is Thr, Ala, Val, Ser, Phe, Asp, Cys, or Gly;

Xaa₃ is Thr, Cys, Arg, Gin, Met, or Lys; Xaa is Ala, Thr, Val, Tyr, Gly, Ser, Lys, Arg, His, or Glu;

Xaas is Asp, Glu, Thr, Ala, Asn, Ser, Gly, or Leu;

Xaa₇ is Cys, Val, He, or Leu;

Xaa₈ is Lys, Leu, Gly, Ala, Met, or Thr;

Xaaio is Leu or no amino acid;

Xaau is Gly or no amino acid;

Xaai₂ is Cys or no amino acid;

Xaai ₃ is Lys or no amino acid; Xaaμ is Phe or no amino acid;

Xaais is Arg, Gin, Leu, or Thr;

Xaaπ is Lys or Pro;

Xaaι₈ is Tyr, His, or Val; Xaa i₉ is Cys;

Xaa₂o is Ala or Gly;

Xaa₂ι is Trp; and

Xaa₂₂ is Asp, Asn, Thr, Ala, Val, His, Cys; a second amino acid sequence that is less than 70% identical to each one of SEQ ID

NOs:24-29, the second amino acid sequence being present C-terminal to the first amino acid sequence; and a third amino acid sequence that is less than 70% identical to each one of SEQ ID NOs:24-29, the third amino acid sequence being present N-terminal to the first amino acid sequence, provided that the second amino acid sequence is not SEQ ID NO:39 and the third amino acid sequence is not SEQ ID NO:38.

10. The polypeptide of claim 9, wherein the first amino acid sequence is selected from the group consisting of SEQ ID NOs:l-14 and 16-23.

11. The polypeptide of claim 9, wherein the second amino acid sequence is less than 50% identical to each one of SEQ ID NOs:24-29.

12. The polypeptide of claim 9, wherein the third amino acid sequence is less than 50% identical to each one of SEQ ID NOs:24-29.

13. A polypeptide consisting of a first amino acid sequence consisting of Xaaι-Xaa₂-Xaa₃-Xaa₄-Xaa5-Xaa6-Xaa -Xaa₈-

Xaa9-Xaa i o-Xaa 11 -Xaa 12-Xaa 13-Xaai ₄-Xaa j 5-Xaa i β-Xaa 17-Xaa 1₈-Xaa 19-Xaa2o-X a21 -Xaa22, wherein

Xaai is Lys, Arg, Thr, Ser, Ala, Glu, Gin, His, Gly or Tyr; Xaa₂ is Thr, Ala, Val, Ser, Phe, Asp, Cys, or Gly; Xaa₃ is Thr, Cys, Arg, Gin, Met, or Lys;

Xa ₄ is Ala, Thr, Val, Tyr, Gly, Ser, Lys, Arg, His, or Glu; Xaas is Asp, Glu, Thr, Ala, Asn, Ser, Gly, or Leu; Xaa6 is Cys; Xaa₇ is Cys, Val, He, or Leu;

Xaa₈ is Lys, Leu, Gly, Ala, Met, or Thr;

Xaa ιo is Leu or no amino acid;

Xaa π is Gly or no amino acid;

Xaa i₂ is Cys or no amino acid;

Xaaι₃ is Lys or no amino acid;

Xaaι₄ is Phe or no amino acid;

Xaa is is Arg, Gin, Leu, or Thr;

Xaai6 is Asp;

Xaaπ is Lys or Pro;

Xaaι₈ is Tyr, His, or Val;

Xaaig is Cys;

Xaa₂o is Ala or Gly;

Xaa₂ι is Trp; and

Xaa₂₂ is Asp, Asn, Thr, Ala, Val, His, Cys; and a second amino acid sequence.

14. The polypeptide of claim 13, wherein the first amino acid sequence is selected from the group consisting of SEQ ID NOs:l-14 and 16-23.

15. The polypeptide of claim 13, wherein the second amino acid sequence is a detectable protein selected from the group consisting of a fluorescent, luminescent, and chromogenic protein.

16. The polypeptide of claim 1, wherein the polypeptide is substantially pure.

17. The polypeptide of claim 4, wherein the polypeptide is substantially pure.

18. An isolated nucleic acid encoding the polypeptide of claim 1.

19. An isolated nucleic acid encoding the polypeptide of claim 4.

20. An isolated nucleic acid encoding the polypeptide of claim 9.

21. An isolated nucleic acid encoding the polypeptide of claim 13.

22. A purified antibody that specifically binds to a hanatoxin-like sequence in a semaphorin polypeptide.

23. A method of identifying a mammalian polypeptide sequence, the method comprising providing a relational database comprising amino acid sequence values, species origin values, and polypeptide identity values, each of the amino acid sequence values being associated with a species origin value and a polypeptide identity value; querying the relational database with an test amino acid sequence value of a polypeptide toxin or toxin domain to obtain a sequence value that is identical, to within a pre-determined threshold percentage, to the test amino acid sequence value; determining the species origin value associated with the amino acid sequence value; determining if the species origin value is associated with a mammalian or non- mammalian species; selecting the species origin value if the species origin value is associated with a mammalian species; and determining the polypeptide identity value associated with selected species origin value.

24. The method of claim 23, wherein the threshold percentage is 50%.

25. The method of claim 23, wherein the threshold percentage is 80%.

26. The method of claim 23, wherein the toxin is produced by an invertebrate, reptile, or fish.

27. The method of claim 23, wherein the toxin is a neurotoxin.