U.S. patent application number 11/580373 was filed with the patent office on 2007-02-08 for semaphorin gene family.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to David R. Bentley, Corey S. Goodman, Alex L. Kolodkin, David Matthes, Timothy O'Connor.
Application Number | 20070033669 11/580373 |
Document ID | / |
Family ID | 22398364 |
Filed Date | 2007-02-08 |
United States Patent
Application |
20070033669 |
Kind Code |
A1 |
Goodman; Corey S. ; et
al. |
February 8, 2007 |
Semaphorin gene family
Abstract
A novel class of proteins, semaphorins, nucleic acids encoding
semaphorins, semaphorin peptides, and methods of using semaphorins
and semaphorin-encoding nucleic acids are disclosed. Semaphorin
peptides and receptor agonists and antagonists provide potent
modulators of nerve cell growth and regeneration. The invention
provides pharmaceutical compositions, methods for screening
chemical libraries for regulators of cell growth/differentiation;
semaphorin gene-derived nucleic acids for use in genetic mapping,
as probes for related genes, and as diagnostic reagents for genetic
neurological disease; specific cellular and animal systems for the
development of neurological disease therapy.
Inventors: |
Goodman; Corey S.;
(Berkeley, CA) ; Kolodkin; Alex L.; (Berkeley,
CA) ; Matthes; David; (Berkeley, CA) ;
Bentley; David R.; (Berkeley, CA) ; O'Connor;
Timothy; (Berkeley, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
The Regents of the University of
California
Oakland
CA
|
Family ID: |
22398364 |
Appl. No.: |
11/580373 |
Filed: |
October 12, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10067632 |
Feb 4, 2002 |
7153936 |
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11580373 |
Oct 12, 2006 |
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09060610 |
Apr 15, 1998 |
6344544 |
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10067632 |
Feb 4, 2002 |
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08835268 |
Apr 8, 1997 |
5807826 |
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09060610 |
Apr 15, 1998 |
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08121713 |
Sep 13, 1993 |
5639856 |
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08835268 |
Apr 8, 1997 |
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Current U.S.
Class: |
800/18 ;
435/320.1; 435/325; 435/69.1; 514/17.7; 514/3.7; 514/8.3; 530/326;
530/327; 530/328; 530/329; 530/330; 530/350; 530/388.1 |
Current CPC
Class: |
C12N 15/8509 20130101;
A61P 35/00 20180101; A01K 2217/075 20130101; A61P 37/00 20180101;
A01K 67/0339 20130101; A01K 2217/05 20130101; A01K 2267/0318
20130101; A61P 31/12 20180101; C07K 14/005 20130101; C07K 14/705
20130101; C07K 14/4703 20130101; A01K 2267/03 20130101; A01K
2267/01 20130101; C12N 2710/24122 20130101; A61K 38/00 20130101;
A61P 25/00 20180101 |
Class at
Publication: |
800/018 ;
530/326; 530/327; 530/328; 530/329; 530/330; 530/388.1; 435/069.1;
435/320.1; 435/325; 530/350; 514/013; 514/014; 514/015; 514/016;
514/017 |
International
Class: |
A01K 67/027 20060101
A01K067/027; A61K 38/10 20070101 A61K038/10; A61K 38/08 20070101
A61K038/08; C07K 14/47 20070101 C07K014/47; C12N 5/06 20070101
C12N005/06; C07K 16/18 20070101 C07K016/18 |
Claims
1. An isolated peptide of at least 5 amino acids comprising a
unique portion of a semaphorin, and said peptide has a semaphorin
binding specificity.
2. An isolated peptide according to claim 1 wherein said semaphorin
comprises a human semaphorin.
3. An isolated peptide according to claim 1 wherein the amino acid
sequence of said unique portion comprises a sequence selected from
the group consisting of: TABLE-US-00007 (a) [DE]C[QKRAN]N[YFV]I
C[QKRAN]N[YFV]I[RKQT] (b) CGT[NG][Asn][YFHG][KRHNQ] CGT[NG][Asn]XXP
CGT[NG]XXXPX[CD] CGTXXXXPX[CD]XX[YI] (c)
[RIQV][GA][LVK][CS]P[FY][DN] [CS]P[FY][DN]P[DERK][HLD]
GX[GA]X[CS]PY[DN]P (d) L[FY]S[GA]T[VNA]A L[FY]SXTXA[DE][FY]
[FY]S[GA]T[VNA]A[DE][FY] (e) L[ND][AK]PNFV (f) FFFRE FF[FY]RE[TN]
FFRE[TN]A F[FY]RE[TN]A YFF[FY]RE [FY]FF[FY]RE [FY][FY][FY]RE[TN]A
[IV][FY]F[FY][FY]RE D[KFY]V[FY][FYIL][FYIL][FY]
[VI][FY][FYIL][FYIL]F[RT]X[TN]
[VI][FY][FYIL][FYIL][FY][RT][EDV][TN] (g) E[FY]IN[CS]GK
[FY]INCGK[AVI] (h) R[VI][AG][RQ][VI]CK R[VI]X[RQ][VI]CXXD
GK[VAI]XXXR[VAI]XXXCK (i) [RKN]W[TAS][TAS][FYL]L[KR]
[FY]L[KR][AS]RL[NI]C [NI]CS[IV][PS]G W[TAS][TAS][FYL]LK[ASVIL]XL
W[TAS][TAS]XLKXXLXC WX[TS]XLKXXLXC (j) [FY][FY][ND]EIQS
[FY]P[FY][FY][FY][ND]E (k) GSA[VIL]CX[FY] SA[VIL]CX[FY]XM (l)
NS[NA]WL[PA]V (m) [VLI]P[EDYSF]PRPG [VLI]PXP[RA]PGXC
P[EDYSF]PRPG[TQS]C (n) DP[HFY]C[AG]W P[HFY]C[AG]WD DPXC[AG]WD
CXXXXDPXCXWD CXXXDPXCXWD CXXDPXCXWD CXXCXXXXDXXCXWD CXXCXXXDXXCXWD
CXXCXXDXXCXWD
4. An isolated peptide according to claim 1 wherein the amino acid
sequence of said unique portion comprises a sequence selected from
the group consisting of: TABLE-US-00008 (a) [DE]C[QKRAN]N[YFV]I
C[QKRAN]N[YFV]I[RKQT] (b) CGT[NG][AS][YFHG][KRHNQ]
CGT[NG][Asn][YFH][KRHNQ] CGT[NG][AS]XXP (c)
[RIQV][GA][LVK][CS]P[FY][DN] [CS]P[FY][DN]P[DERK][HLD]
GX[GA]X[CS]PY[DN]P (d) L[FY]S[GA]T[VNA]A L[FY]SXTXA[DE][FY]
[FY]S[GA]T[VNA]A[DE][FY] (e) L[ND][AK]PNFV (f) FEFRE FF[FY]RE[TN]
FFRE[TN]A F[FY]RE[TN]A YFF[FY]RE [FY]FF[FY]RE [FY][FY][FY]RE[TN]A
[IV][FY]F[FY][FY]RE D[KFY]V[FY][FYL][FYIL][FY]
D[KFY]V[FY][FYIL][FYI][FY] [VI][FY][FYL][FYIL]F[RT]X[TN]
[VI][FY][FYIL][FYI]F[RT]X[TN] [VI][FY][FYIL][FYIL]FRX[TN]
[VI][FY][FYL][FYIL][FY][RT][EDV][TN] (g) E[FY]IN[CS]GK
[FY]INCGK[AVI] (h) R[VI][AG][RQ][VI]CK R[VI]X[RQ][VI]CXXD
GK[VAI]XXXR[VAI]XXXCK (i) [RKN]W[TA][TAS][FYL]L[KR]
[FY]L[KR][AS]RL[NI]C [NI]CS[IV][PS]G W[TA][TAS][FYL]LK[ASVIL]XL
W[TAS][TAS][FYL]LK[ASIL]XL W[TA][TAS]XLKXXLXC (j) [FY][FY][ND]EIQS
[FY]P[FY][FY][FY][ND]E (k) GSA[VIL]CX[FY] SA[VI]CX[FY]XM (l)
NS[NA]WL[PA]V (m) [VLI]P[EDYSF]PRPG [VLI]PXPRPGXC
P[EDYSF]PRPG[TQS]C (n) DP[HFY]C[AG]W P[HFY]C[AG]WD DPXC[AG]WD
CXXXXDPXCXWD CXXXDPXCXWD CXXDPXCXWD CXXCXXXXDXXCXWD CXXCXXXDXXCXWD
CXXCXXDXXCXWD
5. An isolated peptide according to claim 1 wherein the amino acid
sequence of said unique portion comprises a sequence selected from
the group consisting of: TABLE-US-00009 (a) DCQNYI (b)
CGT[NG][AS]XXP (c) GX[SC]PYDP (d) LYSGT[VNA]A (e) LNAPNFV (f)
[FY]FF[FY]RE (g) E[FY]IN[CS]GK (h) R[VI]ARVCK (i)
W[TA][TS][FY]LK[AS]RL (j) PFYF[ND]EIQS (k) GSAVCX[FY] (l)
NSNWL[PA]V (m) P[ED]PRPG[TQS]C (n) DPYC[AG]WD
6. An isolated antibody that specifically binds a peptide according
to claim 1.
7. An isolated nucleic acid comprising a nucleotide sequence
encoding a peptide according to claim 1 wherein said sequence is
joined to a nucleotide not naturally joined to said sequence and
said sequence is other than that of the A39 ORF of vaccinia
virus.
8. A cell comprising a nucleic acid according to claim 7.
9. A transgenic rodent comprising a nucleic acid according to claim
7 wherein said nucleic acid is xenogeneic to said rodent.
10. A process for the production of a recombinant unique portion of
a semaphorin comprising culturing the cell of claim 8 under
conditions suitable for the expression of said peptide, and
recovering said peptide.
11. A method of identifying a pharmacological agent useful in the
diagnosis or treatment of disease associated with the binding of a
semaphorin to a semaphorin receptor, said method comprising the
steps of: contacting a panel of prospective agents with a peptide
according to claim 1; measuring the binding of a plurality of said
prospective agents to said peptide; identifying from said plurality
a pharmacological agent which specifically binds said peptide;
wherein said pharmacological agent is useful in the diagnosis or
treatment of disease associated with the binding of a semaphorin to
a cellular receptor.
12. A method of diagnosing a patient for a predisposition to
neurological disease associated with a genetic locus, said method
comprising the steps of: isolating somatic cells from a patient;
isolating genomic DNA from said somatic cells; contacting said
genomic DNA with a with a probe comprising a DNA sequence encoding
a peptide according to claim 1 under conditions wherein said probe
hybridizes to homologous DNA; identifying a region of said genomic
DNA which hybridizes with said probe; wherein the presence, absence
or sequence of said region correlates with a predisposition to a
neurological disease.
14. A method of treating a patient with neurological injury or
disease or a pathological viral infection, said method comprising
the steps of: administering to a patient a therapeutically
effective dosage of a pharmaceutical composition comprising a
pharmaceutically acceptable carrier and a peptide according to
claim 1; wherein said peptide modulates neural cell growth cone
function or viral pathogenicity in said patient.
15. An isolated polypeptide comprising an amino acid sequence
substantially similar to that of a semaphorin, and said polypeptide
has a semaphorin binding specificity.
16. An isolated peptide of at least about 5 amino acids comprising
a unique portion of a semaphorin receptor, and said peptide has a
semaphorin receptor binding specificity.
17. An isolated antibody that specifically binds a peptide
according to claim 16.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/067,632 filed Feb. 4, 2002, which is a continuation of U.S.
application Ser. No. 09/060,610 filed Apr. 15, 1998, now U.S. Pat.
No. 6,344,544, which is a continuation of U.S. application Ser. No.
08/835,268 filed Apr. 8, 1997, now U.S. Pat. No. 5,807,826, which
is a division of U.S. application Ser. No. 08/121,713 filed Sep.
13, 1993, now U.S. Pat. No. 5,639,856, each of which applications
is incorporated herein by reference.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH OR DEVELOPMENT
[0002] The research carried out in the subject application was
supported in part by grants from the National Institutes of Health.
The government may have rights in any patent issuing on this
application.
INTRODUCTION
[0003] 1. Technical Field
[0004] The technical field of this invention concerns peptides,
polypeptides, and polynucleotides involved in nerve cell
growth.
[0005] 2. Background
[0006] The specificity of the wiring of the nervous system--the
complex pattern of specific synaptic connections--begins to unfold
during development s the growing tips of neurons--the growth
cones--traverse long distances to find their correct targets. Along
their journey, they are confronted by and correctly navigate a
series of choice points in a remarkably unerring way to ultimately
contact and recognize their correct target.
[0007] The identification of growth cone guidance cues is to a
large extent, the holy grail of neurobiology. There are the
compounds that tell neurons when to grow, where to grow, and when
to stop growing. The medical applications of such compounds and
their antagonists are enormous and include modulating neuronal
growth regenerative capacity, treating neurodegenerative disease,
and mapping (e.g. diagnosing) genetic neurological defects.
[0008] Over decades of concentrated research, various hypotheses of
chemo-attractants and repellant, labeled pathways, cell adhesion
molecules, etc. have been evoked to explain guidance. Recently,
several recent lines of experiments suggest repulsion may play an
important role in neuron guidance and two apparently unrelated
factors ("Neurite Growth Inhibitor" and "Collapsin") capable of
inhibiting or collapsing growth cones have been reported.
Relevant Literature
[0009] For a recent review of much of the literature in this field,
see Goodman and Shatz (1993) Cell 72/Neuron 10, 77-98. A
description of grasshopper fasciclin IV (now called G-Semaphorin I)
appears in Kolodkin et al. (1992) Neuron 9, 831-845. Recent reports
on Collapsin and Neurite Growth Inhibitor include Raper and
Kapfhammer (1990) Neuron 4, 21-29, an abstract presented by Raper
at the GIBCO-BRL Symposium on "Genes and Development/Function of
Brain" on Jul. 26, 1993 and Schwab and Caroni (1988) J Neurosci 8,
2381 and Schnell and Schwab (1990) Nature 343, 269,
respectively.
SUMMARY OF THE INVENTION
[0010] A novel class of proteins, semaphorins, nucleic acids
encoding semaphorins, and methods of using semaphorins and
semaphorin-encoding nucleic acids are disclosed. Semaphorins
include the first known family of human proteins which function as
growth cone inhibitors and a family of proteins involved in viral,
particularly pox viral, pathogenesis and oncogenesis. Families of
semaphorin-specific receptors, including receptors found on nerve
growth cones and immune cells are also disclosed.
[0011] The invention provides agents, including semaphorin
peptides, which specifically bind semaphorin receptors and agents,
including semaphorin receptor peptides, which specifically bind
semaphorins. These agents provide potent modulators of nerve cell
growth, immune responsiveness and viral pathogenesis and find use
in the treatment and diagnosis of neurological disease and
neuro-regeneration, immune modulation including hypersensitivity
and graft-rejection, and diagnosis and treatment of viral and
oncological infection/diseases.
[0012] Semaphorins, semaphorin receptors, semaphorin-encoding
nucleic acids, and unique portions thereof also find use variously
in screening chemical libraries for regulators of semaphorin or
semaphorin receptor-mediated cell activity, in genetic mapping, as
probes for related genes, as diagnostic reagents for genetic
neurological, immunological and oncological disease and in the
production of specific cellular and animal systems for the
development of neurological, immunological, oncological and viral
disease therapy.
DESCRIPTION OF SPECIFIC EMBODIMENTS
[0013] The present invention discloses novel families of proteins
important in nerve and immune cell function: the semaphorins and
the semaphorin receptors. The invention provides agents, including
semaphorin peptides, which specifically bind semaphorin receptors
and agents, including semaphorin receptor peptides, which
specifically bind semaphorins. These agents find a wide variety of
clinical, therapeutic and research uses, especially agents which
modulate nerve and/or immune cell function by specifically mimicing
or interfering with semaphorin-receptor binding. For example,
selected semaphorin peptides shown to act as semaphorin receptor
antagonists are effective by competitively inhibiting native
semaphorin association with cellular receptors. Thus, depending on
the targeted receptor, these agents can be used to block semaphorin
mediated neural cell growth cone repulsion or contact inhibition.
Such agents find broad clinical application where nerve cell growth
is indicated. e.g. traumatic injury to nerve cells,
neurodegenerative disease, etc. A wide variety of semaphorin- and
semaphorin receptor-specific binding agents and methods for
identifying, making and using the same are described below.
[0014] Binding agents of particular interest are semaphorin
peptides which specifically bind and antagonize a semaphorin
receptor and semaphorin receptor peptides which specifically bind a
semaphorin and prevent binding to a native receptor. While
exemplified primarily with semaphorin peptides, much of the
following description applies analogously to semaphorin receptor
peptides.
[0015] The semaphorin peptides of the invention comprise a unique
portion of a semaphorin and have semaphorin binding specificity. A
"unique portion" of a semaphorin has an amino acid sequence unique
to that disclosed in that it is not found in any previously known
protein. Thus a unique portion has an amino acid sequence length at
least long enough to define a novel peptide. Unique semaphorin
portions are found to vary from about 5 to about 25 residues,
preferably from 5 to 10 residues in length, depending on the
particular amino acid sequence. Unique semaphorin portions are
readily identified by comparing the subject semaphorin portion
sequences with known peptide/protein sequence data bases. Preferred
unique portions derive from the semaphorin domains (which exclude
the Ig-like. intracellular and transmembrane domains as well as the
signal sequences) of the disclosed semaphorin sequences, especially
regions that bind the semaphorin receptor, especially that of the
human varieties. Preferred semaphorin receptor unique portions
derive from the semaphorin binding domains, especially regions with
residues which contact the semaphorin ligand, especially that of
the human varieties. Particular preferred peptides are further
described herein.
[0016] The subject peptides may be free or coupled to other atoms
or molecules. Frequently the peptides are present as a portion of a
larger polypeptide comprising the subject peptide where the
remainder of the polypeptide need not be semaphorin- or semaphorin
receptor-derived. Alternatively, the subject peptide may be present
as a portion of a "substantially full-length" semaphorin domain or
semaphorin receptor sequence which comprises or encodes at least
about 200, preferably at least about 250, more preferably at least
about 300 amino acids of a disclosed semaphorin/receptor sequence.
Thus the invention also provides polypeptides comprising a sequence
substantially similar to that of a substantially full-length
semaphorin domain or a semaphorin receptor. "Substantially similar"
sequences share at least about 40%, more preferably at least about
60%, and most preferably at least about 80% sequence identity.
Where the sequences diverge, the differences are generally point
insertions/deletions or conservative substitutions, i.e. a
cysteine/threonine or serine substitution, an acidic/acidic or
hydrophobic/hydrophobic amino acid substitution, etc.
[0017] The subject semaphorin peptides/polypeptides are "isolated",
meaning unaccompanied by at least some of the material with which
they are associated in their natural state. Generally, an isolated
peptide/polypeptide constitutes at least about 1%, preferably at
least about 10%, and more preferably at least about 50% by weight
of the total peptide/protein in a given sample. By pure
peptide/polypeptide is intended at least about 90%. preferably at
least 95%, and more preferably at least about 99% by weight of
total peptide/protein. Included in the subject peptide/polypeptide
weight are any atoms, molecules, groups, or polymers covalently
coupled to the subject semaphorin/receptor peptide/polypeptide,
especially peptides, proteins, detectable labels, glycosylations,
phosphorylations, etc.
[0018] The subject peptides/polypeptides may be isolated or
purified in a variety of ways known to those skilled in the art
depending on what other components are present in the sample and to
what, if anything, the peptide/polypeptide is covalently linked.
Purification methods include electrophoretic, molecular,
immunological and chromatographic techniques, especially affinity
chromatography and RP-HPLC in the case peptides. For general
guidance in suitable purification techniques, see Scopes, R.,
Protein Purification, Springer-Verlag, N.Y. (1982).
[0019] The subject peptides/polypeptides generally comprise
naturally occurring amino acids but D-amino acids or amino acid
mimetics coupled by peptide bonds or peptide bond mimetics may also
be used. Amino acid mimetics are other than naturally occurring
amino acids that conformationally mimic the amino acid for the
purpose of the requisite semaphorin/receptor binding specificity.
Suitable mimetics are known to those of ordinary skill in the art
and include .beta.-.gamma.-.delta. amino and imino acids,
cyclohexylalanine, adamantylacetic acid, etc., modifications of the
amide nitrogen, the .alpha.-carbon, amide carbonyl, backbone
modifications, etc. See, generally, Morgan and Gainor (1989) Ann.
Repts. Med. Chem 24, 243-252; Spatola (1983) Chemistry and
Biochemistry of Amino Acids, Peptides and Proteins. Vol VII
(Weinstein) and Cho et. al (1993) Science 261, 1303-1305 for the
synthesis and screening of oligocarbamates.
[0020] The subject semaphorin peptides/polypeptides have a
"semaphorin binding specificity" meaning that the subject
peptide/polypeptide retains a molecular conformation specific to
one or more of the disclosed semaphorins and specifically
recognizable by a semaphorin-specific receptor, antibody, etc. As
such, a semaphorin binding specificity may be provided by a
semaphorin-specific immunological epitope, lectin binding site,
etc., and preferably, a receptor binding site. Analogously, the
semaphorin receptor peptides/polypeptides have a "semaphorin
receptor binding specificity" meaning that these
peptides/polypeptides retain a molecular conformation specific to
one or more of the disclosed semaphorin receptors and specifically
recognizable by a semaphorin, a receptor-specific antibody,
etc.
[0021] "Specific binding" is empirically determined by contacting,
for example a semaphorin-derived peptide with a mixture of
components and identifying those components that preferentially
bind the semaphorin. Specific binding is most conveniently shown by
competition with labeled ligand using recombinant semaphorin
peptide either in vitro or in cellular expression systems as
disclosed herein. Generally, specific binding of the subject
semaphorin has binding affinity of 10.sup.-6M, preferably
10.sup.-8M, more preferably 10.sup.-10M, under in vitro conditions
as exemplified below.
[0022] The peptides/polypeptides may be modified or joined to other
compounds using physical, chemical, and molecular techniques
disclosed or cited herein or otherwise known to those skilled in
the relevant art to affect their semaphorin binding specificity or
other properties such as solubility, membrane transportability,
stability, binding specificity and affinity, chemical reactivity,
toxicity, bioavailability, localization, detectability, in vivo
half-life, etc. as assayed by methods disclosed herein or otherwise
known to those of ordinary skill in the art. For example, point
mutations are introduced by site directed mutagenesis of
nucleotides in the DNA encoding the disclosed semaphorin
polypeptides or in the course of in vitro peptide synthesis.
[0023] Other modifications to further modulate binding
specificity/affinity include chemical/enzymatic intervention (e.g.
fatty acid-acylation, proteolysis, glycosylation) and especially
where the peptide/polypeptide is integrated into a larger
polypeptide, selection of a particular expression host, etc. In
particular, many of the disclosed semaphorin peptides contain
serine and threonine residues which are phosphorylated or
dephosphorylated. See e.g. methods disclosed in Roberts et al.
(1991) Science 253, 1022-1026 and in Wegner et al. (1992) Science
256, 370-373. Amino and/or carboxyl termini may be functionalized
e.g., for the amino group, acylation or alkylation, and for the
carboxyl group, esterification or amidification, or the like. Many
of the disclosed semaphorin peptides/polypeptides also contain
glycosylation sites and patterns which may disrupted or modified,
e.g. by enzymes like glycosidases or used to purify/identify the
receptor, e.g. with lectins. For instance, N or O-linked
glycosylation sites of the disclosed semaphorin peptides may be
deleted or substituted for by another basic amino acid such as Lys
or His for N-linked glycosylation alterations, or deletions or
polar substitutions are introduced at Ser and Thr residues for
modulating O-linked glycosylation. Glycosylation variants are also
produced by selecting appropriate host cells, e.g. yeast, insect,
or various mammalian cells, or by in vitro methods such as
neuraminidase digestion. Useful expression systems include COS-7,
293, BHK, CHO, TM4, CV1, VERO-76, HELA, MDCK, BRL 3A, W138, Hep G2,
MMT 060562. TRI cells, baculovirus systems, for examples. Other
covalent modifications of the disclosed semaphorin
peptides/polypeptides may be introduced by reacting the targeted
amino acid residues with an organic derivatizing (e.g.
methyl-3-[(p-azido-phenyl)dithio]propiolmidate) or crosslinking
agent (e.g. 1,1-bis(diazoacetyl)-2-phenylethane) capable of
reacting with selected side chains or termini. For therapeutic and
diagnostic localization, semaphorins and peptides thereof may be
labeled directly (radioisotopes, fluorescers. etc.) or indirectly
with an agent capable of providing a detectable signal, for
example, a heart muscle kinase labeling site.
[0024] The following are 14 classes of preferred semaphorin
peptides where bracketed positions may be occupied by any one of
the residues contained in the brackets and "X" signifies that the
position may be occupied by any one of the 20 naturally encoded
amino acids (see, Table 1). These enumerated peptides maintain
highly conserved structures which provide important semaphorin
binding specificities; TABLE-US-00001 (a)
[AspGlu]Cys[GlnLysArgAlaAsn]Asn[TyrPheVal]Ile (SEQ ID NO:1)
Cys[GlnLysArgAlaAsn]Asn[TyrPheVal]Ile[ArgLysGlnThr] (SEQ ID NO:2)
(b) CysGlyThr[AsnGly][AlaSerAsn][TyrPheHisGly][LysArgHisAsnGln]
(SEQ ID NO:3) CysGlyThr[AsnGly][AlaSerAsn]XaaXaaPro (SEQ ID NO:4)
CysGlyThr[AsnGly]XaaXaaXaaProXaa[CysAsp] (SEQ ID NO:5)
CysGlyThrXaaXaaXaaXaaProXaa[CysAsp]XaaXaa[TyrIle] (SEQ ID NO:6) (c)
[ArgIleGlnVal][GlyAla][LeuValLys][CysSer]Pro[PheTyr][AspAsn] (SEQ
ID NO:7) [CysSer]Pro[PheTyr][AspAsn]Pro[AspGluArgLys][HisLeuAsp]
(SEQ ID NO:8) GlyXaa[GlyAla]Xaa[CysSer]ProTyr[AspAsn]Pro (SEQ ID
NO:9) (d) Leu[PheTyr]Ser[GlyAla]Thr[ValAsnAla]Ala (SEQ ID NO:10)
Leu[PheTyr]SerXaaThrXaaAla[AspGlu][PheTyr] (SEQ ID NO:11)
[PheTyr]Ser[GlyAla]Thr[ValAsnAla]Ala[AspGlu][PheTyr] (SEQ ID NO:12)
(e) Leu[AsnAsp][AlaLys]ProAsnPheVal (SEQ ID NO:13) (f)
PhePhePheArgGlu (SEQ ID NO:14) PhePhe[PheTyr]ArgGlu[ThrAsn] (SEQ ID
NO:15) PhePheArgGlu[ThrAsn]Ala (SEQ ID NO:16)
Phe[PheTyr]ArgGlu[ThrAsn]Ala (SEQ ID NO:17) TyrPhePhe[PheTyr]ArgGlu
(SEQ ID NO:18) [PheTyr]PhePhe[PheTyr]ArgGlu (SEQ ID NO:19)
[PheTyr][PheTyr][PheTyr]ArgGlu[ThrAsn]Ala (SEQ ID NO:20)
[IleVal][PheTyr]Phe[PheTyr][PheTyr]ArgGlu (SEQ ID NO:21)
Asp[LysPheTyr]Val[PheTyr][PheTyrIleLeu][PheTyrIleLeu][PheTyr] (SEQ
ID NO:22)
[ValIle][PheTyr][PheTyrIleLeu][PheTyrIleLeu]Phe[ArgThr]Xaa[ThrAsn]
(SEQ ID NO:23)
[ValIle][PheTyr][PheTyrIleLeu][PheTyrIleLeu][PheTyr][ArgThr][GluAspVal][T-
hrAsn] (SEQ ID NO:24) (g) Glu[PheTyr]IleAsn[CysSer]GlyLys (SEQ ID
NO:25) [PheTyr]IleAsnCysGlyLys[AlaValIle] (SEQ ID NO:26) (h)
Arg[ValIle][AlaGly][ArgGln][ValIle]CysLys (SEQ ID NO:27)
Arg[ValIle]Xaa[ArgGln][ValIle]CysXaaXaaAsp (SEQ ID NO:28)
GlyLys[ValAlaIle]XaaXaaXaaArg[ValAlaIle]XaaXaaXaaCysLys (SEQ ID
NO:29) (i)
[ArgLysAsn]Trp[ThrAlaSer][ThrAlaSer][PheTyrLeu]Leu[LysArg] (SEQ ID
NO:30) [PheTyr]Leu[LysArg][AlaSer]ArgLeu[AsnIle]Cys (SEQ ID NO:31)
[AsnIle]CysSer[IleVal][ProSer]Gly (SEQ ID NO:32)
Trp[ThrAlaSer][ThrAlaSer][PheTyrLeu]LeuLys[AlaSerValIleLeu]XaaLeu
(SEQ ID NO:33) Trp[ThrAlaSer][ThrAlaSer]XaaLeuLysXaaXaaLeuXaaCys
(SEQ ID NO:34) TrpXaa[ThrSer]XaaLeuLysXaaXaaLeuXaaCys (SEQ ID
NO:35) (j) [PheTyr][PheTyr][AsnAsp]GluIleGlnSer (SEQ ID NO:36)
[PheTyr]Pro[PheTyr][PheTyr][PheTyr][AsnAsp]Glu (SEQ ID NO:37) (k)
GlySerAla[ValIleLeu]CysXaa[PheTyr] (SEQ ID NO:38)
SerAla[ValIleLeu]CysXaa[PheTyr]XaaMet (SEQ ID NO:39) (l)
AsnSer[AsnAla]TrpLeu[ProAla]Val (SEQ ID NO:40) (m)
[ValLeuIle]Pro[GluAspTyrSerPhe]ProArgProGly (SEQ ID NO:41)
[ValLeuIle]ProXaaPro[ArgAla]ProGlyXaaCys (SEQ ID NO:42)
Pro[GluAspTyrSerPhe]ProArgProGly[ThrGlnSer]Cys (SEQ ID NO:43) (n)
AspPro[HisPheTyr]Cys[AlaGly]Trp (SEQ ID NO:44)
Pro[HisPheTyr]Cys[AlaGly]TrpAsp (SEQ ID NO:45)
AspProXaaCys[AlaGly]TrpAsp (SEQ ID NO:46)
CysXaaXaaXaaXaaAspProXaaCysXaaTrpAsp (SEQ ID NO:47)
CysXaaXaaXaaAspProXaaCysXaaTrpAsp (SEQ ID NO:48)
CysXaaXaaAspProXaaCysXaaTrpAsp (SEQ ID NO:49)
CysXaaXaaCysXaaXaaXaaXaaAspXaaXaaCysXaaTrpAsp (SEQ ID NO:50)
CysXaaXaaCysXaaXaaXaaAspXaaXaaCysXaaTrpAsp (SEQ ID NO:51)
CysXaaXaaCysXaaXaaAspXaaXaaCysXaaTrpAsp. (SEQ ID NO:52)
[0025] The following peptides represent particularly preferred
members of each class: TABLE-US-00002 (a) AspCysGlnAsnTyrIle (SEQ
ID NO:67) (b) CysGlyThr[AsnGly][AlaSer]XaaXaaPro (SEQ ID NO:68) (c)
GlyXaa[SerCys]ProTyrAspPro (SEQ ID NO:69) (d)
LeuTyrSerGlyThr[ValAsnAla]Ala (SEQ ID NO:70) (e)
LeuAsnAlaProAsnPheVal (SEQ ID NO:71) (f)
[PheTyr]PhePhe[PheTyr]ArgGlu (SEQ ID NO:19) (g)
Glu[PheTyr]IleAsn[CysSer]GlyLys (SEQ ID NO:25) (h)
Arg[ValIle]AlaArgValCysLys (SEQ ID NO:72) (i)
Trp[ThrAla][ThrSer][PheTyr]LeuLys[AlaSer]ArgLeu (SEQ ID NO:73) (j)
ProPheTyrPhe[AsnAsp]GluIleGInSer (SEQ ID NO:74) (k)
GlySerAlaValCysXaa[PheTyr] (SEQ ID NO:75) (l)
AsnSerAsnTrpLeu[ProAla]Val (SEQ ID NO:76) (m)
Pro[GluAsp]ProArgProGly[ThrGlnSer]Cys (SEQ ID NO:77) (n)
AspProTyrCys[AlaGly]TrpAsp. (SEQ ID NO:78)
[0026] The following 14 classes are preferred peptides which
exclude semaphorin peptides encoded in open reading frames of
Variola major or Vaccinia viruses. TABLE-US-00003 (a)
[AspGlu]Cys[GlnLysArgAlaAsn]Asn[TyrPheVal]Ile (SEQ ID NO:01)
Cys[GlnLysArgAlaAsn]Asn[TyrPheVal]Ile[ArgLysGlnThr] (SEQ ID NO:02)
(b) CysGlyThr[AsnGly][AlaSer][TyrPheHisGly][LysArgHisAsnGln] (SEQ
ID NO:79) CysGlyThr[AsnGly][AlaSerAsn][TyrPheHis][LysArgHisAsnGln]
(SEQ ID NO:80) CysGlyThr[AsnGly][AlaSer]XaaXaaPro (SEQ ID NO:81)
(c) [ArgIleGlnVal][GlyAla][LeuValLys][CysSer]Pro[PheTyr][AspAsn]
(SEQ ID NO:07)
[CysSer]Pro[PheTyr][AspAsn]Pro[AspGluArgLys][HisLeuAsp] (SEQ ID
NO:08) GlyXaa[GlyAla]Xaa[CysSer]ProTyr[AspAsn]Pro (SEQ ID NO:09)
(d) Leu[PheTyr]Ser[GlyAla]Thr[ValAsnAla]Ala (SEQ ID NO:10)
Leu[PheTyr]SerXaaThrXaaAla[AspGlu][PheTyr] (SEQ ID NO:11)
[PheTyr]Ser[GlyAla]Thr[ValAsnAla]Ala[AspGlu][PheTyr] (SEQ ID NO:12)
(e) Leu[AsnAsp][AlaLys]ProAsnPheVal (SEQ ID NO:13) (f)
PhePhePheArgGlu (SEQ ID NO:14) PhePhe[PheTyr]ArgGlu[ThrAsn] (SEQ ID
NO:15) PhePheArgGlu[ThrAsn]Ala (SEQ ID NO:16)
Phe[PheTyr]ArgGlu[ThrAsn]Ala (SEQ ID NO:17) TyrPhePhe[PheTyr]ArgGlu
(SEQ ID NO:18) [PheTyr]PhePhe[PheTyr]ArgGlu (SEQ ID NO:19)
[PheTyr][PheTyr][PheTyr]ArgGlu[ThrAsn]Ala (SEQ ID NO:20)
[IleVal][PheTyr]Phe[PheTyr][PheTyr]ArgGlu (SEQ ID NO:21)
Asp[LysPheTyr]Val[PheTyr][PheTyrLeu][PheTyrIleLeu][PheTyr] (SEQ ID
NO:22) Asp[LysPheTyr]Val[PheTyr][PheTyrIleLeu][PheTyrIle][PheTyr]
(SEQ ID NO:82)
[ValIle][PheTyr][PheTyrLeu][PheTyrIleLeu]Phe[ArgThr]Xaa[ThrAsn]
(SEQ ID NO:83)
[ValIle][PheTyr][PheTyrIleLeu][PheTyrIle]Phe[ArgThr]Xaa[ThrAsn]
(SEQ ID NO:84)
[ValIle][PheTyr][PheTyrIleLeu][PheTyrIleLeu]PheArgXaa[ThrAsn] (SEQ
ID NO:85)
[ValIle][PheTyr][PheTyrLeu][PheTyrIleLeu][PheTyr][ArgThr][GluAspVal][ThrA-
sn] (SEQ ID NO:86) (g) Glu[PheTyr]IleAsn[CysSer]GlyLys (SEQ ID
NO:25) [PheTyr]IleAsnCysGlyLys[AlaValIle] (SEQ ID NO:26) (h)
Arg[ValIle][AlaGly][ArgGln][ValIle]CysLys (SEQ ID NO:27)
Arg[ValIle]Xaa[ArgGln][ValIle]CysXaaXaaAsp (SEQ ID NO:28)
GlyLys[ValAlaIle]XaaXaaXaaArg[ValAlaIle]XaaXaaXaaCysLys (SEQ ID
NO:29) (i) [ArgLysAsn]Trp[ThrAla][ThrAlaSer][PheTyrLeu]Leu[LysArg]
(SEQ ID NO:87) [PheTyr]Leu[LysArg][AlaSer]ArgLeu[AsnIle]Cys (SEQ ID
NO:31) [AsnIle]CysSer[IleVal][ProSer]Gly (SEQ ID NO:32)
Trp[ThrAla][ThrAlaSer][PheTyrLeu]LeuLys[AlaSerValIleLeu]XaaLeu (SEQ
ID NO:88)
Trp[ThrAlaSer][ThrAlaSer][PheTyrLeu]LeuLys[AlaSerIleLeu]XaaLeu (SEQ
ID NO:89) Trp[ThrAla][ThrAlaSer]XaaLeuLysXaaXaaLeuXaaCys (SEQ ID
NO:90) (j) [PheTyr][PheTyr][AsnAsp]GluIleGlnSer (SEQ ID NO:36)
[PheTyr]Pro[PheTyr][PheTyr][PheTyr][AsnAsp]Glu (SEQ ID NO:37) (k)
GlySerAla[ValIleLeu]CysXaa[PheTyr] (SEQ ID NO:38)
SerAla[ValIle]CysXaa[PheTyr]XaaMet (SEQ ID NO:39) (l)
AsnSer[AsnAla]TrpLeu[ProAla]Val (SEQ ID NO:40) (m)
[ValLeuIle]Pro[GluAspTyrSerPhe]ProArgProGly (SEQ ID NO:41)
[ValLeuIle]ProXaaProArgProGlyXaaCys (SEQ ID NO:91)
Pro[GluAspTyrSerPhe]ProArgProGly[ThrGlnSer]Cys (SEQ ID NO:43) (n)
AspPro[HisPheTyr]Cys[AlaGly]Trp (SEQ ID NO:44)
Pro[HisPheTyr]Cys[AlaGly]TrpAsp (SEQ ID NO:45)
AspProXaaCys[AlaGly]TrpAsp (SEQ ID NO:46)
CysXaaXaaXaaXaaAspProXaaCysXaaTrpAsp (SEQ ID NO:47)
CysXaaXaaXaaAspProXaaCysXaaTrpAsp (SEQ ID NO:48)
CysXaaXaaAspProXaaCysXaaTrpAsp (SEQ ID NO:49)
CysXaaXaaCysXaaXaaXaaXaaAspXaaXaaCysXaaTrpAsp (SEQ ID NO:50)
CysxaaXaaCysXaaXaaXaaAspXaaXaaCysXaaTrpAsp (SEQ ID NO:51)
CysXaaXaaCysXaaXaaAspXaaXaaCysXaaTrpAsp. (SEQ ID NO:52)
[0027] The following 2 classes are preferred peptides which exclude
semaphorin peptides encoded in open reading frames of Variola major
or Vaccinia viruses Grasshopper Semaphorin I. TABLE-US-00004 (f)
TyrPhePhe[PheTyr]ArgGlu (SEQ ID NO:18)
Asp[LysTyr]Val[PheTyr][PheTyrLeu][PheTyrIleLeu][PheTyr] (SEQ ID
NO:92) Asp[LysTyr]Val[PheTyr][PheTyrIleLeu][PheTyrIle][PheTyr] (SEQ
ID NO:93)
[VaIIle]Tyr[PheTyrLeu][PheTyrIleLeu]Phe[ArgThr]Xaa[ThrAsn] (SEQ ID
NO:94) [ValIle]Tyr[PheTyrIleLeu][PheTyrIle]Phe[ArgThr]Xaa[ThrAsn]
(SEQ ID NO:95)
[ValIle]Tyr[PheTyrIleLeu][PheTyrIleLeu]PheArgXaa[ThrAsn] (SEQ ID
NO:96)
Val[PheTyr][PheTyrLeu][PheTyrIleLeu][PheTyr][ArgThr][GluAspVal][ThrAsn]
(SEQ ID NO:97)
Val[PheTyr][PheTyrIleLeu][PheTyrIle][PheTyr][ArgThr][GluAspVal][ThrAsn]
(SEQ ID NO:98)
Val[PheTyr][PheTyrIleLeu][PheTyrIleLeu][PheTyr]Arg[GluAspVal][ThrAsn]
(SEQ ID NO:99) (n) CysXaaXaaXaaAspProXaaCysXaaTrpAsp (SEQ ID NO:48)
CysXaaXaaAspProXaaCysXaaTrpAsp (SEQ ID NO:49)
CysXaaXaaCysXaaXaaXaaAspXaaXaaCysXaaTrpAsp (SEQ ID NO:51)
CysXaaXaaCysXaaXaaAspXaaXaaCysXaaTrpAsp. (SEQ ID NO:52)
[0028] The following 5 classes include peptides which encompass
peptides encoded in open reading frames of Variola major or
Vaccinia viruses. Accordingly, in the event that these viral
peptides are not novel per se, the present invention discloses a
hitherto unforseen and unforseeable utility for these peptides as
immunosuppressants and targets of anti-viral therapy.
TABLE-US-00005 (b)
CysGlyThr[AsnGly][AlaSerAsn][TyrPheHisGly][LysArgHisAsnGln] (SEQ ID
NO:03) CysGlyThr[AsnGly][AlaSerAsn]XaaXaaPro (SEQ ID NO:04)
CysGlyThr[AsnGly]XaaXaaXaaProXaa[CysAsp] (SEQ ID NO:05)
CysGlyThrXaaXaaXaaXaaProXaa[CysAsp]XaaXaa[TyrIle] (SEQ ID NO:06)
(f) Asp[LysPheTyr]Val[PheTyr][PheTyrIleLeu][PheTyrIleLeu][PheTyr]
(SEQ ID NO:22)
[ValIle][PheTyr][PheTyrIleLeu][PheTyrIleLeu]Phe[ArgThr]Xaa[ThrAsn]
(SEQ ID NO:23)
Val[PheTyr][PheTyrIleLeu][PheTyrIleLeu][PheTyr][ArgThr][GluAspVal][ThrAsn-
] (SEQ ID NO:100) (i)
[ArgLysAsn]Trp[ThrAlaSer][ThrAlaSer][PheTyrLeu]Leu[LysArg] (SEQ ID
NO:30)
Trp[ThrAlaSer][ThrAlaSer][PheTyrLeu]LeuLys[AlaSerValIleLeu]XaaLeu
(SEQ ID NO:33) Trp[ThrAlaSer][ThrAlaSer]XaaLeuLysXaaXaaLeuXaaCys
(SEQ ID NO:34) TrpXaa[ThrSer]XaaLeuLysXaaXaaLeuXaaCys (SEQ ID
NO:35) (k) SerAla[VaIIleLeu]CysXaa[PheTyr]XaaMet (SEQ ID NO:39) (m)
[ValLeuIle]ProXaaPro[ArgAla]ProGlyXaaCys. (SEQ ID NO:42)
[0029] The disclosed semaphorin sequence data are used to define a
wide variety of other semaphorin- and semaphorin receptor-specific
binding agents using immunologic. chromatographic or synthetic
methods available to those skilled in the art.
[0030] Of particular significance are peptides comprising unique
portions of semaphorin-specific receptors and polypeptides
comprising a sequence substantially similar to that of a
substantially full-length semaphorin receptor. Using semaphorin
peptides, these receptors are identified by a variety of techniques
known to those skilled in the art where a ligand to the target
receptor is known, including expression cloning as set out in the
exemplification below. For other examples of receptor isolation
with known ligand using expression cloning, see, Staunton et al
(1989) Nature 339, 61; Davis et al (1991) Science 253, 59; Lin et
al (1992) Cell 68, 775; Gearing et al (1989) EMBO 8, 3667; Aruffo
and Seed (1987) PNAS 84, 8573 and references therein. Generally,
COS cells are transfected to express a cDNA library or PCR product
and cells producing peptides/polypeptides which bind a
semaphorin/receptor peptide/polypeptide are isolated. For
neurosemaphorin receptors, fetal brain cDNA libraries are
preferred; for immunosemaphorin receptors, libraries derived from
activated lymphoid or myeloid cell lines or tissue derived from
sites of inflammation or delayed-type hypersensitivity are
preferred; and for semaphorin and semaphorin receptor variants used
by tumor cells to evade immune surveillance or suppress an immune
response (oncosemaphorins), libraries derived from cancerous tissue
or tumor cell lines resistant to the host immune system are
preferred. Alternatively, PCR primers based upon known
semaphorin/receptor sequences such as those disclosed herein are
used to amplify PCR product from such tissues/cells. Other
receptor/ligand isolation methods using immobilized ligand or
antibody are known to those skilled in the art.
[0031] Semaphorin receptor peptides with receptor binding
specificity are identified by a variety of ways including having
conserved consensus sequences with other semaphorin receptors, by
crosslinking to ligand or receptor-specific antibody, or
preferably, by screening such peptides for semaphorin binding or
disruption of semaphorin-receptor binding. Methods for identifying
semaphorin receptor peptides with the requisite binding activity
are described herein or otherwise known to those skilled in the
art. By analogous methods, semaphorin receptor peptides are used to
define additional semaphorin peptides with semaphorin binding
specificity, particularly receptor specificity.
[0032] The various semaphorin and semaphorin receptor peptides are
used to define functional domains of semaphorins, identify
compounds that associate with semaphorins, design compounds capable
of modulating semaphorin-mediated nerve and immune cell function,
and define additional semaphorin and semaphorin receptor-specific
binding agents. For example, semaphorin mutants, including deletion
mutants are generated from the disclosed semaphorin sequences and
used to identify regions important for specific protein-ligand or
protein-protein interactions, for example, by assaying for the
ability to mediate repulsion or preclude aggregation in cell-based
assays as described herein. Further. x-ray crystallographic data of
the disclosed protein are used to rationally design binding
molecules of determined structure or complementarity for modulating
growth cone growth and guidance.
[0033] Additional semaphorin- and receptor-specific agents include
specific antibodies that can be modified to a monovalent form, such
as Fab, Fab', or Fv, specifically binding oligopeptides or
oligonucleotides and most preferably, small molecular weight
organic receptor antagonists. For example, the disclosed semaphorin
and receptor peptides are used as immunogens to generate
semaphorin- and receptor-specific polyclonal or monoclonal
antibodies. See, Harlow and Lane (1988) Antibodies, A Laboratory
Manual, Cold Spring Harbor Laboratory, for general methods.
Anti-idiotypic antibody, especially internal imaging anti-ids are
also prepared using the disclosures herein.
[0034] In addition to semaphorin and semaphorin-receptor derived
polypeptides and peptides, other prospective agents are screened
from large libraries of synthetic or natural compounds. For
example, numerous means are available for random and directed
synthesis of saccharide, peptide, and nucleic acid based compounds.
Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant and animal extracts are available or
readily producible. Additionally, natural and synthetically
produced libraries and compounds are readily modified through
conventional chemical, physical, and biochemical means. See. e.g.
Houghten et al. and Lam et al (1991) Nature 354, 84 and 81,
respectively and Blake and Litzi-Davis (1992), Bioconjugate Chem 3,
510.
[0035] Useful agents are identified with a range of assays
employing a compound comprising the subject peptides or encoding
nucleic acids. A wide variety of in vitro, cell-free binding
assays, especially assays for specific binding to immobilized
compounds comprising semaphorin or semaphorin receptor peptide find
convenient use. While less preferred cell-based assays may be used
to determine specific effects of prospective agents on
semaphorin-receptor binding may be assayed. Optionally, the
intracellular C-terminal domain is substituted with a sequence
encoding a oligopeptide or polypeptide domain that provides a
detectable intracellular signal upon ligand binding different from
the natural receptor.
[0036] Useful intracellular domains include those of the human
insulin receptor and the TCR, especially domains with kinase
activity and domains capable of triggering calcium influx which is
conveniently detected by fluorimetry by preloading the host cells
with Fura-2. More preferred assays involve simple cell-free in
vitro binding of candidate agents to immobilized semaphorin or
receptor peptides, or vice versa. See. e.g. Fodor et al (1991)
Science 251, 767 for light directed parallel synthesis method. Such
assays are amenable to scale-up, high throughput usage suitable for
volume drug screening.
[0037] Useful agents are typically those that bind to a semaphorin
or disrupt the association of a semaphorin with its receptor.
Preferred agents are semaphorin-specific and do not cross react
with other neural or lymphoid cell membrane proteins. Useful agents
may be found within numerous chemical classes, though typically
they are organic compounds; preferably small organic compounds.
Small organic compounds have a molecular weight of more than 150
yet less than about 4,500, preferably less than about 1500, more
preferably, less than about 500. Exemplary classes include
peptides, saccharides, steroids, heterocyclics, polycyclics,
substituted aromatic compounds, and the like.
[0038] Selected agents may be modified to enhance efficacy,
stability, pharmaceutical compatibility, and the like. Structural
identification of an agent may be used to identify, generate, or
screen additional agents. For example, where peptide agents are
identified, they may be modified in a variety of ways as described
above, e.g. to enhance their proteolytic stability. Other methods
of stabilization may include encapsulation, for example, in
liposomes, etc.
[0039] The subject binding agents may be prepared in a variety of
ways known to those skilled in the art. For example, peptides under
about 60 amino acids can be readily synthesized today using
conventional commercially available automatic synthesizers.
Alternatively, DNA sequences may be prepared encoding the desired
peptide and inserted into an appropriate expression vector for
expression in a prokaryotic or eukaryotic host. A wide variety of
expression vectors are available today and may be used in
conventional ways for transformation of a competent host for
expression and isolation. If desired, the open reading frame
encoding the desired peptide may be joined to a signal sequence for
secretion, so as to permit isolation from the culture medium.
Methods for preparing the desired sequence, inserting the sequence
into an expression vector, transforming a competent host, and
growing the host in culture for production of the product may be
found in U.S. Pat. Nos. 4,710,473, 4,711,843 and 4,713,339.
[0040] For therapeutic uses, the compositions and agents disclosed
herein may be administered by any convenient way, preferably
parenterally, conveniently in a pharmaceutically or physiologically
acceptable carrier, e.g., phosphate buffered saline, saline,
deionized water, or the like. Typically, the compositions are added
to a retained physiological fluid such as blood or synovial fluid.
For CNS administration, a variety of techniques are available for
promoting transfer of the therapeutic across the blood brain
barrier including disruption by surgery or injection, drugs which
transciently open adhesion contact between CNS vasculature
endothelial cells, and compounds which fascilitate translocation
through such cells. As examples, many of the disclosed therapeutics
are amenable to directly injected or infused, contained within
implants e.g. osmotic pumps, grafts comprising appropriately
transformed cells. Generally, the amount administered will be
empirically determined, typically in the range of about 10 to 1000
.mu.g/kg of the recipient. For peptide agents, the concentration
will generally be in the range of about 50 to 500 .mu.g/ml in the
dose administered. Other additives may be included, such as
stabilizers, bactericides, etc. These additives will be present in
conventional amounts.
[0041] The invention provides isolated nucleic acid sequences
encoding the disclosed semaphorin and semaphorin receptor peptides
and polypeptides, including sequences substantially identical to
sequences encoding such polypeptides. An "isolated" nucleic acid
sequence is present as other than a naturally occurring chromosome
or transcript in its natural state and typically is removed from at
least some of the nucleotide sequences with which it is normally
associated with on a natural chromosome. A complementary sequence
hybridizes to a unique portion of the disclosed semaphorin sequence
under low stringency conditions, for example, at 50.degree. C. and
SSC (0.9 M saline/0.09 M sodium citrate) and that remains bound
when subject to washing at 55.degree. C. with SSC. Regions of
non-identity of complementary nucleic acids are preferably or in
the case of homologous nucleic acids, a nucleotide chance providing
a redundant codon. A partially pure nucleotide sequence constitutes
at least about 5%, preferably at least about 30%, and more
preferably at least about 90% by weight of total nucleic acid
present in a given fraction.
[0042] Unique portions of the disclosed nucleic acid sequence are
of length sufficient to distinguish previously known nucleic acid
sequences. Thus, a unique portion has a nucleotide sequence at
least long enough to define a novel oligonucleotide. Preferred
nucleic acid portions encode a unique semaphorin peptide. The
nucleic acids of the invention and portions thereof, other than
those used as PCR primers, are usually at least about 60 bp and
usually less than about 60 kb in length. PCR primers are generally
between about 15 and 100 nucleotides in length.
[0043] Nucleotide (cDNA) sequences encoding several full length
semaphorins are disclosed herein. The invention also provides for
the disclosed sequences modified by transitions, transversions,
deletions, insertions, or other modifications such as alternative
splicing and also provides for genomic semaphorin sequences, and
gene flanking sequences, including regulatory sequences; included
are DNA and RNA sequences, sense and antisense. Preferred DNA
sequence portions include portions encoding the preferred amino
acid sequence portions disclosed above. For antisense applications
where the inhibition of semaphorin expression is indicated,
especially useful oligonucleotides are between about 10 and 30
nucleotides in length and include sequences surrounding the
disclosed ATG start site, especially the oligonucleotides defined
by the disclosed sequence beginning about 5 nucleotides before the
start site and ending about 10 nucleotides after the disclosed
start site. Other especially useful semaphorin mutants involve
deletion or substitution modifications of the disclosed cytoplasmic
C-termini of transmembrane semaphorins. Accordingly, semaphorin
mutants with semaphorin binding affinities but with altered
intracellular signal transduction capacities are produced.
[0044] For modified semaphorin-encoding sequences or related
sequences encoding proteins with semaphorin-like functions, there
will generally be substantial sequence identity between at least a
segment thereof and a segment encoding at least a portion of the
disclosed semaphorin sequence, preferably at least about 60%, more
preferably at least 80%, most preferably at least 90% identity.
Homologous segments are particularly within semaphorin
domain-encoding regions and regions encoding protein domains
involved in protein-protein, particularly semaphorin-receptor
interactions and differences within such segments are particularly
conservative substitutions.
[0045] Typically, the invention's semaphorin peptide encoding
polynucleotides are associated with heterologous sequences.
Examples of such heterologous sequences include regulatory
sequences such as promoters, enhancers, response elements, signal
sequences, polyadenylation sequences, etc., introns, 5' and 3'
noncoding regions, etc. Other useful heterologous sequences are
known to those skilled in the art or otherwise disclosed references
cited herein. According to a particular embodiment of the
invention, portions of the semaphorin encoding sequence are spliced
with heterologous sequences to produce soluble, secreted fusion
proteins, using appropriate signal sequences and optionally, a
fusion partner such as .beta.-Gal.
[0046] The disclosed sequences are also used to identify and
isolate other natural semaphorins and analogs. In particular, the
disclosed nucleic acid sequences are used as hybridization probes
under low-stringency or PCR primers, e.g. oligonucleotides encoding
functional semaphorin domains are .sup.32P-labeled and used to
screen .lamda.cDNA libraries at low stringency to identify similar
cDNAs that encode proteins with related functional domains.
Additionally, nucleic acids encoding at least a portion of the
disclosed semaphorin are used to characterize tissue specific
expression of semaphorin as well as changes of expression over
time, particularly during organismal development or cellular
differentiation.
[0047] The semaphorin encoding nucleic acids can be subject to
alternative purification, synthesis, modification, sequencing,
expression, transfection, administration or other use by methods
disclosed in standard manuals such as Molecular Cloning, A
Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold
Spring Harbor), Current Protocols in Molecular Biology (Eds.
Aufubel, Brent, Kingston, More, Feidman, Smith and Stuhl. Greene
Publ. Assoc., Wiley-Interscience, NY, N.Y., 1992) or that are
otherwise known in the art. For example, the nucleic acids can be
modified to alter stability, solubility, binding affinity and
specificity, etc. semaphorin-encoding sequences can be selectively
methylated, etc. The nucleic acid sequences of the present
invention may also be modified with a label capable of providing a
detectable signal, either directly or indirectly. Exemplary labels
include radioisotopes, fluorescers, biotinylation, etc.
[0048] The invention also provides vectors comprising nucleic acids
encoding semaphorin peptides, polypeptides or analogs. A large
number of vectors, including plasmid and viral vectors, have been
described for expression in a variety of eukaryotic and prokaryotic
hosts. Advantageously, vectors may also include a promotor operably
linked to the semaphorin-encoding portion. Vectors will often
include one or more replication systems for cloning or expression,
one or more markers for selection in the host, e.g. antibiotic
resistance. The inserted semaphorin coding sequences may be
synthesized, isolated from natural sources, prepared as hybrids,
etc. Suitable host cells may be transformed/transfected/infected by
any suitable method including electroporation, CaCl.sub.2 mediated
DNA uptake, viral infection, microinjection, microprojectile, or
other methods.
[0049] Appropriate host cells include bacteria, archebacteria,
fungi, especially yeast, and plant and animal cells, especially
mammalian cells. Of particular interest are E. coli. B. subtilis,
Saccharomvces cerevisiae, SF9 cells, C129 cells, 293 cells,
Neurospora, and CHO. COS, HeLa cells, immortalized mammalian
myeloid and lymphoid cell lines, and pluripotent cells, especially
mammalian ES cells and zygotes. Preferred replication systems
include M13, ColE1, SV40, baculovirus, lambda, adenovirus, AAV,
BPV, etc. A large number of transcription initiation and
termination regulatory regions have been isolated and shown to be
effective in the transcription and translation of heterologous
proteins in the various hosts. Examples of these regions, methods
of isolation, manner of manipulation, etc. are known in the art.
Under appropriate expression conditions, host cells can be used as
a source of recombinantly produced semaphorins or analogs.
[0050] For the production of stably transformed cells and
transgenic animals, nucleic acids encoding the disclosed
semaphorins may be integrated into a host genome by recombination
events. For example, such a sequence can be microinjected into a
cell, and thereby effect homologous recombination at the site of an
endogenous gene, an analog or pseudogene thereof, or a sequence
with substantial identity to an semaphorin-encoding gene. Other
recombination-based methods such as nonhomologous recombinations,
deletion of endogenous gene by homologous recombination, especially
in pluripotent cells. etc., provide additional applications.
Preferred transgenics and stable transformants over-express the
disclosed receptor gene and find use in drug development and as a
disease model. Alternatively, knock-out cells and animals find use
in development and functional studies. Methods for making
transgenic animals, usually rodents, from ES cells or zygotes are
known to those skilled in the art.
[0051] The compositions and methods disclosed herein may be used to
effect gene therapy. See, e.g. Zhu et al. (1993) Science 261,
209-211; Gutierrez et al. (1992) Lancet 339, 715-721. For example,
cells are transfected with semaphorin sequences operably linked to
gene regulatory sequences capable of effecting altered semaphorin
expression or regulation. To modulate semaphorin translation, cells
may be transfected with complementary antisense polynucleotides.
For gene therapy involving the transfusion of semaphorin
transfected cells, administration will depend on a number of
variables that are ascertained empirically. For example, the number
of cells will vary depending on the stability of the transfused
cells. Transfusion media is typically a buffered saline solution or
other pharmacologically acceptable solution. Similarly the amount
of other administered compositions, e.g. transfected nucleic acid,
protein, etc., will depend on the manner of administration, purpose
of the therapy, and the like.
[0052] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
I. Isolation and Characterization of Grasshopper Semaphorin I (SEQ
ID NOS: 57 and 58 (Previously Referred to as Fasciclin IV)
[0053] In order to identify cell surface molecules that function in
selective fasciculation, a series of monoclonal antibody (MAb)
screens was conducted. The immunogen used for most of these screens
was membranes from the longitudinal connectives (the collection of
longitudinal axons) between adjacent segmental ganglia of the
nervous system of the larval grasshopper. From these screens, MAb
3B11 and 8C6 were used to purify and characterize two surface
glycoproteins, fasciclin I and fasciclin II, see, Bastiani et al.,
1987, the genes encoding both were subsequently cloned, see, Snow
et al. 1989, Zinn et al. 1988. and Harrelson and Goodman, 1988.
[0054] Another MAb isolated during these screens, MAb 6F8, was
chosen for the present study because, just as with fasciclin I and
fasciclin II, the antigen recognized by this MAb is expressed on a
different but overlapping subset of axon pathways in the developing
CNS. The 6F8 antigen appears to be localized on the outside of cell
surfaces, as indicated by MAb binding when incubated both in live
preparations, and in fixed preparations in which no detergents have
been added. Because the 6F8 antigen is a surface glycoprotein
expressed on a subset of axon fascicles (see below), we call it
fasciclin IV.
[0055] Fasciclin IV expression begins early in embryonic
development before axonogenesis. At 29% of development, expression
is seen on the surface of the midline mesectodermal cells and
around 5-7 neuroblasts and associated ectodermal cells per
hemisegment. This expression is reminiscent of the mesectodermal
and neuroblast-associated expression observed with both fasciclin I
and fasciclin II; however, in each case, the pattern resolves into
a different subset of neuroblasts and associated ectodermal
cells.
[0056] At 32% of development, shortly after the onset of
axonogenesis in the CNS, fasciclin IV expression is seen on the
surface of the axons and cell bodies of the three pairs of MP4,
MP5, and MP6 midline progeny, the three U motoneurons, and on
several unidentified neurons in close proximity to the U's. This is
in contrast to fasciclin II, which at this stage is expressed on
the MP1 and dMP2 neurons, and fasciclin I, which is expressed on
the U neurons but not on any midline precursor progeny.
[0057] The expression of fasciclin IV on a subset of axon pathways
is best observed around 40% of development, after the establishment
of the first longitudinal and commissural axon pathways. At this
stage, the protein is expressed on two longitudinal axon fascicles,
a subset of commissural axon fascicles, a tract extending
anteriorly along the midline, and a subset of fascicles in the
segmental nerve (SN) and intersegmental nerve (ISN) roots.
[0058] Specifically, fasciclin IV is expressed on the U fascicle, a
longitudinal pathway (between adjacent segmental neuromeres)
pioneered in part by the U neurons, and on the A/P longitudinal
fascicle (in part an extension of the U fascicle within each
segmental neuromere). In addition, fasciclin IV is also expressed
on a second narrower, medial. and more ventral longitudinal
pathway. The U axons turn and exit the CNS as they pioneer the ISN;
the U's and many other axons within the ISN express fasciclin IV.
The continuation of the U fascicle posterior to the ISN junction is
also fasciclin IV-positive. The specificity of fasciclin IV for
distinct subsets of longitudinal pathways can be seen by comparing
fasciclin IV and fasciclin II expression in the same embryo;
fasciclin IV is expressed on the U and A/P pathways whereas
fasciclin II is expressed on the MP1 pathway.
[0059] The axons in the median fiber tract (MFT) also express
fasciclin IV. The MFT is pioneered by the three pairs of progeny of
the midline precursors MP4, MP5, and MP6. The MFT actually contains
three separate fascicles. The axons of the two MP4 progeny pioneer
the dorsal MFT fascicle and then bifurcate at the posterior end of
the anterior commissure: whereas the axons of the two MP6 progeny
pioneer the ventral MFT fascicle and then bifurcate at the anterior
end of the posterior commissure. Fasciclin IV is expressed on the
cell bodies of the six MP4, MP5, and MP6 neurons, and on their
growth cones and axons as they extend anteriorly in the MFT and
bifurcate in one of the two commissures. However, this expression
is regional in that once these axons bifurcate and begin to extend
laterally across the longitudinal pathways and towards the
peripheral nerve roots, their expression of fasciclin IV greatly
decreases. Thus, fasciclin IV is a label for the axons in the MFT
and their initial bifurcations in both the anterior and posterior
commissures. It appears to be expressed on other commissural
fascicles as well. However, the commissural expression of fasciclin
IV is distinct from the transient expression of fasciclin II along
the posterior edge of the posterior commissure, or the expression
of fasciclin I on several different commissural axon fascicles in
both the anterior and posterior commissure (Bastiani et al., 1987;
Harrelson and Goodman. 1988).
[0060] Fasciclin IV is also expressed on a subset of motor axons
exiting the CNS in the SN. The SN splits into two major branches,
one anterior and the other posterior, as it exits the CNS. Two
large bundles of motoneuron axons in the anterior branch express
fasciclin IV at high levels; one narrow bundle of motoneuron axons
in the posterior branch expresses the protein at much lower levels.
Fasciclin IV is also expressed on many of the axons in the ISN.
[0061] The CNS and nerve root expression patterns of fasciclin IV,
fasciclin I, and fasciclin II at around 40% of embryonic
development are summarized below. Although there is some overlap in
their patterns (e.g., both fasciclin IV and fasciclin I label the U
axons), these three surface glycoproteins label distinct subsets of
axon pathways in the developing CNS.
Fasciclin IV is Expressed on Epithelial Bands in the Developing
Limb Bud
[0062] Fasciclin IV is expressed on the developing limb bud
epithelium in circumferential bands; at 34.5% of development these
bands can be localized with respect to constrictions in the
epithelium that mark presumptive segment boundaries. In addition to
a band just distal to the trochanter/coxa segment boundary, bands
are also found in the tibia, femur, coxa, and later in development
a fifth band is found in the tarsus. Fasciclin IV is also expressed
in the nascent chordotonal organ in the dorsal aspect of the femur.
The bands in the tibia, trochanter, and coxa completely encircle
the limb. However, the femoral band is incomplete, containing a gap
on the anterior epithelia of this segment.
[0063] The position of the Ti1 axon pathway with respect to these
bands of fasciclin IV-positive epithelia suggests a potential role
for fasciclin IV in guiding the Ti1 growth cones. First, the band
of fasciclin IV expression in the trochanter, which is
approximately three epithelial cell diameters in width when
encountered by the Ti1 growth cones, is the axial location where
the growth cones reorient from proximal migration to
circumferential branch extension. The Tr1 cell, which marks the
location of the turn, lies within this band, usually over the
central or the proximal cell tier. Secondly, although there is a
more distal fasciclin IV expressing band in the femur, where a
change in Ti1 growth is not observed, there exists a gap in this
band such that fasciclin IV expressing cells are not traversed by
the Ti1 growth cones. The Ti1 axons also may encounter a fasciclin
IV expressing region within the coxa. where interactions between
the growth cones, the epithelial cells, and the Cx1 guidepost cells
have not yet been investigated. In addition to its expression over
the surface of bands of epithelial cells, fasciclin IV protein, as
visualized with MAb 6F8, is also found on the basal surface of
these cells in a punctate pattern. This punctate staining is not an
artifact of the HRP immunocytochemistry since fluorescent
visualization of MAb 6F8 is also punctate. The non-neuronal
expression of fasciclin IV is not restricted to limb buds.
Circumferential epithelial bands of fasciclin IV expression are
also seen on subesophageal mandibular structures and on the
developing antennae.
MAb Directed Against Fasciclin IV can Alter the Formation of the
Ti1 Axon Pathway in the Limb Bud
[0064] The expression of fasciclin IV on an epithelial band at a
key choice point in the formation of the Ti1 axon pathway led us to
ask whether this protein is involved in growth cone guidance at
this location. To answer this question, we cultured embryos, or
epithelial fillets (e. g., O'Connor et al., 1990), during the 5% of
development necessary for normal pathway formation, either in the
presence or absence of MAb 6F8 or 6F8 Fab fragments. Under the
culture conditions used for these experiments, defective Ti1
pathways are observed in 14% of limbs (Chang et al., 1992); this
defines the baseline of abnormalities observed using these
conditions. For controls we used other MAbs and their Fab fragments
that either bind to the surfaces of these neurons and epithelial
cells (MAb 3B11 against the surface protein fasciclin I) or do not
(MAb 4D9 against the nuclear protein engrailed: Patel et al.,
1989). To assess the impact of MAb 6F8 on Ti1 pathway formation, we
compared the percentage of aberrant pathways observed following
treatment with MAb 6F8 to that observed with MAbs 3B11 and 4D9. Our
cultures began at 32% of development when the Ti1 growth cones have
not yet reached the epithelium just distal to the trochanter/coxa
boundary and therefore have not encountered epithelial cells
expressing fasciclin IV. Following approximately 30 hours in
culture (.about.4% of development), embryos were fixed and
immunostained with antibodies to HRP in order to visualize the Ti1
axons and other neurons in the limb bud. Criteria for scoring the
Ti1 pathway, and the definition of "aberrant", are described in
detail in the Experimental Procedures.
[0065] Although MAb 6F8 does not arrest pathway formation, several
types of distinctive, abnormal pathways are observed. These defects
generally begin where growth cones first contact the fasciclin IV
expressing cells in the trochanter. Normally, the Ti1 neurons each
have a single axon, and the axons of the two cells are fasciculated
in that portion of the pathway within the trochanter. Following
treatment with MAb 6F8, multiple long axon branches are observed
within, and proximal to, the trochanter. Two major classes of
pathways are taken by these branches; in 36% of aberrant limbs,
multiple, long axon branches extend ventrally in the region distal
to the Cx1 cells which contains the band of fasciclin IV expressing
epithelial cells. In the ventral region of the trochanter, these
branches often independently turn proximally to contact the Cx1
cells, and thus complete the pathway in this region.
[0066] In the second major class of pathway defect, seen in 47% of
aberrant limbs, axon branches leave the trochanter at abnormal,
dorsal locations, and extend proximally across the trochanter/coxa
boundary. These axons then veer ventrally, often contacting the Cx1
neurons. The remaining 17% of defects include defasciculation
distal to the trochanter, axon branches that fail to turn
proximally in the ventral trochanter and continue into the
posteirior compartment of the limb, and axon branches which cross
the trochanter/coxa boundary and continue to extend proximally
without a ventral turn.
[0067] When cultured in the presence of MAb 6F8, 43% of limbs
exhibited malformed Ti1 pathways (n=381) as compared to 11% with
MAb 3B11 (n=230) and 5% with MAb 4D9 (n=20). These percentages are
pooled from treatments with MAbs concentrated from hybridoma
supernatant, IgGs isolated from these supernatants, and Fab
fragments isolated from these IgG preparations (see Experimental
Procedures). The frequency of malformed Ti1 pathways and the types
of defects observed showed no significant variation regardless of
the method of antibody preparation or type of antibody used. Since
Fabs show similar results as IgGs, the effects of MAb 6F8 are not
due to cross linking by the bivalent IgG.
[0068] In summary, following treatment with MAb 6F8, the Ti1
pathway typically exhibits abnormal morphology beginning just
distal to the trochanter and at the site of fasciclin IV
expression. The two most common types of Ti1 pathway defects
described above occur in 36% of experimental limbs (treated with
MAb 6F8), but are seen in only 4% of control limbs (treated with
MAbs 3B11 and 4D9).
Fasciclin IV cDNAs Encode a Novel Integral Membrane Protein
[0069] Grasshopper fasciclin IV was purified by passing crude
embryonic grasshopper lysates over a MAb 6F8 column. After affinity
purification, the protein was eluted, precipitated, denatured,
modified at cysteines, and digested with either trypsin or Lys-C.
Individual peptides were resolved by reverse phase HPLC and
microsequenced using standard methods.
[0070] The amino acid sequences derived from these proteolytic
fragments were used to generate oligonucleotide probes for PCR
experiments, resulting in products that were used to isolate cDNA
clones from the Zinn embryonic grasshopper cDNA library (Snow et
al., 1988). Sequence analysis of these cDNAs reveals a single open
reading frame (ORF) encoding a protein with two potential
hydrophobic stretches of amino acids: an amino-terminal signal
sequence of 20 residues and (beginning at amino acid 627) a
potential transmembrane domain of 25 amino acids. Thus, the deduced
protein has an extracellular domain of 605 amino acids, a
transmembrane domain, and a cytoplasmic domain of 78 amino acids.
The calculated molecular mass of the mature fasciclin IV protein is
80 kd and is confirmed by Western blot analysis of the affinity
purified and endogenous protein as described below. The
extracellular domain of the protein includes 16 cysteine residues
that fall into three loose clusters but do not constitute a
repeated domain and are not similar to other known motifs with
cysteine repeats. There are also six potential sites for N-linked
glycosylation in the extracellular domain. Treatment of affinity
purified fasciclin IV with N-Glycanase demonstrates that fasciclin
IV does indeed contain N-linked oligosaccharides. Fasciclin IV
shows no sequence similarity when compared with other proteins in
the PIR data base using BLASTP (Altschul et al., 1990), and is
therefore a novel type I integral membrane protein.
[0071] A polyclonal antiserum directed against the cytoplasmic
domain of the protein encoded by the fasciclin IV cDNA was used to
stain grasshopper embryos at 40% of development. The observed
staining pattern was identical to that seen with MAb 6F8. On
Western blots, this antiserum recognizes the protein we affinity
purified using MAb 6F8 and then subjected to microsequence
analysis. Additionally, the polyclonal serum recognizes a protein
of similar molecular mass from grasshopper embryonic membranes.
Taken together these data indicate that the sequence we have
obtained is indeed fasciclin IV.
[0072] Four other cell surface proteins that label subsets of axon
pathways in the insect nervous system (fasciclin I, fasciclin II,
fasciclin III, and neuroglian) are capable of mediating homophilic
cell adhesion when transfected into S2 cells in vitro (Snow et al.,
1989: Elkins et al., 1990b; Grenningloh et al., 1990). To ask
whether fasciclin IV can function as a homophilic cell adhesion
molecule, the fasciclin IV cDNA with the complete ORF was placed
under the control of the inducible metallothionein promoter (Bunch
et al., 1988). transfected into S2 cells, and assayed for its
ability to promote adhesion in normally non-adhesive S2 cells.
Following induction with copper, fasciclin IV was synthesized in
these S2 cells as shown by Western blot analysis and cell surface
staining of induced S2 cells with the polyclonal antiserum
described above.
[0073] We observed no evidence for aggregation upon induction of
fasciclin IV expression, thus suggesting that, in contrast to the
other four proteins, fasciclin IV does not function as a homophilic
cell adhesion molecule. Alternatively, fasciclin IV-mediated
aggregation might require some further posttranslational
modification, or co-factor, not supplied by the S2 cells, but
clearly this protein acts differently in the S2 cell assay than the
other four axonal glycoproteins previously tested. This is
consistent with the pattern of fasciclin IV expression in the
embryonic limb since only the epithelial cells and not the Ti1
growth cones express fasciclin IV, and yet antibody blocking
experiments indicate that fasciclin IV functions in the epithelial
guidance of these growth cones. Such results suggest that fasciclin
IV functions in a heterophilic adhesion or signaling system.
Discussion
[0074] Fasciclin IV is expressed on groups of axons that
fasciculate in the CNS,. suggesting that, much like other insect
axonal glycoproteins, it functions as a homophilic cell adhesion
molecule binding these axons together. Yet, in the limb bud,
fasciclin IV is expressed on a band of epithelium but not on the
growth cones that reorient along this band, suggesting a
heterophilic function. That fasciclin IV functions in a
heterophilic rather than homophilic fashion is supported by the
lack of homophilic adhesion in S2 cell aggregation assays. In
contrast, fasciclin I, fasciclin II, fasciclin III, and neuroglian
all can function as homophilic cell adhesion molecules (Snow et
al., 1989; Elkins et al., 1990b; Grenningloh et al., 1990).
[0075] cDNA sequence analysis indicates that fasciclin IV is an
integral membrane protein with a novel sequence not related to any
protein in the present data base. Thus, fasciclin IV represents a
new type of protein that functions in the epithelial guidance of
pioneer growth cones in the developing limb bud. Given its
expression on a subset of axon pathways in the developing CNS,
fasciclin IV functions in the guidance of CNS growth cones as
well.
[0076] The results from the MAb blocking experiments illuminate
several issues in Ti1 growth cone guidance and axon morphogenesis
in the limb. First, the most striking change in growth cone
behavior in the limb is the cessation of proximal growth and
initiation of circumferential extension of processes upon
encountering the trochanter/coxa boundary region (Bentley and
Caudy, 1983; Caudy and Bentley, 1987). This could be because the
band of epithelial cells within the trochanter promotes
circumferential growth, or because the cells comprising the
trochanter/coxa boundary and the region just proximal to it are
non-permissive or aversive for growth cone migration, or both. The
extension of many axon branches across the trochanter/coxa boundary
following treatment with MAb 6F8 suggests that the trochanter/coxa
boundary cells, which do not express fasciclin IV, are not aversive
or non-permissive. Thus the change in behavior at the boundary
appears to be due to the ability of fasciclin IV expressing
epithelial cells to promote circumferential extension of processes
from the Ti1 growth cones.
[0077] Secondly, treatment with MAb 6F8 results in frequent
defasciculation of the axons of the two Ti1 neurons, and also
formation of abnormal multiple axon branches, within the trochanter
over fasciclin IV-expressing epithelial cells. Previous studies
have shown that treatment with antibodies against ligands expressed
on non-neural substrates (Landmesser et al., 1988), or putative
competitive inhibitors of substrate ligands (Wang and Denburg,
1992) can promote defasciculation and increased axonal branching.
Our results suggest that Ti1 axon:axon fasciculation and axon
branching also are strongly influenced by interactions with
substrate ligands, and that fasciclin IV appears to be a component
of this interaction within the trochanter.
[0078] Thirdly, despite the effects of MAb 6F8 on axon branching,
and on crossing the trochanter/coxa boundary, there remains a
pronounced tendency for branches to grow ventrally both within the
trochanter and within the distal region of the coxa. Consequently,
all signals which can promote ventral migration of the growth cones
have not been blocked by MAb 6F8 treatment. Antibody treatment may
have a threshold effect in which ventral growth directing
properties of fasciclin IV are more robust, and less incapacitated
by treatment, than other features; alternatively, guidance
information promoting ventral migration may be independent of
fasciclin IV. Time lapse video experiments to determine how the
abnormal pathways we observe actually form can resolve these
issues.
[0079] These results demonstrate that fasciclin IV functions as a
guidance cue for the Ti1 growth cones just distal to the
trochanter/coxa boundary, is required for these growth cones to
stop proximal growth and spread circumferentially, and that the
function of fasciclin IV in Ti1 pathway formation result from
interactions between a receptor/ligand on the Ti1 growth cones and
fasciclin IV on the surface of the band of epithelial cells results
in changes in growth cone morphology and subsequent reorientation.
Fasciclin IV appears to elicit this change in growth cone
morphology and orientation via regulation of adhesion, a signal
transduction function, or a combination of the two.
Experimental Procedures
Immunocytochemistry
[0080] Grasshopper embryos were obtained from a colony maintained
at the U. C. Berkeley and staged by percentage of total embryonic
development (Bentley et al., 1979). Embryos were dissected in PBS,
fixed for 40 min in PEM-FA [0.1 M PIPES (pH 6.95), 2.0 mM EGTA. 1.0
mM MgSO.sub.4, 3.7% formaldehyde], washed for 1 hr with three
changes in PBT (1.times.PBS. 0.5% Triton X-100, 0.2% BSA), blocked
for 30 min in PBT with 5% normal goat serum, and incubated
overnight at 4.degree. C. in primary antibody. PBSap (1.times.PBS,
0.1% Saponin, 0.2% BSA) was used in place of PBT with MAb 8G7.
Antibody dilutions were as follows: MAb 6F8 1:1. polyclonal
antisera directed against a fasciclin IV bacterial fusion protein
(#98-3) 1:400: MAb 8G7 1:4; MAb 8C6 1:1. The embryos were washed
for one hour in PBT with three changes, blocked for 30 min, and
incubated in secondary antibody for at least 2 hr at room
temperature. The secondary antibodies were HRP-conjugated goat
anti-mouse and anti-rat IgG (Jackson Immunoresearch Lab), and were
diluted 1:300. Embryos were washed in PBT for one hour with three
changes and then reacted in 0.5% diaminobenzidine (DAB) in PBT. The
reaction was stopped with several washes in PBS and the embryos
were cleared in a glycerol series (50%, 70%, 90%), mounted and
viewed under Nomarski or bright field optics. For double-labelled
preparations the first HRP reaction was done in PBT containing
0.06% NiCl, followed by washing, blocking, and incubation overnight
in the second primary antibody. The second antibody was visualized
with a DAB reaction as described above. Embryos cultured in the
presence of monoclonal antibodies were fixed and incubated
overnight in goat anti-HRP (Jackson Immunoresearch Labs) conjugated
to RITC (Molecular Probes), washed for one hour in PBT with three
changes, mounted in 90% glycerol, 2.5% DABCO (Polysciences), and
viewed under epifluorescence. S2 cells were stained with polyclonal
sera #98-3 diluted 1:400 and processed as described previously
(Snow et al., 1989).
Monoclonal Antibody Blocking Experiments
[0081] In order to test for functional blocking, monoclonal
antibody reagents were prepared as follows. Hybridoma supernatant
was brought to 20% with H.sub.2O-saturated (NH.sub.4),SO.sub.4,
incubated in ice 1 hr, and spun at 15,000 g at 4.degree. C. for 20
min. The supernatant was brought to 56% with H.sub.2O-saturated
(NH.sub.4).sub.2SO.sub.4, incubated overnight at 4.degree. C., spun
as above. The pellet was resuspended in PBS using approximately
1/40 volume of the original hybridoma supernatant (often remaining
a slurry) and dialyzed against 1.times.PBS overnight at 4.degree.
C. with two changes. This reagent is referred to as "concentrated
hybridoma supernatant." Purified IgG was obtained by using
Immunopure Plus Immobilized Protein A IgG Purification Kit (Pierce)
to isolate IgG from the concentrated hybridoma supernatant. Fab
fragments were obtained using the Immunopure Fab Preparation Kit
(Pierce) from the previously isolated IgGs. For blocking
experiments each reagent was diluted into freshly made supplemented
RPMI culture media (O'Connor et al., 1990) and dialyzed overnight
at 4.degree. C. against 10 volumes of the same culture media.
Dilutions were as follows: concentrated hybridoma supernatant 1:4;
purified IgG 150 mg/ml; Fab 75 mg/ml.
[0082] Embryos for culture experiments were carefully staged to
between 31 and 32% of development. As embryos in each clutch
typically differ by less that 1% of embryonic development from each
other, the growth cones of the Ti1 neurons at the beginning of the
culture period were located approximately in the mid-femur, well
distal to the trochanter/coxa segment boundary. From each clutch at
least two limbs were filleted and the Ti1 neurons labelled with the
lipophillic dye Di I (Molecular Probes) as described (O'Connor et
al., 1990) in order to confirm the precise location of the Ti1
growth cones. Prior to culturing, embryos were sterilized and
dissected (Chang et al., 1992). The entire amnion and dorsal
membrane was removed from the embryo to insure access of the
reagents during culturing. Embryos were randomly divided into
groups and cultured in one of the blocking reagents described
above. Cultures were incubated with occasional agitation at
30.degree. C. for 30 hrs. At the end of the culture period embryos
were fixed and processed for analysis as described above in
immunocytochemistry.
[0083] For each culture experiment, the scoring of the Ti1 pathway
in each limb was confirmed independently by a second observer.
There was no statistically significant variation between the two
observers. Limbs from MAb cultured embryos were compared to
representative normal limbs from non-MAb cultured embryos and were
scored as abnormal if any major deviation from the normal Ti1
pathway was observed. The Ti1 pathway was scored as abnormal for
one or more of the following observed characteristics: (1)
defasciculation for a minimum distance of approximately 25 mm
anywhere along the pathway, (2) multiple axon branches that
extended ventrally within the trochanter, (3) presence of one or
more axon branches that crossed the trochanter/coxa boundary dorsal
to the Cx1 cells, but then turned ventrally in the coxa and
contacted the Cx1 cells, (4) the presence of axon branches that
crossed the trochanter/coxa segment boundary, did not turn
ventrally, but continued proximally toward the CNS, and (5) failure
of ventrally extended axons within the trochanter to contact and
reorient proximally to the Cx1 cells. For each MAb tested, the data
are presented as a percentage of the abnormal Ti1 pathways
observed.
Protein Affinity Purification and Microsequencing
[0084] Grasshopper fasciclin IV was purified by passing crude
embryonic grasshopper lysate (Bastiani et al., 1987) over an
Affi-Gel 15 column (Bio Rad) conjugated with the monoclonal
antibody 6F8. Protein was eluted with 50 mM DEA (pH 11.5), 0.1%
Lauryldimethylamine oxide (Cal Bio Chem), and 1 mM EDTA. Protein
was then precipitated, denatured, modified at cysteines, and
digested with either trypsin or Lys-C (Boehringer-Mannheim).
Individual peptides were resolved by RP-HPLC and microsequenced
(Applied Biosystems 4771 Microsequencer) using standard
chemistry.
PCR Methods
[0085] DNA complementary to poly(A)+RNA from 45%-50% grasshopper
embryos was prepared (Sambrook et al., 1989). PCR was performed
using Perkin Elmer Taq polymerase (Saiki et al., 1988), and
partially degenerate (based on grasshopper codon bias)
oligonucleotides in both orientations corresponding to a portion of
the protein sequence of several fasciclin IV peptides as determined
by microsequencing. These oligonucleotides were designed so as not
to include all of the peptide-derived DNA sequence, leaving a
remaining 9-12 base pairs that could be used to confirm the correct
identity of amplified products. All possible combinations of these
sequences were tried. 40 cycles were performed, the parameters of
each cycle as follows: 96.degree. for one min; a sequentially
decreasing annealing temperature (2.degree. C./cycle, starting at
65.degree. C. and ending at 55.degree. C. for remaining 35 cycles)
for 1 min; and at 72.degree. C. for one min. Reaction products were
cloned into the Sma site of M13 mp 10 and sequenced. Two products,
1074 bp and 288 bp in length, contained DNA 3' to the
oligonucleotide sequences encoded the additional amino acid
sequence of the fasciclin IV peptide from which the oligonuceotides
were derived.
cDNA Isolation and Sequence Analysis
[0086] Both PCR products were used to screen 1.times.10.sup.6
clones from a grasshopper embryonic cDNA library (Snow et al.,
1988). 21 clones that hybridized to both fragments were recovered,
and one 2600 bp clone was sequenced using the dideoxy chain
termination method (Sanger et al., 1977) and Sequenase (US
Biochemical Corp.). Templates were made from M13 mp 10 vectors
containing inserts generated by sonication of plasmid clones. One
cDNA was completely sequenced on both strands using
Oligonucleotides and double strand sequencing of plasmid DNA
(Sambrook et al., 1989) to fill gaps. Two additional cDNAs were
analyzed by double strand sequencing to obtain the 3' 402 bp of the
transcript. All three cDNAs were used to construct a plasmid
containing the entire transcript. The complete transcript sequence
is 2860 bp in length with 452 bp of 5' and 217 bp of 3'
untranslated sequences containing stop codons in all reading
frames. The predicted protein sequence was analyzed using the
FASTDB and BLASTP programs (Intelligenetics). The fasciclin IV ORF
unambiguously contains 10 of the 11 peptide sequences determined by
microsequencing the fasciclin IV trypsin and Lys-C peptides.
Generation of Polyclonal Antibodies from Bacterial Fusion
Proteins
[0087] Bacterial trpE fusion proteins were constructed using pATH
(Koerner et al., 1991) vectors, three restriction fragments
encoding extracellular sequences, and one fragment (770 bp
HindII/Eco R1, which includes amino acids 476-730) encoding both
extracellular and intracellular sequences (designated #98-3).
Fusion proteins were isolated by making an extract of purified
inclusion bodies (Spindler et al., 1984), and rats were immunized
with .about.70 mg of protein emulsified in RIBI adjuvant
(Immunochem Research). Rats were injected at two week intervals and
serum was collected 7 days following each injection. Sera were
tested histologically on grasshopper embryos at 45% of development.
Construct #98-3 showed a strong response and exhibited a staining
pattern identical to that of MAb 6F8. Two of the extracellular
constructs responded weakly but also showed the fasciclin IV
staining pattern. All pre-immune sera failed to stain grasshopper
embryos.
S2 Cell Transfections, Aggregation Assays, and Western Analysis
[0088] A restriction fragment containing the full length fasciclin
IV cDNA was cloned into pRmHa-3 (Bunch et al, 1988) and
co-transformed into Drosophila S2 cells (Schneider, 1972) with the
plasmid pPC4 (Jokerst et al., 1989), which confers a-amanitin
resistance. S2 cells were transformed using the Lipofectin Reagent
and recommended protocol (BRL) with minor modifications. All other
S2 cell manipulations are essentially as described (Snow et al.,
1989), including adhesion assays. Fasciclin IV expression in
transformed cell lines was induced for adhesion assays and
histology by adding CuSO.sub.4 to 0.7 mM and incubating for at
least 48 hrs. Northern analysis confirmed transcription of
fasciclin IV and surface-associated staining of the S2 cells with
polyclonal serum #98-3 strongly suggests fasciclin IV is being
transported to the cell surface. Preparation of membranes from S2
cells and from grasshopper embryos, PAGE, and Western blot were
performed as previously described (Elkins et al., 1990b) except
that signal was detected using the enhanced chemiluminescence
immunodetection system kit (Amersham). Amount of protein per lane
in each sample loaded: fasciclin IV protein, .about.5 ng; S2 cell
membranes, 40 mg; grasshopper membranes 80 mg. Amounts of protein
loaded were verified by Ponceau S staining of the blot prior to
incubation with the antibody.
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P. M., Bieber, A. J., and Goodman, C. S. (1989). Fasciclin III: a
novel homophilic adhesion molecule in Drosophila. Cell. 59,
313-323. [0124] Snow, P. M., Zinn, K., Harrelson, A. L.,
McAllister, L., Schilling, J., Bastiani. M. J., Makk, G., and
Goodman, C. S. (1988). Characterization and cloning of fasciclin I
and fasciclin II glycoproteins in the grasshopper. Proc. Natl.
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cultured embryos of the cockroach. (1992). Neuron. 8, 701-714.
[0127] Wang, L. S., Feng, Y., and Denburg, J. L. (1992). A
multifunctional cell surface developmental stage-specific antigen
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and Goodman, C. S. (1988). Sequence analysis and neuronal
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577-587. [0129] Genbank Accession Number: [0130] The accession
number for the sequence reported in this paper is L00709.
II. Isolation and Characterization of Tribolium (SEQ ID NOS:63 and
64) and Drosophila (SEQ ID NOS:59 and 60) Semaphorin I, Drosophila
Semaphorin II (SEQ ID NOS:61 and 62), Human Semaphorin III (SEQ ID
NOS:53 and 54) and Vaccinia Virus Semaphorin IV (SEQ ID NOS:55 and
56) and Variola Major (smallpox) Virus Semaphorin V (SEQ ID NOS:67
and 68)
[0131] We used our G-Semaphorin I cDNA in standard low stringency
screening methods (of both cDNA and genomic libraries) in an
attempt to isolate a potential Semaphorin I homologue from
Drosophila. We were unsuccessful in these screens. Since the
sequence was novel and shared no similarity to anything else in the
data base, we then attempted to see if we could identify a
Semaphorin I homologue in other, more closely related insects. If
possible, we would then compare these sequences to find the most
conserved regions, and then to use probes (i.e., oligonucleotide
primers for PCR) based on these conserved regions to find a
Drosophila homologue.
[0132] In the process, we used the G-Semaphorin I cDNA in low
stringency screens to clone Semaphorin I cDNAs from libraries made
from locust Locusta migratoria embryonic RNA and from a cDNA
embryonic library from the cricket Acheta domestica. We used PCR to
clone genomic fragments from genomic DNA in the beetle Tribolium,
and from the moth Manduca. We then used the Tribolium genomic DNA
fragment to isolate cDNA clones and ultimately sequenced the
complete ORF for the Tribolium cDNA.
[0133] In the meantime, we used the partial Tribolium and Manduca
sequences in combination with the complete grasshopper sequence to
identify conserved regions that allowed us to design primers for
PCR in an attempt to clone a Drosophila Semaphorin I homologue.
Several pairs of primers generated several different bands, which
were subcloned and sequenced and several of the bands gave partial
sequences of the Drosophila Semaphorin homologue. One of the bands
gave a partial sequence of what was clearly a different, more
divergent gene, which we call D-Semaphorin II.
[0134] Based on the sequence of PCR products, we knew we had
identified two different Drosophila genes, one of which appeared to
be the Semaphorin I homologue, and the other a second related gene.
The complete ORF sequence of the D-Semaphorin I homologue revealed
an overall structure identical to G-Semaphorin I: a signal
sequence, an extracellular domain of around 550 amino acids
containing 16 cysteines, a transmembrane domain of 25 amino acids,
and a cytoplasmic domain of 117 amino acids. When we had finished
the sequence for D-Semaphorin II, we were able to begin to run
homology searches in the data base, which revealed some of its
structural features further described herein. The Semaphorin II
sequence revealed a different structure: a signal sequence of 16
amino acids, a .about.525 amino acid domain containing 16
cysteines, with a single immunoglobulin (Ig) domain of 66 amino
acids, followed by a short unique region of 73 amino acids. There
is no evidence for either a transmembrane domain or a potential
phospholipid linkage in the C-terminus of this protein. Thus, it
appears that the D-Semaphorin II protein is secreted from the cells
that produce it. The grasshopper, Tribolium, and Drosophila
Semaphorin I cDNA sequences, as well as the sequence of the
D-Semaphorin II cDNA, are shown herein. In addition, we used this
same technique to identify Semaphorin I genes in a moth, Manduca
sexta, a locust, Locusta migratoria, and a cricket, Acheta
domestica.
[0135] With this large family of insect Semaphorin genes, we
identified a number of good stretches of the right amino acids
(with the least degeneracy based on their codons) with strong
homology for designing primers for PCR to look for human genes. We
designed a set of oligonucleotide primers, and plated out several
human cDNA libraries: a fetal brain library (Stratagene), and an
adult hippocampus library. We ultimately obtained a human cDNA PCR
bands of the right size that did not autoprime and thus were good
candidates to be bonafide Semaphorin-like cDNAs from humans. These
bands were purified, subcloned, and sequenced.
[0136] Whole-mount in situ hybridization experiments showed that
D-Semaphorin I and II are expressed by different subsets of neurons
in the embryonic CNS. D-Semaphorin I is expressed by certain cells
along the midline as well as by other neurons, whereas D-Semaphorin
II is not expressed at the midline, but is expressed by a different
subset of neurons. In addition, D-Semaphorin II is expressed by a
subset of muscles prior to and during the period of innervation by
specific motoneuron. On the polytene chromosomes, the D-Semaphorin
I gene maps to (gene-band-chromosome) 29E1-22L and that of
D-Semaphorin II to 53C9-102R. We have identified loss of function
mutations in the D-Semaphorin I gene and a pair of P-element
transposon insertions in the D-Semaphorin II gene which appear to
cause severe phenotypes.
[0137] When we lined up the G-Semaphorin I, T-Semaphorin I,
D-Semaphorin I, and D-Semaphorin II sequences and ran the sequences
through a sequence data base in search of other sequences with
significant similarity, we discovered a curious finding: these
Semaphorins share sequence similarity with the A39R open reading
frame (ORF) from Vaccinia virus and the A43R ORF from Variola Major
(smallpox) virus and we discovered that the amino acids shared with
the virus ORF were in the same regions where the insect proteins
shared their greatest similarity. The viral ORF began with a
putative signal sequence, continued for several hundred amino acids
with sequence similarity to the Semaphorin genes, and then ended
without any membrane linkage signal (suggesting that the protein as
made by the infected cell would likely be secreted).
[0138] We reasoned that the virus semaphorins were appropriated
host proteins advantageously exploited by the viruses, which would
have host counterparts that most likely function in the immune
system to inhibit or decrease an immune response, just as in the
nervous system they appear to function by inhibiting growth cone
extension. Analogous to situations where viruses are thought to
encode a secreted form of a host cellular receptor, here the virus
may cause the infected cell to make a lot of the secreted ligand to
mimic an inhibitory signal and thus help decrease the immune
response.
III. Isolation and Characterization of Murine CNS Semaphorin III
Receptor using Epitope Tagged Human Semaphorin III (hSIII)
[0139] mRNA was isolated from murine fetal brain tissue and used to
construct a cDNA library in a mammalian exprssion vector, pCMX,
essentially as in Davis et al. (1991) Science 253, 59.
[0140] The transfection and screening procedure is modified from
Lin et al (1992) Cell 68. 775. COS cells grown on glass slide
flaskettes are transfected with pools of the cDNA clones, allowed
to bind radioiodinated hSIII truncated at the C-terminus end of the
semaphorin domain. In parallel, similarly treated COS cells are
allowed to bind unlabelled human semaphorin III truncated at the
C-terminus end of the semaphorin domain and there joined to a
10-amino acid extension derived from the human c-myc proto-oncogene
product. This modified hSIII allows the identification of hSIII
receptors with the use of the tagged ligand as a bridge between the
receptor and a murine monoclonal antibody which is specific for an
epitope in the c-myc tag. Accordingly, after binding unlabelled
hSIII the cells are exposed to the monoclonal which may be labeled
directly or subsequently decorated with a secondary anti-mouse
labeled antibody for enhanced signal amplification.
[0141] Cells are then fixed and screened using dark-field
microscopy essentially as in Lin et al. (supra). Positive clones
are identified and sequence analysis of murine CNS Semphorin III
receptor cDNA clones by the dideoxy chain termination method is
used to construct full-length receptor coding sequences.
[0142] It is evident from the above results that one can use the
methods and compositions disclosed herein for making and
identifying diagnostic probes and therapeutic drugs. It will also
be clear to one skilled in the art from a reading of this
disclosure that advantage can be taken to effect alterations of
semaphorin responsiveness in a host.
[0143] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. Although
the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding,
it will be readily apparent to those of ordinary skill in the art
in light of the teachings of this invention that certain changes
and modifications may be made thereto without departing from the
spirit or scope of the appended claims.
Table 1. Deduced Amino-Acid Sequence of Semaphorin Gene Family.
[0144] Approximate position of enumerated peptide classes are
indicated by parenthetical (a) through (o); semaphorin domains are
bounded by arrows; G: grasshopper semaphorin I (SEQ ID NO:58), T:
Tribolium semaphorin I (SEQ ID NO:64), D1: Drosophila semaphorin I
(SEQ ID NO:60), D2: Drosophila semaphorin II (SEQ ID NO:62), H3:
Human semaphorin I (SEQ ID NO:54), V4: Vaccinia virus semaphorin IV
(SEQ ID NO:56), V5: Variola virus (human small pox) semaphorin IV
(SEQ ID NO:66); small case residues: conserved residues: underline:
signal sequence; solid bar: transmembrane domain; double dashes:
immunoglobulin domain. TABLE-US-00006 G
MRAALVAVAALLWVVALHAAAWVNDVSPKMYVQFGEERVQR T
MVVKILVWSICLIALCHAWMPDSSSKLINHFKSVESKS D1 D2
MSLLQLSPLLALLLLLCSSVSETAADYENTWNFYYERPCCTGNDQGNNNYGKHGADHVRE H3
MGWLTRIVCLFWGVLLTARANYQNGKNNVPRLKLSYKEMLESNNVIT V4
MMVLLHAVYSIVFVDVIIIKVQRYINDI .dwnarw. G
f-Lg--nESHKDHfKLLeKDHNSlLvga--rNI---VYnISLRDLTEFTEQRiEwHSsGAHRELcY
T
fT-g--nATFPDHfIVLNQDETSILvgG--rNR---VYnLSIFDLSERKGGRiDwPSsDAHGQLcI
D1 D2
fNCgKLY---YRTfHMNeDRDT-lYvgaMDrVF---RVnLQNISSSNCNRDAiNLEPTRDDVVScV
H3
fN-gLAnSSSYHTfLLDeERSR-lYvgaKDHIFSFDLVnI-------KDFQKiVwPVsYTRRDEcK
V4
LTLDIFYLFKKMIPLLFILFYFANGIEWHKFETSEEIISTYLLDDV------LYTGVNGAVYTFSN
A F V DDCQN-YIR(a) ICGTN(b) G
LkgkS-Eddcqn-yir--VlAKIDDDrVLIcgtnaYKpLcRHyALKd-----GDyVVeKEYEgRg----
T
LkgkT-Dddcqn-yir--ilYSSEPGKlVICgtnSYKpLcRTyaFKE-----GKyLVeKEVEgIg----
D1 Eddcqn-yir--iMVVPSPGrlFvcgtnSFRpMcNTyIISd-----SNyTLeATKNgQA----
D2
SkgkSQIFdcKnHViQ--SMDQGD--rlYvcgtnaHNpKDYViYANL-----THLPRSEYVIgVgLGIA
H3
WAgkDILKEcAn-FiK--VlKAYNQTHlYAcgtGaFHpIcTYiEIGHPEDNIFKLENSHFENgRg----
VR
NkLNKTGLTNNn-yiTTSiKVEDADKDTLvcgtnNGNpKcWKiDGSd----------------------
S F A CPYDP(c) TVADFSG(d) G
LcpFdpDh---------nstAIYSEgQ-------lysAtvadfsgTdpLiyrGpl------------
T
LcpyNpEh---------nstsVSYNgQ-------lFsAtvadfsgGdpLiyrEpQ------------
D1
VcpydpRh---------nstsVLADNE-------lysgtvadfsgSdpIiyrEpl------------
D2
KcpydpLD---------nstAIYVENGNPGGLPGlysgtNaEfTKAdTViFrTDlYNTSAKRLEYKF
H3
KSpydpKL---------LTAsLLIDgE-------lysgtAadfMgRdFAiFrT-lGHHHPIRTEQHD
V4
----dpKhRGRGYAPYQnsKVTIISHNGcYLSDINIsKEGIKRWRRFDGPcGYDl------------
V5 MIYl------------ N LNAPNFV(e) (f)FFFRETA EYINCGK(g) (h)DKGG G
-rteRSdLkQ-lnapnfv-NTMEyNdFIFfffretaveyincgkaiysrvarvckHdkgg T
-rteLSdLkQ-lnapnfv-NsVAygdYIFffYretaveyMncgkViysrvarvckDdkgg D1
-QteQYdSLS-lnapnfv-SsFtQgdFvyfffretaveFincgkaiysrvarvckWdkgg D2
KrtLKYdSkW-lDKpnfv-GsFDIgEYvyfffretaveyincgkaVysriarvckKdVgg H3
-SRWLNdpkF-ISaHLISESdNPEDdkvyfffreNaIDGEHSgkaTHAriGQIckndFgg V4
YTADNVIpkDGlRGA-fvDKdGty-dkvyILfTDtIG-SKRIVkIPy--iaQMcLndEgg V5
YTADNVIpkDGlQGA-fvDKdGty-dkvyILfTVtIG-SKRIVkIPy--iaQMcLndECg SSY(i)
V PH WTTFLKAR NCSIPG(j) G
phQF-GDrwtsflkSrlncsVpgDypfyf---neiqs---tsdIIegNyGGQVEkliygv T
phQ-SRnrwtsflkarlncsipgEypfyf---Deiqs---tsdIvegRyNsDDskIiygI D1
phRF-RNrwtsflkSrlncsipgDypfyf---neiqs---AsNLvegQyGsMSskliygv D2
KNLl-AhNwAtYlkarlncsiSgEFpfyf---neiqs---VYQL-----PsDKsRF-FAT H3
-hRSLVNKwttflkarlIcsVpgPNGIDTHf-DeLq------dVFLMNFKDPKNPVVygv V4
pSSlSShrwStflkVElEcDiDgRSYRQIIHSRTiKTDNDtILYvF--FDsPYsk----- V5
pSSlSShrwStLlkVElEcDiDgRSYSQINHSKTiKQIMIRYYMYSLIVLFQVRIMYLFY V
GSAVC(k) NSNWLPV(l) PRPGTCVND(m) G
fttpVnSiGgsavcafsmKSiLESfDgPfkeqETMnsnwlAvPSLKvpeprpgQcvndsr T
LttpVnAiGgsaIcayQmAdiLRVfEgSfkHqETInsnwlpvPQNLvpeprpgQcvRdsr D1
fNtpSnSiPgsavcafALQdiADTfEgQfkeqTGInsnwlpvNNAKvpDprpgScHndsr D2
fttSTnGLIgsavcSfHINEiQAAfNgKfkeqSSSnsAwlpvLNSRvpeprpgTcvndTS H3
fttSSnIFKgsavcMysmSdVRRVfLgPYAHRDGPnYQwVp-YQGRvpYprpgTc--PsK V4
----------saLcTysmNTiKQSfSTSKLeg----------YTKQLpSpApgIcLPAGK V5 EYH
G
---------TlpdVSVnfV-kShTlmdEAvpaFfTRpilIrIslQyrftKiAvdQqvRtPDgKAYdvLf
T
---------IlpdKNVnfi-kThSlmED-vpaLfGKpVlVrVslQyrftAiTvdPqvKtINNQYLdvLY
D1
---------AlpdPTLnfi-kThSlmdENvpaFfSQpilVrTsTIyrftQiAvdAqIKtPGgKTYdvIf
D2
---------NlpdTVLnfi-RShPlmdKAvNHEHnNpVYYKRDlVFTK-LVVDKIRIDILNQEYI-vYY
H3
TFGGFDSTKDlpdDVITfA-rshPAmYNPvFPMNnRpiVIKTDVNyQftQiVvd-RvDAEDgQY-dvMf
V4
---------VVpHTTFEViEKYNVlDdIIKp-LSnQpiFEGPSGVKWFDIKEKENEHREYRIYFIKENS
G igtddgkvIkALnSAsFDSSDTvDSvVIeeLQvLPPGVpVKnlYVvr-------Mdg--d T
igtddgkvLkAvnIPKRHAKALLYRKYRTSVHPHGA--pVKQlKIAP------------G D1
VgtdHgkIIkSvnAEsADSADKvTSvVIeeIDvLTKSEpIRnlEIvrTMQYDQPKdgSYd D2
VgtNLgRIYkIvnGEsLSKLLDIFEvAPNeAIQVMEISQTR------------------- H3
igtdVgTvLkVvSIPKETWY-DLEEvLLeeMTvFREPTAISA------------MELSTK V4
iYSFdTkSKQTRSSQVDARLFSvMVTSKPLFIADIGIGVGMPQMKKILKM* DPYCAWD(n) G
dSklVVvSdDEiLAiKlhrcGSdkItNcRecvSlqdpycawdNVELKcTAVgSpDwSAG T
YGkVVVvGKDEiRLANlNHcAs-k-tRcKDcvElqdpHcawdAKQNLcVSIDTVTSY-- D1
dGklIivTdSQVVAiQlhrcHNdkItScSecvAlqdpycawdKIAGKcRSHgApRw-LE D2
-KSlYiGTdHRiKQiDlAMc-NRRYDNcFRcv--RdpycGwdKEANTcRPY-------- H3
QQQlYiGSTAGVAQLPlhrcDIYG-KAcAecCLARdpycawd--GSAcS---RYFPTAK
.dwnarw. G
KrRFIqNISLgEH-KAcGGRPQTEIVASPVPTQPTTKSSGDPVHSIHQAEFEpeiDNEiVI T
-rFLIqdvVRgDD-NKcWsPQTDKkTVIKNKPSEVENEITNSIDEKDLDsSdpLiKTGLdD D
ENYFYqNvATgQH-AAcPsGKINSkDANAGEGKGFRNDMDLLDSRRQ--sKdQeiIDNidK D2
ELDLLqdvANETS-DIcDsSVLKKk H3 RrTRRqdIRNgDPLTHcSDLHDNHH G
GVddSNVIPNTLAEINHAGSKLPSSQEKlPiytaetlTiaIvTSCLGAlVvgfIsgFLFS T
DSdcDPVSENSIGGcAV--------RQQlViytaGtlHiVvvVVsiVGlFSWLYsgLSVF D
NFEdD---------------------IINAQytVetlVMavLAGsiFSlLvgfFTgYFCG G
rrcRGEDYTDMpFpdQRHQLNRLTEAGlNADsPYLPPCANnkAAInlvLNv-----PpkN T
AKFHSd----SQypEAPFIEQHNHLERlsANQTGYLTPRAnk-AVnlvvKvSSSTPRpkK D
rrcHKdEDDNLpypdTEYEYFEQRQNVNsFPsSCRIQQEPKLLPQVEEvTYAEPVLLpQP
KKTYI(o) G AnGKNANsSAENKP----IQkktyi* T DnLDVSKDLNIASDGTLQKIkktyi*
D PPPNKMHsPKNTLRKPPMHQMHQGPNSETLFQFHVTATTPSSRIVVATTSEHCVPTR* D2
-----IVVTyg---QsVHlGcFVkIPEVlKENQvTwYHHSKDKG H3
GHSPEERIIygVENSsTFlEcSPkSQRAl----vYwQFQRRNEE
============================ D2
rYeIRYSPTKYiETtERglVVVsVNEAdGgRyDchLGGSLLcSYNITVDAHRcTPPNKSN H3
rKeE-IRVDDHiIRtDQglLLRsLQQKdSgNyLchAVEHGFIQTLLKVTLEVIDTEHLEE
====================================== D2
DYQKIYSDWcHEFEKYKTAMKSWEKKQGQcSTRQNFScNQHPNEIFRKPNV* H3
LLHKDDDGDGSKTKEMSNSMTPSQKVWYRDFMQLINHPNLNTMDEFcEQVWKRDRKQRRQ H3
RPGHTPGNSNKHLQENKKGRNRRTHEFERAPRSV*
[0145]
Sequence CWU 1
1
* * * * *