U.S. patent application number 10/925970 was filed with the patent office on 2005-11-10 for novel semaphorin gene: semaphorin w.
This patent application is currently assigned to SUMITOMO PHARMACEUTICALS COMPANY, LIMITED. Invention is credited to Kikuchi, Kaoru, Kimura, Toru.
Application Number | 20050249741 10/925970 |
Document ID | / |
Family ID | 17719801 |
Filed Date | 2005-11-10 |
United States Patent
Application |
20050249741 |
Kind Code |
A1 |
Kimura, Toru ; et
al. |
November 10, 2005 |
Novel Semaphorin gene: Semaphorin W
Abstract
The present invention provides Semaphorin W inhibiting neurite
outgrowth, and a gene therefor, as well as other Semaphorins
hybridizing to said Semaphorin W gene, modified proteins or partial
peptides of said Semaphorin W, antibodies against said Semaphorin
W, antisense nucleotides against said Semaphorin W gene, and the
use of such substances as pharmaceutical or diagnostic agents or
laboratory reagents. The present invention further provides a
method of screening for Semaphorin W antagonists employing said
Semaphorin W, Semaphorin W antagonists obtained by said screening
method, pharmaceutical agents comprising such antagonists, and
transgenic animals involving said Semaphorin W.
Inventors: |
Kimura, Toru; (Kusatsu-shi,
JP) ; Kikuchi, Kaoru; (Takarazuka-shi, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
SUMITOMO PHARMACEUTICALS COMPANY,
LIMITED
|
Family ID: |
17719801 |
Appl. No.: |
10/925970 |
Filed: |
August 26, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10925970 |
Aug 26, 2004 |
|
|
|
09284180 |
Jun 9, 1999 |
|
|
|
6902730 |
|
|
|
|
09284180 |
Jun 9, 1999 |
|
|
|
PCT/JP97/03549 |
Oct 3, 1997 |
|
|
|
Current U.S.
Class: |
424/185.1 ;
435/320.1; 435/325; 435/69.1; 530/350; 530/388.25; 536/23.5 |
Current CPC
Class: |
C07K 14/4703 20130101;
A01K 2217/05 20130101; A61K 38/00 20130101 |
Class at
Publication: |
424/185.1 ;
435/069.1; 435/320.1; 435/325; 530/350; 536/023.5; 530/388.25 |
International
Class: |
A61K 039/00; C07H
021/04; C12N 015/09; C07K 014/47; C07K 016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 9, 1996 |
JP |
287636 |
Claims
1. An isolated polypeptide comprising the amino acid sequence shown
in SEQ ID NO:3.
2. An isolated polypeptide encoded by a nucleotide sequence
selected from the group consisting of: (a) a nucleotide sequence
comprising SEQ ID NO:1; and (b) a nucleotide sequence comprising
SEQ ID NO:2.
3. An isolated polypeptide encoded by a polynucleotide having a
nucleotide sequence selected from the group consisting of: (a) the
nucleotide sequence of SEQ ID NO:5; and (b) the nucleotide sequence
of SEQ ID NO:10, wherein said polynucleotide specifically
hybridizes with a complement of a polynucleotide having a
nucleotide sequence selected from the group consisting of: (a) the
nucleotide sequence of SEQ ID NO:1, and (b) the nucleotide sequence
of SEQ ID NO:2; under conditions of a buffer comprising 45% (v/v)
formamide, 5.times.SSPE, at 42.degree. C., and washing after
hybridization with a buffer comprising 2.times.SSPE at 42.degree.
C., and said polynucleotide encodes a protein having the biological
activity of inhibiting neurite outgrowth from dorsal root ganglion
cells.
4. An isolated polypeptide encoded by a polynucleotide having a
nucleotide sequence selected from the group consisting of: (a) the
nucleotide sequence of SEQ ID NO:5; and (b) the nucleotide sequence
of SEQ ID NO:10, wherein said polynucleotide specifically
hybridizes with a complement of a polynucleotide having a
nucleotide sequence selected from the group consisting of: (a) the
nucleotide sequence of SEQ ID NO:1, and (b) the nucleotide sequence
of SEQ ID NO:2; under conditions of a buffer comprising 45% (v/v)
formamide, 5.times.SSPE, at 42.degree. C., and washing after
hybridization with a buffer comprising 2.times.SSPE at 42.degree.
C., and said polynucleotide encodes a protein having the biological
activity of collapsing growth cones of retinal ganglion cells.
5. A polypeptide obtained by expressing in a cell a nucleic acid
comprising a polynucleotide having a nucleotide sequence selected
from the group consisting of: (a) the nucleotide sequence of SEQ ID
NO:1; (b) the nucleotide sequence of SEQ ID NO:2; and (c) a
nucleotide sequence encoding the amino acid sequence of SEQ ID
NO:3.
6. A polypeptide obtained by expressing in a cell a nucleic acid
comprising a polynucleotide having a nucleotide sequence selected
from the group consisting of: (a) the nucleotide sequence of SEQ ID
NO:5; and (b) the nucleotide sequence of SEQ ID NO:10, wherein said
polynucleotide specifically hybridizes with a complement of a
polynucleotide having a nucleotide sequence selected from the group
consisting of: (a) the nucleotide sequence of SEQ ID NO:1, and (b)
the nucleotide sequence of SEQ ID NO:2; under conditions of a
buffer comprising 45% (v/v) formamide, 5.times.SSPE, at 42.degree.
C., and washing after hybridization with a buffer comprising
2.times.SSPE at 42.degree. C., and said polynucleotide encodes a
protein having the biological activity of inhibiting neurite
outgrowth from dorsal root ganglion cells.
7. A polypeptide obtained by expressing in a cell a nucleic acid
comprising a polynucleotide having a nucleotide sequence selected
from the group consisting of: (a) the nucleotide sequence of SEQ ID
NO:5; and (b) the nucleotide sequence of SEQ ID NO:10, wherein said
polynucleotide specifically hybridizes with a complement of a
polynucleotide having a nucleotide sequence selected from the group
consisting of: (a) the nucleotide sequence of SEQ ID NO: 1, and (b)
the nucleotide sequence of SEQ ID NO:2; under conditions of a
buffer comprising 45% (v/v) formamide, 5.times.SSPE, at 42.degree.
C., and washing after hybridization with a buffer comprising
2.times.SSPE at 42.degree. C., and said polynucleotide encodes a
protein having the biological activity of collapsing growth cones
of retinal ganglion cells.
8. A polypeptide comprising a fragment consisting of at least 6
contiguous amino acids of any one of the polypeptides of claims 1
to 7.
9. An isolated antibody or a fragment thereof which specifically
binds to the polypeptide of claim 1.
Description
TECHNICAL FIELD
[0001] The present invention relates to Semaphorin W, a novel
Semaphorin belonging to the Semaphorin family, and use of
Semaphorin W for pharmaceutical or diagnostic agents or laboratory
reagents. More particularly, it relates to Semaphorin W inhibiting
neurite outgrowth, and a gene therefor, as well as other
Semaphorins hybridizing to said Semaphorin W gene, modified
proteins or partial peptides of said Semaphorin W, antibodies
against said Semaphorin W, antisense nucleotides against said
Semaphorin W gene, antagonists of said Semaphorin W, transgenic
animals, and use of such substances as pharmaceutical or diagnostic
agents or laboratory reagents.
BACKGROUND ART
[0002] It is widely known that a central nervous system
(CNS)-neuron in higher organisms such as human is not capable of
regeneration once injured. Therefore, one who has received an
injury on his (her) spinal cord due to, for example, a traffic
accident, is compelled to spend the rest of his (her) life in a
hemiplegic state. On the contrary, it is known that a peripheral
nervous system (PNS)-neuron retains a vigorous regeneration ability
even in those higher organisms, and therefore, neurons in a limb,
when disconnected, can gradually regenerate with a concomitant
recovery of their function.
[0003] In the early nineteen-eighties, a group of Aguayo et al.
found that when PNS-neuron is experimentally grafted into an
injured CNS-neuron in a higher organism, axon growth of CNS-neuron
is induced. This observation demonstrated that CNS-neuron in higher
organisms which had been generally considered not to have a
regeneration ability can regenerate if a suitable environment is
provided (Nature, 284, 264-265 (1980), Science, 214, 931-933
(1981)). That report suggests a possibility that in CNS of higher
organisms, there may exist a factor, namable "CNS-neuron
regeneration inhibitor", which inhibits the regeneration of
CNS-neuron, and that a release from such inhibition may allow the
regeneration of CNS-neurons. This suggestion paved the way for a
CNS-neuron regeneration therapy.
[0004] In 1988, a group of Schwab et al., demonstrated that there
existed such CNS-neuron regeneration inhibitor among proteins
derived from CNS myelin. They also succeeded in purifying, though
partially, a protein having said CNS-neuron regeneration inhibition
activity, and named this protein fraction NI35/250 (Annu. Rev.
Neurosci., 16, 565-595 (1993)), although no one has succeeded in
its isolation, identification and gene cloning yet. In addition,
they immunized animals with the partially purified NI35/250, and
succeeded in obtaining an antibody (IN-1) having a neutralizing
activity. This antibody is capable of recognizing the band for
NI35/250 in Western blotting, and capable of staining, in an
immunostaining, the region to which NI35/250 is supposed to be
distributed. Furthermore, they demonstrated that administration of
this antibody to an animal experimentally received an injury on its
spinal cord has promoted regeneration of axons in spinal cord,
though partially, within 2-3 weeks, and restored its function
within 2-3 months (Nature, 343, 269-272 (1990), Nature, 378,
498-501 (1995)). These findings are of great value, because they
experimentally demonstrated that there existed a CNS-neuron
regeneration inhibitor as suggested by Aguayo et al. (supra) and
that CNS-neuron can be regenerated by inhibiting the activity of
said inhibitor. The above antibody is, however, directed not to
human but to rat NI35/250, and exhibits a low stability and
specificity. In addition, although regeneration of CNS-neuron was
observed as described above by administering said antibody, its
effect was so partial and incomplete that not all of the motor
functions could be restored. It is, therefore, believed essential
in solving these problems to identify the gene for NI35/250 or
similar CNS-neuron regeneration inhibitor, and, based on knowledges
of molecular biology, neuroscience and the like, develop an
antagonist more effectively inhibiting the CNS-neuron regeneration
inhibition activity, or develop a method for inhibiting the
expression of the gene for said regeneration inhibitor.
[0005] Apart from the above, the nervous system, whether it is
central or peripheral, requires formation of a complicated neural
network among neurons or between neurons and peripheral receivers
or effectors during development, that is, in the stage of embryo or
fetus, in order to precisely carry out its principal functions,
i.e., to transfer and process the information. To establish the
neural network, an ingenious mechanism is necessary, which
precisely guides a growing neurite to the target site locating
remote therefrom.
[0006] It has been hitherto believed that a factor which positively
controls the neurite outgrowth, such as neurite growth promoter and
neurite growth attractant may play a major role in the formation of
the neural network. However, it is now being demonstrated by recent
studies on the mechanism of the network formation that the opposite
factor, that is, a negative factor having an outgrowth inhibition
activity is important for an accurate guidance (Cell, 78, 353-356
(1994)).
[0007] A representative factor having such an outgrowth inhibition
activity is a protein called "Semaphorin". Semaphorin firstly
discovered is Fasciclin IV found in grasshopper. Collapsin
(latterly named Collapsin I) was subsequently discovered in chick
(Cell, 75, 217-227 (1993); Neuron, 9, 831-845 (1992)). To date,
more than 10 genes belonging to the Semaphorin family have been
reported in a wide range of species covering insects such as
drosophila and beetle, human, and viruses (Cell, 81, 471-474
(1995)). These Semaphorins characteristically contain in their
amino acid sequences similar structures called semaphorin domains
each consisting of about 500 amino acids (Neuron, 14, 941-948
(1995); Cell, 75, 1389-1399 (1993)). However, the homologies of the
primary amino acid sequences in semaphorin domains among these
Semaphorin genes are 80-20%, and not necessarily high.
[0008] Of these Semaphorins, functions have been verified for only
a few, including, for example, Fasciclin IV of grasshopper,
Semaphorins I and II of drosophila, Collapsin of chick, and
Semaphorin III which corresponds to Collapsin in mammals. All of
these Semaphorins are known to inhibit neurite outgrowth or
synapsis formation. In particular, Semaphorin III has been reported
to have an activity collapsing in a short time the growth cone of
cultured neuron (growth-cone collapse activity) in vitro (Neuron,
14, 941-948 (1995); Neuron, 14, 949-959 (1995); Cell, 81, 631-639
(1995); Cell, 75, 1389-1399 (1993); Cell, 75, 217-227 (1993);
Neuron, 9, 831-845 (1992)).
[0009] Although it is now being demonstrated, as described above,
that known Semaphorins have a growth-cone collapse activity and a
neurite outgrowth inhibition activity during development, and play
a role in giving an accurate guidance to neuron, it is not evident
at present whether or not the Semaphorins exert some function not
only during development but also in the adult, and less evident
whether or not Semaphorins play a role as a CNS-neuron regeneration
inhibitor. Of course, since known Semaphorins have been shown to be
a negative guidance factor inhibiting neurite outgrowth, it would
not be unreasonable to consider said Semaphorins as a candidate for
a CNS-neuron regeneration inhibitor (Nature, 378, 439-440 (1995)).
However, it has been shown by in vitro experiments that Semaphorin
III (Sema III), only one Semaphorin of higher organisms of which
function has been analyzed, exerts its neurite-outgrowth inhibition
activity on a sensory neuron and sympathetic neuron both of which
are peripheral, but not on a retinal neuron which is central (Cell,
75, 217-227 (1993)). In addition, Northern analysis on the
distribution of Sema III expression in the adult carried out by the
present inventors has revealed that it is expressed mainly in
peripheral tissues (see Reference example 2 below). It is therefore
hardly believed that Sema III having such features has a function
as a CNS-neuron regeneration inhibitor.
PROBLEM TO BE SOLVED BY THE INVENTION
[0010] The present invention aims to provide Semaphorin W, a novel
Semaphorin belonging to the Semaphorin family, and a gene therefor,
and to provide pharmaceutical agents for neural diseases, in
particular for regeneration of CNS-neuron, and related diagnostic
agents or laboratory reagents. More specifically, the present
invention aims to provide Semaphorin W which inhibits neurite
outgrowth and a gene therefor, as well as other Semaphorins
hybridizing to said Semaphorin W gene, modified proteins or partial
peptides of said Semaphorin W, antibodies against said Semaphorin
W, antisense nucleotides against said Semaphorin W gene, and use of
such substances as pharmaceutical or diagnostic agents or
laboratory reagents. The present invention further aims to provide
a method of screening for Semaphorin W antagonists employing said
Semaphorin W, Semaphorin W antagonists obtained by said screening
method, pharmaceutical agents comprising such antagonists, and
transgenic animals regarding said Semaphorin W.
MEANS OF SOLVING THE PROBLEM
[0011] In order to provide pharmaceutical agents for neural
diseases, in particular for regeneration of CNS-neuron, and related
diagnostic agents or laboratory reagents, the present inventors
have planed to identify a novel Semaphorin which has not yet been
cloned. In particular, the present inventors have paid their
attention to the similarity between the in vitro activities of the
above-described NI35/250 and the negative guidance factor
Semaphorin, i.e., to the fact that NI35/250 has a growth-cone
collapse activity and a neurite-growth inhibition activity in vitro
(J. Neurosci., 8, 2381-2393 (1988); Science, 259, 80 (1993)), while
known Semaphorins similarly possess a neurite-growth inhibition
activity, and particularly Semaphorin III has also a growth-cone
collapse activity. This suggested to the inventors the possibility
that unknown Semaphorins which have not yet been identified may
include the one inhibiting regeneration of CNS-neuron.
Specifically, the present inventors' idea was that Semaphorin
having those characteristics that 1) it is highly expressed in the
CNS of the adult, but 2) it is poorly expressed in other tissues
where regeneration of neuron (or neurite outgrowth) is not
inhibited, such as peripheral tissues in the adult and fetus
tissues, has not been identified yet, and if one can identify a new
unknown Semaphorin having such characteristics, the Semaphorin
might be involved in inhibition of regeneration of CNS-neuron.
[0012] First of all, the inventors have searched EST (Expressed
Sequence Tags) database for DNA sequences encoding the amino acids
relatively well conserved among previously reported Semaphorin
genes. As a consequence, a DNA fragment T09073 was identified,
which encodes, as a partial sequence, a sequence:
Gln-Asp-Pro-Val-Cys-Ala-Trp, similar to that consisting of seven
amino acids extremely well conserved among Semaphorins (Gln (or
Arg)-Asp-Pro-Tyr-Cys-Ala (or Gly)-Trp).
[0013] The T09073 gave a sequence information as to only 364 bp and
contained undetermined bases, and further, the open reading frame
could not be determined. It was, therefore, utterly impossible at
that stage to conclude that T09073 corresponds to part of a gene
encoding "Semaphorin". Furthermore, although T09073 has been
submitted to the database as a sequence derived from a human child
brain cDNA library, it was unknown whether or not the sequence is
expressed in the fetus or peripheral tissues of the adult, and
therefore, it could not be concluded that the sequence corresponds
to part of a novel Semaphorin gene specifically expressed in the
CNS.
[0014] Thus, the present inventors firstly carried out Northern
analysis using a DNA fragment consisting of 196 base pairs from the
5' region of T09073 as a probe, in order to check the distribution
of expression of a gene containing T09073. As a result, it was
found that the gene corresponding to T09073 was highly and widely
expressed in CNS tissues in the adult, whereas among the other
tissues, it was expressed only in the lung and spleen of the adult
throughout the fetal and postnatal periods. It was thus
demonstrated that the gene exhibited a distribution pattern of
expression expected for a novel Semaphorin gene at which the
present inventors aimed.
[0015] Next, the present inventors cloned the gene containing
T09073 in full length in order to ascertain whether it is a novel
Semaphorin or not. Specifically, a rat cDNA library was screened
using the above DNA fragment consisting of 196 base pairs as a
probe. As a result, the gene thus cloned proved to be a novel
Semaphorin gene having a sequence characteristic to Semaphorins. We
named this novel Semaphorin "Semaphorin W".
[0016] Further analysis revealed that Semaphorin W has an effect as
a novel Semaphorin gene at which the present inventors aimed, that
is, an inhibition activity against neurite outgrowth, especially an
inhibition activity for CNS-neurons.
[0017] Semaphorin W of the present invention appears to be involved
in inhibition of regeneration of CNS-neuron in the adult, since it
is highly expressed in CNS in the adult and has an inhibitory
effect on CNS-neurons as described above. Semaphorin W may be used
to screen for Semaphorin W antagonists, and those antagonists
identified in such screening system are expected to promote
regeneration of CNS-neuron. Similarly, antisense DNAs or RNAs
against Semaphorin W gene are also expected to promote regeneration
of CNS-neuron as well as the above antagonists.
[0018] In addition, in view of the fact that Semaphorin W of the
present invention has also an inhibitory effect on PNS-neurons, it
may be used as a therapeutic or diagnostic agent for pains or
immune diseases such as atopic dermatitis, by administering it to
peripheral tissues, which results in the inhibition of neurite
outgrowth of PNS-neuron. Furthermore, Semaphorin W is a novel
Semaphorin belonging to the Semaphorin family which has
unprecedented features regarding its distribution of expression and
its effect as described above. Semaphorin W, therefore, serves as
an important research material or a laboratory reagent.
[0019] The present invention has been completed on the basis of the
above findings.
[0020] Thus, the gist of the present invention relates to:
[0021] (1) a gene encoding the following protein (a) or (b):
[0022] (a) Semaphorin W protein comprising the amino acid sequence
shown in SEQ ID NO: 3,
[0023] (b) a protein which comprises an amino acid sequence wherein
one or more amino acids are deleted, substituted and/or added in
the amino acid sequence shown in SEQ ID NO: 3, and which protein
inhibits neurite outgrowth;
[0024] (2) a gene comprising the following DNA (c), (d), or
(e):
[0025] (c) Semaphorin W DNA comprising the base sequence shown in
SEQ ID NO: 1 or 2,
[0026] (d) DNA which hybridizes under stringent conditions to DNA
comprising the base sequence shown in SEQ ID NO: 1 or 2, and which
encodes a protein inhibiting neurite outgrowth,
[0027] (e) DNA of the above item (d) which contains the base
sequence shown in SEQ ID NO: 4 or 5 and/or the base sequence shown
in SEQ ID NO: 10;
[0028] (3) a gene comprising DNA which hybridizes under stringent
conditions to DNA comprising the base sequence shown in SEQ ID NO:
7, and which encodes a protein having a semaphorin domain;
[0029] (4) a protein obtained by expressing a gene of any one of
the above items (1) to (3);
[0030] (5) a gene encoding a protein comprising an amino acid
sequence wherein one or more amino acids are deleted, substituted
and/or added in the amino acid sequence shown in SEQ ID NO: 3, and
which protein promotes neurite outgrowth;
[0031] (6) a protein obtained by expressing a gene of the above
item (5);
[0032] (7) DNA which is cloned from a human cDNA library or a human
genomic library, and which hybridizes under stringent conditions to
DNA comprising at least part of DNA consisting of the base sequence
shown in SEQ ID NO: 1, 4, or 10;
[0033] (8) an expression plasmid which expresses either a gene of
any one of the above items (1) to (3) and (5), or DNA of the above
item (7);
[0034] (9) a transformant transformed with an expression plasmid of
the above item (8);
[0035] (10) a process for producing a recombinant protein, which
process comprises culturing a transformant of the above item (9),
and recovering the recombinant protein expressed;
[0036] (11) a peptide comprising a segment of at least six or more
amino acids of a protein of the above item (4) or (6);
[0037] (12) a pep tide of the above item (11) which promotes
neurite outgrowth;
[0038] (13) a peptide of the above item (11) characterized in that
it contains the glutamic acid residue at position 204 of the amino
acid sequence shown in SEQ ID NO: 3 or an amino acid residue
corresponding to the position of said glutamic acid residue;
[0039] (14) an antisense nucleotide, or chemically modified variant
thereof, which is directed against a segment of at least eight or
more bases of a gene of any one of the above items (1) to (3), or
of DNA of the above item (7);
[0040] (15) an antisense nucleotide, or chemically modified variant
thereof, of the above item (14), characterized in that it inhibits
expression of a protein of the above item (4);
[0041] (16) an antibody against a protein of the above item (4) or
(6), or against a peptide of any one of the above items (11) to
(13);
[0042] (17) a pharmaceutical agent comprising, as an active
ingredient, a gene of any one of the above items (1) to (3) and
(5), DNA of the above item (7), a protein of the above item (4) or
(6), a peptide of any one of the above items (11) to (13), an
antisense nucleotide or chemically modified variant thereof of the
above item (14) or (15), or an antibody of the above item (16);
[0043] (18) a method of screening for Semaphorin W antagonists,
characterized in that it employs a protein of the above item
(4);
[0044] (19) Semaphorin W antagonist obtained by the screening
method of the above item (18);
[0045] (20) Semaphorin W antagonist of the above item (19) which
comprises a protein of the above item (6), a peptide of any one of
the above items (11) to (13), or an antibody of the above item
(16);
[0046] (21) a CNS-neuron regeneration promoter, characterized in
that it contains at least one of the antisense nucleotides or
chemically modified variants thereof of the above item (14) or
(15), or Semaphorin W antagonists of the above item (19) or
(20);
[0047] (22) a neurite outgrowth inhibitor for PNS-neuron,
characterized in that it contains at least one of the proteins of
the above item (4); and
[0048] (23) a transgenic animal in which either a gene of any one
of the above items (1) to (3) and (5), or DNA of the above item (7)
has been artificially inserted into its chromosome, or has been
knocked out.
MODE FOR CARRYING OUT THE INVENTION
[0049] The 1st embodiment of the present invention is (a) a gene
encoding Semaphorin W protein which comprises the amino acid
sequence shown in SEQ ID NO: 3, or (b) a gene encoding a protein
which comprises an amino acid sequence wherein one or more amino
acids are deleted, substituted and/or added in the above amino acid
sequence shown in SEQ ID NO: 3 and which protein inhibits neurite
outgrowth. The 2nd embodiment of the present invention is (c) a
gene comprising Semaphorin W DNA which comprises the base sequence
shown in SEQ ID NO: 1 or 2, or (d) a gene comprising DNA which
hybridizes under stringent conditions to the above DNA comprising
the base sequence shown in SEQ ID NO: 1 or 2 and which encodes a
protein inhibiting neurite outgrowth, or (e) a gene comprising DNA
of the above item (d) which comprises the base sequence shown in
SEQ ID NO: 4 or 5 and/or the base sequence shown in SEQ ID NO: 10.
These genes are explained below in order.
[0050] 1) Gene Encoding Semaphorin W (Semaphorin W Gene)
[0051] Of the above-mentioned genes, "a gene encoding Semaphorin W
protein which comprises the amino acid sequence shown in SEQ ID NO:
3" or "a gene comprising Semaphorin W DNA which comprises the base
sequence shown in SEQ ID NO: 1 or 2" is a gene encoding rat
Semaphorin W. Among these genes, the DNA comprising the base
sequence shown in SEQ ID NO: 2 corresponds to the open reading
frame of the rat Semaphorin W gene shown in SEQ ID NO: 1. These
genes may be cloned, as described in Example 3, by screening a cDNA
library derived from rat CNS tissues or a genomic library using a
probe (for example, a DNA probe having the base sequence shown in
SEQ ID NO: 7) prepared on the basis of the sequence of "T09073"
found in EST database. Particular techniques for such cloning may
be found in the standard texts such as "Molecular Cloning", 2nd
ed., Cold Spring Harbor Laboratory Press (1989). The base sequence
of the cloned DNA may also be determined by conventional methods,
for example, using a sequencing kit commercially available.
[0052] Alternatively, after publication of the base sequence of rat
Semaphorin W cDNA of the present invention, one skilled in the art
can also easily clone the gene encoding rat Semaphorin W by using
part of said cDNA as a probe or PCR primer, without using cloning
methods described above.
[0053] 2) Gene Encoding Modified Protein of Semaphorin W
[0054] Of the above-mentioned genes, "a gene encoding a protein
which comprises an amino acid sequence wherein one or more amino
acids are deleted, substituted and/or added in the amino acid
sequence shown in SEQ ID NO: 3 and which protein inhibits neurite
outgrowth" refers to a gene encoding a so-called "modified
proteins" of Semaphorin W which inhibits neurite outgrowth. Those
skilled in the art may easily obtain a gene encoding such protein,
for example, by site-directed mutagenesis (Methods in Enzymology,
100, 448- (1983)) or PCR method ("Molecular Cloning", 2nd ed.,
Chapter 15, Cold Spring Harbor Laboratory Press (1989); "PCR A
Practical Approach", IRL Press, 200-210 (1991)). In this context,
the number of amino acid residues to be deleted, substituted and/or
added is to be such a number that permits the deletion,
substitution and/or addition by well-known methods such as
site-directed mutagenesis described above.
[0055] For the purpose of the present invention, the phrase
"inhibiting neurite outgrowth" means that the protein has the
collapse activity for growth cone of neuron, or that the protein
has the neurite-outgrowth inhibition activity. These activities may
be measured with a test substance such as an expression product of
DNA encoding Semaphorin W or modified protein thereof, for example,
in the following manner.
[0056] Since Semaphorin W is a membrane protein, it exists in the
cell membrane of the cells transformed with Semaphorin W gene. The
activities of the above test substance may, therefore, easily be
measured by using the membrane fraction of the transformed cells as
a test material.
[0057] Examples of activity measurement include measurement of
collapse activity for growth cone of neuron (M. Igarashi et al.,
Science, vol. 259, pp. 77-79 (1993)), or measurement of
neurite-outgrowth inhibition activity (e.g., J. A. Davies et al.,
Neuron, vol. 2, pp. 11-20 (1990) and M. Bastmeyer, J. Neurosci.,
vol. 11, pp. 626-640 (1991)). A method of measuring the growth-cone
collapse activity is described in detail in literature (M. Igarashi
et al., Science, vol. 259, pp. 77-79 (1993)). Briefly, the
measurement may be carried out by a method in which cells
expressing a test substance such as Semaphorin W is homogenized,
and the homogenate containing the cell membrane fraction or the
purified membrane fraction is used (E. C. Cox et al., Neuron, vol.
2, pp. 31-37 (1990)), or by a method in which a protein extracted
from the membrane fraction is used as such (Neuron, vol. 2, pp.
21-29 (1990)) or is used as a test material after reconstituting it
in a liposome (C. E. Bandtlow, Science, vol. 259, pp. 80-84
(1993)), or by a method in which a solubilized protein modified not
to bind the cell membrane is used (Neuron vol. 18, pp. 383-396
(1997)). In order to measure the growth-cone collapse activity in
practice using these materials, a test substance such as Semaphorin
W in one of the forms as describe above is added to neurons
cultured under conventional conditions (e.g., "Culturing, Nerve
Cells" edited by Banker et al., MIT Press (1991)) in a chamber
coated with a substance promoting the neurite outgrowth and the
growth-cone formation such as laminin, collagen, polylysine or
polyornithine. After the addition, when a sufficient time has
passed to induce collapse of growth cone (typically from 30 minutes
to one hour after the addition), those neurons are fixed with 1%
glutaraldehyde or the like, and the number of the growth cones
which have been collapsed is counted under a microscope. In this
measurement, it is important that another sample is used as a
control, which is prepared from cells not expressing the test
substance such as Semaphorin W according to the completely same
procedures as those used for the test substance-expressing cells.
Typically, normalization of the samples is conducted on the basis
of the total amounts of protein included within the samples. To
measure the neurite-outgrowth inhibition activity, part of the
surface of a micropore filter or a culture container made of glass
or plastics is coated with a test substance such as Semaphorin W
prepared as described above, and the activity is indicated, for
example, by the inability of neurons cultured under conventional
conditions to adhere to the coated area, or by a remarkable
decrease in the rate of neurite outgrowth on the coated area, or by
the inability of invasion of growing neurites from the outside of
the coated area into the coated area because of its stopping on the
border between the coated and non-coated areas or its avoidance
from the coated area. Furthermore, a stripe assay (Development 101,
685-696 (1987)), a modification of the above method in which the
surface is coated in stripes alternately using two kinds of test
substances, may also be used to measure the neurite outgrowth
inhibition activity. When a cluster of cells expressing a test
substance is co-cultured with neurons in a collagen gel, the
inability of outgrowing neurite to enter the cluster of cells
expressing the test substance may also be used as an indicator (A.
Sophia et al., Cell, vol. 81, 621-629 (1995)).
[0058] Both of CNS and PNS neurons may be used as the cells for the
above activity measurements. As described in the section
"BACKGROUND ART", CNS in adult mammals naturally contains a large
amount of regeneration (outgrowth) inhibitor. It is, therefore,
extremely difficult to measure in vivo an inhibitory effect on
neurite outgrowth of CNS-neuron, and such inhibitory effect is
usually measured by an in vitro method as described above. Since
these in vitro methods each have individual characteristics, it is
preferred to use more than one method to confirm the activity.
Although preferred neurons used for a measurement of the activity
are CNS-neurons such as motor neurons in spinal cord or motor
cortex, or retinal ganglion cells, PNS-neurons of superior cervical
ganglion and dorsal root ganglion may also be used because NI35/250
known as a CNS-neuron regeneration inhibitor has proved to have
effects such as neurite-growth inhibition and growth-cone collapse
activities also on such PNS-neurons (J. Cell Biol., 106, 1281-1288
(1988); Science, 259, 80-83 (1993)).
[0059] Examples of the modified proteins of this embodiment may
include modified proteins of human or rat Semaphorin W, and more
particularly, include the modified proteins as described below.
[0060] Based on the structural comparison of known Semaphorins,
most of the conserved amino acids are located in the semaphorin
domain, suggesting that these conserved amino acids are essential
for expression of the Semaphorin activity. Furthermore, the present
inventors have found that a modified Sema III protein in which
aspartic acid residue at position 198 in its semaphorin domain has
been substituted with glycine did not have the growth-cone collapse
activity (see Reference example 1 below). Accordingly, the aspartic
acid residue at position 198 of Sema III is believed essential for
expression of the activity. When the amino acid residues
corresponding to this position were compared among known
Semaphorins, it was shown that they are extremely well conserved
and are all aspartic acid residue with a few exceptions in which
glutamic acid residue is located at this position. It is,
therefore, believed that the amino acid residue at this position is
also essential for expression of the activity of Semaphorins other
than Sema III. In Semaphorin W of the present invention, the amino
acid residue corresponding to the position 198 of Sema III is
presumed to be glutamic acid residue at position 204 of the amino
acid sequence shown in SEQ ID NO: 3, while in the after-mentioned
amino acid sequence of human Semaphorin W shown in SEQ ID NO: 6, it
is presumed to be glutamic acid residue at position 16.
[0061] Considering the above information, it is desirable to make
the above-described deletions, substitutions and/or additions of
amino acids at positions other than those conserved among
Semaphorins, in order to retain the activity of Semaphorin W in
modified proteins. Particularly, it is desirable not to modify the
glutamic acid residue at position 204 in rat Semaphorin W shown in
SEQ ID NO: 3 and the glutamic acid residue at position 16 in human
Semaphorin W shown in SEQ ID NO: 6. On the other hand, in order to
displace the amino acid residues conserved among Semaphorins while
retaining the activity of Semaphorin W, it is desirable to
substitute an amino acid residue having a similar side chain for
the amino acid residue to be substituted. By substituting such
amino acid having a similar side chain for a conserved amino acid,
it may be possible to produce a modified protein which has an
enhanced activity of Semaphorin W. Such modified protein having the
enhanced activity is highly suitable as a neurite-outgrowth
inhibitor for PNS-neuron as will be described hereinafter in the
section of the 22nd embodiment of the present invention.
[0062] In the above-noted embodiment, "a conserved amino acid"
refers to an amino acid located at a position at which more than
50% of Semaphorin genes shown in FIG. 2 of Cell, 75, 1389-1399
(1993) or FIG. 1 of Neuron, 14, 941-948 (1995) encode the same
amino acid.
[0063] 3) DNA Hybridizing Under Stringent Conditions to Semaphorin
W Gene
[0064] Of the above-mentioned DNAs, "a gene comprising DNA which
hybridizes under stringent conditions to DNA comprising the base
sequence shown in SEQ ID NO: 1 or 2 and which encodes a protein
inhibiting neurite outgrowth" refers to a gene which hybridizes
under stringent conditions to rat Semaphorin W gene comprising the
base sequence shown in SEQ ID NO: 1 or 2, including all the
Semaphorin W genes derived from mammals such as human and
mouse.
[0065] As used herein, "a gene which hybridizes under stringent
conditions" refers to such a gene that hybridizes to the above rat
Semaphorin W gene, for example, when subjected to hybridization at
a formamide concentration of about 45% (v/v) and a salt
concentration of about 5.times.SSPE and at a temperature around
42.degree. C., and washed at a salt concentration of about
2.times.SSPE and at a temperature around 42.degree. C. Cloning of
such genes may be achieved, for example, by screening cDNA or
genomic libraries prepared from various animal tissues using all or
part of DNA shown in SEQ ID NO: 1 as a probe. An example of cDNA
library used herein may be a cDNA library prepared from mRNAs
derived from human CNS tissues. Such screening may be carried out
consulting to the standard texts such as "Molecular Cloning" (2nd
ed., Cold Spring Harbor Laboratory Press (1989)).
[0066] Specific examples of the gene of this embodiment may include
all the Semaphorin W genes of mammal and avian. Between mammals or
between mammal and avian, homologous genes have quite similar
sequences, and usually more than 80%, in many cases more than 90%,
of the base sequence are common to each other. All the mammal and
avian Semaphorin W genes, therefore, correspond to this embodiment.
In other words, those genes which have a homology of 80% or above
are included in this embodiment, and those having a homology of 90%
or above are preferred.
[0067] One specific example of a gene of this embodiment is human
Semaphorin W gene containing the base sequence shown in SEQ ID NO:
4 or 5 and/or the base sequence shown in SEQ ID NO: 10. The base
sequences shown in SEQ ID NOs: 4 and 5 represent DNAs encoding 587
amino acids (SEQ ID NO: 6) of the C-terminal region of human
Semaphorin W, and the base sequence shown in SEQ ID NO: 5
corresponds to the open reading frame of the base sequence shown in
SEQ ID NO: 4. The base sequence shown in SEQ ID NO: 10 represents
DNA encoding 111 amino acids (SEQ ID NO: 11) of the N-terminal
region of human Semaphorin W. Cloning of these human Semaphorin W
DNA may be achieved by the screening method described above, or may
also be achieved by, for example, synthesizing primers on the basis
of the base sequence of rat Semaphorin W shown in SEQ ID NO: 1 and
carrying out PCR reaction using cDNAs prepared from mRNAs derived
from a human CNS tissue as template (see "PCR" (1991) edited by
McPherson et. al., IRL Press). Similarly, the same may be cloned by
using an EST clone (Genome Systems, USA).
[0068] As described above, "the base sequence shown in SEQ ID NO: 4
or 5" and "the base sequence shown in SEQ ID NO: 10" are partial
base sequences of human Semaphorin W. However, one skilled in the
art can easily clone the human Semaphorin W gene in full length by
screening a human cDNA or human genomic library with DNA comprising
part or all of said base sequence as a probe, or by using PCR
method, and determine its base sequence in the same method as
described above. Accordingly, such full-length human Semaphorin W
gene may be one specific example of the gene of this
embodiment.
[0069] The 3rd embodiment of the present invention is a gene
comprising DNA which hybridizes under stringent conditions to DNA
comprising the base sequence shown in SEQ ID NO: 7, and which
encodes a protein having a semaphorin domain.
[0070] In the above description, "DNA comprising the base sequence
shown in SEQ ID NO: 7" is DNA of which complete base sequence was
determined on the basis of the sequence information as to the DNA
"T09073" encoding, as a partial sequence, a sequence
(Gln-Asp-Pro-Val-Cys-Ala-Trp) similar to the sequence consisting of
seven amino acids well conserved among Semaphorins (Gln (or
Arg)-Asp-Pro-Tyr-Cys-Ala (or Gly)-Trp), and is a DNA fragment
corresponding to the region from position 1561 to position 1756 in
the base sequence of rat Semaphorin W shown in SEQ ID NO: 1, or the
region from position 922 to position 1117 in the base sequence of
human Semaphorin W shown in SEQ ID NO: 4.
[0071] The "stringent conditions" as used herein refers to those
conditions described above in the section of the 2nd embodiment of
the present invention.
[0072] Cloning of DNAs of this embodiment is achieved by, for
example, hybridization with DNA of SEQ ID NO: 7, and specifically
may be carried out, for example, according to the procedures
described in TINS, 15, 319-323 (1992) and references cited therein,
and more specifically according to the following procedures.
[0073] That is, the cloning may be achieved by screening cDNA or
genomic libraries prepared from various animal tissues using DNA
comprising the base sequence shown in SEQ ID NO: 7 as a probe. The
screening may be carried out according to, for example, the
procedures as those described in Example 3. Preferred cDNA
libraries are those derived from an adult tissue of CNS, and cDNA
libraries derived from hippocampus, corpus striatum, and cerebellum
are more preferred. As described above, the conditions shown in
Example 3 or those described in TINS, 15, 319-323 (1992) and
references cited therein may be used for the hybridization.
[0074] The gene of this embodiment is also "DNA which encodes a
protein having a semaphorin domain". As used herein, "semaphorin
domain" refers to a domain consisting of 300-600 amino acid
residues more than 20% of which are identical to those amino acids
constituting the semaphorin domain of any one of ten known
Semaphorins (G-Sema I, T-Sema, I, D-Sema II, H-Sema III,
C-Collapsin, Sem A, Sem B, Sem C, Sem D, Sem E) described in, for
example, Cell, 75, 1389-1399 (1993) or Neuron, 14, 941-948 (1995).
Those proteins having a semaphorin domain more than 30% of which
amino acids are identical to those amino acids in any one of the
known Semaphorins are particularly preferred. The identity of amino
acids is determined by comparison using, for example, DNASIS Ver.
2.0 (HITACHI Software Engineering) under conditions of ktup=1 and
cutoff=1. More preferred proteins are those in which ten or more
cysteines, particularly twelve or more cysteines, of the thirteen
cysteines conserved in semaphorin domains of the ten known
Semaphorins (for example, those cysteines marked in FIG. 1 on page
942 of Neuron, 14, 941-948 (1995)) are conserved.
[0075] Examples of such gene of this embodiment may include
Semaphorin genes which hybridize under stringent conditions to DNA
comprising the base sequence shown in SEQ ID NO: 7 and which
contain semaphorin domains and exhibit the neurite-outgrowth
inhibition activity, or all of the Semaphorin W genes of mammal and
avian like the 2nd embodiment described above.
[0076] The 4th embodiment of the present invention is a protein
obtained by expressing a gene of any one of the 1st to 3rd
embodiments described above.
[0077] Typical examples of protein included in this embodiment are
rat Semaphorin W having the amino acid sequence shown in SEQ ID NO:
3, and human Semaphorin W having the amino acid sequence shown in
SEQ ID NO: 6 or 11. Semaphorin W has a signal sequence at its
N-terminus, and said signal sequence undergoes processing to be
removed during its transfer to membrane, resulting in mature
Semaphorin W. The mature form of rat Semaphorin W is presumed to
consist of the amino acid sequence beginning at amino acid 40 of
the amino acid sequence shown in SEQ ID NO: 3, and the mature form
of human Semaphorin W is presumed to consist of the amino acid
sequence beginning at amino acid 28 of the amino acid sequence
shown in SEQ ID NO: 11. Since such mature form of Semaphorin W or
modified protein thereof may also be obtained by expressing a gene
of any one of the 1st to 3rd embodiments described above, it is
also included in this embodiment.
[0078] Preparation of the proteins of this embodiment may be
achieved, for example, by ligating a cloned rat Semaphorin W cDNA
into a known expression vector such as pET or pCDM8, and
introducing it into appropriate host cells to express and produce
Semaphorin W. The host cells may be prokaryotic or eukaryotic. For
example, Escherichia coli strains or animal cell lines are already
conventionally used for such purpose and are commercially or
publicly available. Examples of animal host cells include COS-1,
COS-7, CHO cells and the like.
[0079] To transform appropriate animal host cells with an
expression plasmid, a known procedure such as DEAE-dextran method
(Current Protocols in Molecular Biology, F. M. Ausubel et al. ed.,
John Wiley & Sons (1987)) may be used. As confirmed in Examples
6 and 7, Semaphorin W exists in the cell membrane faction which
contains a sufficient amount of Semaphorin W to be directly used in
various assays. Therefore, various assays for activities of a
protein of this embodiment may easily be carried out using a cell
membrane fraction prepared from appropriate cells.
[0080] Furthermore, a protein of this embodiment may be purified
by, for example, affinity purification using Semaphorin
W-recognizing antibodies described hereinafter in the section of
the 16th embodiment of the present invention, or conventional
column chromatography.
[0081] The 5th embodiment of the present invention is a gene
encoding a protein which comprises an amino acid sequence wherein
one or more amino acids are deleted, substituted and/or added in
the amino acid sequence shown in SEQ ID NO: 3 and which protein
promotes neurite outgrowth. The 6th embodiment of the present
invention is a protein obtained by expressing a gene of the 5th
embodiment of the present invention.
[0082] In the genes of the 5th embodiment described above,
deletions, substitutions and/or additions may be introduced in the
procedures similar to those used for a gene encoding a modified
protein of the 1st embodiment of the present invention. Similarly,
the promotion effect on neurite outgrowth may easily be measured,
for example, by adding Semaphorin W in an assay system for
Semaphorin W activity described above in the section of the 1st
embodiment of the present invention and further adding thereto a
test substance (i.e., a candidate modified Semaphorin W protein).
For details, see the descriptions in the section of the 18th
embodiment of the present invention.
[0083] Specific examples of the proteins of the 6th embodiment of
the present invention may be modified rat or human Semaphorin W
proteins of which neurite-outgrowth inhibition activity has been
eliminated. Such modified protein lacking the activities of
Semaphorin W is expected to exert the promotion effect on
neurite-outgrowth, when it binds to receptors for Semaphorin W in
place of Semaphorin W, or to Semaphorin W itself, by inhibiting the
binding of Semaphorin W to the receptors. As described above in the
section of the 1st embodiment of the present invention, it has been
suggested that the active site of Semaphorin may be located in the
semaphorin domain, and particularly, it may probably be located at
glutamic acid residue at position 204 in rat Semaphorin W shown in
SEQ ID NO: 3, or glutamic acid residue at position 16 in human
Semaphorin W shown in SEQ ID NO: 6. Accordingly, in order to
eliminate the Semaphorin W activity from the modified protein, it
is desirable to introduce the deletions, substitutions and/or
additions to the conserved amino acids in said semaphorin domain,
preferably to the glutamic acid residue at position 204 of the
amino acid sequence shown in SEQ ID NO: 3, or to the glutamic acid
residue at position 16 of the amino acid sequence shown in SEQ ID
NO: 6. In such cases, those substitutions in which an amino acid
having a side chain of a distinct nature is substituted for the
original amino acid are desirable. Also in the cases of Semaphorin
W other than that from human or rat, modifications are preferably
made on the amino acid residue at the position corresponding to
this position 204, that is, on the amino acid residue at the
position which corresponds to position 204 in the amino acid
sequence shown in SEQ ID NO: 3, or to position 16 of Semaphorin W
shown in SEQ ID NO: 6 when the amino acid sequence of said
Semaphorin W is aligned with that of rat or human Semaphorin W so
as to give the maximum identity.
[0084] Since the proteins of the 6th embodiment of the present
invention promote neurite outgrowth as described above, some of
these proteins will serve as CNS-neuron regeneration promoters as
described hereinafter in the section of the 21st embodiment.
[0085] The 7th embodiment of the present invention is DNA which is
cloned from a human cDNA library or a human genomic library, and
which hybridizes under stringent conditions to DNA comprising at
least part of rat or human Semaphorin W DNA shown in SEQ ID NO: 1,
4, or 10.
[0086] Methods of cloning are described in detail in, for example,
"Molecular Cloning", 2nd ed., Cold Spring Harbor Laboratory Press
(1989), and specifically include, for example, methods employing
hybridization or PCR. Although a preferred library used herein is a
genomic library derived from human, a cDNA library derived from
CNS-neuron in adult human may also be used. Those methods employing
hybridization may be carried out according to, for example, TINS,
15, 319-323 (1992) and references cited therein. Those methods
employing PCR may be carried out according to, for example, "PCR"
edited by McPherson et al., IRL Press (1991).
[0087] The DNAs thus cloned include not only the full length DNA
but also its DNA fragments comprising more than 200 bases, or
single-stranded forms (coding strands or complementary stands
thereof) of said DNA fragments. Examples of DNA of the 7th
embodiment of the present invention may include chromosomal DNAs
containing 5' and/or 3' transcriptional control regions, noncoding
sequences of exons, introns or the like, in addition to regions
encoding amino acids. Such sequences which do not encode any amino
acids are also quite useful, for example, in developing a medicine
using antisense techniques described hereinafter.
[0088] The 8th embodiment of the present invention is an expression
plasmid which expresses either a gene of the 1st, 2nd, 3rd or 5th
embodiment, or DNA of the 7th embodiment of the present invention.
The 9th embodiment of the present invention is a transformant
transformed with an expression plasmid of the 8th embodiment.
Furthermore, the 10th embodiment of the present invention is a
process for producing a recombinant protein which process comprises
culturing a transformant of the 9th embodiment and recovering the
recombinant protein expressed. As described above in the section of
the 4th embodiment of the present invention, methods of preparing
an expression plasmid and a transformant, and methods of producing
a recombinant protein, per se, are all well known to those skilled
in the art.
[0089] The 11th embodiment of the present invention is a peptide
comprising a segment of at least 6 amino acids in a protein of the
4th or 6th embodiment of the present invention. In this context,
the limitation "at least 6 amino acids" is based on the fact that a
minimal size of peptide capable of forming a stable structure
consists of 6 amino acids, and preferred peptides are those
consisting of 8 or more amino acids, more preferably of about 10-20
amino acids. A short peptide such as those consisting of about
10-20 amino acids can be synthesized on a peptide synthesizer,
while a longer peptide can be obtained by preparing DNA through
conventional genetic engineering, and expressing it in, for
example, animal cells as described above. The peptide thus prepared
can also be modified by conventional methods.
[0090] These peptides can be applied to pharmaceutical agents
described hereinafter in the section of the 12th and 13th
embodiments, and can also be used for producing antibodies.
[0091] The 12th embodiment of the present invention is a peptide of
the 11th embodiment of the present invention which promotes neurite
outgrowth. Such peptide may be prepared by the methods described
above in the section of the 11th embodiment of the present
invention. The promotion effect on neurite outgrowth can also be
easily measured as described above in the section of the 5th
embodiment of the present invention by adding Semaphorin W to an
activity assay system described above in the section of the 1st
embodiment of the present invention and further adding thereto a
test substance (i.e., a candidate peptide of Semaphorin W). For
details, see the descriptions in the section of the 18th embodiment
of the present invention.
[0092] Examples of these peptides may be peptides which have lost
the neurite-outgrowth inhibition activity of Semaphorin W. A
peptide lacking Semaphorin W activity is expected to exert its
neurite-outgrowth promotion effect, when it binds to receptors for
Semaphorin W or to Semaphorin W itself, by inhibiting the binding
of Semaphorin W to the receptors. Some of such peptides will serve
as CNS-neuron regeneration promoters as described hereinafter in
the section of the 21st embodiment.
[0093] The 13th embodiment of the present invention is a peptide of
the 11th embodiment of the present invention, characterized in that
it contains the glutamic acid residue at position 204 of the amino
acid sequence shown in SEQ ID NO: 3 or an amino acid residue
corresponding to the position of said glutamic acid residue. Such
peptides may be prepared by the methods described above in the
section of the 11th embodiment.
[0094] As described above in the section of the 1st embodiment of
the present invention, the glutamic acid residue at position 204 of
rat Semaphorin W shown in SEQ ID NO: 3 (in the case of human
Semaphorin W shown in SEQ ID NO: 6, the glutamic acid residue at
position 16) seems essential for expression of the Semaphorin W
activity. Since this amino acid residue may possibly be involved in
the binding between Semaphorin W and its receptors, a peptide of
this embodiment containing said amino acid residue or an amino acid
residue at the position corresponding to that of said amino acid
residue may interfere the neurite-outgrowth inhibition activity of
Semaphorin W by binding to receptors for Semaphorin W or to
Semaphorin W itself and thereby inhibiting the binding of
Semaphorin W to the receptors, resulting in promotion of neurite
outgrowth. Some of the peptides having such effect will serve as
CNS-neuron regeneration promoters as described hereinafter in the
section of the 21st embodiment. Such neurite-outgrowth promotion
activity can easily be measured as described above in the section
of the 5th embodiment of the present invention by adding Semaphorin
W to an activity assay system described in the section of the 1st
embodiment of the present invention, and further adding thereto a
test substance (i.e., a candidate peptide of Semaphorin W). For
details, see the descriptions in the section of the 18th embodiment
of the present invention.
[0095] In this embodiment, "an amino acid corresponding to the
position of said glutamic acid residue" refers to an amino acid
residue which is located at the position corresponding to position
204 in rat Semaphorin W, when the amino acid sequence of the
protein of the 4th or 6th embodiment of the present invention is
aligned with the amino acid sequence of rat Semaphorin W shown in
SEQ ID NO: 3 so as to give the maximum identity. Accordingly, "a
peptide characterized in that it contains an amino acid residue
corresponding to the position of said glutamic acid residue" refers
to a peptide which comprises such amino acid residue at the
position corresponding to position 204 in rat Semaphorin W as well
as flanking amino acids on either side thereof.
[0096] The 14th embodiment of the present invention is an antisense
nucleotide, or chemically modified variant thereof, which is
directed against a segment of at least eight or more bases in a
gene of any one of the 1st to 3rd embodiments, or in DNA of the 7th
embodiment of the present invention.
[0097] As used herein, "antisense nucleotide" refers to a so-called
antisense oligonucleotide, antisense RNA, or antisense DNA, and it
may be artificially prepared using a synthesizer, or may be
obtained by, for example, expressing a gene in the direction
opposite to the usual case (i.e., in the antisense direction). For
details, see the descriptions in the section of the 21st embodiment
of the present invention.
[0098] These antisense nucleotides are used for inhibiting the
expression of Semaphorin W as described hereinafter in the section
of the 15th embodiment of the present invention, and are also
useful as laboratory reagents for, for instance, in situ
hybridization. In the present invention, "a chemically modified
variant" specifically refers to such a variant that is chemically
modified so as to enhance the transferability of the antisense
nucleotide into cells or the stability of the antisense nucleotide
in the cells. Examples of such chemically modified variant are
phosphorothioate, phosphorodithioate, alkyl phosphotriester, alkyl
phosphonate, alkyl phosphoamidate and the like derivatives
("Antisense RNA and DNA", WILEY-LISS, 1992, pp. 1-50, J. Med.
Chem., 36, 1923-1937 (1993)). The chemically modified variant may
be prepared according to, for example, the reference cited just
above.
[0099] The 15th embodiment of the present invention is an antisense
nucleotide, or chemically modified variant thereof, of the 14th
embodiment described above, characterized in that it inhibits the
expression of a protein of the 4th embodiment of the present
invention.
[0100] mRNAs produced by usual gene transcription are
sense-strands, and the antisense nucleotides or chemically modified
variants thereof can bind to such sense-strand mRNAs in cells to
inhibit the expression of those particular genes. Therefore, the
above antisense nucleotides or chemically modified variants thereof
can inhibit the expression of Semaphorin W, and can thereby inhibit
the activity of Semaphorin W. Some of antisense nucleotides or
chemically modified variants thereof having such effect will serve
as CNS-neuron regeneration promoters as described hereinafter in
the section of the 21st embodiment of the present invention.
[0101] It can easily be determined whether a particular antisense
nucleotide prepared, or a chemically modified variant thereof, has
a desired inhibitory effect or not, by directly introducing the
antisense oligonucleotide itself or by introducing a gene which
produces said antisense RNA when transcribed, into cells expressing
Semaphorin W, and then determining whether the amount of the
expressed Semaphorin W is decreased or not.
[0102] Examples of antisense nucleotide having such inhibitory
effect are those oligonucleotides having sequences complementary to
either the coding region or the 5' noncoding region of Semaphorin
gene of the above-described embodiments, and those antisense
nucleotides having sequences complementary to the transcription
initiation site, translation initiation site, 5' noncoding region,
exon-intron junction region, or 5' CAP region are desirable.
[0103] The 16th embodiment of the present invention is an antibody
against a protein of the 4th or 6th embodiment, or against a
peptide of any one of the 11th to 13th embodiments. Such antibody
can easily be produced by using mouse or rabbit according to the
procedures described in, for example, "Current Protocols in
Immunology", pp. 2.4.1-2.6.6 (1992, J. E. Coligan ed.). Monoclonal
antibodies can also be easily produced by the methods described in
the above-mentioned reference. Such antibodies may be used in
affinity chromatography or screening of cDNA libraries, and as
pharmaceutical or diagnostic agents, or laboratory reagents. Some
of such antibodies have the activity of neutralizing Semaphorin W.
Such neutralizing activity can easily be determined, as described
above in the section of the 5th embodiment of the present
invention, by adding Semaphorin W to an activity assay system
described in the section of the 1st embodiment of the present
invention, and further adding thereto a test substance (i.e., a
candidate antibody against Semaphorin W). Some of such neutralizing
antibodies will serve as CNS-neuron regeneration promoters as
described hereinafter in the section of the 21st embodiment of the
present invention.
[0104] The 17th embodiment of the present invention is a
pharmaceutical agent comprising, as an active ingredient, any one
of all of the genes (DNAs), proteins, peptides, antisense
nucleotides or chemically modified variants thereof, and antibodies
of the present invention.
[0105] Among such pharmaceutical agents, CNS-neuron regeneration
promoters and neurite-outgrowth inhibitors for PNS-neuron will be
described in the sections of the 21st and 22nd embodiments of the
present invention, respectively. See, therefore, the sections of
the 21st and 22nd embodiments for such applications.
[0106] It is being demonstrated in recent years that certain
Semaphorins play important roles not only in the nervous system but
also in non-nervous systems. For example, it has been suggested
that Semaphorin may probably act in inhibiting the growth of
cardiac muscles (Nature, 383, 525-528 (1996)). Also in the immune
system, a certain Semaphorin has been suggested to be involved in
aggregation and survival of B lymphocytes (Proc. Natl. Acad. Sci.
USA, 93, 11780-11785 (1996)). It has also been suggested more
recently that a certain Semaphorin may play some role in the immune
reactions in rheumatism (B.B.R.C., 234, 153-156 (1997)).
Furthermore, involvement of Semaphorins in lung cancer has also
been suggested (Proc. Natl. Acad. Sci. USA, 93, 4120-4125
(1996)).
[0107] Accordingly, Semaphorin W of the present invention or its
modified proteins, peptides, antisense nucleotides and the like are
expected to be useful as antiallergic agents, immunosuppressive
agents, or anti-tumor agents. For specific directions for use,
dosage and the like of such pharmaceutical agents, see the sections
of the 21st and 22nd embodiments.
[0108] The 18th embodiment of the present invention is a method of
screening for Semaphorin W antagonists, characterized in that it
employs a protein of the 4th embodiment of the present invention.
As used herein, "Semaphorin W antagonist" refers to a substance
which inhibits, for example, the neurite-outgrowth inhibition
activity of Semaphorin W.
[0109] The screening is conducted by adding Semaphorin W to an
assay system for Semaphorin W activity described in the section of
the 1st embodiment of the present invention, and further adding
thereto a test substance. In particular, inhibition of the
Semaphorin W activity resulted from the addition of the test
substance to the culture medium throughout the incubation period or
only temporarily in the incubation period can be used as an
indicator in the Semaphorin W activity assay carried out with added
Semaphorin W. It is also important to confirm that the test
substance alone does not influence the survival, neurite outgrowth
and the like of neurons at the same concentration. When both of
these requirements are fulfilled, one can consider the test
substance as a Semaphorin W antagonist. Although it is preferred to
prepare in advance the test substance in the form of aqueous
solution, an organic solvent such as DMSO may also be used as a
solvent. In any cases, it is important to minimize the volume of
solvent so as to exclude any effects of the solvent on neurons.
Specifically, the volume to be added should be less than an equal
volume, preferably less than {fraction (1/10)} volume, and more
preferably less than {fraction (1/100)} volume relative to the
culture medium. Some of Semaphorin W antagonists thus obtained will
serve as CNS-neuron regeneration promoters as described hereinafter
in the section of the 21st embodiment of the present invention.
[0110] The 19th embodiment of the present invention is Semaphorin W
antagonist obtained by the screening method of the 18th embodiment
of the present invention. Such antagonist may have any structure
and any form, provided that it inhibits the activity of Semaphorin
W.
[0111] The 20th embodiment of the present invention is Semaphorin W
antagonist of the 19th embodiment which comprises a protein of the
6th embodiment, a peptide of any one of the 11th to 13th
embodiments, or an antibody of the 16th embodiment of the present
invention. In other words, it is a protein of the 6th embodiment, a
polypeptide of any one of the 11th to 13th embodiments, or an
antibody of the 16th embodiment of the present invention which has
an effect of inhibiting the activity of Semaphorin W. Such
antagonists can be identified by subjecting one of the above
substances to the screening system of the 18th embodiment of the
present invention, and some of the antagonists thus identified will
serve as CNS-neuron regeneration promoters as described below in
the section of the 21st embodiment of the present invention.
[0112] The 21st embodiment of the present invention is a CNS-neuron
regeneration promoter, characterized in that it contains at least
one of the antisense nucleotides or chemically modified variants
thereof of the 14th or 15th embodiment, or Semaphorin W antagonists
of the 19th or 20th embodiment of the present invention. Since this
embodiment relates to use of such substances in "regeneration
therapy for CNS-neuron", specific directions for use, dose and the
like are described below.
[0113] 1) Antisense Nucleotide or Chemically Modified Variant
Thereof.
[0114] Application of antisense nucleotides has been attempted in
various diseases, and in recent years, it is also considered to be
applicable in neurological disorders (TINS 20, No. 8, 321-322
(1997)).
[0115] As described above in the section of the 14th or 15th
embodiment of the present invention, the antisense nucleotide or
chemically modified variant thereof of the 14th or 15th embodiment
of the present invention can be used for inhibiting expression of
Semaphorin W gene. Accordingly, such antisense nucleotide may
decrease the abundance of the Semaphorin protein, and promote
regeneration of CNS-neurons. Therapeutic methods using the
nucleotide or the variant include those in which the antisense
oligonucleotide or its chemically modified variant is administered
as such, and those in which antisense RNA is produced in cells.
[0116] In the method in which the antisense oligonucleotide or its
chemically modified variant is administered as such, a preferred
antisense oligonucleotide has a length of, for example, about 5-200
bases, more preferably 8-25 bases, and especially preferably 12-25
bases. Antisense oligonucleotide or its chemically modified variant
may be formulated by mixing it with stabilizing agent, buffer,
solvent and the like prior to its administration. Such formulation
may be co-administered with, for example, an antibiotic,
anti-inflammatory, or anesthetic agent. Although the formulation
thus prepared may be administered via various routes, it is
preferred to topically administered at a site in which neurons are
notably disordered. Usually, regeneration of neuron takes several
days to several months, and the formulation is administered every
day or every several days to several weeks during the period. To
avoid such frequent administrations, a sustained-release
mini-pellet formulation may be prepared and embedded near the
affected site. Alternatively, a formulation may be gradually and
continuously administered to the affected site by means of, for
example, an osmotic pump. The dose is typically adjusted so that
the concentration at the site of action will be 0.1 nM to 10
.mu.M.
[0117] In the method in which antisense RNA is produced in cells, a
preferred antisense RNA has a length of, for example, more than 100
bases, preferably more than 300 bases, and more preferably 500
bases or more.
[0118] The methods by which a gene expressing an antisense RNA is
introduced into a patient include an in vivo method in which the
gene is directly introduced into cells in a living body, and an ex
vivo method in which the gene is introduced into particular cells
ex vivo and the cells are returned into the body (Nikkei Science,
April, 1994, pp. 20-45; Gekkan-Yakuji, 36 (1), 23-48 (1994);
Jikkenn-Igaku-Zokan, 12 (15), 1994; and references cited therein).
An in vivo method is more preferred.
[0119] Such in vivo methods include a method employing recombinant
viruses and other methods (Nikkei Science, April, 1994, pp. 20-45;
Gekkan-Yakuji, 36 (1), 23-48 (1994); Jikken-Igaku-Zokan, 12 (15),
in its entirety (1994); and references cited therein).
[0120] The methods employing recombinant viruses may include the
methods in which a gene producing an antisense RNA is incorporated
into a virus genome of, for example, retrovirus, adenovirus,
adeno-associated virus, herpesvirus, vaccinia virus, poliovirus, or
sindbis virus, and the recombinant virus is introduced into a
living body. Among these methods, those employing retrovirus,
adenovirus or adeno-associated virus are particularly
preferred.
[0121] Other methods may include a liposome method or a lipofectin
method. The liposome method is particularly preferred.
[0122] For the ex vivo methods, a micro-injection method, the
calcium phosphate method, electroporation and the like may also be
used, besides those techniques described above.
[0123] Administration of the gene to a patient is carried out via
appropriate routes depending on, for example, the particular
disease or symptom to be treated. For example, it may be
administered intravenously, intraarterially, subcutaneously, or
intramuscularly, or directly administered into an affected site
such as neuron. For example, when spinal cord is infected with the
recombinant viruses, the expression of Semaphorin gene is inhibited
exclusively in the spinal cord. Expression of antisense
oligonucleotide of the present invention typically lasts several
days to several months, and such single infection is sufficient to
allow regeneration of neuron. The gene may also be re-infected,
when weakly expressed. When administered by an in vivo method, the
gene may be formulated in the form of, for example, a solution, and
typically it is formulated in the form of an injection containing
an antisense nucleotide as an active ingredient to which
conventional carrier and the like may be added, if necessary. In
the case of liposomes or membrane-fused liposomes (such as Sendai
virus (HVJ)-liposomes) containing an antisense nucleotide, the
liposome preparations may be in the form of a suspension, a frozen
preparation, a centrifugally-concentrated frozen preparation or the
like.
[0124] Although the amount of antisense nucleotide in the
formulation may vary depending on, for example, the disease to be
treated, the age and weight of the patient, it is typically
0.0001-100 mg, and preferably 0.001-10 mg. Such formulation is
administered once or more, and when administered more than twice,
it is desirable to administer it every day or repeatedly at
appropriate intervals.
[0125] 2) Modified Protein of Semaphorin W
[0126] As described above in the sections of the 5th and 6th
embodiments of the present invention, one can prepare a modified
Semaphorin W of which neurite-outgrowth inhibition activity on
CNS-neuron has been eliminated. When administered into a living
body, such modified protein is expected to bind to receptors for
Semaphorin W in place of Semaphorin W, resulting in inhibition of
Semaphorin W activity and promotion of regeneration of
CNS-neuron.
[0127] Such modified protein of Semaphorin W is formulated with
stabilizer, buffer, and/or diluent, and administered to a patient
for therapy. Such formulation may be administered via various
routes, and it is preferred to topically administer to the focal
site. Since regeneration of neuron typically takes several days to
several months, the formulation is administered once or more in
order to continuously inhibit Semaphorin W activity throughout the
period. When administered more than once, it is desirable to
administer it every day or repeatedly at appropriate intervals.
When administered to CNS by injection, for example, into the spinal
cord, several hundreds .mu.g to 2 g, preferably less than several
tens mg, are used per administration. To reduce the administration
frequency, it may be administered using a sustained-release
formulation or gradually administered over a long period by means
of, for example, an osmotic pump. Alternatively, it may be
administered by grafting cells expressing such modified Semaphorin
W protein into a living body.
[0128] 3) Peptide of Semaphorin W
[0129] Some of the peptides of any one of the embodiments from 11th
to 13th embodiments of the present invention suppress the neurite
outgrowth inhibition activity of Semaphorin W on CNS-neuron by
inhibiting the binding of Semaphorin W to its receptors, resulting
in promotion of CNS-neuron regeneration. Examples of peptide having
such effect include a peptide characterized in that it contains
glutamic acid residue at position 204 of rat Semaphorin W shown in
SEQ ID NO: 3 or an amino acid residue at the position corresponding
to that of said glutamic acid residue, as described above in the
section of the 13th embodiment of the present invention. The
suppression may be any one of competitive, noncompetitive,
uncompetitive, and allosteric inhibitions.
[0130] As for the methods of formulating or administering such
polypeptides, and their doses, see the above section "2) Modified
protein of Semaphorin W".
[0131] 4) Antibody Against Semaphorin W
[0132] A neutralizing antibody which neutralizes the activity of
Semaphorin W is expected to promote the regeneration therapy of
CNS-neuron by inhibiting Semaphorin W activity, when administered
into a living body.
[0133] The methods of formulating or administering such
neutralizing antibodies and their doses may be the same as
described in the above section "2) Modified protein of Semaphorin
W". Alternatively, a method in which cells producing such
monoclonal antibody are grafted directly into CNS may also be used,
as described in Nature, 343, 269-272 (1990).
[0134] The 22nd embodiment of the present invention is a neurite
outgrowth inhibitor for PNS-neuron, characterized in that it
contains at least one of the proteins of the 4th embodiment of the
present invention. Although the proteins of the 4th embodiment of
the present invention may inhibit the neurite outgrowth of
CNS-neuron, they are also expected to inhibit the neurite outgrowth
of PNS-neuron, since PNS-neuron also probably expresses receptors
for Semaphorin W, and receptors for other Semaphorins also probably
react with Semaphorin W. Accordingly, they may serve as therapeutic
agents for atopic dermatitis, pain or other diseases by virtue of
their inhibition activity on neurite outgrowth of PNS-neuron.
[0135] As for the methods of formulating or administering such
proteins, and their doses, see the above section "2) Modified
protein of Semaphorin W".
[0136] The 23rd embodiment of the present invention is a transgenic
animal in which either a gene of any one of the 1st to 3rd and 5th
embodiments, or DNA of the 7th embodiment of the present invention
has been artificially inserted into its chromosome, or has been
knocked out.
[0137] In the light of the gene information on Semaphorin W of the
present invention, one skilled in the art can quite easily produce
a transgenic animal which expresses the gene of the 1st, 4th, 7th,
or 9th embodiment of the present invention, as apparent from the
following references: "Manipulation of Mouse Embryo" edited by B.
Hogan et al., 1986, Cold Spring Harbor Laboratory; Shinichi Aizawa,
"Gene Targeting", 1995, Yodosha, etc. Accordingly, the transgenic
animal thus produced is naturally included within the scope of the
present invention. The transgenic animal thus produced is very
useful as an animal model for developing pharmaceuticals or as an
animal used for screening of pharmaceuticals. Furthermore, a
so-called knockout animal in which the gene of the 1st, 4th, 7th,
or 9th embodiment of the present invention has been deleted is
characterized in that it does not contain such gene. As described
in literatures, or as apparent from the common knowledge in the
art, such knockout animals cannot be produced without the gene
information on Semaphorin W of the present invention. It goes
without saying, therefore, that such knockout animals are included
within the scope of the present invention.
[0138] While Semaphorin W has an important in vivo function
relating to regeneration of neurons as described above, it has been
also suggested as mentioned above that Semaphorin W may have other
unknown functions such as immunosuppression (Cell, 75, 1389-1399
(1993)). Accordingly, it is quite important to investigate the
expression of Semaphorin W gene or the distribution and function of
Semaphorin W protein for studying this technical field or for
diagnosing patients with neurological disorders or other diseases.
The present invention can also provide gene probes, antibodies,
recombinant proteins, transgenic animals and the like which can be
used for such purposes.
BRIEF DESCRIPTION OF DRAWINGS
[0139] FIG. 1 shows a picture of electrophoresis indicating
distribution of Semaphorin W expression among various tissues
determined by Northern analysis.
[0140] Total RNAs were extracted from various tissues of six-weeks
old rats, electrophoresed on 1% agarose-formamide gel, blotted onto
a filter, and hybridized with a .sup.32P-labeled rat Semaphorin W
DNA probe to determine the distribution of Semaphorin W mRNA
expression. Fifteen .mu.g of RNA was loaded in each lane. The upper
panel shows the result of autoradiography. The positions
corresponding to 18S and 28S ribosomal RNAs are indicated at the
left margin of the panel. The lower panel shows the ethidium
bromide staining of the gel. The upper and lower bands correspond
28S and 18S ribosomal RNAs, respectively.
[0141] FIG. 2 shows a picture of electrophoresis indicating
distribution of Semaphorin W expression in fetus and CNS tissues of
the adult determined by Northern analysis.
[0142] Total RNAs were extracted from rat tissues at various ages,
electrophoresed on 1% agarose-formamide gel, blotted onto a filter,
and hybridized with a .sup.32P-labeled rat Semaphorin W DNA probe
to determine the distribution of Semaphorin W mRNA expression. In
this figure, E12, E15, E18, and P0 indicate the results for those
samples at embryonic-days 12, 15, 18, and immediately after birth,
respectively. The distribution among the CNS tissues (nine lanes at
the left side) was obtained with those samples all at 6-weeks old.
Fifteen .mu.g of RNA was loaded in each lane. The upper panel shows
the result of autoradiography. The positions corresponding to 18S
and 28S ribosomal RNAs are indicated at the left margin of the
panel. The lower panel shows the ethidium bromide staining of the
gel. The upper and lower bands correspond 28S and 18S ribosomal
RNAs, respectively.
[0143] FIG. 3 shows a picture of electrophoresis indicating
expression of Semaphorin W protein in COS 7 cells.
[0144] An expression plasmid for Semaphorin W
(pUCSR.alpha.-rSWsense) was constructed, and introduced into COS 7
cells for transient expression (indicated as "S"). A plasmid
containing Semaphorin W gene in the opposite direction
(pUCSR.alpha.-rSWanti-sense) was used as control (indicated as
"AS"). Three days after introducing plasmids, the cells were
harvested, and the membrane fraction was prepared. The membrane
fraction was fractionated by SDS-PAGE, and then subjected to
Western blotting using an anti-Semaphorin W antibody. The antibody
was obtained by immunizing rabbit with a partial peptide within the
intracellular region of Semaphorin W (710-731:
APPSGTTSYSQDPPSPSPEDE). The position of the band for Semaphorin W
protein is indicated at the right margin of the figure. Positions
and molecular weights of molecular weight makers are indicated in
kilodaltons (kDa) at the left margin of the figure.
[0145] FIG. 4 shows a photomicrograph indicating that Semaphorin W
inhibits neurite outgrowth.
[0146] A. The arrangement of membranes blotted on a polycarbonated
filter. The membranes of COS cells containing Semaphorin W (W) and
of control COS cells (C) were each blotted so that they formed the
vertical stripes shown in the panel. The interval between the
stripes was about 0.1 mm.
[0147] B. Subsequently, a dorsal root ganglion removed from chick
embryo at embryonic-day 7 was placed on the membrane described
above in A., incubated, fixed, stained, and then photographed under
a fluorescence microscope. The white region corresponds to the
neurites.
[0148] C. A schematic representation of B. DRG indicate the
position of the tissue piece of dorsal root ganglion. Another
tissue piece can be seen at the left side. As apparent from this
figure, the neurite outgrowth was inhibited on the membrane
containing Semaphorin W.
[0149] FIG. 5 is a graph indicating the growth-cone collapse
activity of Semaphorin W. Retinal ganglion cells removed from chick
embryo at embryonic-day 6 were cultured overnight, and a membrane
extract (filled bars) prepared from COS cells transfected with a
Semaphorin W-expressing plasmid or a membrane extract (unfilled
bars) prepared from COS cells transfected with the vector alone was
added at the final concentrations indicated on the horizontal axis
of the graph. After culturing for additional 45 minutes, the cells
were fixed, and the ratio (%; the vertical axis) of collapsed
growth cone was measured under a microscope.
[0150] FIG. 6 shows a picture of electrophoresis indicating the in
vivo distribution of Semaphorin III expression among various
tissues determined by Northern analysis.
[0151] Total RNAs were extracted from various tissues of adult
rats, electrophoresed on 1% agarose-formamide gel, blotted onto a
filter, and hybridized with .sup.32P-labeled mouse Semaphorin III
DNA probe to determine the distribution of Semaphorin III mRNA
expression. Fifteen .mu.g of RNA was loaded in each lane. The upper
panel shows the result of autoradiography. The positions of 18S and
28S ribosomal RNAs are indicated at the left margin of the figure.
The lower panel indicates the ethidium bromide staining of the gel.
The upper and lower bands correspond to 28S and 18S ribosomal RNAs,
respectively.
EXAMPLES
[0152] Fundamental procedures for experiments are described in
detail in many publications such as "Molecular Cloning, 2nd Ed."
edited by Maniatis et al. (Cold Spring Harbor Laboratory Press,
1989), "Current Protocols in Molecular Biology" edited by Ausubel
et al., (John Wiley & Sons, 1987), and "Saibo-Kogaku-Jikken
Protocols" edited by Department of Oncology, The Institute of
Medical Science, The University of Tokyo (Shujunsha, 1991). The
present invention is not intended to be limited by the following
examples, and the examples may be of course modified as usual.
Example 1
[0153] Search Through Database for a Novel Semaphorin Gene
[0154] Using the dbEST database of the National Center for
Biotechnology Research (Bethesda, Md., USA), DNA sequence which
encodes an amino acid sequence well conserved in known Semaphorin
genes was searched. As a result, T09073 proved to encode, as a
partial sequence, the sequence: Gln-Asp-Pro-Val-Cys-Ala-Trp, which
is similar to the sequence consisting of seven amino acids
extremely well conserved among known Semaphorin genes: Gln (or
Arg)-Asp-Pro-Tyr-Cys-Ala (or Gly)-Trp. It was, however, impossible
to conclude that the sequence is part of a novel Semaphorin gene,
partly because this sequence consisting of 364 bp is so short and
partly because the reading frame could not be determined due to the
presence of undetermined bases. In addition, distribution of a gene
containing this sequence was not known. Thus, the present inventors
adopted the following strategy. Firstly, we confirmed by
determining distribution of the gene expression that it is mainly
expressed in the adult CNS in agreement with the aim of the present
invention. The full length gene was then cloned in order to judge
whether or not it corresponds to a novel Semaphorin gene.
Example 2
[0155] Distribution of Expression of Gene Containing T09073
[0156] Distribution of expression of a gene containing T09073 as a
partial sequence was studied by Northern method. Although T09073 is
presumed to be a human gene sequence since it has been submitted to
the database as a sequence derived from a human child brain cDNA
library, Northern analysis was carried out with rat samples in the
light of the ease of the sample preparation. RNAs were prepared
from various tissues of adult and fetal rats according to AGPC
method (Takashi Tuji and Toshikazu Nakamura, Jikken-Igaku, vol. 9,
1991, pp. 1937-1940; M. F. Ausubel et al., ed., "Current Protocols
in Molecular Biology", 1989, pp. 4.2.4-4.2.8, Greene Pub.
Associates & Wiley-Interscience). Briefly, 10 ml of a
denaturing solution (4M guanidine thiocyanate, 25 mM sodium citrate
(pH 7.0), 0.5% sarkosyl, 0.1 M 2-mercaptoethanol) was added to each
1 g of excised tissues, and quickly homogenized using a Polytron
homogenizer. To the homogenate, 0.1 volume of 2 M sodium acetate
(pH 4.0), 1 volume of water-saturated phenol, and 0.2 volumes of
chloroform-isoamyl alcohol (49:1) were added, and the mixture was
vigorously stirred. After centrifugation, the aqueous layer was
isolated, an equal volume of isopropyl alcohol was added thereto,
and the mixture was allowed to stand at -20.degree. C. for 1 hour.
The precipitate was recovered by centrifugation, and dissolved
again in 2-3 ml of the denaturing solution per 1 g tissue. An equal
volume of isopropyl alcohol was added, and the mixture was allowed
to stand at -20.degree. C. for 1 hour, and then RNA was
centrifuged. The precipitate was washed with 75% ethyl alcohol,
dried briefly, and then dissolved in an appropriate amount of
water.
[0157] Subsequently, electrophoresis and Northern blotting of RNAs
were carried out by conventional methods described below. RNAs
prepared from various tissues were firstly electrophoresed on 1%
agaroseose gel containing formaldehyde. The gel was shaken in 50 mM
NaOH for 20 min, and then in 10.times.SSPE (1.times.SSPE consists
of 0.15 M sodium chloride, 10 mM sodium dihydrogenphosphate, and 1
mM ethylenediaminetetraacetic acid disodium salt, adjusted to pH
7.0) for 40 min. The RNAs were then blotted onto a nylon membrane
(Biodyne B, Nippon Pall) by means of capillary transfer, and fixed
using a UV cross-linker (Stratagene) (0.6 J/cm.sup.2) for use in
hybridization. As a probe, a DNA fragment consisting of 196 base
pairs at the 5' region of T09073 was synthesized, and labeled with
.sup.32P using Megaprime DNA Labeling System (Amersham).
Hybridization was carried out by placing the nylon membrane onto
which RNAs were blotted and the probe DNA in the same hybridization
buffer as that described above in (2) and allowing them to stand at
42.degree. C. for 48 hours. After the reaction, the nylon membrane
was washed 2-3 times in 2.times.SSPE, 0.5% (w/v) SDS at 42.degree.
C. for 10 min, and then 2-3 times in 2.times.SSPE, 0.5% SDS (w/v)
at 55.degree. C. for 10 min. Radioactivity on the membrane was then
analyzed using BAS 2000 Bio-Imaging Analyzer. As shown in FIGS. 1
and 2, the results confirmed that the gene was widely expressed in
the adult CNS tissues, whereas among the other tissues, it was
expressed only in the lung and spleen of the adult throughout the
fetal and postnatal periods. It was thus demonstrated that the gene
exhibited a distribution of expression expected for a gene for
CNS-neuron regeneration inhibitor.
Example 3
[0158] Cloning of Rat Semaphorin W Gene
[0159] In order to determine whether or not the above gene
containing T09073 sequence actually corresponds to a novel
Semaphorin gene, the inventors cloned the gene in full length using
the 196 bp DNA fragment prepared in the above Example 2 as a probe,
and determined the sequence. Because of the ease of sample
preparation, the rat gene was firstly cloned. cDNA libraries were
prepared by a conventional method described in the above-mentioned
laboratory manual, using mRNAs prepared from rat brain and muscle
by conventional procedures with Lambda Zap II (.lambda.ZapII) cDNA
Library Preparation Kit (Stratagene). About 150 thousand plaques
were then generated on agarose plates using the cDNA library, and
the plaques were transferred onto nylon membranes (Nippon Pall).
After denaturing and neutralizing the DNAs, they were fixed with
ultraviolet rays of 0.6 J/cm.sup.2, and used in hybridization. The
hybridization was carried out by placing the nylon membrane and the
196 bp DNA fragment labeled with .sup.32P (prepared using Megaprime
DNA Labeling System (Amersham) according to the manufacturer's
protocol) as a probe in a hybridization buffer (45% (v/v)
formamide, 5.times.SSPE, 2.times. Denhardt's solution (Wako Pure
Chemical Industries), 0.5% (w/v) SDS, 20 .mu.g/ml salmon sperm DNA
(Wako Pure Chemical Industries)) and allowing them to stand at
42.degree. C. for 48 hours. After the reaction, the nylon membrane
was washed 2-3 times in 2.times.SSPE, 0.5% (w/v) SDS at room
temperature for 10 min, and then 2-3 times in 2.times.SSPE, 0.5%
(w/v) SDS at 42.degree. C. for 10 min. The filters thus prepared
were analyzed using BAS 2000 Bio-Imaging Analyzer (Fuji Film), and
six positive signals were obtained. Plaques located at the
positions of the positive signals were excised from the agarose
plates, placed in 500 .mu.l of SM buffer (100 mM sodium chloride,
15 mM magnesium sulfate, 50 mM Tris, pH 7.5, and 0.01% gelatin)
supplemented with 20 .mu.l of chloroform, and left stand overnight
at 4.degree. C. to elute the phages. The recombinant lambda phages
thus obtained were subjected to a secondary screening according to
the same procedures as those described above, and single plaques
were isolated. The phages thus obtained were treated in the
following manner for in vivo excision of a phagemid containing the
cDNA insert, according to the protocol of Stratagene. Agarose gels
containing the four single plaques obtained in the secondary
screening were each placed in 500 .mu.l of SM buffer, supplemented
with 20 .mu.l of chloroform, and then allowed to stand overnight at
4.degree. C. Two hundred fifty .mu.l of the phage solution
obtained, 200 .mu.l of E. coli XL-1 Blue MRF' suspended in 10 mM
magnesium chloride at OD.sub.600=1.0, and 1 .mu.l of ExAssist
helper phage (>1.times.10.sup.6 pfu/ml) were mixed, and
incubated at 37.degree. C. for 1.5 min. Then, 3 ml of LB medium
(prepared by mixing 0.5% (w/v) sodium chloride, 1% (w/v)
Bactotrypton (Difco), and 0.5% (w/v) yeast extract (Difco) followed
by adjusting the pH to 7.0 with 5 M sodium hydroxide) was added,
and the mixture was shaken at 37.degree. C. for 2-3 hours. The
cells were removed by centrifugation at 2000.times.g for 15 min,
and the supernatant was heat-treated at 70.degree. C. for 15 min.
The supernatant was then centrifuged again at 2000.times.g for 15
min, and recovered as a stock solution of a phagemid containing the
cDNA insert. An aliquot (10-100 .mu.l) of the phagemid stock
solution was mixed with 200 .mu.l of E. coli SOLR (OD.sub.600=1.0),
incubated at 37.degree. C. for 15 min. Then, 10-50 .mu.l of the
mixture was plated onto an ampicillin plate, and incubated
overnight at 37.degree. C. to obtain E. coli strain which contained
the double-stranded phagemid into which the gene fragment of
interest has been inserted.
[0160] The base sequence of the cDNA clone thus obtained was then
analyzed on Perkin-Elmer Model 377 DNA Sequencer to determine the
complete base sequence. The reaction was carried out using PRISM
Dye termination kit (Perkin-Elmer). The DNA base sequence thus
determined (4008 bases), the putative open reading frame (2331
bases), and the amino acid sequence (776 amino acids) are shown in
SEQ ID NOs: 1, 2, and 3, respectively.
[0161] Since the gene encoded an amino acid sequence having a
so-called semaphorin domain at positions 62 to 567, definitely
confirming that the protein belongs to the Semaphorin family, it
was designated Semaphorin W. In addition, the segment of T09073
consisting of 196 base pairs at its 5' terminus, used as a probe in
Examples 2 and 3, proved to correspond to the base sequence from
position 1561 to position 1756 (SEQ ID NO: 7) of Semaphorin W gene
shown in SEQ ID NO: 1. Furthermore, the base sequence from position
1561 to position 1924 of Semaphorin W gene shown in SEQ ID NO: 1
had 87% identity with the whole sequence of T09073 consisting of
364 bp, it was confirmed for the first time that T09073 is a
partial sequence of human Semaphorin W gene.
Example 4
[0162] Cloning of Human Semaphorin W
[0163] Human hippocampus and forebrain cDNA libraries purchased
from Stratagene were screened as described in Example 3 using the
full-length rat Semaphorin W cDNA cloned in Example 3 as a probe to
obtain a clone #103. Determination of the base sequence of the
clone #103 by the same procedures as those described in Example 3
revealed that this clone comprised the cDNA sequence consisting of
333 base pairs shown in SEQ ID NO: 10 at its 5' region and the cDNA
sequence consisting of 2315 base pairs shown in SEQ ID NO: 4 at its
3' region. In these sequences, all the base sequence shown in SEQ
ID NO: 10 and the segment of the base sequence shown in SEQ ID NO:
4 from position 1 through position 1761 (SEQ ID NO: 5) are presumed
to be parts of an open reading frame. The base sequence shown in
SEQ ID NO: 10 could be thus translated into a peptide consisting of
contiguous 111 amino acids (SEQ ID NO: 11), and the base sequence
shown in SEQ ID NO: 5 could be translated into a peptide consisting
of contiguous 587 amino acids (SEQ ID NO: 6). Since these amino
acid sequence had 82% (SEQ ID NO: 11) and 92% (SEQ ID NO: 6)
identities to the sequences at the corresponding region in rat
Semaphorin W, it was definitely confirmed that they are parts of
human Semaphorin W gene. In addition, the sequence T09073 found in
the EST database corresponded to a partial sequence of clone #103
from position 922 to position 1285 in SEQ ID NO: 4, and within this
region the base sequences were 98% identical to each other.
[0164] E. coli strain SOLR (hSW103), a transformant obtained by
introducing a plasmid hSW103, which incorporates the insert of the
above clone #103 (a region corresponding to cDNA for human
Semaphorin W) in a vector pBluescript, into E. coli strain SOLR,
has been deposited at the National Institute of Bioscience and
Human Technology (1-1-3 Higashi, Tsukuba, Ibaraki, Japan) under
Deposit No. FERM BP-6089 on Aug. 29, 1997.
Example 5
[0165] E. coli Expression and Purification of Partial Protein of
Semaphorin W
[0166] The intracellular domain of Semaphorin W (the part
corresponding to position 687 through position 776 of the amino
acid sequence shown in SEQ ID NO: 3) was expressed in E. coli
cells, and purified.
[0167] A 310 bp fragment encoding the sequence of the intracellular
domain was firstly obtained by carrying out PCR under usual
conditions using two primers, 5'-GATAAGGATCCGGGTCGCCGTCAGCAGCGT-3'
(SEQ ID NO: 8) and 5'-GGCTGGAATTCATTTTCCCCGGCTTTA-3' (SEQ ID NO:
9), with Semaphorin W cDNA (SEQ ID NO: 1) as a template. This
fragment was then cleaved with restriction enzymes BamHI and EcoRI,
and incorporated into an expression plasmid pRSETB (Invitrogen)
which had been also cleaved at BamHI and EcoRI sites to obtain an
expression plasmid pRSWinc.
[0168] The plasmid pRSWinc thus obtained was used to transform E.
coli. strain BL21(DE3)pLysS (Stratagene), and the cells were
cultured overnight on LB plate containing 50 .mu.g/ml ampicillin to
obtain transformants. By analyzing the base sequence of a plasmid
prepared from the transformant, it was confirmed that the
transformant carried a plasmid having the desired structure. The
transformant thus obtained was cultured with shaking in LB broth
containing 50 .mu.g/ml ampicillin. When OD.sub.600 of the culture
reached 0.5, IPTG was added at a final concentration of 1 mM, and
the cells were further cultured overnight. The culture medium was
then centrifuged at 5000.times.g for 15 min to harvest the cells.
All proteins of the harvested cells were analyzed by SDS-PAGE, and
it was confirmed that a protein having an expected molecular weight
was produced.
[0169] The Sema W partial protein expressed in E. coli cells was
then affinity-purified by taking advantage of affinity between the
His tag at the amino terminus of this protein and a
nickel-Sepharose. This procedure is described in detail in the
protocol supplied by Qiagen. Briefly, Sema W protein was expressed
in E. coli cells as described above, and 5 ml of Solution A (6M
guanidine-HCl, 0.1 M sodium phosphate, 0.01 M Tris-HCl, pH=8.0) was
added to each 1 g of the cell. The cells were suspended well in the
solution, and then stirred at room temperature for more than 1 hour
to be solubilized. Subsequently, this protein solution was mixed
with Ni-NTA resin (Qiagen) pre-equilibrated with Solution A, gently
stirred at room temperature for more than 2 hours to allow the
binding of proteins to the resin, and the resin was then packed
into a column. The column was washed with 10 volumes of Solution A,
then with 5 volumes of Solution B (8M urea, 0.1 M sodium phosphate,
0.01 M Tris-HCl, pH=8.0), and further with 5 volumes of Solution C
(8M urea, 0.1 M sodium phosphate, 0.01 M Tris-HCl, pH=6.3) to elute
the bound proteins. During the elution, the eluate was collected in
one column volume fractions, and subjected to SDS-PAGE to check the
proteins eluted. The desired fractions were then concentrated, and
stored at -20.degree. C. until use.
[0170] The N-terminal amino acid sequence of the protein thus
obtained was analyzed to confirm that it was the desired protein.
This partial protein of Semaphorin W can be used, for example, as
an antigen in producing antibodies.
[0171] Example 6
[0172] Production of Anti-Semaphorin W Antibody
[0173] In order to produce anti-Semaphorin W antibodies, a
polyantigenic peptide having the N- and C-terminal sequences of rat
Semaphorin W, ALTLPFSGERPRRID and APPSGTTSYSQDPPSPSPEDER, was
synthesized by a conventional method (Seikagaku, vol. 63 (1991) pp.
1345-1348), and used to immunize a rabbit. Immunization of rabbit
was achieved according to conventional procedures. Specifically,
0.4 mg of the antigen was mixed with Freund's complete adjuvant,
and used to immunize a rabbit subcutaneously. Subsequently, the
rabbit was further subcutaneously immunized 4 times at 2 weeks
intervals with 0.2 mg of the antigen mixed with Freund's incomplete
adjuvant. One week after the last immunization, whole blood was
collected from the rabbit. Purification was then carried out by a
conventional method using a protein A column to obtain a purified
monoclonal antibody. It was concluded that the antibody obtained
was an anti-Semaphorin W antibody properly recognizing Semaphorin W
protein, because of the reasons, for example, that this antibody
recognized a protein having a molecular weight expected for
Semaphorin W in Western blotting as described below in Example 7,
that when Semaphorin W having a myc tag attached thereto was
immunoprecipitated with an anti-myc antibody, the protein
recognized by this antibody was also co-precipitated, and that both
of an antibody produced using a N-terminal peptide as an antigen
and an antibody produced using a C-terminal peptide as an antigen
recognized a protein having the same molecular weight.
Example 7
[0174] Expression of Semaphorin W in Animal Cells
[0175] A DNA fragment encoding Myc tag having the sequence
Asp-Ile-Gly-Gly-Glu-Gln-Lys-Lue-Ile-Ser-Glu-Glu-Asp-Leu was
inserted just before the stop codon of rat Semaphorin W gene shown
in SEQ ID NO: 1, and the recombinant gene was introduced into an
expression plasmid pUCSR.alpha. (pUCSR.alpha.-rSWMYC).
[0176] This expression plasmid containing Semaphorin W gene was
then introduced into COS 7 cells according to conventional
procedures, and the cells were harvested after 3 days in order to
prepare the membrane fraction in the following manner. The cells
were harvested using a cell scraper, and the harvested cells were
homogenized in the presence of protease inhibitors. The homogenate
was separated into precipitate and supernatant by high-speed
centrifugation at 12,000 g for 10 min. The supernatant was further
subjected to ultracentrifugation at 100,000 g for 30 min, and the
soluble fraction was recovered as the cytoplasmic fraction (S100).
The cytoplasmic fraction obtained was stored at -80.degree. C.
until use. On the other hand, the precipitate from the above
high-speed centrifugation was washed twice with the homogenizing
solution, suspended in 2 volumes of 2.25 M sucrose/PBS, and
overlaid onto 2.25 M sucrose/PBS. After 0.8 M sucrose/PBS was
further overlaid onto the top, it was centrifuged at 12,000 g for
20 min. The membrane fraction was recovered from the lower
interface, then washed twice with the homogenizing solution, and
stored at -80.degree. C. until use.
[0177] The membrane and cytoplasmic fractions thus obtained were
each separated by SDS-PAGE, and then subjected to Western blotting
in a conventional manner using anti-Myc antibody 9E10 (Calbiochem).
An alkali phosphatase-labeled anti-mouse IgG antibody (Biosource)
was used as the secondary antibody. In the Western blotting, a
specific band was observed at the position corresponding to about
110 kDa only when the plasmid containing Semaphorin W gene was
introduced, confirming that the Myc-tagged rat Semaphorin W protein
was expressed in COS cells and existed in the membrane.
[0178] Similarly, another membrane fraction was prepared using an
expression plasmid for Semaphorin W having no Myc tag
(pUCSR.alpha.-rSWsense), and subjected to Western blotting as above
using the anti-Semaphorin W antibody produced in Example 6. As a
result, a specific band of about 100 kDa was recognized, confirming
that Semaphorin W protein was expressed in COS cells and existed in
the membrane in consistent with the above result (FIG. 3).
Example 8
[0179] Effect of Semaphorin W on Neurite Outgrowth
[0180] A stripe assay was carried out in order to evaluate the
effect of Semaphorin W on neurite outgrowth. The method of stripe
assay is described in detail in literature (Development 101,
685-696 (1987)). Briefly, an expression plasmid for Semaphorin W
was firstly introduced into COS cells as described above in Example
7, and the membrane fraction was prepared from the COS cells
expressing Semaphorin W on their cell membrane. In parallel,
another membrane fraction was also prepared in the same manner from
COS cells not expressing Semaphorin W (that is, COS cells
transfected with a vector carrying no Semaphorin gene) as a
control. The membrane fractions obtained were blotted onto a
micropore polycarbonated filter (Coaster) using a silicone matrix
striped with slits so as to form alternating stripes each having a
width of about 0.1 mm. The filter was then transferred into a
neuron culture medium (F12, 10% FCS, 20 ng/ml NGF), and a dorsal
root ganglion removed by a conventional method from chick embryo at
embryonic-day 7 was carefully placed on the filter, and cultured in
a CO.sub.2 incubator at 37.degree. C. for 48 hours. After
completion of the cultivation, the cells were fixed with 1%
glutaraldehyde. DiI (Molecular Probe) was then inserted with
caution into the dorsal root ganglion, and the filter was incubated
in PBS (-) at 37.degree. C. for 2 to 3 days. Subsequently,
elongation of neurite outgrowing from the dorsal root ganglion on
the membrane containing Semaphorin W was compared with that on the
control membrane to evaluate the effect of Semaphorin W on neurite
outgrowth. The result thus obtained is shown in FIG. 4. In the
figure, "W" indicates the regions onto which the membrane of COS
cells containing Semaphorin W was blotted, and "C" indicates the
regions onto which the membrane of control COS cells was blotted.
The neurites appear in white because they were stained with DiI. As
can be seen at a glance, it was demonstrated that neurite outgrowth
was inhibited on the cell membrane containing Semaphorin W. In
living bodies, Semaphorin W is widely expressed in CNS of the adult
(Example 2), and therefore, neurite outgrowth in the CNS is
believed to be inhibited.
Example 9
[0181] Growth-Cone Collapse Activity of Semaphorin W
[0182] In order to determine whether or not Semaphorin W has a
growth-cone collapse activity, retinal ganglion cells were cultured
as described below, and the morphological change of growth cone was
evaluated following addition of Semaphorin W protein solubilized
from the membrane fraction using a surfactant.
[0183] Specifically, retinal ganglion removed from chick embryo at
embryonic-day 6 was placed on a dish coated with polylysine and
laminin by a conventional method, and cultured overnight in a
medium (F12, 10% FCS, 20 ng/ml BDNF) in a CO.sub.2 incubator at
37.degree. C. Next day, a membrane fraction prepared as described
above in Example 7 was solubilized with a surfactant, added to the
medium, and further incubated for 30 min to one hour. Subsequently,
the sample was fixed with 1% glutaraldehyde, and the morphological
change of the growth cone was observed. In this procedure, the
membrane fraction may be solubilized, for example, according to the
method described in literature (Cell 75, 217-227 (1993)).
Specifically, COS cells transfected with an expression plasmid for
Semaphorin W and expressing Semaphorin W, and COS cells transfected
with a control vector plasmid and not expressing Semaphorin W were
used as row materials, and the cell membrane fractions prepared as
described above in Example 7 were each solubilized with 1-2% CHAPS,
and then dialyzed against F12 to remove CHAPS. After centrifuging
the dialyzed material to remove insoluble, the protein
concentration was measured by a conventional method using BCA kit
(Pierce), and used in the above measurement of growth-cone collapse
activity. All the preparations were carried out in practice at
4.degree. C.
[0184] As shown in FIG. 5, the results indicated that collapse of
growth cone was observed at a rate 1.6- to 4.6-fold higher than the
control when the extract of COS cells transfected with the
Semaphorin W expressing plasmid was added. Accordingly, Semaphorin
W is believed to have a neurite-outgrowth inhibition activity on
retinal ganglion cells which are CNS-neuron.
Reference Example 1
[0185] Identification of the Site Essential to the Semaphorin
Activity Using Semaphorin III
[0186] PCR was conducted on the basis of the sequence information
on Semaphorin III described in Neuron, 14, 941-948 (1995), and the
structural gene of Semaphorin III was incorporated into an
expression plasmid pUCSR.alpha.. The expression plasmid was then
introduced into COS 7 cells by DEAE-dextran method. After 2 days,
the Semaphorin III activity contained in the culture supernatant
was determined by a method similar to that described in Cell, 75,
217-227 (1993), using the growth-corn collapse activity on chick
dorsal root ganglion cells as an indicator. As a result, one clone
which did not exhibit any activity was found. The base sequencing
of the clone revealed that aspartic acid residue at position 198
was substituted by glycine. When compared with other known animal
Semaphorins, the regions before and after the position 198 were not
markedly conserved, although the position corresponding to aspartic
acid residue was highly conserved among Semaphorins with a few
exceptions in which glutamic acid residue was located at that
position. This suggested that the aspartic acid residue is
essential to expression of the activity. The gene was then
subjected to site-directed mutagenesis by a conventional method to
replace the glycine residue with aspartic acid. Since this
mutagenesis restored the strong collapse activity, it was confirmed
that all of the regions in the expression plasmid normally function
except for that position. In conclusion, the aspartic acid at
position 198 of Semaphorin III appears essential for the expression
of the Semaphorin function. The amino acid residue corresponding to
this aspartic acid residue is glutamic acid residue at position 204
in the amino acid sequence of Semaphorin W shown in SEQ ID NO:
3.
Reference Example 2
[0187] Tissue-Specific Gene Expression of Semaphorin III Determined
by Northern Analysis
[0188] To determine the distribution of Semaphorin III gene
expression among mouse tissues, RNAs were prepared from various
adult mouse tissues, and subjected to Northern analysis. The
procedures for preparation, blotting, and hybridization of RNA were
the same as those described in Example 2. The 560 bp MspI fragment
of mouse Semaphorin III DNA described in Reference example 1 was
used as a probe. As a result, it was demonstrated as shown in FIG.
6 that the expression of Semaphorin III in the adult is extremely
high in the lung which is peripheral, while it is rather low in the
CNS.
EFFECTS OF THE INVENTION
[0189] The present invention provides Semaphorin W inhibiting
neurite outgrowth, and a gene therefor, as well as other
Semaphorins hybridizing to said Semaphorin W gene, modified
proteins or partial peptides of said Semaphorin W, antibodies
against said Semaphorin W, antisense nucleotides against said
Semaphorin W gene, and the use of such substances as pharmaceutical
or diagnostic agents or laboratory reagents. The present invention
further provides a method of screening for Semaphorin W antagonists
employing said Semaphorin W, Semaphorin W antagonists obtained by
said screening method, pharmaceutical agents comprising such
antagonists, and transgenic animals regarding said Semaphorin W.
Sequence CWU 1
1
16 1 4008 DNA Rattus norvegicus 5'UTR (1)..(75) misc_feature
(76)..(2406) Coding region 1 gccgaggccc gcgcagtagc ggtactaagt
agaggctgct ggacgcgccc cacccggcac 60 caggcggagc cagagatgct
tgccagggcc gagcggcccc gcccgggccc ccggccgcct 120 ccggtctttc
ccttcccgcc gccgctgtcg ctgctgctgc tgctggcgat actaagcgcc 180
ccggtgtgcg gccgcgtccc ccgctcagtg cccagaacct cgctgcccat ctccgaggct
240 gactcctatc tcacccggtt tgcagcgtct catacgtaca attactctgc
tctccttgtg 300 gatcctgcct cccacacact ttacgtcggt gcacgggata
gcatcttcgc tttaaccctc 360 cccttctctg gggaaagacc ccgaaggatc
gactggatgg tacctgagac tcacagacag 420 aactgcagga agaaaggcaa
gaaagaggac gaatgtcaca attttatcca gattctcgcc 480 attgtcaatg
cctctcacct cctcacgtgc ggcaccttcg cttttgatcc gaagtgcggg 540
gttattgatg tgtccagttt ccagcaggtt gaaagacttg agagcggccg ggggaaatgt
600 ccttttgagc cagctcaacg gtcagcagct gtaatggctg ggggcgtcct
ctacaccgcc 660 actgtgaaga acttcctggg gactgagccc atcatctccc
gagctgtggg tcgagctgag 720 gactggattc gaacagagac cttgtcatcc
tggcttaatg ctccagcctt tgtcgcagct 780 atggtcctga gcccagctga
gtggggggat gaagatggag acgatgaaat cttttttttc 840 ttcacggaga
cctcccgagt gttggactcc tatgagcgca tcaaggtccc aagagtggcc 900
cgagtgtgtg cgggggacct tgggggcagg aagacccttc agcagagatg gacgacgttt
960 ctgaaggctg acctgctgtg cccagggccc gagcatggcc gggcctccgg
ggttctgcag 1020 gctatggcag agcttcggcc tcagcctgga gcgggaaccc
ccatctttta tgggatcttt 1080 tcctcccagt gggaaggagc tgccatctct
gctgtgtgtg ccttccgacc ccaagacatc 1140 cgggcagtgc tgaatggtcc
ctttagagag ctaaaacatg actgcaacag gggactgcct 1200 gtcatggaca
acgaggtgcc ccagcccaga cctggagagt gcatcgccaa caacatgaag 1260
ctccagcagt ttggatcctc actctccctg ccagaccgcg tgctcacctt tatcagagac
1320 caccctctca tggacaggcc cgtgttcccg gctgacggcc gccccctgct
ggtcactaca 1380 gatacagcct atctcagagt cgtggcccac agggtgacca
gcctctcagg gaaagaatat 1440 gacgtgctct acctggggac agaggatgga
cacctccacc gggctgtgcg cattggagct 1500 cagctcagtg tcttggagga
tctggccttg ttcccagaac cacagccggt tgagagcatg 1560 aaattgtacc
acgattggct cctggtgggc tcccatactg aggtgacaca agtgaacacc 1620
agcaactgtg gccgtctcca gagctgctcg gagtgtatcc tggcccagga ccccgtgtgc
1680 gcctggagct tccggcttga tgcttgtgtg gcccacgccg gcgagcaccg
cgggatggtt 1740 caagatatag agtcagcgga tgtctcttct ttgtgtccaa
aagaacctgg agaacatccc 1800 gtagtgtttg aagttccggt ggctactgtg
ggccacgtgg tcctgccatg ttcccccagt 1860 tctgcctggg catcctgtgt
gtggcaccag cccagtggag tgactgcgct cactccccgg 1920 agggatggac
tagaggtggt ggtgacccca ggggccatgg gggcttatgc ttgcgagtgt 1980
caggagggtg gagccgcccg cgtggtggct gcttatagct tggtgtgggg cagccagcgg
2040 ggaccctcaa accgggccca caccgttgtg ggggctggat tggttggctt
tctcctgggt 2100 gttcttgcag catccctcac tctcctcctg attggtcgcc
gtcagcagcg tcggcgacag 2160 agggagcttc tagctagaga caaggtgggc
ttagatctgg gggctccacc ttctgggacc 2220 acaagctata gtcaggaccc
tccctctcct tcgcctgaag atgaacggct gcccctggcc 2280 ctgggtaagc
ggggcagtgg ttttggtggc ttccctccac ccttcctgct ggattcttgc 2340
ccaagcccag cccacatccg gctcactggg gcgcctctag ccacgtgtga tgagacctcc
2400 atctaaagcc ggggaaaatg actgccagcc atgagcagtc tctggaacta
gtggctacca 2460 agaccatgat catggctgct cctttctctt ggagtctgtg
tgttcacaca ttagtgtctg 2520 tcctctggac ctggacctgg cctttgccca
gattcctgat tctcatgaga gatcaaccct 2580 gtaaccttct gcgatggcct
cttgtcttgg gcccatcagc ttgtggggtg gagtaaggac 2640 ataggccccg
gaaagggaat cagtgtggag gtagttgggg cgtgtgtgcc ctgcgtcctt 2700
gtggtggctg tatgatttcc cagtctgctg actctgggga gcgcatgatc ccctgactgc
2760 cttgagatct ctcccaactc agtttcccct tgctctggaa gagtgtgtgt
ctatacactg 2820 gtgtgcctag aaggcctgtc catgtgtgca tggacgacag
ggccggtgcc tcggtgcttt 2880 tggggagtcg gagagaaagg ttggaatggg
ggacaactta accctcggta gccagtgagg 2940 gaaaccacat gcccgtcccc
atcaccccac agcgcttctt taactttgag caaagttccc 3000 aaagtgacct
tctgggtggg aagggcagca ggacatgtgg cccccgtcct tctccttgtc 3060
tttcccttct ggctgccaac cactgccgtg ccaccgctgc gctttccctg gctggagtgg
3120 aggctgagtc ctctgtcctt ggtttccatt taaaatgaac ttcacaacat
tctaaatatt 3180 gggggatgac aaatgacttt tttccccaga aaagtgtgta
ggaaatacaa gcaggttaaa 3240 gaagatttgc ctcagtgact ttcacccttg
ccctaaagca ggagtccctc agctagcgtc 3300 tgtggactcc ctgaaattgt
atgcgtctgt ggactccctg aaattgtatg caaagtgtct 3360 gtgtgtgtgt
gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtttgcgtgc atgtgtgcat 3420
gtgtgtttga tggctttcat cagattctca aggccttaat gaggttaaag gaccacggcc
3480 tatagtcacc acacttgggc cacatggagg aggtgttgct ctctgaggca
gttcctccct 3540 ggcctgcctg aggccagccc ctggacacat tgctgctgga
gaccccacat ctctccagaa 3600 cttggaagct aggctctgcg cgtgcttgaa
ggcaccacca tctcccttct tgcttcattc 3660 tcctgtgtgc tctgcctctg
ctcagtcctg ctcttggcct gtgaatgtgc ctcgcccgtc 3720 cctggtgggg
gacctcaaac cccagtgctg atgctaccct ttccagtggg agtttctgtt 3780
ctgctttcct tgacagcagc ctgtgaacta ctcacgagtc cccttggttt ggagttcccg
3840 gtggctttga gtaggatctt tggcgtggca tctaacctag cagcattgat
cgttcattgt 3900 aaagtgggga tatacctacc tcagggttgc tgcaaggatc
aaacgaggaa acgtataaat 3960 aaagcattac ccacagcaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaa 4008 2 2331 DNA Rattus norvegicus misc_feature
(1)..(2331) Coding region 2 atgcttgcca gggccgagcg gccccgcccg
ggcccccggc cgcctccggt ctttcccttc 60 ccgccgccgc tgtcgctgct
gctgctgctg gcgatactaa gcgccccggt gtgcggccgc 120 gtcccccgct
cagtgcccag aacctcgctg cccatctccg aggctgactc ctatctcacc 180
cggtttgcag cgtctcatac gtacaattac tctgctctcc ttgtggatcc tgcctcccac
240 acactttacg tcggtgcacg ggatagcatc ttcgctttaa ccctcccctt
ctctggggaa 300 agaccccgaa ggatcgactg gatggtacct gagactcaca
gacagaactg caggaagaaa 360 ggcaagaaag aggacgaatg tcacaatttt
atccagattc tcgccattgt caatgcctct 420 cacctcctca cgtgcggcac
cttcgctttt gatccgaagt gcggggttat tgatgtgtcc 480 agtttccagc
aggttgaaag acttgagagc ggccggggga aatgtccttt tgagccagct 540
caacggtcag cagctgtaat ggctgggggc gtcctctaca ccgccactgt gaagaacttc
600 ctggggactg agcccatcat ctcccgagct gtgggtcgag ctgaggactg
gattcgaaca 660 gagaccttgt catcctggct taatgctcca gcctttgtcg
cagctatggt cctgagccca 720 gctgagtggg gggatgaaga tggagacgat
gaaatctttt ttttcttcac ggagacctcc 780 cgagtgttgg actcctatga
gcgcatcaag gtcccaagag tggcccgagt gtgtgcgggg 840 gaccttgggg
gcaggaagac ccttcagcag agatggacga cgtttctgaa ggctgacctg 900
ctgtgcccag ggcccgagca tggccgggcc tccggggttc tgcaggctat ggcagagctt
960 cggcctcagc ctggagcggg aacccccatc ttttatggga tcttttcctc
ccagtgggaa 1020 ggagctgcca tctctgctgt gtgtgccttc cgaccccaag
acatccgggc agtgctgaat 1080 ggtcccttta gagagctaaa acatgactgc
aacaggggac tgcctgtcat ggacaacgag 1140 gtgccccagc ccagacctgg
agagtgcatc gccaacaaca tgaagctcca gcagtttgga 1200 tcctcactct
ccctgccaga ccgcgtgctc acctttatca gagaccaccc tctcatggac 1260
aggcccgtgt tcccggctga cggccgcccc ctgctggtca ctacagatac agcctatctc
1320 agagtcgtgg cccacagggt gaccagcctc tcagggaaag aatatgacgt
gctctacctg 1380 gggacagagg atggacacct ccaccgggct gtgcgcattg
gagctcagct cagtgtcttg 1440 gaggatctgg ccttgttccc agaaccacag
ccggttgaga gcatgaaatt gtaccacgat 1500 tggctcctgg tgggctccca
tactgaggtg acacaagtga acaccagcaa ctgtggccgt 1560 ctccagagct
gctcggagtg tatcctggcc caggaccccg tgtgcgcctg gagcttccgg 1620
cttgatgctt gtgtggccca cgccggcgag caccgcggga tggttcaaga tatagagtca
1680 gcggatgtct cttctttgtg tccaaaagaa cctggagaac atcccgtagt
gtttgaagtt 1740 ccggtggcta ctgtgggcca cgtggtcctg ccatgttccc
ccagttctgc ctgggcatcc 1800 tgtgtgtggc accagcccag tggagtgact
gcgctcactc cccggaggga tggactagag 1860 gtggtggtga ccccaggggc
catgggggct tatgcttgcg agtgtcagga gggtggagcc 1920 gcccgcgtgg
tggctgctta tagcttggtg tggggcagcc agcggggacc ctcaaaccgg 1980
gcccacaccg ttgtgggggc tggattggtt ggctttctcc tgggtgttct tgcagcatcc
2040 ctcactctcc tcctgattgg tcgccgtcag cagcgtcggc gacagaggga
gcttctagct 2100 agagacaagg tgggcttaga tctgggggct ccaccttctg
ggaccacaag ctatagtcag 2160 gaccctccct ctccttcgcc tgaagatgaa
cggctgcccc tggccctggg taagcggggc 2220 agtggttttg gtggcttccc
tccacccttc ctgctggatt cttgcccaag cccagcccac 2280 atccggctca
ctggggcgcc tctagccacg tgtgatgaga cctccatcta a 2331 3 776 PRT Rattus
norvegicus 3 Met Leu Ala Arg Ala Glu Arg Pro Arg Pro Gly Pro Arg
Pro Pro Pro 1 5 10 15 Val Phe Pro Phe Pro Pro Pro Leu Ser Leu Leu
Leu Leu Leu Ala Ile 20 25 30 Leu Ser Ala Pro Val Cys Gly Arg Val
Pro Arg Ser Val Pro Arg Thr 35 40 45 Ser Leu Pro Ile Ser Glu Ala
Asp Ser Tyr Leu Thr Arg Phe Ala Ala 50 55 60 Ser His Thr Tyr Asn
Tyr Ser Ala Leu Leu Val Asp Pro Ala Ser His 65 70 75 80 Thr Leu Tyr
Val Gly Ala Arg Asp Ser Ile Phe Ala Leu Thr Leu Pro 85 90 95 Phe
Ser Gly Glu Arg Pro Arg Arg Ile Asp Trp Met Val Pro Glu Thr 100 105
110 His Arg Gln Asn Cys Arg Lys Lys Gly Lys Lys Glu Asp Glu Cys His
115 120 125 Asn Phe Ile Gln Ile Leu Ala Ile Val Asn Ala Ser His Leu
Leu Thr 130 135 140 Cys Gly Thr Phe Ala Phe Asp Pro Lys Cys Gly Val
Ile Asp Val Ser 145 150 155 160 Ser Phe Gln Gln Val Glu Arg Leu Glu
Ser Gly Arg Gly Lys Cys Pro 165 170 175 Phe Glu Pro Ala Gln Arg Ser
Ala Ala Val Met Ala Gly Gly Val Leu 180 185 190 Tyr Thr Ala Thr Val
Lys Asn Phe Leu Gly Thr Glu Pro Ile Ile Ser 195 200 205 Arg Ala Val
Gly Arg Ala Glu Asp Trp Ile Arg Thr Glu Thr Leu Ser 210 215 220 Ser
Trp Leu Asn Ala Pro Ala Phe Val Ala Ala Met Val Leu Ser Pro 225 230
235 240 Ala Glu Trp Gly Asp Glu Asp Gly Asp Asp Glu Ile Phe Phe Phe
Phe 245 250 255 Thr Glu Thr Ser Arg Val Leu Asp Ser Tyr Glu Arg Ile
Lys Val Pro 260 265 270 Arg Val Ala Arg Val Cys Ala Gly Asp Leu Gly
Gly Arg Lys Thr Leu 275 280 285 Gln Gln Arg Trp Thr Thr Phe Leu Lys
Ala Asp Leu Leu Cys Pro Gly 290 295 300 Pro Glu His Gly Arg Ala Ser
Gly Val Leu Gln Ala Met Ala Glu Leu 305 310 315 320 Arg Pro Gln Pro
Gly Ala Gly Thr Pro Ile Phe Tyr Gly Ile Phe Ser 325 330 335 Ser Gln
Trp Glu Gly Ala Ala Ile Ser Ala Val Cys Ala Phe Arg Pro 340 345 350
Gln Asp Ile Arg Ala Val Leu Asn Gly Pro Phe Arg Glu Leu Lys His 355
360 365 Asp Cys Asn Arg Gly Leu Pro Val Met Asp Asn Glu Val Pro Gln
Pro 370 375 380 Arg Pro Gly Glu Cys Ile Ala Asn Asn Met Lys Leu Gln
Gln Phe Gly 385 390 395 400 Ser Ser Leu Ser Leu Pro Asp Arg Val Leu
Thr Phe Ile Arg Asp His 405 410 415 Pro Leu Met Asp Arg Pro Val Phe
Pro Ala Asp Gly Arg Pro Leu Leu 420 425 430 Val Thr Thr Asp Thr Ala
Tyr Leu Arg Val Val Ala His Arg Val Thr 435 440 445 Ser Leu Ser Gly
Lys Glu Tyr Asp Val Leu Tyr Leu Gly Thr Glu Asp 450 455 460 Gly His
Leu His Arg Ala Val Arg Ile Gly Ala Gln Leu Ser Val Leu 465 470 475
480 Glu Asp Leu Ala Leu Phe Pro Glu Pro Gln Pro Val Glu Ser Met Lys
485 490 495 Leu Tyr His Asp Trp Leu Leu Val Gly Ser His Thr Glu Val
Thr Gln 500 505 510 Val Asn Thr Ser Asn Cys Gly Arg Leu Gln Ser Cys
Ser Glu Cys Ile 515 520 525 Leu Ala Gln Asp Pro Val Cys Ala Trp Ser
Phe Arg Leu Asp Ala Cys 530 535 540 Val Ala His Ala Gly Glu His Arg
Gly Met Val Gln Asp Ile Glu Ser 545 550 555 560 Ala Asp Val Ser Ser
Leu Cys Pro Lys Glu Pro Gly Glu His Pro Val 565 570 575 Val Phe Glu
Val Pro Val Ala Thr Val Gly His Val Val Leu Pro Cys 580 585 590 Ser
Pro Ser Ser Ala Trp Ala Ser Cys Val Trp His Gln Pro Ser Gly 595 600
605 Val Thr Ala Leu Thr Pro Arg Arg Asp Gly Leu Glu Val Val Val Thr
610 615 620 Pro Gly Ala Met Gly Ala Tyr Ala Cys Glu Cys Gln Glu Gly
Gly Ala 625 630 635 640 Ala Arg Val Val Ala Ala Tyr Ser Leu Val Trp
Gly Ser Gln Arg Gly 645 650 655 Pro Ser Asn Arg Ala His Thr Val Val
Gly Ala Gly Leu Val Gly Phe 660 665 670 Leu Leu Gly Val Leu Ala Ala
Ser Leu Thr Leu Leu Leu Ile Gly Arg 675 680 685 Arg Gln Gln Arg Arg
Arg Gln Arg Glu Leu Leu Ala Arg Asp Lys Val 690 695 700 Gly Leu Asp
Leu Gly Ala Pro Pro Ser Gly Thr Thr Ser Tyr Ser Gln 705 710 715 720
Asp Pro Pro Ser Pro Ser Pro Glu Asp Glu Arg Leu Pro Leu Ala Leu 725
730 735 Gly Lys Arg Gly Ser Gly Phe Gly Gly Phe Pro Pro Pro Phe Leu
Leu 740 745 750 Asp Ser Cys Pro Ser Pro Ala His Ile Arg Leu Thr Gly
Ala Pro Leu 755 760 765 Ala Thr Cys Asp Glu Thr Ser Ile 770 775 4
2315 DNA Homo sapiens misc_feature (1)..(1764) Coding region 4
gggggtgtcc tctatgctgc cactgtgaaa aactacctgg ggacggagcc aattatcacc
60 agagcagtgg gtcgtgccga ggactggatt cggacagata ccttgccttc
ctggctgaac 120 gccccagcct ttgtcgcagc cgtggccttg agcccagccg
aatgggggga tgaagatgga 180 gacgacgaaa tctacttctt ctttacggag
acttcccgag catttgactc atacgagcgc 240 attaaagtcc cacgggtggc
ccgtgtgtgt gcgggggacc tcgggggccg gaagaccctc 300 cagcagagat
ggacgacgtt tttgaaagct gacctgctct gtccagggcc tgagcatggc 360
cgggcctcca gtgtcctgca ggatgttgct gtgcttcgac ctgagcttgg ggcagggact
420 cccatctttt atggcatctt ttcttcccag tgggaggggg ctactatctc
tgctgtctgt 480 gccttccgac cacaagacat tcggacagtg ctgaatggtc
ccttcagaga actaaaacat 540 gactgcaaca gaggactgcc tgtcgtggac
aatgatgtgc cccagcccag acctggagag 600 tgcatcacca acaacatgaa
gctccggcac tttggctcat ctctctccct gcctgaccgc 660 gtactcacct
tcatccggga ccacccactc atggacaggc cagtgtttcc agctgatggc 720
caccccctgc tggtcactac agatacagcc tatctcagag tcgtggccca cagggtgacc
780 agcctctcag ggaaagagta tgatgtgctc tacctgggga cagaggatgg
acacctccac 840 cgagcagtgc ggatcggagc tcagctcagc gttcttgaag
atctggcctt attcccagag 900 ccacagccag ttgagaacat gaaattgtac
cacagctggc tcctggttgg ctcccgtact 960 gaggtgacac aagtgaatac
aaccaactgt ggccgtctcc agagctgctc agagtgcatc 1020 ctggcccagg
acccagtctg tgcctggagc ttccggctgg atgagtgtgt ggcccatgcc 1080
ggggagcacc gagggttggt ccaagacata gagtcagcag atgtctcctc tttgtgtcct
1140 aaagagcctg gagaacgtcc agtagtgttt gaagttcccg tggctacagc
tgcgcatgtg 1200 gtcttgccat gttctccaag ctcagcatgg gcatcctgtg
tgtggcacca gcccagtgga 1260 gtgactgcac tcaccccccg gcgggatgga
ctggaggtgg tggtgacccc aggggccatg 1320 ggcgcttatg cctgtgaatg
tcaggagggt ggggcagccc atgtggtagc agcttacagc 1380 ttggtatggg
gcagccagcg agatgctccg agccgggccc acacagtggg ggcgggactg 1440
gctggcttct tcttggggat tctcgcagca tccctgactc tcattctgat tggtcggcgt
1500 cagcagcgac ggcgacagag ggaacttctg gctagagaca aggtgggcct
ggacctgggg 1560 gctccacctt ctgggaccac aagctacagc caagaccctc
cctccccctc tcctgaagat 1620 gagcggttgc cgctggccct ggccaagagg
ggcagtggct ttggtggatt ctcaccaccc 1680 ttcctgcttg atccttgccc
aagcccagcc cacattcggc taactggggc tcctctagcc 1740 acatgtgatg
aaacatccat ctagagctgg gcaaatgacc actagtgtat aagtgatcac 1800
tggaacggag tgaccactga gatgctgggg gtcactgggc ctggaagacc atcccagcct
1860 ctgagttctc tttgagtatg agtgattact tggattttag tatctgttct
ctctgagcct 1920 ggatgggctt ggggccagac ctttgcctga ttcctgattc
ccatgagaaa tcagaactgc 1980 tttctgcagc aaatcagggc ttccccctaa
catctgaact cctgtaaacc ttcatccctg 2040 gccccctatc ttgggcccat
tagttttggg gatggggcac agggcatagc tatgactttg 2100 ctttctggtt
ggagcctggc cggaaggaag agccctggag gtggttgggg gcaaatgtgc 2160
cctgagtcct tggggtggtt ctgcttattc ttcaagttta tctgaatctg tggggagtgc
2220 atgatcccca tgttgcaata tggagtctct gccctgagat cttccccatc
tcagttttcc 2280 ttccatgaaa gagtacgtgt aaatacatag tgttc 2315 5 1761
DNA Homo sapiens misc_feature (1)..(1761) Coding region 5
gggggtgtcc tctatgctgc cactgtgaaa aactacctgg ggacggagcc aattatcacc
60 agagcagtgg gtcgtgccga ggactggatt cggacagata ccttgccttc
ctggctgaac 120 gccccagcct ttgtcgcagc cgtggccttg agcccagccg
aatgggggga tgaagatgga 180 gacgacgaaa tctacttctt ctttacggag
acttcccgag catttgactc atacgagcgc 240 attaaagtcc cacgggtggc
ccgtgtgtgt gcgggggacc tcgggggccg gaagaccctc 300 cagcagagat
ggacgacgtt tttgaaagct gacctgctct gtccagggcc tgagcatggc 360
cgggcctcca gtgtcctgca ggatgttgct gtgcttcgac ctgagcttgg ggcagggact
420 cccatctttt atggcatctt ttcttcccag tgggaggggg ctactatctc
tgctgtctgt 480 gccttccgac cacaagacat tcggacagtg ctgaatggtc
ccttcagaga actaaaacat 540 gactgcaaca gaggactgcc tgtcgtggac
aatgatgtgc cccagcccag acctggagag 600 tgcatcacca acaacatgaa
gctccggcac tttggctcat ctctctccct gcctgaccgc 660 gtactcacct
tcatccggga ccacccactc atggacaggc cagtgtttcc agctgatggc 720
caccccctgc tggtcactac agatacagcc tatctcagag tcgtggccca cagggtgacc
780 agcctctcag ggaaagagta tgatgtgctc tacctgggga cagaggatgg
acacctccac 840 cgagcagtgc ggatcggagc tcagctcagc gttcttgaag
atctggcctt attcccagag 900 ccacagccag ttgagaacat gaaattgtac
cacagctggc tcctggttgg ctcccgtact 960 gaggtgacac aagtgaatac
aaccaactgt ggccgtctcc agagctgctc agagtgcatc 1020 ctggcccagg
acccagtctg tgcctggagc ttccggctgg atgagtgtgt ggcccatgcc 1080
ggggagcacc gagggttggt ccaagacata gagtcagcag atgtctcctc tttgtgtcct
1140 aaagagcctg gagaacgtcc agtagtgttt gaagttcccg tggctacagc
tgcgcatgtg 1200 gtcttgccat gttctccaag ctcagcatgg gcatcctgtg
tgtggcacca gcccagtgga 1260 gtgactgcac tcaccccccg gcgggatgga
ctggaggtgg tggtgacccc
aggggccatg 1320 ggcgcttatg cctgtgaatg tcaggagggt ggggcagccc
atgtggtagc agcttacagc 1380 ttggtatggg gcagccagcg agatgctccg
agccgggccc acacagtggg ggcgggactg 1440 gctggcttct tcttggggat
tctcgcagca tccctgactc tcattctgat tggtcggcgt 1500 cagcagcgac
ggcgacagag ggaacttctg gctagagaca aggtgggcct ggacctgggg 1560
gctccacctt ctgggaccac aagctacagc caagaccctc cctccccctc tcctgaagat
1620 gagcggttgc cgctggccct ggccaagagg ggcagtggct ttggtggatt
ctcaccaccc 1680 ttcctgcttg atccttgccc aagcccagcc cacattcggc
taactggggc tcctctagcc 1740 acatgtgatg aaacatccat c 1761 6 587 PRT
Homo sapiens 6 Gly Gly Val Leu Tyr Ala Ala Thr Val Lys Asn Tyr Leu
Gly Thr Glu 1 5 10 15 Pro Ile Ile Thr Arg Ala Val Gly Arg Ala Glu
Asp Trp Ile Arg Thr 20 25 30 Asp Thr Leu Pro Ser Trp Leu Asn Ala
Pro Ala Phe Val Ala Ala Val 35 40 45 Ala Leu Ser Pro Ala Glu Trp
Gly Asp Glu Asp Gly Asp Asp Glu Ile 50 55 60 Tyr Phe Phe Phe Thr
Glu Thr Ser Arg Ala Phe Asp Ser Tyr Glu Arg 65 70 75 80 Ile Lys Val
Pro Arg Val Ala Arg Val Cys Ala Gly Asp Leu Gly Gly 85 90 95 Arg
Lys Thr Leu Gln Gln Arg Trp Thr Thr Phe Leu Lys Ala Asp Leu 100 105
110 Leu Cys Pro Gly Pro Glu His Gly Arg Ala Ser Ser Val Leu Gln Asp
115 120 125 Val Ala Val Leu Arg Pro Glu Leu Gly Ala Gly Thr Pro Ile
Phe Tyr 130 135 140 Gly Ile Phe Ser Ser Gln Trp Glu Gly Ala Thr Ile
Ser Ala Val Cys 145 150 155 160 Ala Phe Arg Pro Gln Asp Ile Arg Thr
Val Leu Asn Gly Pro Phe Arg 165 170 175 Glu Leu Lys His Asp Cys Asn
Arg Gly Leu Pro Val Val Asp Asn Asp 180 185 190 Val Pro Gln Pro Arg
Pro Gly Glu Cys Ile Thr Asn Asn Met Lys Leu 195 200 205 Arg His Phe
Gly Ser Ser Leu Ser Leu Pro Asp Arg Val Leu Thr Phe 210 215 220 Ile
Arg Asp His Pro Leu Met Asp Arg Pro Val Phe Pro Ala Asp Gly 225 230
235 240 His Pro Leu Leu Val Thr Thr Asp Thr Ala Tyr Leu Arg Val Val
Ala 245 250 255 His Arg Val Thr Ser Leu Ser Gly Lys Glu Tyr Asp Val
Leu Tyr Leu 260 265 270 Gly Thr Glu Asp Gly His Leu His Arg Ala Val
Arg Ile Gly Ala Gln 275 280 285 Leu Ser Val Leu Glu Asp Leu Ala Leu
Phe Pro Glu Pro Gln Pro Val 290 295 300 Glu Asn Met Lys Leu Tyr His
Ser Trp Leu Leu Val Gly Ser Arg Thr 305 310 315 320 Glu Val Thr Gln
Val Asn Thr Thr Asn Cys Gly Arg Leu Gln Ser Cys 325 330 335 Ser Glu
Cys Ile Leu Ala Gln Asp Pro Val Cys Ala Trp Ser Phe Arg 340 345 350
Leu Asp Glu Cys Val Ala His Ala Gly Glu His Arg Gly Leu Val Gln 355
360 365 Asp Ile Glu Ser Ala Asp Val Ser Ser Leu Cys Pro Lys Glu Pro
Gly 370 375 380 Glu Arg Pro Val Val Phe Glu Val Pro Val Ala Thr Ala
Ala His Val 385 390 395 400 Val Leu Pro Cys Ser Pro Ser Ser Ala Trp
Ala Ser Cys Val Trp His 405 410 415 Gln Pro Ser Gly Val Thr Ala Leu
Thr Pro Arg Arg Asp Gly Leu Glu 420 425 430 Val Val Val Thr Pro Gly
Ala Met Gly Ala Tyr Ala Cys Glu Cys Gln 435 440 445 Glu Gly Gly Ala
Ala His Val Val Ala Ala Tyr Ser Leu Val Trp Gly 450 455 460 Ser Gln
Arg Asp Ala Pro Ser Arg Ala His Thr Val Gly Ala Gly Leu 465 470 475
480 Ala Gly Phe Phe Leu Gly Ile Leu Ala Ala Ser Leu Thr Leu Ile Leu
485 490 495 Ile Gly Arg Arg Gln Gln Arg Arg Arg Gln Arg Glu Leu Leu
Ala Arg 500 505 510 Asp Lys Val Gly Leu Asp Leu Gly Ala Pro Pro Ser
Gly Thr Thr Ser 515 520 525 Tyr Ser Gln Asp Pro Pro Ser Pro Ser Pro
Glu Asp Glu Arg Leu Pro 530 535 540 Leu Ala Leu Ala Lys Arg Gly Ser
Gly Phe Gly Gly Phe Ser Pro Pro 545 550 555 560 Phe Leu Leu Asp Pro
Cys Pro Ser Pro Ala His Ile Arg Leu Thr Gly 565 570 575 Ala Pro Leu
Ala Thr Cys Asp Glu Thr Ser Ile 580 585 7 196 DNA Homo sapiens
misc_feature (1)..(196) Coding region 7 aaattgtacc acagctggct
cctggttggc tcccgtactg aggtgacaca agtgaataca 60 accaactgtg
gccgtctcca gagctgctca gagtgcatcc tggcccagga cccagtctgt 120
gcctggagct tccggctgga tgagtgtgtg gcccatgccg gggagcaccg agggttggtc
180 caagacatag agtcag 196 8 30 DNA Artificial Sequence PCR primer
used to obtain the sequence encoding the intracellular domain of
Semaphorin W 8 gataaggatc cgggtcgccg tcagcagcgt 30 9 27 DNA
Artificial Sequence Anti-sense PCR primer sequence used to obtain
the sequence encoding the intracellular domain of Semaphorin W 9
ggctggaatt cattttcccc ggcttta 27 10 333 DNA Homo sapiens
misc_feature (1)..(333) Coding region 10 ccccggccgg gtcccgggca
gcctacagcc tcgcccttcc cgctactgct gctggcggtg 60 ctgagcggcc
cggtatccgg ccgcgtcccc cgctcggtgc ccagaacctc gcttccaatc 120
tctgaggctg acttctgtct cacccggttc gcagtccctc acacatacaa ttactctgtt
180 ctccttgtgg atcctgcctc ccacacactt tatgttggcg cccgggacac
catcttcgct 240 ttatccctgc ccttctcagg ggagagaccc cgcaggattg
actggatggt tcctgaggct 300 cacagacaga actgtaggaa gaaaggcaag aaa 333
11 111 PRT Homo sapiens 11 Pro Arg Pro Gly Pro Gly Gln Pro Thr Ala
Ser Pro Phe Pro Leu Leu 1 5 10 15 Leu Leu Ala Val Leu Ser Gly Pro
Val Ser Gly Arg Val Pro Arg Ser 20 25 30 Val Pro Arg Thr Ser Leu
Pro Ile Ser Glu Ala Asp Phe Cys Leu Thr 35 40 45 Arg Phe Ala Val
Pro His Thr Tyr Asn Tyr Ser Val Leu Leu Val Asp 50 55 60 Pro Ala
Ser His Thr Leu Tyr Val Gly Ala Arg Asp Thr Ile Phe Ala 65 70 75 80
Leu Ser Leu Pro Phe Ser Gly Glu Arg Pro Arg Arg Ile Asp Trp Met 85
90 95 Val Pro Glu Ala His Arg Gln Asn Cys Arg Lys Lys Gly Lys Lys
100 105 110 12 7 PRT Homo sapiens 12 Gln Asp Pro Val Cys Ala Trp 1
5 13 7 PRT Homo sapiens misc_feature (1)..(1) Xaa = Gln or Arg 13
Xaa Asp Pro Tyr Cys Xaa Trp 1 5 14 14 PRT Unknown Description of
Unknown Organism Myc tag 14 Asp Ile Gly Gly Glu Gln Lys Leu Ile Ser
Glu Glu Asp Leu 1 5 10 15 517 DNA Homo sapiens misc_feature
(1)..(517) sequence of GenBank Accession No R54387 15 gaccacaaga
cattcggaca gtgctgaatg gtcccttcag agaactaaaa catgactgca 60
acagaggact gcctgtcgtg gacaatgatg tgccccagcc cagacctgga gagtgcatca
120 ccaacaacat gaagctccgg cactttggct catctctctc cctgcctgac
cgcgtactca 180 ccttcatccg ggancaccca ctcatggaca ggccagtntt
tccagctgat ggccaccccc 240 tgntggtcac tacagataca gnctatctca
gagtcgtggc ccacagggtg accagcctct 300 cagggaaaga gtatgatgtg
ctctacctgg gggacagagg atgggacaac ttcaccgagc 360 agtgcggatt
cggagctcag ttcagcgttt ctttgaagat cttgggctta tttnccagag 420
tcacagncag tttnaggaac ntgaaatttg ttacccacag ttnggttcng gggttggttt
480 ccgttatttt agggtnacac aagtggatta caaccca 517 16 364 DNA Homo
sapiens misc_feature (1)..(364) sequence of GenBank Accession No
T09073 16 aaattgtacc acagctggct cctggttggc tcccgtactg aggtgacaca
agtgaataca 60 acnaactgtg gccgtctcca gagctgctca gagtgcatcc
tggcccagga cccagtctgt 120 gcctggagct tccggctgga tgagtgtgtg
gcccatgccg gggagcaccg agggttggtc 180 caagacatag agtcagcaga
tgtctcctct ttgtgtccta aagagcctgg agaacgtcca 240 gtagtgtttg
aagttcccgt ggctacagnt gcgcatgtgg tcttnccatg ttctccaagc 300
tcagcatggg catcctgtgt gtggcaccag cccagtggag ttacttcact taccccccgg
360 cggg 364
* * * * *