U.S. patent application number 10/144031 was filed with the patent office on 2002-10-10 for novel semaphorin genes (i).
This patent application is currently assigned to Sumitomo Pharmaceuticals Company, Limited. Invention is credited to Furuyama, Tatsuo, Inagaki, Shinobu.
Application Number | 20020146775 10/144031 |
Document ID | / |
Family ID | 18128454 |
Filed Date | 2002-10-10 |
United States Patent
Application |
20020146775 |
Kind Code |
A1 |
Inagaki, Shinobu ; et
al. |
October 10, 2002 |
Novel semaphorin genes (I)
Abstract
The present invention provides a novel Semaphorin having
neurite-outgrowth inhibition activity or proteins analogous
thereto, peptide fragments of, or antibodies against, such
proteins, genes encoding such proteins, expression vectors for said
genes, transformed cells into which said expression vectors have
been introduced, methods for producing a recombinant protein which
employ said transformed cells, antisense nucleotides against the
above genes, transgenic animals involving insertion or deletion of
the above genes, and screening methods for antagonists of the above
proteins, all of which are useful mainly in diagnoses, treatments,
or studies relating to neurological diseases. The present invention
further provides use of such proteins, peptides, antibodies, genes,
or antisense nucleotides as pharmaceutical or diagnostic agents or
laboratory reagents.
Inventors: |
Inagaki, Shinobu;
(Ibaraki-shi, JP) ; Furuyama, Tatsuo;
(Ibaraka-shi, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
Sumitomo Pharmaceuticals Company,
Limited
|
Family ID: |
18128454 |
Appl. No.: |
10/144031 |
Filed: |
May 14, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10144031 |
May 14, 2002 |
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09308179 |
May 14, 1999 |
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09308179 |
May 14, 1999 |
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PCT/JP97/04111 |
Nov 12, 1997 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
Current CPC
Class: |
A01K 2217/05 20130101;
A61K 38/00 20130101; C07K 14/4703 20130101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
International
Class: |
C12P 021/02; C12N
005/06; C07H 021/04; C07K 014/435 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 15, 1996 |
JP |
321068/1996 |
Claims
1. An isolated polypeptide comprising the amino acid sequence show
in SEQ ID NO:1.
2. An isolated polypeptide comprising six or more contiguous amino
acids of SEQ ID NO:1.
3. 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:2; (b) the nucleotide sequence of SEQ ID NO:2 from nucleotide
370 to nucleotide 2694; and (c) a nucleotide sequence encoding the
amino acid sequence of SEQ ID NO:1.
4. A polypeptide obtained by expressing in a cell a nucleic acid
comprising a polynucleotide that specifically hybridizes with the
complement of a polynucleotide having a nucleotide sequence
selected from the group consisting of: (a) the nucleotide sequence
of SEQ ID NO:2; and (b) the nucleotide sequence of SEQ ID NO:2 from
nucleotide 370 to nucleotide 2694; under hybridization conditions
of a buffer comprising 45%(v/v) formamide, 5.times.SSPE at
42.degree. C., and washing after hybridization with a buffer
comprising 0.1.times.SSC, 0.1% SDS at 65.degree. C., wherein said
polypeptide has the biological activity of inhibiting neurite
outgrowth from dorsal root ganglion cells.
5. A polypeptide obtained by expressing in a cell a nucleic acid
comprising a polynucleotide that specifically hybridizes with the
complement of a polynucleotide having a nucleotide sequence
selected from the group consisting of: (a) the nucleotide sequence
of SEQ ID NO:2; and (b) the nucleotide sequence of SEQ ID NO:2 from
nucleotide 370 to nucleotide 2694; under hybridization conditions
of a buffer comprising 45%(v/v) formamide, 5.times. SSPE at
42.degree. C., and washing after hybridization with a buffer
comprising 0.1.times. SSC, 0.1% SDS at 65.degree. C., wherein said
polypeptide has the biological activity of collapsing growth cones
of neuron cells.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel Semaphorin
belonging to the Semaphorin family and a gene therefor. More
particularly, it relates to a novel Semaphorin having neurite
outgrowth inhibition activity and proteins analogous thereto, or
peptide fragments of, or antibodies against, such proteins, genes
(DNAs) encoding such proteins, expression vectors for said genes,
transformed cells into which said expression vectors have been
introduced, methods for producing a recombinant protein which
employ said transformed cells, antisense nucleotides against the
above genes, transgenic animals involving insertion or deletion of
the above genes, or screening methods for antagonists of the above
proteins, and it further relates to use of such proteins, peptides,
antibodies, genes, antisense nucleotides or the like as
pharmaceutical or diagnostic agents or laboratory reagents.
BACKGROUND ART
[0002] In 1992, Fasciclin IV (latterly called G-Sema I) was cloned
as one of the genes involved in guidance for neuron in grasshopper.
The next year, the existence of a gene family of which members
encode analogous domains and are distributed in a wide range of
species covering insects, viruses, nematodes, and human was
revealed, and the gene family members were designated "Semaphorin
genes". To date, more than ten genes belonging to the Semaphorin
family have been reported (Cell, 81, 471-474 (1995)).
[0003] These Semaphorin genes characteristically contains, in the
amino acid sequences which they encode, similar structures called
semaphorin domain each consisting of about 500 amino acids (Neuron,
14, 941-948 (1995); Cell, 75, 1389-1399 (1993)). Although the
homologies of the above amino acid sequences among Semaphorins are
80-20% and are thus not always high, some of the amino acid
residues are extremely well conserved as exemplified by thirteen
cysteine residues located at conserved positions. In the regions
other than semaphorin domains, Semaphorins are highly varied one
another. Specifically, Semaphorins include both of secretory and
membrane-bound forms, and have various structures including those
having Ig domains, thrombospondin domains, or a cluster of basic
amino acids at its carboxy terminus.
[0004] Among such 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, however, known to inhibit axon outgrowth and
synapsis formation during the stage of ontogenesis, that is, in the
course of the neural network formation at the embryonic or fetal
stage (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); and Neuron, 9, 831-845 (1992))
[0005] Although Semaphorin genes are known to perform its function
at the ontogenetic stage as described above, it has not yet been
ascertained whether or not they perform any function also in the
adult. However, some Semaphorin genes are known to be expressed
also in the adult in which formation of the neural net work has
been already completed, suggesting that they may have some function
also in said adult. For example, the central nervous system (CNS)
in the adult is widely known to lack regenerating ability, and some
Semaphorins which inhibit neurite outgrowth may conceivably
function as a CNS-neuron regeneration inhibitor in the adult
(Nature, 378, 439-440 (1995)). In addition, it has been suggested,
by a recently reported study on Semaphorin III-knockout mouse, that
a certain Semaphorin may probably act in inhibiting the growth of
cardiac muscles (Nature, 383, 525-528 (1996)). Furthermore, a
certain Semaphorin has been suggested to be involved in survival
and aggregation of B lymphocytes (Proc. Natl. Acad. Sci. USA, 93,
11780-11785 (1996))
[0006] It is thus being demonstrated that Semaphorins play
important roles not only in the nervous system but also in
non-nervous systems, and therefor attracting great interest in
studies on said Semaphorins.
[0007] Problem to be Solved by the Invention
[0008] Accordingly, an object of the present invention is to
provide a novel Semaphorin and proteins analogous thereto, peptide
fragments of, or antibodies against, such proteins, genes (DNAs)
encoding such proteins, expression vectors for said genes,
transformed cells into which said expression vectors have been
introduced, methods for producing a recombinant protein which
employ said transformed cells, antisense nucleotides against the
above genes, transgenic animals involving insertion or deletion of
the above genes, or screening methods for antagonists of the above
proteins, all of which are useful for medical treatments,
diagnoses, or studies of neurological diseases, and to further
provide use of such proteins, peptides, antibodies, genes,
antisense nucleotide or the like as pharmaceutical or diagnostic
agents or laboratory reagents.
[0009] Means of Solving the Problem
[0010] Despite the increasing interest in studies on Semaphorin as
described above, not all structures of the presumably more than
twenty kinds of Semaphorin genes have been elucidated. The present
inventors have planed to clone unknown Semaphorins by making use of
gene regions homologous among known Semaphorins. Firstly, we aimed
at a region homologous between Collapsin derived from chick and
G-Sema I derived from grasshopper, and used it to prepare synthetic
primers for amplifying a fragment corresponding to this region.
Although it was uncertain whether or not the use of these synthetic
primers may result in successful cloning of any novel Semaphorin
other than Collapsin and G-Sema I, we performed PCR using the
synthetic primers with cDNAs derived from mouse embryo as a
template. As a result, we have succeeded in cloning a novel
Semaphorin gene.
[0011] Analysis revealed that the novel Semaphorin of the present
invention contains no transmembrane regions in its structure,
indicating that it belongs to the secretory Semaphorin subfamily.
Although five to six Semaphorins of the secretory type have been
hitherto known, only one of such Semaphorins has demonstrated
activities. The Semaphorin of the present invention is of the
secretory type, and therefore, is characterized in that it may
serve, as such, as a pharmaceutical or diagnostic agent or
laboratory reagent in the art.
[0012] It was also shown, by further investigations on the
Semaphorin of the present invention, that it inhibits neurite
outgrowth, and that expression of the gene begins at the embryonal
stage, and in the adult, the gene is characteristically expressed
in a wide range of central and peripheral tissues in a localized
manner.
[0013] Thus, the gist of the present invention relates to:
[0014] (1) a gene encoding the following protein (a) or (b):
[0015] (a) a protein comprising the amino acid sequence shown in
SEQ ID NO: 1,
[0016] (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: 1, and which protein
has neurite-outgrowth inhibition activity;
[0017] (2) a gene comprising the following DNA (a) or (b):
[0018] (a) DNA comprising the base sequence shown in SEQ ID NO:
2,
[0019] (b) DNA which hybridizes under stringent conditions to DNA
comprising the base sequence shown in SEQ ID NO: 2, and which
encodes a protein having neurite-outgrowth inhibition activity;
[0020] (3) 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: 2;
[0021] (4) a protein obtained by expressing either a gene of the
above item (1) or (2), or DNA of the above item (3);
[0022] (5) an expression vector which contains either a gene of the
above item (1) or (2), or DNA of the above item (3);
[0023] (6) a transformant obtained by introduction of an expression
vector of the above item (5);
[0024] (7) a transformant of the above item (6) in which a gene of
the above item (1) or (2), or DNA of the above item (3), existing
in an expression vector of the above item (5) is stably
retained;
[0025] (8) a process for producing a recombinant protein, which
process comprises culturing a transformant of the above item (6) or
(7), and recovering the recombinant protein expressed;
[0026] (9) a peptide comprising a segment of at least six or more
amino acids of a protein of the above item (4);
[0027] (10) 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 the above item (1) or (2), or of DNA of the
above item (3);
[0028] (11) an antisense nucleotide, or chemically modified variant
thereof, of the above item (10), which inhibits expression of a
protein of the above item (4) when introduced into cells;
[0029] (12) an antibody or its fragment which specifically binds to
a protein of the above item (4) or to a peptide of the above item
(9);
[0030] (13) a pharmaceutical agent comprising, as an active
ingredient, a gene of the above item (1) or (2), DNA of the above
item (3), a protein of the above item (4), a peptide of the above
item (9), an antisense nucleotide or chemically modified variant
thereof of the above item (10) or (11), or an antibody or its
fragment of the above item (12);
[0031] (14) a neurite outgrowth inhibitor for PNS-neurons,
characterized in that it contains at least one of the proteins of
the above item (4);
[0032] (15) a screening method for antagonists of a protein
comprising the amino acid sequence shown in SEQ ID NO: 1,
characterized in that it employs a protein of the above item (4);
and
[0033] (16) a transgenic animal in which either a gene of the above
item (1) or (2), or DNA of the above item (3) has been artificially
inserted into its chromosome, or has been knocked out.
[0034] Mode for Carrying Out the Invention
[0035] For the purpose of the present invention, "gene" is also
referred to as "DNA". The gene is not specifically restricted so
far as it encodes a protein having the neurite-outgrowth inhibition
activity according to the novel Semaphorin. Examples are novel
mouse Semaphorin genes such as a gene encoding a protein which
comprises the amino acid sequence shown in SEQ ID NO: 1 of the
sequence listing or a gene comprising the base sequence shown in
SEQ ID NO: 2 of the sequence listing. Also included in the genes of
the present invention are those genes 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: 1, and those genes which hybridize under
stringent conditions to DNA comprising the base sequence shown in
SEQ ID NO: 2, provided that they encode proteins having
neurite-outgrowth inhibition activity. These genes are explained
below in order.
[0036] 1) Gene Encoding A Novel Mouse Semaphorin
[0037] Of the above-mentioned genes, "a gene which encodes a
protein comprising the amino acid sequence shown in SEQ ID NO: 1"
or "a gene comprising the base sequence shown in SEQ ID NO: 2" is a
novel mouse Semaphorin gene cloned in the present invention.
[0038] Such genes may be cloned, for example, as follows: PCR may
be performed using single-stranded cDNAs derived from mouse embryo
as a template with oligonucleotide primers which can amplify the
region homologous between two known Semaphorins, in this case
Collapsin and G-Sema I, and the resulting amplified fragment may be
used as a probe for screening cDNA libraries such as those derived
from adult mouse brain to clone the desired gene. Particular
techniques for such cloning may be found in the standard texts such
as "Molecular Cloning", 2nd ed., Cold Spring Harbor Laboratory
Press (1989). PCR may be performed, for example, by making
reference to "PCR", IRL Press (1991). The base sequence of cloned
DNA may be determined by conventional methods, for example, using a
sequencing kit commercially available.
[0039] When compared with previously reported Semaphorin genes, the
novel Semaphorin gene of the present invention exhibits 54% overall
identity with mouse Semaphorin D (a mouse homolog of chick
Collapsin) at the amino acid level, and exhibits 85% identity with
chick Collapsin-5, of which sequence is known only partially, in
their semaphorin domains.
[0040] 2) Gene Encoding Modified Protein of The Novel
Semaphorin
[0041] 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: 1 and which protein has
neurite-outgrowth inhibition activity" refers to a gene encoding a
so-called "modified protein" of the novel Semaphorin of the present
invention which has neurite-outgrowth inhibition activity. 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 a well-known method such as
site-directed mutagenesis described above.
[0042] For the purpose of the present invention, a protein "which
has neurite-outgrowth inhibition activity" refers to such a protein
that has collapse activity on growth cone of neuron or that has
neurite-outgrowth inhibition activity. Specifically, these
activities may be measured by the following procedures.
[0043] The collapse activity on growth cone of neuron may be
measured by making reference to M. Igarashi et al., Science, vol.
259, pp. 77-79 (1993)), while the neurite-outgrowth inhibition
activity may be measured by making reference to, for example, J. A.
Davies et al., Neuron, vol. 2, pp. 11-20 (1990) or M. Bastmeyer, J.
Neurosci., vol. 11, pp. 626-640 (1991)).
[0044] Briefly, the former activity is measured by adding an
expression product derived from the gene of the present invention
to neurons cultured under conventional conditions (e.g.,
"Culturing, Nerve Cells" edited by Banker et al., MIT Press (1991))
in a culture container 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. Normalization of the samples is typically
carried out on the basis of the total amounts of protein included
within the samples.
[0045] On the other hand, to measure the latter activity, for
example, a mass of cells expressing a gene of the present invention
described above is co-cultured with neurons in a collagen matrix,
and inability of the neurons to outgrow towards said mass of cells
expressing the gene or inhibition of the neurite outgrowth is used
as an indicator.
[0046] Alternatively, a nervous tissue may be trypsinized, and the
single cells thus obtained are treated with culture supernatant
from cells expressing a gene of the present invention. Significant
decrease in the number of neurons having outgrown neurites relative
to the control may be used as an indicator.
[0047] Examples of modified proteins obtained above are the human
and mouse types of modified protein of Semaphorin of the present
invention.
[0048] 3) Gene Hybridizing Under Stringent Conditions To The Novel
Semaphorin Gene
[0049] Of the above-mentioned genes, "a gene comprising DNA which
hybridizes under stringent conditions to DNA comprising the base
sequence shown in SEQ ID NO: 2 and which encodes a protein having
neurite-outgrowth inhibition activity" may refer to any mammal type
of the novel Semaphorin such as the human or rat type of Semaphorin
of the present invention. In addition, any genes which hybridize
under stringent conditions to DNA comprising the base sequence
shown in SEQ ID NO: 2 are included in the genes of the present
invention, provided that they have neurite-outgrowth inhibition
activity. Based on the results of a search through EST database,
the human type of novel Semaphorin of the present invention
obtained by cloning techniques is believed to comprise, as a
partial sequence, the sequence from position 1 to position 200 of
Accession Number W16752 or to comprise a sequence extremely
analogous (more than 95%, and preferably more than 98% identical)
to said sequence. Accordingly, the human type of novel Semaphorin
gene of the present invention can be easily cloned on the basis of
the EST sequence described above. Libraries used for such screening
may be human genomic libraries or human cDNA libraries, and cDNA
libraries derived from tissues of human nervous system are
preferably used.
[0050] As used herein, a gene "which hybridizes under stringent
conditions" refers to such a gene that hybridizes under conditions
described below in Example 1 wherein the salt concentration is
5.times.SSPE and the temperature is around 42.degree. C.
[0051] Also included within the scope of DNA of the present
invention are those DNAs cloned from a human cDNA library or a
human genomic library which hybridize under stringent conditions to
DNA comprising at least part of DNA consisting of the base sequence
shown in SEQ ID NO: 2.
[0052] Method for cloning such genes may be found, for example, in
"Molecular Cloning", 2nd ed., Cold Spring Harbor Laboratory Press
(1989), or "PCR" edited by McPherson et al., IRL Press (1991). A
preferred library used herein is a human genomic library. DNAs of
the present invention may also be cloned based on the sequence from
position 1 to position 200 of Accession Number W16572 of the EST
database, as described above.
[0053] The DNAs cloned as above include full-length DNAs, DNA
fragments consisting of about 100 bases or more, and a
single-stranded forms (coding strands or complementary stands
thereof) of said DNA fragments. In some preferred embodiments, they
may be genomic DNA such as 5' transcriptional control region, 3'
transcriptional control region, noncoding sequence of exons,
introns or the like. Such sequences which don't encode any amino
acids are also quite useful, for example, in developing a medicine
using antisense nucleotides described below.
[0054] In the present invention, "proteins" refers to those
proteins which may be obtained by expressing the genes (DNAs) of
the present invention. A specific example is a novel mouse
Semaphorin having the amino acid sequence shown in SEQ ID NO: 1
which is encoded by the longest open reading frame having the base
sequence from position 370 to position 2694 of SEQ ID NO: 2. This
Semaphorin has a semaphorin domain which corresponds to the amino
acid sequence from position 49 to position 572 of SEQ ID NO: 1. The
above novel mouse Semaphorin also contains a signal sequence at its
N-terminus, and said signal sequence may undergo processing to be
removed during its transfer to membrane, resulting in a mature
protein. In the case of SEQ ID NO: 1, the mature form is presumed
to be a protein consisting of the amino acid sequence beginning at
position 20. Since such mature form or modified protein thereof may
also be obtained by expressing a gene of the present invention
described above, it is also included within the proteins of the
present invention.
[0055] Expression vectors used in the present invention are not
specifically restricted so far as they are expressible vectors into
which a gene or DNA of the present invention is inserted, and
specific examples are known expression vectors such as pET, pCDM8
and the like.
[0056] A transformant of the present invention is obtained by
introducing an expression vector described above into desired host
cells. Host cells may be prokaryotic or eukaryotic, and are
selected depending on the expression vector used. Those
transformants in which the foreign gene existing in the expression
vector is stably retained in the cells are more preferred.
[0057] The expression vector may be suitably introduced into cells
by any one of known methods such as the calcium phosphate method,
DEAE-dextran method, or electroporation ("Current Protocols in
Molecular Biology", F. M. Ausubel et al. ed., John Wiley & Sons
(1987)).
[0058] A recombinant protein of the present invention is a protein
which is obtained by culturing the above transformants and
recovering the recombinant protein expressed and which has
neurite-outgrowth inhibition activity. Since Semaphorin of the
present invention is presumed from its structure to be a secretory
protein, the culture supernatant from transformed cells may
probably contain the Semaphorin. Therefore, activity measurement of
said Semaphorin can be easily carried out as described above by
using the culture supernatant as such.
[0059] In addition, the recombinant protein produced may be easily
purified by a method such as conventional column chromatography or
affinity purification using an antibody of the present invention
described below.
[0060] "Peptide", as used herein, refers to a peptide fragment
comprising a segment of at least six amino acids in the amino acid
sequence of a protein of the present invention. In this context,
the limitation "at least six amino acids" is based on the fact that
a minimal size of peptide capable of forming a stable structure
consists of six amino acids, and preferred peptides are those
consisting of eight 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 (for example, using
treatments with restriction enzymes), and expressing it in, for
example, animal cells. The peptide thus prepared may also be
modified by conventional methods.
[0061] These peptides can be used as pharmaceutical agents as
described below, and can also be used for producing antibodies.
[0062] "Antisense nucleotide" which is directed against a segment
of at least eight or more bases in a gene or DNA of the present
invention 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). Antisense nucleotides are used
for inhibiting expression of Semaphorin of the present invention,
and can also be used as laboratory reagents for, for instance, in
situ hybridization.
[0063] A "chemically modified variant" of the above antisense
nucleotide refers to such a chemically modified variant that can
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, and such variants may be
prepared in accordance with known references ("Antisense RNA and
DNA", WILEY-LISS, 1992, pp. 1-50; J. Med. Chem., 36, 1923-1937
(1993)).
[0064] When introduced into cells, the above antisense nucleotides
or chemically modified variants thereof can inhibit expression of a
protein of the present invention. mRNAs produced by usual gene
transcription are sense-strands, and the antisense nucleotides or
chemically modified variants thereof can bind to such a
sense-strand mRNA in cells to inhibit translation of the particular
mRNA, resulting in inhibition of production of Semaphorin of the
present invention. Because of such an effect of the antisense
nucleotides or chemically modified variants thereof, they may serve
as CNS-neuron regeneration promoters as described below.
[0065] It can easily be determined whether a particular antisense
nucleotide prepared, or a chemically modified variant thereof, has
a desired inhibitory effect or not, for example, by one of the
following two methods. In one method, an antisense oligonucleotide
or chemically modified variant thereof itself is directly
introduced into cells expressing a novel Semaphorin of the present
invention, and change in expression of said Semaphorin is then used
as an indicator. In the other method, a gene capable of producing,
when transcribed, said antisense RNA is introduced into the above
Semaphorin-expressing cells, and change in expression of said
Semaphorin is then used as an indicator.
[0066] Preferred examples of antisense nucleotides having such an
expression-inhibiting effect are those oligonucleotides having
sequences complementary to the transcription initiation site,
translation initiation site, 5' noncoding region, exon-intron
junction region, or 5' CAP region.
[0067] Antibodies of the present invention may be either polyclonal
or monoclonal antibodies which specifically bind to a protein or
peptide of the present invention. Such antibodies can easily be
produced by immunizing an animal such as mouse or rabbit using a
protein or peptide of the present invention in whole or in part
according to the procedures described in, for example, "Current
Protocols in Immunology", pp. 2.4.1-2.6.6 (J. E. Coligan ed.,
1992). Fragments of antibodies obtained by purifying the above
antibodies and degrading them with a peptidase are also included
within the antibodies of the present invention. Such antibodies may
be used, for example, in affinity chromatography or screening of
cDNA libraries, and as pharmaceutical or diagnostic agents, or
laboratory reagents.
[0068] The screening method for antagonists of a novel Semaphorin
of the present invention refers to, for example, such a screening
method of searching for substances which inhibits the
neurite-outgrowth inhibition activity of a novel Semaphorin of the
present invention. Such screening can be easily carried out by
adding the above Semaphorin of the present invention to an assay
system for Semaphorin activity described above, and further adding
thereto a test substance (for example, a peptide, modified protein,
low molecular weight compound or the like). In particular,
inhibition of an activity (for example, the neurite-outgrowth
inhibition activity) of Semaphorin of the present invention
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 activity assay
carried out with an added protein such as Semaphorin of the present
invention. 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
an antagonist of Semaphorin of the present invention. 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 antagonists of a novel Semaphorin of the
present invention thus obtained will serve as CNS-neuron
regeneration promoters as described below.
[0069] A transgenic animal of the present invention is produced by
artificially inserting a gene or DNA of the present invention into
the chromosome, or by knocking out said gene or DNA. Accordingly,
the transgenic animals of the present invention include not only
animals which expresses DNA of the present invention, but also
so-called knockout animals in which DNA of the present invention is
destroyed and dysfunctional. Destruction of DNA may be achieved by
a known method (Shinichi Aizawa, "Gene Targeting", Yodosha,
1995).
[0070] Animals used herein may be those animals other than human,
for example, laboratory animals such as mouse, rat, hamster, and
rabbit, or domestic animals such as bovine and swine, and mouse is
suitably used because of remarkable development of cell technology
for mouse.
[0071] Methods for producing a transgenic animal are briefly
described below for a transgenic mouse by way of illustration. For
example, in the first method, DNA is microinjected into a
pronucleus of an fertilized ovum of mouse. In the second method,
DNA is introduced by infecting an eight cell stage embryo with a
recombinant retrovirus. In the third method, DNA is introduced into
an embryonic stem cell (ES cell) having totipotency, for example,
by electroporation, and the cell is injected into another blastula
to produce a chimera. See, "Manipulation of Mouse Embryo", B. Hogan
et al. ed., 1986, Cold Spring Harbor Laboratory, and Shinichi
Aizawa, "Gene Targeting", 1995, Yodosha.
[0072] Transgenic animals produced by the above methods are quite
useful as an animal model for developing pharmaceutical agents or
an animal used for a screening of pharmaceutical agents.
[0073] The usefulness of the novel Semaphorin and others of the
present invention (proteins, DNAs, or peptides of the novel
Semaphorin, antisense nucleotides or antibodies against the
Semaphorin, and transgenic animals) is described below.
[0074] 1) Usefulness as Reagents in Relevant Area of
Investigation
[0075] While Semaphorin of the present invention inhibits
neurite-outgrowth as described above, it has been also suggested
that Semaphorin gene may have other unknown functions such as
immunosuppression (Cell, 75, 1389-1399 (1993)). Accordingly, it is
quite important for studies in relevant field to investigate
expression of Semaphorin gene or distribution and function of
Semaphorin protein. The present invention can provide DNAs,
proteins, peptides, antibodies, antisense nucleotide, transgenic
animals and the like which can be used for such purposes. Since the
novel Semaphorin of the present invention is a secretory protein,
it may be advantageously used as laboratory reagents.
[0076] 2) Usefulness as Pharmaceutical or Diagnostic Agents
[0077] One embodiment of the protein of the present invention is a
protein which inhibits neurite outgrowth. Accordingly, such a
protein may serve as therapeutic agent for immune diseases such as
atopic dermatitis, pain or other diseases by virtue of their
inhibition activity on neurite outgrowth of PNS-neurons.
[0078] In addition, as described in the "Background Art" section,
it has been recently suggested that a certain Semaphorin may play
an important role also in peripheral non-nervous systems. In
particular, it has been suggested that a certain Semaphorin may 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 survival and aggregation 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)). Involvement of Semaphorin in lung cancer has
also been suggested (Proc. Natl. Acad. Sci. USA, 93, 4120-4125
(1996)).
[0079] Furthermore, it is believed because of the following reasons
that Semaphorin of the present invention can inhibit cell movement
such as cell migration or infiltration. While actin cytoskeleton
plays an important role in movement of cell itself, it has been
demonstrated that actin cytoskeleton similarly plays an important
role also in movement of growth cone of neurons. It has been also
demonstrated that formation of actin cytoskeleton in both cases is
regulated by similar mechanisms in which G protein belonging to the
Rho family is involved (Genes Develop. 8, 1787-1802 (1994); Cell,
81, 53-62 (1995)). Semaphorin of the present invention is presumed
to inhibit elongation of growth cone via depolymerization of actin,
and it is also conceivable that the same can inhibit movement (such
as migration or infiltration) of cells expressing receptors for
Semaphorin through similar mechanism. This argument is also
supported by an observation that Ephrin known as a neuron guidance
factor causing collapse of growth cone as Semaphorin did inhibit
migration of neural crest cells (Neuron, 18383-396 (1997)).
[0080] In view of the above findings taken together, it is expected
that the novel Semaphorin proteins, DNAs and the like of the
present invention may inhibit infiltration or migration of cancer
cells or immunocytes, and therefore, may be used as antiallergic
agents, immunosuppressive agents, or anti-tumor agents.
[0081] In addition, as described below in Example 2, the novel
Semaphorin of the present invention is expressed in a wide range of
peripheral tissues and in a localized manner, suggesting that it
may play some role at these sites or in the neighborhood thereof.
In that case, the novel Semaphorin of the present invention may
serve as therapeutic or diagnostic agent for diseases at the sites
of expression indicated in Example 2.
[0082] As described in the above section 1), since the novel
Semaphorin of the present invention is a secretory protein, it may
be advantageously used as pharmaceutical or diagnostic agents.
[0083] On the other hand, it is suggested that a substance which
inhibits the binding of natural Semaphorin to Semaphorin receptors
may act as an antagonist inhibiting the neurite-outgrowth
inhibition effect of Semaphorin. Since Semaphorin of the present
invention is expressed also in the central nervous system, it is
also presumed to be a CNS-neuron regeneration inhibitor. In that
case, peptides, antibodies, or antisense nucleotides having an
ability as an antagonist may promote regeneration of CNS-neurons,
and therefore, may serve as therapeutic agents for spinal cord
injury etc. Such antagonists include those substances found by the
screening method described above.
[0084] The novel Semaphorin and other substances of the present
invention, of which usefulness as pharmaceutical or diagnostic
agents was described above, may be administered in doses and by
administration methods as described below. For example, Semaphorin
proteins, peptides, or antibodies may be formulated with an
appropriate stabilizing agent, buffer and/or diluent, and used in
an amount of several hundreds .mu.g to 2 g, and preferably of
several tens mg or less, per administration. To reduce the
administration frequency, it is possible to use a sustained release
preparation, or to administer a formulation by portions over a
prolonged period by means of, for example, an osmotic pump.
Alternatively, cells expressing said Semaphorin protein or other
substances may also be implanted into a living body for that
purpose.
[0085] In the case in which an antisense nucleotide is used as
pharmaceutical agents, such an antisense oligonucleotide or
chemical variant thereof may be administered as such or an
antisense RNA may be produced in cells, with doses and
administration methods as described below.
[0086] In the method in which an antisense oligonucleotide or
chemically modified variant thereof is administered as such, the
antisense oligonucleotide preferably has a length of, for example,
5-200 bases, more preferably 8-25 bases, and especially preferably
12-25 bases. Antisense oligonucleotide or chemically modified
variant thereof may be formulated by mixing it with a stabilizing
agent, buffer, solvent and the like prior to its administration.
Such formulation may optionally 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 administer at the site
disordered. To avoid frequent administrations, a sustained release
mini-pellet preparation 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.
[0087] In the method in which an antisense RNA is produced in
cells, the antisense RNA preferably has a length of, for example,
more than 100 bases, preferably more than 300 bases, and more
preferably 500 bases or more.
[0088] 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.
[0089] 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).
[0090] The methods employing recombinant viruses may include a
method in which Semaphorin gene 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.
[0091] Other methods may include a liposome method or a lipofectin
method. The liposome method is particularly preferred.
[0092] For the ex vivo methods, microinjection, the calcium
phosphate method, electroporation and the like may also be used,
besides those techniques described above.
[0093] 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 the affected site
such as nerve. For example, when spinal cord is infected with the
recombinant virus, the expression of Semaphorin gene is inhibited
exclusively in the spinal cord. Expression of an antisense
nucleotide of the present invention typically lasts several days to
several months, and such single infection is sufficient to allow
regeneration of neuron. Re-infection may also be possible, when the
antisense nucleotide is 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 or other additives may be
added as needed. 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.
[0094] 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
preferably administered once every several days to several
months.
BRIEF DESCRIPTION OF DRAWINGS
[0095] FIG. 1 shows a picture of electrophoresis obtained using
Northern blotting technique described in Example 3. In this figure,
lanes E10, E12, E14, E16, P0, P7, P14 and AD indicate the results
for embryonic day-10, embryonic day-12, embryonic day-14, embryonic
day-16, postnatal day-0, postnatal day-7, postnatal day-14 and the
adult, respectively. The upper arrow indicates the band for the
novel Semaphorin of the present invention, and the lower arrow
indicates the band for GAPDH [2].
[0096] FIG. 2 shows a photomicrograph indicating the result of the
activity measurement of the novel Semaphorin (2) described in
Example 5. A tissue piece of dorsal root ganglion can be seen at
the center of this figure. The dark area at the left side indicates
a cell mass of NIH 3T3 cells expressing a fusion protein of the
novel Semaphorin with alkaline phosphatase, and the dark area at
the right side indicates a cell mass of control NIH 3T3 cells
expressing alkaline phosphatase. As can be seen from this figure,
the neurite outgrowth towards the Semaphorin-expressing cells was
inhibited compared with that towards the control cells.
EXAMPLES
[0097] The present invention is further illustrated by the
following Examples. The present invention is not, however, limited
by such Examples in any way.
Example 1
[0098] Cloning of a Novel Semaphorin Gene
[0099] 1) Amplification of cDNA Fragment Encoding Semaphorin
Domain
[0100] From an embryonic day-14 embryo of ICR mouse (Kiwa Jikken
Dobutsu), mRNA was isolated using FastTrack (Invitrogen), and
single-stranded cDNA was synthesized using said mRNA as a template
together with MLV reverse transcriptase and random hexamer primer.
In order to amplify the region homologous between known Semaphorin
genes Collapsin and G-Sema I (Cell, 75, 217-227 (1993)), PCR was
carried out under the conditions described in FEBS. Lett., 370,
269-272 (1995) using the above single-stranded cDNA as a template
together with the following two synthetic oligonucleotide
primers:
1 5'-TACGACGTN(A/C/T)TNTT(C/T)AT(A/C/T)GG-3' (SEQ ID NO:3) and
5'-TCCCAIGC(A/G)CA(A/G)TAIGG(A/G)TC-3' (SEQ ID NO:4).
[0101] The resulting PCR product was subjected to 1.5% agarose gel
electrophoresis to separate and purify a DNA fragment having an
expected size of about 300 bp. This DNA fragment was inserted into
pCRscript SK(+) (Stratagene), and used as a probe in the following
cDNA library screening.
[0102] 2) Isolation of cDNA Clone
[0103] The cDNA fragment of about 300 bp obtained in the above
section 1) was labeled with .sup.32P using Random Priming
(Pharmacia), and used as a probe in screening of an adult mouse
brain cDNA library (Stratagene) as follows. The phage DNAs
constituting said eDNA library were transferred onto nylon filters
(Hybond-N; Amersham), denatured, neutralized, and then fixed on the
filters. These filters were then hybridized with the above probe to
obtain positive clones. The hybridization was carried out in a
hybridization buffer (5.times.SSPE (0.9M NaCl, 50 mM
NaH.sub.2PO.sub.4 (pH 7.7), 5 mM EDTA), 45% formaldehyde,
5.times.Denhardt's solution, 0.5% SDS, 20 .mu.g/ml denatured salmon
sperm DNA) for 16 hours at 42.degree. C.
[0104] 3) DNA Sequencing
[0105] Of the clones obtained in the above section 2), the base
sequence of a clone containing the longest cDNA was determined by
the dideoxy chain termination method using Taq Dye Primer cycle
sequencing kit or Taq Dye Terminator cycle sequencing kit and ABI
373 A DNA sequencer (Applied Biosystems). The base sequence of cDNA
thus determined is shown in SEQ ID NO: 2. The longest open reading
frame corresponds to the region from position 370 to position 2694
of SEQ ID NO: 2, and the amino acid sequence corresponding to said
ORF is shown in SEQ ID NO: 1.
[0106] Comparison of the base sequence shown in SEQ ID NO: 2 with
known base sequences from databases revealed that the gene is a
novel one. In addition, the region from position 49 to position 572
of the amino acid sequence shown in SEQ ID NO: 1 has homology with
a so-called semaphorin domain sequence, and the amino acid sequence
contains thirteen cysteines well conserved among Semaphorin genes,
definitely confirming that it is a novel Semaphorin belonging to
the Semaphorin family.
[0107] Since the novel Semaphorin of the present invention has an
immunoglobulin-like domain at positions 591 to 659 of its amino
acid sequence but has no transmembrane regions, it presumably
belongs to the secretory Semaphorin subfamily.
[0108] E. coli strain JM109 (pSR.alpha.mS(I)), a transformant
obtained by introducing a plasmid pSR.alpha.mS(I), which
incorporates the full-length cDNA for the novel Semaphorin gene of
the present invention in pUCSR.alpha., into E. coli strain JM109,
has been deposited at the National Institute of Bioscience and
Human Technology (1-1-3 Higashi, Tsukuba, Ibaraki, Japan) under
Deposit No. FERM BP-6157 on Oct. 24, 1997.
Example 2
[0109] Analysis of Distribution of the Novel Semaphorin Gene
Expression by in Situ Hybridization
[0110] 1) Method
[0111] 1. Slide Preparation
[0112] Various organs sampled from ddY mouse (purchased from SLC)
were frozen with dry ice powder, and sliced into 14-.mu.m thick
sections at -15.degree. C. using a cryostat to obtain prepared
slides.
[0113] 2. Preparation of Probe
[0114] A BamH I-EcoR I fragment (about 680 bp) of the cDNA obtained
in Example 1-3) was inserted into Bluescript KS+ (Stratagene), and
the resultant construct was then cut with BamH I. The DNA thus
obtained was used as a template to prepare an antisense RNA probe
labeled with .sup.35S-UTP using T3 RNA polymerase.
[0115] 3. Pretreatment of Slides
[0116] The sections obtained in the above step 1 were fixed with
10% formalin solution for 20 minutes, washed twice with PBS, and
treated with 50 mM Tris-HCl (pH 8.0)/5 mM EDTA solution containing
10 .mu.g/ml protease K for 5 minutes. After washing once with PBS,
these sections were fixed again with 10% formalin solution for 5
minutes, and treated with 0.1 M triethanolamine solution containing
0.25% acetic anhydride for 10 minutes. Furthermore, these sections
were washed once with PBS, and successively treated with 70%, 90%
and 100% alcohol for dehydration.
[0117] 4. Hybridization
[0118] The probe prepared in the above step 2 was added to a
hybridization buffer (50% formamide, 20 mM Tris-HCl (pH 8.0), 5 mM
EDTA, 0.3 M NaCl: 1% Denhardt's solution, 0.2% Sarkosyl, 200
.mu.g/ml yeast tRNA, 200 .mu.g/ml salmon sperm DNA) so that an
amount of the buffer to be used for each slide contained
1.times.10.sup.6 cpm probe. The buffer was placed onto the slides,
incubated overnight at 55.degree. C., and the slides were then
subjected to the following procedures:
[0119] (1) incubation for 15 minutes at 55.degree. C. in
5.times.SSC, 1% 2-mercaptoethanol solution;
[0120] (2) incubation for 30 minutes at 65.degree. C. in
2.times.SSC, 50% formamide, 5% 2-mercaptoethanol solution;
[0121] (3) incubation for 30 minutes at 37.degree. C. in 10 mM
Tris-HCl, 1 mM EDTA, 0.5 M NaCl solution;
[0122] (4) incubation for 30 minutes at 37.degree. C. in the
solution described in the above step (3) containing 1 mg/ml RNase
A;
[0123] (5) incubation for 30 minutes at 65.degree. C. in
2.times.SSC, 50% formamide, 5% 2-mercaptoethanol solution;
[0124] (6) incubation for 10 minutes at room temperature in
2.times.SSC; and
[0125] (7) incubation for 10 minutes at room temperature in
0.1.times.SSC.
[0126] At the end, the slides were successively treated with 70%,
90%, and 100% alcohol for dehydartion.
[0127] 5. Autoradiography
[0128] Microautoradiography was carried out using a photographic
emulsion (Ilford K-5, Ilford).
[0129] 2) Results
[0130] The novel Semaphorin gene of the present invention is mainly
distributed at the following sites (the distribution in mature
mouse, unless indicated as "neonatal mouse").
[0131] (1) Peripheral Tissues
[0132] Tooth (expression in neonatal mouse)
[0133] odontoblast Inner ear (expression in neonatal mouse)
[0134] spiral ganglion
[0135] epithelium of semicircular canal
[0136] macula of utricle
[0137] macula of saccule
[0138] Lung
[0139] trachea
[0140] epithelium to smooth muscle layer of bronchus and
bronchiole
[0141] Kidney
[0142] glomerulus or visceral layer of Bowman's capsule
[0143] Ovary
[0144] granular layer of ovarian follicle
[0145] Skin
[0146] hair matrix
[0147] Cartilage of face, backbone and the like
[0148] Hair root of palp and its periphery
[0149] (2) Central Nervous System
[0150] Eyeball
[0151] lamina ganglionaris and inner granular layer of retina
[0152] Hippocampus
[0153] Cerebral cortex
[0154] lamina profunda (layer 5/6)
[0155] basal ganglia (corpus striatum, globus pallidus, diagonal
band nucleus)
[0156] reticular nucleus of thalamus
[0157] Cerebellum
[0158] granule cell layer
[0159] Olfactory bulb
[0160] glomerular layer
[0161] mitral cell layer
[0162] inner granular layer
[0163] Nucleus pontis
[0164] nucleus of the spinal tract of the trigeminal nerve
[0165] main sensory nucleus of the trigeminus
[0166] Neurons in part of dorsal root ganglion (expression in
neonatal mouse)
[0167] Tractus solitarius nucleus
[0168] Reticular formation
Example 3
[0169] Analysis of Distribution of the Novel Semaphorin Gene
Expression by Northern Analysis
[0170] 1) Method
[0171] From pregnant ddY mice, fetal mice at embryonic days-10, 12,
14, and 16 (considering the day on which the presence of vaginal
plug was confirmed as embryonic day-0) were sampled. mRNAs from the
whole body of embryonic day-10 embryo, the head of embryonic day-12
embryo, or the brain of embryonic day-14 or 16 embryo, postnatal
day-0, 7, 14, or mature mouse were purified using QuickPrep Micro
mRNA Purification Kit (Pharmacia). Each 4 .mu.g per lane of mRNAs
was subjected to electrophoresis on 1% agarose-formaldehyde gel,
and then transferred onto a nylon membrane (Hybond-N, Amersham). A
probe labeled with [.alpha.-.sup.32P]dCTP was prepared using a Xba
I-Hind III fragment (about 790 bp) of cDNA obtained in Example 1-3)
as a template together with Random Labeling Kit (Pharmacia). In the
same manner, GAPDH [2] was also labeled as an internal
standard.
[0172] The above probe and GAPDH [2] were added to a hybridization
solution (50% formamide, 5.times.SSC, 5.times.Denhardt's solution,
25 mM sodium phosphate buffer (pH 6.8), 5 mM EDTA, 0.1% SDS, 20
.mu.g/ml salmon sperm DNA) so that the solution contained each
1.times.10.sup.6 cpm/ml of the labeled compounds, and hybridized
overnight at 42.degree. C. with the above nylon membrane. The nylon
membrane was finally washed three times for 10 minutes and three
times for 20 minutes in 0.1.times.SSC, 0.1% SDS at 65.degree. C.,
and then exposed overnight on Bio-Imaging Analyzer BAS 1000 (Fuji
Film) to detect labeled bands.
[0173] 2) Results
[0174] The results are shown in FIG. 1. Expression of the novel
Semaphorin gene of the present invention can be observed at all
stages studied.
Example 4
[0175] Expression of the Novel Semaphorin in Animal Cells
[0176] In order to express the novel Semaphorin of the present
invention, the novel Semaphorin gene of the present invention
obtained in Example 1-2) was introduced into NIH 3T3 cells.
[0177] First of all, the coding region of the novel Semaphorin cDNA
obtained in Example 1-2) was inserted into AP (alkaline
phosphatase)-1 vector (Flanagan, J. G. and Leader, P., Cell, vol.
63, pp. 185-194 (1990)) to construct a vector for expressing a
novel Semaphorin-alkaline phosphatase fusion protein.
[0178] Two .mu.g of the above vector DNA was introduced into NIH
3T3 cells by the calcium phosphate co-precipitation method
according to the procedures described in Flanagan, J G. and Leder
P., Cell, vol. 63, pp. 185-194 (1990). After 24 hours, the cells
were trypsinized to detach, and suspended in DMEM containing 10%
FCS (Gibco BRL). The cell suspension was plated into a 98-well
dish, and the incubation was continued to screen for transformed
cells which stably express alkaline phosphatase activity in the
presence of Geneticin. Measurement of alkaline phosphatase activity
was achieved as follows: the culture supernatant from the
above-mentioned transformed cells was sampled; p-nitrophenyl
phosphate, a substrate of alkaline phosphatase, was added thereto
to develop the color; and the absorbance at 405 nm (OD.sub.405) was
then measured (Flanagan, J G. and Leder, P., Cell, vol. 63, pp.
185-194 (1990)).
[0179] As a result, transformed cells stably expressing alkaline
phosphatase activity were obtained, and these cells were subjected
to the activity measurement described below in Example 5 as the
cells expressing a fusion protein of a novel Semaphorin of the
present invention with alkaline phosphatase. In parallel, the same
experiment as described above was repeated using AP-1 vector having
no inserted Semaphorin cDNA to prepare control transformed cells
expressing only alkaline phosphatase.
Example 5
[0180] Activity measurement of the novel Semaphorin (1)
[0181] The cDNA obtained in Example 1-2) is inserted into a plasmid
pUCSR.alpha. to construct an expression plasmid. This expression
plasmid is transfected into COS 7 cells by DEAE-dextran method, and
the culture supernatant is recovered after one day and after two
days. The culture supernatant thus obtained and a control culture
supernatant from untransfected COS 7 cells are each added to
culture medium of neurons which have been overnight-cultured in
vitro and thus have well-outgrown neurites, and the growth-cone
collapse activity is measured by the method described in M.
Igarashi et al., Science, vol. 259, pp. 77-79 (1993). As a result,
it is demonstrated that the culture supernatant from COS 7 cells
expressing Semaphorin gene of the present invention has
significantly higher growth-cone collapse activity.
[0182] Activity Measurement of the Novel Semaphorin (2)
[0183] The cDNA obtained in Example 1-2) is inserted into a plasmid
pUCSR.alpha. to construct an expression plasmid. This expression
plasmid is transfected into COS 7 cells by DEAE-dextran method.
After allowing the expressing cells to form a cell mass, it is
enclosed in a collagen gel matrix containing an appropriate culture
medium at an appropriate distance from neurons. In parallel, a mass
of untransfected COS 7 cells as a control is also enclosed
similarly. By examining the neurite outgrowth after culturing for
several days, it is shown that the neurite outgrowth towards COS 7
cells expressing Semaphorin gene of the present invention is
inhibited.
[0184] The same experiment may also be carried out using the novel
Semaphorin-expressing NIH 3T3 transformed cells prepared in Example
4.
[0185] In particular, a tissue piece of dorsal root ganglion
removed from mouse embryo at embryonic day-12.5 to 14.5 by a
convention method is firstly placed into a culture medium (Hanks'
solution; Nissui Pharmaceutical Co.). The tissue piece of dorsal
root ganglion is positioned at a distance of 400-800 .mu.m from
cell masses of the NIH 3T3 cells expressing the novel Semaphorin of
the present invention (cells expressing a fusion protein of the
novel Semaphorin with alkaline phosphatase) prepared in Example 4,
and of control NIH 3T3 cells (cells expressing only alkaline
phosphatase) prepared in Example 4, and all the cell masses and
tissue piece are enclosed in a collagen gel matrix. The collagen
gel matrix is prepared according to the method of Tessier-Iavigne,
M. and Goodman, C. S., Science, 274, 1123-1133 (1996) by mixing
collagen, 10.times.BME and NaHCO.sub.3/NaOH at the ratio of 8:1:1,
and incubating the mixture for 30 minutes at 37.degree. C. to
polymerize, and 2 ml of a culture medium (DMEM containing 10% FCS
(Gibco BRL)) supplemented with 50 ng/ml mouse NGF (Boehringer
Mannheim) is added to the collagen gel matrix.
[0186] After culturing 24-46 hours, the above dorsal root ganglion
and NIH 3T3 cells are fixed with a phosphate buffer containing 4%
paraformaldehyde, and observed under Olympus IMT-2 inverted
phase-contrast microscope. Neurite outgrowth is determined by
measuring the distance from the outer edge of dorsal root ganglion
tissue piece to the rim of outspread neurites (Messersmith et al.,
Neuron, 14, pp. 949-959 (1995)).
[0187] As a result, it is shown that the neurite outgrowth towards
the side on which the novel Semaphorin-expressing NIH 3T3 cells are
placed is significantly decreased compared to that towards the side
on which the control NIH 3T3 cells are placed, demonstrating that
the novel Semaphorin of the present invention inhibits neurite
outgrowth (FIG. 2).
[0188] Activity Measurement of the Novel Semaphorin (3)
[0189] A tissue piece of dorsal root ganglion at embryonic day-11.5
to 12.5 is dissociated using trypsin. The dorsal root ganglion
cells thus isolated using trypsin are inoculated at a low density
into a conditioned medium from control culture (culture medium
obtained by culturing NIH 3T3 cells transformed with AP-1 vector
for four days, and supplemented with 50 ng/ml mouse NGF (Boehringer
Mannheim)), or into a conditioned medium obtained by culturing NIH
3T3 cells expressing a fusion protein of the novel Semaphorin of
the present invention with alkaline phosphatase for four days to
which 50 ng/ml mouse NGF has been added. After culturing at
37.degree. C. for 18 hours, the cells are fixed for one hour with
4% paraformaldehyde. The neurites which have outgrown to the length
at least 2-times longer than the diameter of the cell is defined as
"neurite", and the number of cells having neurites is counted.
[0190] As a result, it is shown that only 12.9% of the dorsal root
ganglion cells treated with the culture medium containing the novel
Semaphorin of the present invention have neurites whereas 71.9% of
the dorsal root ganglion cells treated with the control culture
medium have neurites, demonstrating that the novel Semaphorin of
the present invention inhibits neurite outgrowth.
[0191] Effect of the Invention
[0192] The present invention provides a novel Semaphorin having
neurite outgrowth inhibition activity and proteins analogous
thereto, or peptide fragments of, or antibodies against, such
proteins, genes encoding such proteins, expression vectors for said
genes, transformed cells into which-said expression vectors have
been introduced, methods for producing a recombinant protein which
employ said transformed cells, antisense nucleotides against the
above genes, transgenic animals involving insertion or deletion of
the above genes, or screening methods for antagonists of the above
proteins. Furthermore, pharmaceutical or diagnostic agents or
laboratory reagents employing the above proteins, genes or the like
is expected to promote a dramatic advance mainly in diagnoses,
treatments, or studies relating to neurological diseases.
Sequence CWU 1
1
4 1 775 PRT Mus sp. 1 Met Ala Pro Ala Gly His Ile Leu Thr Leu Leu
Leu Trp Gly His Leu 1 5 10 15 Leu Glu Leu Trp Thr Pro Gly His Ser
Ala Asn Pro Ser Tyr Pro Arg 20 25 30 Leu Arg Leu Ser His Lys Glu
Leu Leu Glu Leu Asn Arg Thr Ser Ile 35 40 45 Phe Gln Ser Pro Leu
Gly Phe Leu Asp Leu His Thr Met Leu Leu Asp 50 55 60 Glu Tyr Gln
Glu Arg Leu Phe Val Gly Gly Arg Asp Leu Val Tyr Ser 65 70 75 80 Leu
Asn Leu Glu Arg Val Ser Asp Gly Tyr Arg Glu Ile Tyr Trp Pro 85 90
95 Ser Thr Ala Val Lys Val Glu Glu Cys Ile Met Lys Gly Lys Asp Ala
100 105 110 Asn Glu Cys Ala Asn Tyr Ile Arg Val Leu His His Tyr Asn
Arg Thr 115 120 125 His Leu Leu Thr Cys Ala Thr Gly Ala Phe Asp Pro
His Cys Ala Phe 130 135 140 Ile Arg Val Gly His His Ser Glu Glu Pro
Leu Phe His Leu Glu Ser 145 150 155 160 His Arg Ser Glu Arg Gly Arg
Gly Arg Cys Pro Phe Asp Pro Asn Ser 165 170 175 Ser Phe Val Ser Thr
Leu Val Gly Asn Glu Leu Phe Ala Gly Leu Tyr 180 185 190 Ser Asp Tyr
Trp Gly Arg Asp Ser Ala Ile Phe Arg Ser Met Gly Lys 195 200 205 Leu
Gly His Ile Arg Thr Glu His Asp Asp Glu Arg Leu Leu Lys Glu 210 215
220 Pro Lys Phe Val Gly Ser Tyr Met Ile Pro Asp Asn Glu Asp Arg Asp
225 230 235 240 Asp Asn Lys Met Tyr Phe Phe Phe Thr Glu Lys Ala Leu
Glu Ala Glu 245 250 255 Asn Asn Ala His Thr Ile Tyr Thr Arg Val Gly
Arg Leu Cys Val Asn 260 265 270 Asp Met Gly Gly Gln Arg Ile Leu Val
Asn Lys Trp Ser Thr Phe Leu 275 280 285 Lys Ala Arg Leu Val Cys Ser
Val Pro Gly Met Asn Gly Ile Asp Thr 290 295 300 Tyr Phe Asp Glu Leu
Glu Asp Val Phe Leu Leu Pro Thr Arg Asp Pro 305 310 315 320 Lys Asn
Pro Val Ile Phe Gly Leu Phe Asn Thr Thr Ser Asn Ile Phe 325 330 335
Arg Gly His Ala Val Cys Val Tyr His Met Ser Ser Ile Arg Glu Ala 340
345 350 Phe Asn Gly Pro Tyr Ala His Lys Glu Gly Pro Glu Tyr His Trp
Ser 355 360 365 Leu Tyr Glu Gly Lys Val Pro Tyr Pro Arg Pro Gly Ser
Cys Ala Ser 370 375 380 Lys Val Asn Gly Gly Lys Tyr Gly Thr Thr Lys
Asp Tyr Pro Asp Asp 385 390 395 400 Ala Ile Arg Phe Ala Arg Met His
Pro Leu Met Tyr Gln Pro Ile Lys 405 410 415 Pro Val His Lys Lys Pro
Ile Leu Val Lys Thr Asp Gly Lys Tyr Asn 420 425 430 Leu Arg Gln Leu
Ala Val Asp Arg Val Glu Ala Glu Asp Gly Gln Tyr 435 440 445 Asp Val
Leu Phe Ile Gly Thr Asp Thr Gly Ile Val Leu Lys Val Ile 450 455 460
Thr Ile Tyr Asn Gln Glu Thr Glu Trp Met Glu Glu Val Ile Leu Glu 465
470 475 480 Glu Leu Gln Ile Phe Lys Asp Pro Ala Pro Ile Ile Ser Met
Glu Ile 485 490 495 Ser Ser Lys Arg Gln Gln Leu Tyr Ile Gly Ser Ala
Ser Ala Val Ala 500 505 510 Gln Val Arg Phe His His Cys Asp Met Tyr
Gly Ser Ala Cys Ala Asp 515 520 525 Cys Cys Leu Ala Arg Asp Pro Tyr
Cys Ala Trp Asp Gly Ile Ser Cys 530 535 540 Ser Arg Tyr Tyr Pro Thr
Gly Ala His Ala Lys Arg Arg Phe Arg Arg 545 550 555 560 Gln Asp Val
Arg His Gly Asn Ala Ala Gln Gln Cys Phe Gly Gln Gln 565 570 575 Phe
Val Gly Asp Ala Leu Asp Arg Thr Glu Glu Arg Leu Ala Tyr Gly 580 585
590 Ile Glu Ser Asn Ser Thr Leu Leu Glu Cys Thr Pro Arg Ser Leu Gln
595 600 605 Ala Lys Val Ile Trp Phe Val Gln Lys Gly Arg Asp Val Arg
Lys Glu 610 615 620 Glu Val Lys Thr Asp Asp Arg Val Val Lys Met Asp
Leu Gly Leu Leu 625 630 635 640 Phe Leu Arg Val Arg Lys Ser Asp Ala
Gly Thr Tyr Phe Cys Gln Thr 645 650 655 Val Glu His Asn Phe Val His
Thr Val Arg Lys Ile Thr Leu Glu Val 660 665 670 Val Glu Glu His Lys
Val Glu Gly Met Phe His Lys Asp His Glu Glu 675 680 685 Glu Arg His
His Lys Met Pro Cys Pro Pro Leu Ser Gly Met Ser Gln 690 695 700 Gly
Thr Lys Pro Trp Tyr Lys Glu Phe Leu Gln Leu Ile Gly Tyr Ser 705 710
715 720 Asn Phe Gln Arg Val Glu Glu Tyr Cys Glu Lys Val Trp Cys Thr
Asp 725 730 735 Lys Lys Arg Lys Lys Leu Lys Met Ser Pro Ser Lys Trp
Lys Tyr Ala 740 745 750 Asn Pro Gln Glu Lys Arg Leu Arg Ser Lys Ala
Glu His Phe Arg Leu 755 760 765 Pro Arg His Thr Leu Leu Ser 770 775
2 2898 DNA Mus sp. misc_feature (1)..(2898) Strandedness
Double-stranded 2 ctgcaggatg gccctgtcct gttcggtgac tcggctctgc
ttcttgcggc cgaacaggtt 60 gcccatggcg gccccgcgcc ggctcgtgcc
gaattcggca cgagccagcc cggacctggc 120 tctcaagacg cgctccttgg
acggtctctt gctccgcgct tctaaccacc gggcccaagg 180 acagaaaggc
ttagcggatc caaatattgc ccggcaaatg gcacttggga atggtatttt 240
ctgatgacaa ccccttctgt ttgtgacaaa gcctgtcgcc cgcagttgcc cctggaggga
300 agtactaagt aaaactcaat cctgtcttaa agtgtggctg caggggccag
aggagagcca 360 gcacgcacca tggcaccggc cggacacatc ctcaccttgc
tgctctgggg tcacctgctg 420 gaactctgga ccccaggtca ctccgcgaac
ccctcctacc ccaggctacg cctgtcacat 480 aaagaacttt tggaactgaa
taggacttca atatttcaaa gcccccttgg atttcttgat 540 ctccatacaa
tgctgctgga tgagtatcaa gaacggctct ttgtgggagg cagagacctt 600
gtctattccc tgaacttgga acgagtcagt gacggctaca gagagatata ctggccgagc
660 acagcagtaa aggtagaaga atgcataatg aaaggaaaag acgcaaatga
gtgtgccaat 720 tatatccggg ttttgcatca ctacaacagg acacaccttc
tgacctgtgc tactggagct 780 tttgatccac actgtgcctt catcagagtc
gggcaccatt cagaggaacc cctgtttcac 840 ctggagtcac acagatctga
gagaggaagg ggcagatgtc cttttgaccc caactcctcc 900 tttgtgtcca
cgctagttgg gaatgagctg tttgctggac tctacagtga ctattggggc 960
agagactcgg cgatcttccg cagcatgggg aagttaggcc atattcgcac tgagcatgac
1020 gatgagcggc tcctgaaaga accaaaattt gtaggttcat atatgattcc
tgataacgaa 1080 gaccgagatg acaacaaaat gtactttttc tttactgaga
aggcgctgga ggcggagaac 1140 aacgcccaca cgatctacac ccgagtgggg
cggctgtgcg tgaatgacat gggaggacag 1200 agaatcctgg tgaacaagtg
gagcactttc cttaaagcgc ggctggtttg ctcagtgccg 1260 ggaatgaatg
gaatcgacac atactttgac gaactagagg atgtgttttt actgccgacc 1320
agagatccta agaatccagt gatatttgga ctgtttaata ctaccagcaa tatatttaga
1380 ggccatgctg tatgtgtgta tcacatgtca agtatccggg aagcctttaa
tggcccatat 1440 gctcataaag aaggccctga ataccactgg tcactatatg
aaggaaaagt cccctaccca 1500 aggcctggtt cctgtgccag caaagtaaac
ggaggcaagt atggaaccac caaagattac 1560 cccgatgacg ccatccggtt
cgcaaggatg catcctctaa tgtatcagcc cataaaacct 1620 gttcataaaa
aaccaatact ggtaaaaaca gatggaaaat acaacctgag gcaacttgcc 1680
gtggatcggg tggaagcgga ggatggccag tatgacgtct tatttattgg gacagacaca
1740 ggaattgtgc tgaaagtaat cacaatttac aaccaagaaa cagagtggat
ggaggaagtc 1800 attctagagg aacttcaaat attcaaggat ccagccccta
tcatttctat ggaaatttct 1860 tcaaagagac aacagcttta cattggatca
gcctctgctg tggcacaagt cagattccat 1920 cactgcgaca tgtatggcag
tgcttgtgct gactgctgcc tggctcgaga cccgtactgt 1980 gcctgggatg
gcatatcctg ctccaggtac tacccaacag gtgcacacgc aaagaggagg 2040
ttccgcaggc aggacgttcg gcatggcaac gccgcccaac agtgctttgg acagcaattt
2100 gttggagacg cgttggacag gactgaagag aggctggctt atggcataga
gagcaacagt 2160 actctgttgg aatgcacccc gcgatcacta caagcaaaag
tcatctggtt tgtacagaag 2220 ggacgcgacg taagaaaaga agaggtgaag
acggatgaca gagttgtcaa gatggacttg 2280 ggcttgctct tcctcagagt
acgcaagtca gatgcaggga cctatttttg ccagacagta 2340 gaacacaatt
ttgtccatac tgtgcgtaaa atcaccttgg aggtggtcga agagcataaa 2400
gtggagggca tgtttcataa ggaccatgaa gaggaaagac atcacaagat gccctgccct
2460 cccttaagcg gtatgtctca ggggacaaaa ccgtggtaca aggaattctt
gcagctgatt 2520 ggctacagca acttccagag agtggaagaa tactgcgaaa
aggtgtggtg tacagataag 2580 aagaggaaaa agcttaaaat gtctccctcc
aagtggaagt atgccaaccc ccaggaaaag 2640 aggcttcgct ctaaagctga
gcacttccgc ctgcccaggc acacgctgct ctcctgaggg 2700 cgccctctgc
cggcggctga ggaacctagg atggaaacat ttttttaaag ggggggaaaa 2760
aaaaaagact gaaagcatgc agtctctttc cattacttca aagaactttc tgtagtactn
2820 agaggctggg aaggtgtttt aagttattct gcatattcat ctgactgtac
aaacatctct 2880 ctcacagtac ttggtact 2898 3 20 DNA Artificial
Sequence Description of Artificial Sequence synthetic
oligonucleotide primer 3 tacgacgtnh tnttyathgg 20 4 20 DNA
Artificial Sequence Description of Artificial Sequence synthetic
oligonucleotide primer 4 tcccangcrc artanggrtc 20
* * * * *