Semaphorin genes (I)

Details Semaphorin genes (I) Semaphorin genes (I)

1999-05-14

2002-08-20

Clark, Deborah J. R. (Department: 1633)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S320100, C435S325000, C435S455000, C536S023100, C536S023500, C424S093200, C424S093210

Reexamination Certificate

active

06436669

ABSTRACT:

This application is the national phase under 35 U.S.C. §371 of PCT International Application No. PCT/JP97/04111 which has an International filing date of Nov. 12, 1997, which designated the United States of America.
TECHNICAL FIELD
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
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)).
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.
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))
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 network 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)).
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.
PROBLEM TO BE SOLVED BY THE INVENTION
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.
MEANS OF SOLVING THE PROBLEM
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.
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.
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.
Thus, the gist of the present invention relates to:
(1) a gene encoding the following protein (a) or (b):
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 1,
(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;
(2) a gene comprising the following DNA (a) or (b):
(a) DNA comprising the base sequence shown in SEQ ID NO: 2,
(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;
(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;
(4) a protein obtained by expressing either a gene of the above item (1) or (2), or DNA of the above item (3);
(5) an expression vector which contains either a gene of the above item (1) or (2), or DNA of the above item (3);
(6) a transformant o

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