Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Hormones – e.g. – prolactin – thymosin – growth factors – etc.
Reexamination Certificate
1999-02-25
2003-09-02
Kunz, Gary (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Hormones, e.g., prolactin, thymosin, growth factors, etc.
C530S350000, C930S010000
Reexamination Certificate
active
06613887
ABSTRACT:
This Application is the National Stage of International Application Serial No. PCT/JP97/03194, filed Sep. 10, 1997.
TECHNICAL FIELD
The present invention relates to a novel, protein showing a physiological activity on the central nervous system such as nerve-extending activity, neuroregenerative activity, gliacyte (glial cells) stimulating activity and so on.
BACKGROUND ART
The engram or trace of memory in the brain can be divided into two phases, namely acquisition of a short-term memory and fixation of a long-term memory. It is known that electroshocks and anesthesia may destroy the short-term memory but have no effect on the long-term memory once established. On the other hand, it is known that the biochemical reactions and structural changes leading to formation of a long-term memory as the antithesis of a short-term memory can be blocked by means of protein synthesis inhibitors such as puromycin and cycloheximide (Flexner, J. B. et al., Science, 141, 57-59 (1963); Agranoff, B. W. and Klinger, P. D., Science, 146, 952-953 (1964); Agranoff, B. W. et al., Brain Research 1, 300-309 (1966)). Therefore, it has been suspected over the past twenty years that certain protein metabolisms including de novo synthesis of proteins are involved in the formation of long-term memories in the brain. However, few proteins have been identified to this day which show an increased metabolic turnover following acquisition of a new behavior (Hyden, H and Lange, P. W., Proceedings of the National Academy of Sciences of the U.S.A., 65, 898-904 (1970); Shashoua, V. E., Science, 193, 1264-1266 (1976)).
Ependymin, among such proteins, was first reported as an entity comprising two kinds of proteins whose expression was increased after learning in the brain of goldfish as assayed by a double-labeling technique using [
3
H]valine and [
14
C]valine (Shashoua, V. E., Science, 193, 1264-1266 (1976)). Early immunohistological distribution studies (Shashoua, V. E., Brain Research, 122, 113-124 (1977), Benowitz, L. I. and Shashoua, V. E., Brain Research, 136, 227-242 (1977)) revealed that those proteins occurred in high concentrations in the ependymal zone (the cellular membrane lining the brain ventricles) and accordingly they were named ependymin &bgr; and ependymin &ggr;, respectively. However, it was subsequently suggested that those proteins were secretory proteins which were secreted into the cerebrospinal fluid (Shashoua, V. E. Brain Research, 166, 349-358 (1979); Shashoua, V. E. Neurochem. Res., 6, 1129-1147 (1981)) and more detailed immunohistological investigation endorsed the suggestion by the detection of ependymin in high concentrations in the mesencephalic structures and cerebrospinal fluid (Schmidt, R. and Lapp, H., Neurochem. Int., 10, 383-390 (1987).
Ependymin &bgr; and ependymin &ggr; were initially considered to be mutually distinct proteins because they gave molecular masses of 35 kDa and 30 kDa, respectively, on SDS-PAGE but it was later discovered that they are proteins identical in amino acid sequence and only dissimilar in sugar chain content (Schmidt, R. and Shashoua, V. E., Journal of Neurochemistry, 40, 652-660, (1983)). Moreover, it was reported that those proteins formed dimers and have a sugar chain content of at least 5% (Shashoua, V. E., Cell. Mol. Neurobiol., 5, 183-207 (1985)).
Based on the above series of research findings, ependymin had come to be considered to be associated with the learning and memory processes. For example, when an anti-ependymin antibody was injected into the cerebral ventricles, the integration of memories was blocked (Shashoua, V. E., Proceedings of the National Academy of Sciences of the U.S.A., 74, 1743-1747 (1977); Shashoua, V. E. and Moore, M. E. Brain Research, 148, 441-449 (1978); Schmidt, R. Adv. Biosci., 59, 213-222 (1986); Piront, M. L. and Schmidt, R., Brain Research, 442, 53-62 (1988)) and also in conditional experiments, the amount of expression of ependymin and its concentration had significant influences on the test results (Schmidt, R., Journal of Neurochemistry, 48, 1870-1878, (1987); Shashoua, V. E. and Hesse, G. W. Brain Research, 484, 333-339 (1989); Schmidt, R. et al., Progress in Brain Research, 91, 7-12 (1992)). Recently it has been discovered that ependymin is involved in the regeneration of the optic nerve of goldfish (Thormodsson, F. R. et al., Society Neuroscience Abstract, 14, 805 (1988)). It has also been found that ependymin is synthesized in the process of optic nerve regeneration (Schmidt, J. T. and Shashoua, V. E., Brain Research, 446, 269-284 (1988); Thormodsson, F. R. et al., Experimental Neurology, 118, 275-283 (1992)). This protein has, therefore, come to be a focus of attention. In the most recent researches using immunoelectron microscopic and in situ hybridization techniques, it was found that the mRNAs were expressed to synthesize the proteins, in reticulofibroblast cells of the inner endomeningeal cell layer but are not expressed in neurons and glial cells. However, after learning, the proteins are found in nerve cells and glial cells where the mRNAs are not detected, suggesting that the takeup of those proteins from the meninx by the neurons and glicytes has a functional importance in the plastic adaptations of the central nervous system (Rother, S. et al. Journal of Neurochemistry, 65, 1456-1464 (1995); Schmidt, R. et al., Journal of Neurochemistry, 65, 1465-1471 (1995)).
However, those studies were invariably undertaken in fish such as goldfish and the cDNA of ependymin has been found only in fish such as zebrafish, salmon, rainbow trout, herring, and carp in addition to goldfish (Koenigstorfer, A. et al., Journal of Neurochemistry, 52, 310-312 (1989); Koenigstorfer, A., Journal of Biological Chemistry, 264, 13689-13692 (1989); Sterrer, S. et al., Neuroscience, 37, 277-284 (1990); Muller-Schmid, A. et al., Gene, 118, 189-196 (1992); Muller-Schmid, A. et al., Journal of Molecular Evolution, 36, 578-585 (1993)). Meanwhile, it has been suspected that some protein corresponding to this ependymin exists in mammals as well, and including the report that an ependymins-like immune reaction is found in rat hippocampal neurons (Schmidt, R. et al., Brain Research, 386, 245-257 (1986)), a large number of immunohistological studies have been reported (Fazei, M. S. et al., European Journal of Neuroscience, Suppl. 1, 90 (1988); Shashoua, V. E. et al., Brain Research, 522, 181-190 (1990); Shashoua, V. E. et al., Journal of Neuroscience Research, 32, 239-244 (1992)). However, it remains to be established, biochemically or molecular biologically, whether there actually exists a mammalian counterpart of ependymin.
As the result of intensive research, the inventors of the present invention succeeded in cloning a cDNA having a novel nucleotide sequence from human placenta-, rat brain- or mouse spinal cord derived cDNA library and found that the protein encoded thereby is an ependymin-like protein having a physiological activity such as nerve-extending activity or neuroregenerative activity on the central nervous system, or gliacyte stimulating activity. As a result of continued investigations based on such findings, the present inventors have now completed the present invention.
DISCLOSURE OF INVENTION
The present invention provides:
(1) An ependymin-like protein derived from a mammal, or a salt thereof;
(2) A protein comprising an amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or a substantial equivalent thereto, or a salt thereof;
(3) The protein according to (2), wherein the substantially equivalent amino acid sequence to the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 is an amino acid sequence comprising an amino acid sequence represented by SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7;
(4) The protein according to (2), wherein the substantially equivalent amino acid sequence to the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 is an amino acid sequence having an identity of not less than about 95% to the am
Ogi Kazuhiro
Onda Haruo
Chao Mark
Hayes Robert C.
Kunz Gary
Ramesh Elaine M.
Takeda Chemical Industries Ltd.
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