Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Patent
1996-03-22
2000-05-09
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
530350, 536 231, 536 232, 536 235, 435 691, 435196, 435198, 435199, 43525233, 4353201, C07K 1400, C12N 1563, C12N 120, C12N 1500
Patent
active
06060302&
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to human phospholipase C-.alpha., which is a secretory protein having an oxidation-reduction activity; genes encoding the human phospholipase C-.alpha.; an expression vector containing the genes; and a transformant having the expression vector.
BACKGROUND ART
Phospholipase C-.alpha. (hereinafter referred to as PLC-.alpha.) has conventionally been considered to belong to the superfamily of various isozymes of phospholipase C (hereinafter referred to as PLC).
PLC is an enzyme hydrolyzing (hereinafter referred to as a PLC activity) glycerophospholipid and sphingophospholipid; which is known to be present in the spleen, the tunica mucosa interstini tenuis, and the placenta, etc. of mammals and to play an important role in a living body. For example, phosphatidylinositol-specific phospholipase C (PI-PLC), which is widely present in a living body, hydrolyzes phosphatidylinositol 4,5-diphosphate to generate 1,2-diacylglycerol and inositol 1,4,5-triphosphate (Rhee, S.G. et al., Science 244, pp. 546-550 (1989)). There are a number of reports on various isozymes of PLC, for example, as follows: C. F. Bennett et al., Nature 334, pp. 268-270 (1988); Y. Emori et al., J. Biol. Chem. 264, pp. 21885-21890 (1989); M. Katan et al., Cell 54, pp. 171-177 (1988); S. Ohta et al., FEBS Lett. 242, pp. 31-35 (1988); M. L. Stahl et al., Nature 332, pp. 269-272 (1988); P. G. Suh et al., Cell 54, pp. 161-169 (1988); and R. W. Kritz et al., CIBA Found. Symp. 150, pp. 112-127 (1990).
In terms of function, the above-mentioned PLC-.alpha. is not well known; however, the expression thereof is known to increase in a living body being subject to stress and having cancerous tissue.
Regarding the PLC-.alpha. of mammals; those of mice (W. M. Hempel et al., J. Immunol. 146, pp. 3713-3720 (1991)), rats (C. F. Bennett et al., Nature 334, pp. 268-270 (1988)), and bovines (Hirano et al., Extended Abstracts, 4L-23, The 15th Annual Meeting of The Japanese Society of Molecular Biology, 1992), have been reported. The PLC-.alpha. of these mammals does not have significant homology with any of known sequences of the other known PLC family members.
The PLC-.alpha. of the above-mentioned mammals has a conserved amino acid sequence, Trp-Cys-Gly-His-Cys-Lys,[SEQ ID NO:5] at two places. This conserved amino acid sequence is identical with or very similar to the amino acid sequence of an active site for oxidation-reduction activity of protein disulfide isomerase (PDI) and thioredoxin. PDI and thioredoxin are both multifunctional proteins which function as protein disulfide reductase and isomerase, and catalyze the conversion of a thiol group to a disulfide group. PDI of mammals has two conserved sequences in its amino acid sequence, which are homologous to the above-mentioned PLC-.alpha.. Thioredoxin of prokaryotes and eukaryotes has one sequence, Trp-Cys-Gly-Pro-Cys-Lys, [SEQ ID NO:6]in its amino acid sequence which is similar to the conserved sequence as shown above. Cys residues in these sequences are respectively assumed to be active sites of catalytic activity (A. Holmgren, J. Biol. Chem. 264, pp. 13963-13966; R. B. Freedman, Cell 57, pp. 1069-1702).
The inventors of the present invention have found that bovine PLC-.alpha. expressed in E. coli is a secretory protein which does not functionally have PLC activity and has the activity of reducing a disulfide bond of protein such as insulin (Hirano et al., Cloning of bovine PLC-.alpha. and biological significance thereof, Extended Abstracts, 4L-23, The 15th Annual Meeting of The Japanese Society of Molecular Biology, 1992). Thus, PLC-.alpha. is not supposed to belong to the PLC superfamily but to the oxidation-reduction control protein family; and its nomenclature has accordingly been changed to thymuredoxin (Hirano et al. supra). This is supported by the above-mentioned consensus of the sequence.
The oxidation-reduction control protein family including PLC-.alpha., thioredoxin, PDI, etc. is considered to be required for maintaining the normal function of a li
REFERENCES:
C. F. Bennett et al., "Molecular Cloning and Complete Amino-Acid Sequence of Form-I Phosphoinositide-specific Phospholipase C," Nature, 334, pp. 268-270 (1988).
R. B. Freedman, "Protein Disulfide Isomerase: Multiple Roles in the Modification of Nascent Secretory Proteins," Cell, 57, pp. 1069-1072 (1989).
W. M. Hempel et al., "Expression of Phospholipase C Isozymes By Murine B Lymphocytes," J. Immunology, 146, pp. 3713-3720 (1991).
H. Hirai et al., "SH2 Mutants of c-src That Are Host Dependent For Transformation Are trans-Dominant Inhibitors of Mouse Cell Transformation By Activated c-src," Genes & Development, 4, pp. 2342-2352 (1990).
H. Hirai et al., "Site-Directed Mutagenesis of the SH2- and SH3-Coding Domains of c-src Produces Varied Phenotypes, Including Oncogenic Activation of p60.sup.c-src," Molecular & Cellular Biol., 10, pp. 1307-1318 (1990).
H. Hirai et al., "Mutations in src Homology Regions 2 and 3 of Activated Chicken c-src That Result in Preferential Transformation of Mouse or Chicken Cells," Proc. Natl. Acad. Sci., 87, 8592-8596 (1990).
N. Hirano et al., "Cloning of Bovine PLC-.alpha. and Biological Significance Thereof," Extended Abstracts: The 15th Annual Meeting of the Japanese Society of Molecular Biology, 4L-23 (1993). (partial English translation provided).
A. Holmgren, "Thioredoxin Glutaredoxin Systems," J. Biol. Chem., 264, pp. 13963-13966 (1989).
Y. Takagi et al., "Confirmation of Gene," Experimentation Methods for Gene Manipulation, p. 167 (1980). (partial English translation provided).
Hirai Hisamaru
Hirano Naoto
Haley, Jr. James F.
Hirano Naoto
Saidha Tekchand
Shionogi & Co. Ltd.
Wax Robert A.
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