| Budget Amount *help |
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1994: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1993: ¥3,900,000 (Direct Cost: ¥3,900,000)
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| Research Abstract |
1. ANALYSIS OF SECRETION OF FIVE RECOMBINANT ARG 15 MUTANTS OF PROTEIN C :
Protein C (PC) deficiency is classified into two types. Type I shows equivalent reductions of both enzymatic activity and antigen concentration and type II shows only reduction of functional activity. More than 40 mutations have been identified in all over the PC molecule and most of them are caused by a single amino acid replacement through a single base exchange. Among these mutations, the replacement of Arg 15 to Gly in the Gla-domain is known as type II (PC-Yonago) , while the Arg-15 to Trp replacement was reported as either type I or II,and the Arg-15 to Gln mutation as type I.These indicate that amino acid mutations at the 15th position affect both the secretion and function of PC.To elucidate this hypothesis, we mutated Arg-15 to one base changeable amino acids, i.e.G,W,Q,L,or P by the PCR cassette mutation method and constructed pcD2-SRa-PC expression vectors. These vectors were transfected transiently CO
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S-7 cells (2x10^5 cells) by calcium phosphate coprecipitation. The recombinant PC antigen amounts of intracellular and secreted fractions were determined by ELISA (4experiments each) . The amounts of wild type rPC in the intracellular and secreted fractions were 8.4 and 131.3 ng/dish/48h, respectively. The relative amounts of R15G mutant were determined as 133.3? B130.8% (intracellular) and 67.1? B15.9% (secreted) of those of wild type. This suggests that R15G mutant was expressed and secreted nearly equal to normal, which agrees with type II deficiency of the manifestation of this case. The relative amounts of R15W mutant were 55.9? B16.1% (intracellular) and 0.4? B10.1% (secreted) , which strongly suggests that this mutation causes type I deficiency. The R15Q mutant showed 92.2? B16.2% (intracellular) and 75.4? B113.3% (secreted) . Unlike type I manifestation of R15Q,the recombinant was secretory. The R15L and R15P mutants were also secreted by 54.1 and 55.0%, respectively, of that of wild type in the medium.Thus, these two unrecognized mutations may be predicted to cause type II deficiency. Other kidney cell lines derived from human (293 cells) and hamster (BHK-21/C13 cells) also showed similar tendency with that of COS-7 cells.
ANALYSIS OF SECRETION OF WARFERIN-TREATED PROTEIN C :
Warfarin, an antagonist of vitamin K,is known to disrupt a microsomal vitamin K cycle, which results in a decrease of the plasma level of protein C as well as some other vitamin K-dependent coagulation factors. We studied the effect of warfarin on secretion of recombinant protein C expressed in 293 or BHK cells, or on the endogenous secretion from HepG2 cells. Warfarin caused a two-to four-fold decrease in the quantity of protein C secreted, compared to findings with vitamin K-treated cells. Pulse-chase experiments using three cell lines showed that, although protein C was fully secreted in the presence of vitamin K,the decrease in the total amount of the radioactivity in the warfarin-treated cells suggested intracellular degradation. This degradation depended on the concentration of warfarin and was not inhibited by an endoplasmic reticulum (ER) -Golgi transport inhibitor (brefeldin A) nor lysosonotropic inhibitors, suggesting that the degradation occurs in a pre-Golgi, nonlysosomal compartment. Among protease inhibitors tested, N-acetyl-Leu-Leu-methioninal and N-acetyl-Leu-Leu-norleucinal blocked the degradation of protein C precursor synthesized in the presence of warefarin and the precursor accumulated intracellularly, in a dose-dependent manner. Both inhibitors, however, did not disturb the secretion from vitamin K-treated cells. Thus, a cysteine protease (s) appeared to be responsible for the degradation. Inhibitors for intracellular glucosidase I and II,or for mannosidase showed no effect on the degradation. Protein C synthesized in the presence of warfarin was located in the same organelle with protein disulfide isomerase, an ER-resident protein, and the intracellular protein C was sensitive to endoglycosidase H digestion. Moreover, cross-link experiments using DPS suggested that intracellular protein C precursor associated with 100kDa, 80kDa, 40kDa, and 35kDa proteins in the warfarin-treated cells. From these results we conclude that protein C synthesized in the presence of warfarin was selectively degraded by a cysteine protease (s) in the ER,through a "quality control" mechanism. Less
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