• Search Research Projects
  • Search Researchers
  • How to Use
  1. Back to previous page

Analyzes of Molecular Mechanism of Protein Secretion Using Abnormal Protein C as a Model Protein

Research Project

Project/Area Number 05454624
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field Structural biochemistry
Research InstitutionHimeji Institute of Technology

Principal Investigator

KOIDE Takehiko  Himeji Institute of Technology, Department of Life Science, Professor, 理学部, 教授 (60018695)

Co-Investigator(Kenkyū-buntansha) TOKUNAGA Fuminori  Himeji Institute of Technology, Dept.of Life Science, Research Associate, 理学部, 助手 (00212069)
WAKABAYASHI Sadao  Himeji Institute of Technology, Dept.of Life Science, Associate Professor, 理学部, 助教授 (80148436)
Project Period (FY) 1993 – 1994
Project Status Completed (Fiscal Year 1994)
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)
KeywordsThrombosis / Protein C / Protein C deficiency / Recombinant Mutants / Vitamin K / Warfarin / Endoplasmic reticulum-associated protein degradation / Quality control / 小胞体内分解 / プロテインC欠乏症 / 異常プロテインC / 遺伝子発現 / 蛋白質の膜透過 / 蛋白質の細胞内移行
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 … More 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

Report

(3 results)
  • 1994 Annual Research Report   Final Research Report Summary
  • 1993 Annual Research Report
  • Research Products

    (14 results)

All Other

All Publications (14 results)

  • [Publications] Tokunaga,F.,: "Analysis of protein C deficiency by expression and characterization of five recombinant Arg‐15 mutants." Thrombosis and Haemostasis. 69. 979 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] 徳永文稔: "哺乳類細胞を用いた組換え体の発現によるプロテインC異常・欠乏症の分子機構解析" 生化学. 65. 922 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Tokunaga,F.,: "Warfarin causes the degradation of protein C precursor in the endoplasmic reticulum." Biochemistry. 34. 1163-1170 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] 小出武比古: "プロテインC異常症と欠乏症の分子メカニズム-アミノ酸置換とその分泌への影響-.第1回血液アゴラ" メジカルジャーナル社, 137 145 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Tokunaga,F.,: "Endoplasmic reticulum‐associated degradation of protein C precursor synthesized in the presence of an anticoagulant warfarin.in Blood Coagulation,Fibrinolysis and Platelet." Spring‐Verlag(in press), (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Tokunaga, F.: "Analysis of protein C deficiency by expression and characterization of five recombinant Arg-15 mutants" Thrombosis and Haemostasis. 69. 979 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Tokunaga, F.: "Analysis of molecular mechanism of protein C deficiency by expression of recombinant mutants using mammalian cell lines" Seikagaku. 65 (8). 922 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Koide, T.: "Molecular mechanism for protein C abnormality and deficiency. Amino-acid substitutions and their effects on the secretion" First Blood Agora.Medical Journal. 137-145 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Tokunaga, F.: "Warfarin causes the degradation of protein C precursor in the endoplasmic reticulum" Biochemistry. 34 (4). 1163-1170 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Tokunaga, F.: "Endoplasmic reticulum-associated degradation of protein C precursor synthesized in the presence of an anticoagulant, warfarin" Blood Coagulation, Fibrinolysis and Platelet (ed by E.Kakishita) Springer-Verlag. (in press). (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Tokunaga,F.et al: "Warfarin causes the degradation of protein C precursor in the endoplasmic reticulum." Biochemistry. 34. 1163-1170 (1995)

    • Related Report
      1994 Annual Research Report
  • [Publications] Tokunaga,F.et al: "Endoplasmic reticulum-associated degradation of protein C synthesized in the presence of an anticoagulant warfarin. in Blood Coagulation,Fibriolysis and Platelet." Springer-Verlag(in press), (1995)

    • Related Report
      1994 Annual Research Report
  • [Publications] Tokunaga,F.et al: "Analysis of protein C deficiency by expression and characterization of five recombinant Arg-15 mutants." Thrombosis and Haemostasis. 69. 979- (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] 徳永 文稔 ほか: "哺乳類細胞を用いた組換え体の発現によるプロテインC異常・欠乏症の分子機構解析" 生化学. 65. 922- (1993)

    • Related Report
      1993 Annual Research Report

URL: 

Published: 1993-04-01   Modified: 2016-04-21  

Information User Guide FAQ News Terms of Use Attribution of KAKENHI

Powered by NII kakenhi