Proteasome degradation of protein C and plasmin inhibitor mutants

Miwako Nishio; Takatoshi Koyama; Masako Nakahara; Nagisa Egawa; Shinsaku Hirosawa

doi:10.1160/TH08-04-0252

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2008; 100(03): 405-412
DOI: 10.1160/TH08-04-0252

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Proteasome degradation of protein C and plasmin inhibitor mutants

Authors

Miwako Nishio

¹Laboratory Molecular Genetics of Hematology, Graduate School of Health Sciences, Tokyo Medical and Dental University, Tokyo, Japan
Takatoshi Koyama

¹Laboratory Molecular Genetics of Hematology, Graduate School of Health Sciences, Tokyo Medical and Dental University, Tokyo, Japan
Masako Nakahara

²School of Allied Health Sciences, Kitasato University, Sagamihara, Kanagawa, Japan
Nagisa Egawa

¹Laboratory Molecular Genetics of Hematology, Graduate School of Health Sciences, Tokyo Medical and Dental University, Tokyo, Japan
Shinsaku Hirosawa

¹Laboratory Molecular Genetics of Hematology, Graduate School of Health Sciences, Tokyo Medical and Dental University, Tokyo, Japan

Abstract

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Summary

Protein C (PC) deficiency and plasmin inhibitor (PI) deficiency are inherited thrombotic and haemorrhagic disorders. We investigated the intracellular degradation of mutant proteins, using naturally occurring PC and PI mutants that lead to congenital deficiencies. To examine the necessity of N-linked glycosylation for the proteasomal degradation of PC and PI, PC178 and PC331 mutants treated with tunicamycin and N-glycosylation-lacking mutants, PC92Stop and PI-America were pulse chased. The analysis revealed that the speed of degradation of the tunicamycin-treated PC mutants, PC92Stop and PI-America lacking glycosylation, was slower than that of N-glycosylated mutants. Immunoprecipitation and immunoblot analysis showed that PC178 and PC331 mutants were associated with molecular chaperones, Bip, GRP94, and calreticulin. PI-America was associated with only Bip. Although degradation of mutants was mediated by proteasomes, no association with ubiquitin was detected. Co-transfection of endoplasmic reticulum (ER) degradation enhancing α-mannosidase-like protein (EDEM) accelerated the degradation of N-glycosylated PC. In the absence of autophagy using Atg5-deficient cell lines, the degradation of the PC331 mutant was mildly accelerated but that of PC178, PI-America and PI-Okinawa mutants was not influenced. While the degradation of the PC and PI mutants was facilitated by N-glycosylation moieties, they were ubiquitin-independently degraded by proteasomes, irrespective of the presence or absence of N-glycosylation. Molecular chaperone binding was influenced by the presence of N-glycosylation moieties. When the misfolded or truncated mutant proteins are functionally active, proteasome inhibitors such as bortezomib may have therapeutic potential for treatment of protein deficiencies.

Keywords

Endoplasmic reticulum-associated degradation - N-linked glycosylation - protein C - plasmin inhibitor - proteasome inhibitor

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References

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