title: Proteasome degradation of protein C and plasmin inhibitor mutants.
abstract: 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. Cotransfection of endoplasmic reticulum (ER) degradation enhancing alpha-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.
authors.affiliation: Laboratory Molecular Genetics of Hematology, Graduate School of Health Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
authors.forename: Miwako
authors.initials: M
authors.lastname: Nishio
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Koyama
authors.forename: Masako
authors.initials: M
authors.lastname: Nakahara
authors.forename: Nagisa
authors.initials: N
authors.lastname: Egawa
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Hirosawa