Characterization of endoplasmic reticulum-associated degradation of a protein S mutant identified in a family of quantitative protein S deficiency.

Abstract

INTRODUCTION:

Misfolded and unassembled glycoproteins are eliminated from the endoplasmic reticulum (ER) lumen by the ER-associated degradation (ERAD). We previously identified a Tyr595Cys (Y595C) mutation of protein S (PS) in a family of a quantitative PS deficiency. The mutation causes intracellular degradation and decreased secretion of the Y595C mutant PS. The aim of the present study was to further characterize the molecular basis of the intracellular degradation of the mutant.

MATERIALS AND METHODS:

We stably expressed the mutant in mammalian cells, and analyzed the intracellular localization of the protein. The intracellular degradation pathway was determined by pulse-chase analyses in the presence of various inhibitors of ERAD.

RESULTS AND CONCLUSIONS:

Endoglycosidase H digestion and immunofluorescence staining revealed the mutant being retained in the ER. Epoxomicin, a potent and specific proteasome inhibitor, and Ala-Ala-Phe-CH(2)Cl (AAF), an inhibitor of tripeptidyl peptidase II (TPPII), suppressed the intracellular degradation of the mutant by about 65% and 50%, respectively. When epoxomicin was combined with AAF, the inhibitory effect was substantially enhanced. Although castanospermine, an inhibitor of glucosidases I and II, did not affect the degradation, kifunensine, an inhibitor of ER mannosidase I, suppressed it. Thus, it appears that the Y595C mutant is degraded through more than one pathway of ERAD, including the proteasome-dependent pathway and an alternate proteasome-independent pathway where proteases such as TPPII may be involved. Production of the critical B isoform of Man(8)GlcNAc(2) targets the mutant for ERAD, however, the interaction with calnexin/calreticulin through monoglucosylated oligosaccharides may not be required for the degradation of the mutant.

• Authors: Hiroko Tsuda, Fuminori Tokunaga, Hiroshi Nagamitsu, Takehiko Koide
• Journal: Thrombosis research (Thromb Res) Vol. 117 Issue 3 Pg. 323-31 ( 2006) ISSN: 0049-3848 [Print] United States
• PMID: 15893367 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Calreticulin
• Culture Media
• Culture Media, Conditioned
• DNA, Complementary
• Indolizines
• Oligopeptides
• Oligosaccharides
• Protease Inhibitors
• Proteasome Inhibitors
• Protein S
• Recombinant Proteins
• Calnexin
• Glycoside Hydrolases
• Mannosidases
• mannosyl-oligosaccharide 1,2-alpha-mannosidase
• castanospermine
• epoxomicin

Topics

• Animals
• Calnexin (metabolism)
• Calreticulin (metabolism)
• Cell Line
• Culture Media (pharmacology)
• Culture Media, Conditioned (pharmacology)
• DNA, Complementary (metabolism)
• Endoplasmic Reticulum (metabolism)
• Glycoside Hydrolases (metabolism)
• Humans
• Immunoprecipitation
• Indolizines (metabolism)
• Mannosidases (antagonists & inhibitors)
• Microscopy, Fluorescence
• Models, Statistical
• Mutation
• Oligopeptides (pharmacology)
• Oligosaccharides (chemistry)
• Protease Inhibitors (pharmacology)
• Proteasome Inhibitors
• Protein Denaturation
• Protein Folding
• Protein S (genetics)
• Protein S Deficiency (genetics)
• Recombinant Proteins (chemistry)
• Time Factors