Secretion, gamma-carboxylation, and endoplasmic reticulum-associated degradation of chimeras with mutually exchanged Gla domain between human protein C and prothrombin.
Warfarin, an antagonist of vitamin K, causes diminution of vitamin K-dependent coagulation factors in the circulation. Although all vitamin K-dependent factors have Gla domains, the warfarin-induced decrease in their plasma concentration differs among factors. In warfarin-treated HepG2 cells, we found modest and severe intracellular degradation of prothrombin and protein C, respectively. To investigate the structural features of these proteins that contribute to their warfarin sensitivity, chimeric prothrombin containing the prepropeptide and Gla domain of protein C was expressed in baby hamster kidney (BHK) cells. This chimera showed similar secretion kinetics and warfarin sensitivity to those of wild-type prothrombin, demonstrating that the Gla domain cannot solely explain the warfarin sensitivity of protein C. In contrast, two chimeric protein Cs containing either the Gla domain alone or the prepropeptide and Gla domain of prothrombin showed impaired secretion. Even though gamma-carboxylation proceeded normally, both chimeras were degraded intracellularly by the proteasome. From these results, we conclude that not only the folding of the Gla domain, but the entire structure and conformation of protein C and prothrombin, contribute to their quality control and susceptibility to warfarin-induced ER (endoplasmic reticulum)-associated degradation.
Tokunaga
F
F
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Garden City, Hyogo, Japan.
Takeuchi
S
S
Omura
S
S
Arvan
P
P
Koide
T
T
D000251:Adenosine Triphosphatases / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000378:metabolism / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D011486:Protein C / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011516:Prothrombin / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D016415:Sequence Alignment; D014162:Transfection; D014407:Tumor Cells, Cultured; D014812:Vitamin K / Q000494:pharmacology; D014859:Warfarin / Q000494:pharmacology
D009097:Multienzyme Complexes; D011486:Protein C; D011993:Recombinant Fusion Proteins; D014812:Vitamin K; D014859:Warfarin; D011516:Prothrombin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases