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Secretion, gamma-carboxylation, and endoplasmic reticulum-associated degradation of chimeras with mutually exchanged
F Tokunaga1, S Takeuchi, S Omura
1Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Garden City, Hyogo, Japan.
Abstract:
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.
Insights
Warfarin sensitivity in vitamin K-dependent factors like prothrombin and protein C is complex. Protein C is more susceptible to warfarin-induced degradation due to its overall structure, not just the Gla domain.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Warfarin, a vitamin K antagonist, reduces vitamin K-dependent coagulation factors.
- Different factors exhibit varying sensitivity to warfarin, despite sharing Gla domains.
Purpose of the Study:
- Investigate structural elements influencing warfarin sensitivity of prothrombin and protein C.
- Determine the role of Gla domains and overall protein structure in warfarin-induced degradation.
Main Methods:
- Expression of chimeric proteins (prothrombin with protein C domains, protein C with prothrombin domains) in cell lines (HepG2, BHK).
- Analysis of protein secretion, gamma-carboxylation, and intracellular degradation pathways (proteasome, ER-associated degradation).
Main Results:
- Chimeric prothrombin (with protein C's Gla domain) retained prothrombin's warfarin sensitivity, indicating the Gla domain alone doesn't dictate protein C's sensitivity.
- Chimeric protein Cs (with prothrombin's Gla domain or prepropeptide/Gla domain) showed impaired secretion and intracellular degradation, despite normal gamma-carboxylation.
- Warfarin-induced degradation is linked to the overall structure and conformation of these proteins.
Conclusions:
- The Gla domain is insufficient to explain differential warfarin sensitivity.
- Protein C's susceptibility to warfarin-induced ER-associated degradation involves its entire structure and conformation, impacting quality control mechanisms.