The Inhibitory Effect of Calumenin on the Vitamin K-dependent γ-Carboxylation System

Abstract

The vitamin K-dependent γ-carboxylation system is responsible for post-translational modification of vitamin K-dependent proteins, converting them to Gla-containing proteins. The system consists of integral membrane proteins located in the endoplasmic reticulum membrane and includes the γ-carboxylase and the warfarin-sensitive enzyme vitamin K1 2,3-epoxide reductase (VKOR), which provides γ-carboxylase with reduced vitamin K1 cofactor. In this work, an in vitro γ-carboxylation system was designed and used to understand how VKOR and γ-carboxylase work together as a system and to identify factors that can regulate the activity of the system. Results are presented that demonstrate that the endoplasmic reticulum chaperone protein calumenin is associated with γ-carboxylase and inhibits its activity. Silencing of the calumenin gene with siRNA resulted in a 5-fold increase in γ-carboxylase activity. The results provide the first identification of a protein that can regulate the activity of the γ-carboxylation system. The propeptides of vitamin K-dependent proteins stimulate γ-carboxylase activity. Here we show that the factor X and prothrombin propeptides do not increase reduced vitamin K1 cofactor production by VKOR in the system where VKOR is the rate-limiting step for γ-carboxylation (Wallin, R., Sane, D. C., and Hutson, S. M. (2002) Thromb. Res. 108, 221–226). These findings put calumenin in a central position concerning regulation of γ-carboxylation of vitamin K-dependent proteins. Reduced vitamin K1 cofactor transfer between VKOR and γ-carboxylase is shown to be significantly impaired in the in vitro γ-carboxylation system prepared from warfarin-resistant rats. Furthermore, the sequence of the 18-kDa subunit 1 of the VKOR enzyme complex (Rost, S., Fregin, A., Ivaskeviclus, V., Conzelmann, E., Hortnagel, K., Pelz, H-J., Lappegard K., Seifried, E., Scharrer, I., Tuddenham, E. G. D., Muller, C. R., Storm, T. M., and Oldennburg, J. (2004) Nature 427, 537–541) was found to be identical in the two rat strains. This finding supports the notion that different forms of genetic warfarin resistance exist.