The inhibitory effect of calumenin on the vitamin K-dependent γ-carboxylation system -: Characterization of the system in normal and warfarin-resistant rats

The vitamin K-dependent gamma-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 gamma-carboxylase and the warfarin-sensitive enzyme vitamin K-1 2,3-epoxide reductase (VKOR), which provides gamma-carboxylase with reduced vitamin K-1 cofactor. In this work, an in vitro gamma-carboxylation system was designed and used to understand how VKOR and gamma-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 gamma-carboxylase and inhibits its activity. Silencing of the calumenin gene with siRNA resulted in a 5-fold increase in gamma-carboxylase activity. The results provide the first identification of a protein that can regulate the activity of the gamma-carboxylation system. The propeptides of vitamin K-dependent proteins stimulate gamma-carboxylase activity. Here we show that the factor X and prothrombin propeptides do not increase reduced vitamin K-1 cofactor production by VKOR in the system where VKOR is the rate-limiting step for gamma-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 gamma-carboxylation of vitamin K-dependent proteins. Reduced vitamin K-1 cofactor transfer between VKOR and gamma-carboxylase is shown to be significantly impaired in the in vitro gamma-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.