Abstract 289: Regulation of Vitamin K-Dependent Epoxide Reductase by Protein Disulphide Isomerase
The vitamin K-dependent γ-carboxylation system is a multi-component system of proteins residing in the endoplasmic reticulum (ER). The system modifies proteins posttranslationally by converting certain glutamic acid residues into Gla, calcium binding residues. Calcium binding by Gla residues is essential for the proteins to carry out their normal physiological functions, including blood coagulation, and bone mineralization. The vitamin K-dependent gamma-carboxylation system consists of two essential components
the vitamin K-dependent gamma-carboxylase, an integral membrane protein of 92 kDa which requires reduced vitamin K (vitamin K1H2) as cofactor and
the warfarin sensitive enzyme vitamin K1 2,3-epoxide reductase (VKORC1), which produces the reduced cofactor.
VKORC1 is recently cloned, contains three predicted hydrophobic transmembrane domains and CXXC thioredoxin like motif. Previously by site directed mutagenesis we have shown that CXXC thioredoxin like motif in VKORC1 is the redox center of the enzyme. During gamma-carboxylation cycle VKORC1 must be reduced to provide electrons to convert epoxide form of vitamin K (KO) to reduced form of vitamin K (K1H2). To better understand how the VKORC1 enzyme is kept reduced during the gamma-carboxylation cycle, we have identified a protein associated with VKORC1 which provides electrons to keep VKORC1 in reduced state during the cycle. The associated protein is protein disulphide isomerase (PDI). Using co-immunoprecipitations and biochemical studies, we have demonstrated that PDI co-immunoprecipitates with VKORC1. Stably transfected HEK 293 cells with rat VKORC1 and rat microsomes incubated with reduced RNAase increases the VKOR activity, increased VKOR activity was inhibited by PDI antagonism with bacitracin and PDI siRNA. Our results suggest that PDI is the physiological reductant of VKORC1 which kept VKORC1 in reduced state during the gamma-carboxylation cycle.