Disulfide-dependent Protein Folding Is Linked to Operation of the Vitamin K Cycle in the Endoplasmic Reticulum: A PROTEIN DISULFIDE ISOMERASE-VKORC1 REDOX ENZYME COMPLEX APPEARS TO BE RESPONSIBLE FOR VITAMIN K1 2,3-EPOXIDE REDUCTION

Nadeem Wajih,Susan M Hutson,Reidar Wallin

doi:10.1074/jbc.m608954200

Abstract

Gamma-carboxylation of vitamin K-dependent proteins is dependent on formation of reduced vitamin K1 (Vit.K1H2) in the endoplasmic reticulum (ER), where it works as an essential cofactor for gamma-carboxylase in post-translational gamma-carboxylation of vitamin K-dependent proteins. Vit.K1H2 is produced by the warfarin-sensitive enzyme vitamin K 2,3-epoxide reductase (VKOR) of the vitamin K cycle that has been shown to harbor a thioredoxin-like CXXC center involved in reduction of vitamin K1 2,3-epoxide (Vit.K>O). However, the cellular system providing electrons to the center is unknown. Here data are presented that demonstrate that reduction is linked to dithiol-dependent oxidative folding of proteins in the ER by protein disulfide isomerase (PDI). Oxidative folding of reduced RNase is shown to trigger reduction of Vit.K>O and gamma-carboxylation of the synthetic gamma-carboxylase peptide substrate FLEEL. In liver microsomes, reduced RNase-triggered gamma-carboxylation is inhibited by the PDI inhibitor bacitracin and also by small interfering RNA silencing of PDI in HEK 293 cells. Immunoprecipitation and two-dimensional SDS-PAGE of microsomal membrane proteins demonstrate the existence of a VKOR enzyme complex where PDI and VKORC1 appear to be tightly associated subunits. We propose that the PDI subunit of the complex provides electrons for reduction of the thioredoxin-like CXXC center in VKORC1. We can conclude that the energy required for gamma-carboxylation of proteins is provided by dithiol-dependent oxidative protein folding in the ER and thus is linked to de novo protein synthesis.

Highlights

Vitamin K-dependent proteins are modified post-translationally to contain ␥-carboxyglutamic acid, Ca2ϩ binding amino acid residues [1,2,3]
Vit.K1H2 is produced by the warfarin-sensitive enzyme vitamin K 2,3-epoxide reductase (VKOR) of the vitamin K cycle that has been shown to harbor a thioredoxin-like CXXC center involved in reduction of vitamin K1 2,3epoxide (Vit.K>O)
We propose that the protein disulfide isomerase (PDI) subunit of the complex provides electrons for reduction of the thioredoxin-like CXXC center in VKORC1

Summary

EXPERIMENTAL PROCEDURES

The oligonucleotides used for rat VKORC1 PCR were as follows. The sense primer was 5Ј-AAA AAA AAG CTT GCC GCC ACC ATG GGC ACC ACC TGG AGG-3Ј (underlined bases were used to generate HindIII site; bases in italics were used to generate a Kozak sequence). Portions of the various buffer D cell extracts with the same protein content were prepared for SDS-PAGE and Western blotting with PDI antibodies. Stripping of the PVDF membrane and reprobing with an ␣-actin antibody was used to verify equal protein loading in each lane In this experiment, we brought extracted microsomes into solution with buffer B (25 mM phosphate, 25 mM KCl, 20% glycerol, 0.75% CHAPS, pH 7.85, containing 10 ␮g/ml Sigma protease inhibitor mixture), which we [5] previously have reported partially solubilizes the VKOR enzyme complex in an active form. The magnetic bead-containing tubes were allowed to rotate for 30 min at 4 °C followed by five washes in buffer C in a magnet rack as described by the supplier (Pierce). VKOR activity was measured in each suspension present in tubes 1, 2, 3, and 4

Enzyme Assays

RESULTS

The Amount of PDI Present in Extracted Microsomes