Human Vitamin K 2,3-Epoxide Reductase Complex Subunit 1-like 1 (VKORC1L1) Mediates Vitamin K-dependent Intracellular Antioxidant Function

Philipp Westhofen,Matthias Watzka,Milka Marinova,Moritz Hass,Gregor Kirfel,Jens Müller,Carville G Bevans,Clemens R Müller,Johannes Oldenburg

doi:10.1074/jbc.m110.210971

Abstract

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1), expressed in HEK 293T cells and localized exclusively to membranes of the endoplasmic reticulum, was found to support both vitamin K 2,3-epoxide reductase (VKOR) and vitamin K reductase enzymatic activities. Michaelis-Menten kinetic parameters for dithiothreitol-driven VKOR activity were: K(m) (μM) = 4.15 (vitamin K(1) epoxide) and 11.24 (vitamin K(2) epoxide); V(max) (nmol·mg(-1)·hr(-1)) = 2.57 (vitamin K(1) epoxide) and 13.46 (vitamin K(2) epoxide). Oxidative stress induced by H(2)O(2) applied to cultured cells up-regulated VKORC1L1 expression and VKOR activity. Cell viability under conditions of no induced oxidative stress was increased by the presence of vitamins K(1) and K(2) but not ubinquinone-10 and was specifically dependent on VKORC1L1 expression. Intracellular reactive oxygen species levels in cells treated with 2,3-dimethoxy-1,4-naphthoquinone were mitigated in a VKORC1L1 expression-dependent manner. Intracellular oxidative damage to membrane intrinsic proteins was inversely dependent on VKORC1L1 expression and the presence of vitamin K(1). Taken together, our results suggest that VKORC1L1 is responsible for driving vitamin K-mediated intracellular antioxidation pathways critical to cell survival.

Highlights

ROS as a result of normal aerobic metabolism including oxidative phosphorylation in mitochondria and oxidative protein folding in the endoplasmic reticulum (ER)
VKORC1L1 Catalyzes Both De-epoxidation of Vitamin K-Epoxide and Reduction of Vitamin K Quinone—To directly assess VKORC1L1 function, we began by exploring whether VKORC1L1 could support vitamin K epoxide reductase (VKOR) and vitamin K quinone reductase (VKR) enzymatic activities similar to those catalyzed by VKORC1 and known to be critical for hemostasis [11, 26]
We hypothesized that the specific function of VKORC1L1 is to keep the intracellular intramembranous pool of K vitamins in the reduced, antioxidant-active hydroquinone form

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Transfection—HEK 293T human embryonic kidney cells (ATCC cell line CRL-11268) were cultured in APRIL 29, 2011 VOLUME 286 NUMBER 17. Vitamin K hydroquinone was detected by flow-cell fluorometry (L-2485 fluorescence detector; ␭ex 240 nm, ␭em 430 nm, telution ϳ2.4 min). VKR activity of VKORC1L1 was semiquantitatively confirmed by comparison of reaction products separated by C18 RP-HPLC monitored by flow-cell fluorometry detection of the K1 hydroquinone product (LaChrom Elite L-2485 fluorometer, ␭ex ϭ 246 nm, ␭em ϭ 430 nm; VWR-Hitachi). Intracellular Protein Carbonylation Quantitation—Protein carbonyl moieties were measured in crude membranes of HEK 293T cells, with or without VKORC1L1 overexpression or knockdown and/or 1 ␮M supplemented K1, using the BioCell protein carbonyl assay kit (Cell Biolabs, Inc., San Diego) according to the manufacturer’s instructions for low protein level and fluorescence plate reader (Fluoroscan Acent FL). Unpaired Student’s t test (for time course data) and analysis of variance with post hoc Fisher’s least significant difference test (for matched data groups) were used to assess statistical significance between data mean values as indicated by calculated p values

RESULTS

Although the viability of unstressed cells that overexpress

DISCUSSION