Biochemical characterization of spontaneous mutants of rat VKORC1 involved in the resistance to antivitamin K anticoagulants Ahmed Hodroge 1 , Christiane Longin-Sauvageon 1 , Isabelle Fourel, Etienne Benoit, Virginie Lattard ⇑ USC 1233 INRA/Vetagro Sup, Mycotoxines et Toxicologie Comparée des Xénobiotiques, Veterinary School of Lyon, 1 av. Bourgelat, 69280 Marcy l’Etoile, France article info Article history: Received 12 May 2011 and in revised form 24 August 2011 Available online 31 August 2011 Keywords: VKORC1 Antivitamin K Resistance Mutation Kinetic analysis abstract Antivitamin K anticoagulants have been commonly used to control rodent pest all over the world for more than 50 years. These compounds target blood coagulation by inhibiting the vitamin K epoxide reductase (VKORC1), which catalyzes the reduction of vitamin K 2,3-epoxide to vitamin K. Resistance to anticoagulants has been reported in wild rat populations from different countries. From these popula- tions, several mutations of the rVkorc1 gene have been reported. In this study, rat VKORC1 and its most frequent mutants L120Q–, L128Q–, Y139C–, Y139S– and Y139F–VKORC1 were expressed as membrane- bound proteins in Pichia pastoris and characterized by the determination of kinetic and inhibition param- eters. The recombinant rVKORC1 showed similar properties than those of the native proteins expressed in the rat liver microsomes, validating the expression system as a good model to study the consequences of VKORC1 mutations. The determination of the inhibition parameters towards various antivitamin K anti- coagulants demonstrated that mutations at Leu-120, Leu-128 and Tyr-139 confer the resistance to the first generation AVKs observed in wild rat populations. Ó 2011 Elsevier Inc. All rights reserved. Introduction Antivitamin K (AVK), 2 derivatives of either 4-hydroxycoumarin or indane-1,3-dione are widely used as therapeutic agent for treatment and prophylaxis of thrombotic diseases in humans, and as rodenti- cides for pest control. AVK are non-competitive inhibitors of the vita- min K epoxide reductase enzyme (VKORC1). The function of VKORC1 is to regenerate vitamin K and vitamin K hydroquinone (K and KH2) from vitamin K 2,3-epoxide (K > O), a byproduct of the vitamin K-dependent gamma carboxylation reaction [1] (Fig. 1). Inhibition of VKORC1 by AVK limits the amount of KH2 available for the carboxylation reaction and results in partially carboxylated vitamin K-dependent blood clotting factors. In 2004, the gene Vkorc1, encoding for the pharmacological target of AVKs, was simultaneously described by Rost et al. [2] and Li et al. [3]. Mutations in this gene were immediately considered as linked to resistance to AVKs [2,4]. Several mutations in this gene were observed in wild rat populations from different west European coun- tries and these mutated rats were resistant to AVKs [4,5]. Five point mutations (i.e., Y139F, Y139C, Y139S, L120Q and L128Q) were mainly observed in wild rats. Prevalence of such mutations in wild rat popu- lations are still unknown. However, Y139C mutation was described to be very abundant in Denmark and in north-east Germany with fo- cus of 100% resistant rats [4]. In France, about 40% of the samples from 91 locations all over the country carried one of the five mutations with highest prevalence for Y139F mutation [5]. Importance of this point was reinforced by the observation of a 20 MB part of the chro- mosome 1, centered on the Vkorc1 gene in the rat, that was associated with complete resistance phenotype similar to the one observed in wild trapped homozygous Y139F rats [6]. High prevalence of these mutations renders the use of first generation AVKs unefficient and thus becomes a primary public health concern. A better knowledge of the structure of the mammalian VKORC1 enzyme could help to design new AVKs. Recent determination by Li et al. [7] of a three-dimensional structure of a bacterial homolog of VKORC1 was an important step in this way. The VKORC1 is a 163- amino acid integral membrane protein that contains a C 132 XXC 135 redox motif characteristic of the thioredoxin family of enzymes. The site directed mutations of one of these cysteines to alanine or the mutation of both of these cysteines abolish the activity [8]. This redox center is located in the fourth transmembrane domain of the bacterial homolog of VKORC1 [7] (Fig. 2). Electrons needed for the reduction of the C 132 XXC 135 redox motif are trans- ferred from two conserved cysteines (i.e., Cys43 and Cys51) [9] located in the luminal loop of the bacterial homolog of VKORC1 [7]. The physiological reductant, allowing the reduction of the 0003-9861/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2011.08.010 ⇑ Corresponding author. Fax: +33 (0) 4 78 87 05 16. E-mail address: v.lattard@vetagro-sup.fr (V. Lattard). 1 These authors contributed equally to this work. 2 Abbreviations used: AVK, antivitamin K; VKORC1, vitamin K epoxide reductase enzyme; K > O, vitamin K 2,3-epoxide; K, vitamin K; KH2, vitamin K hydroquinone; LC, liquid chromatography; MS, mass spectrometry; GGCX, gamma-glutamyl-carboxylase; NQO1, NADPH quinone oxidoreductase 1. Archives of Biochemistry and Biophysics 515 (2011) 14–20 Contents lists available at SciVerse ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi