Biochemical characterization of spontaneous mutants of rat VKORC1 involved in the resistance to antivitamin K anticoagulants
Highlights
► Rat VKORC1 and its five major mutants were expressed in Pichia pastoris. ► Properties of the recombinant proteins were similar to those of the native proteins. ► Mutations at Leu120 and Tyr139 dramatically affect the VKOR activity. ► Mutations at Leu120, Leu128 and Tyr139 confer the resistance to AVKs observed in wild rats. ► This expression system will be a good model to study the consequences of human VKORC1 mutations.
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 rodenticides for pest control. AVK are non-competitive inhibitors of the vitamin 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 countries 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 populations are still unknown. However, Y139C mutation was described to be very abundant in Denmark and in north-east Germany with focus 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 chromosome 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 C132XXC135 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 C132XXC135 redox motif are transferred 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 VKORC1 loop cysteines remains still unclear. In vitro, the DTT reductor agent is used to reduce the disulfide form of the enzyme and to produce the sulfydril involved in the reduction of vitamin K > O. The main point mutations found in wild rats resistant to AVKs are close to the binding site of the quinone and thus, close to the CXXC redox motif. Indeed, in the three-dimensional structure of the bacterial homolog of VKORC1, Leu120 and Leu128 were located at the end of the third transmembrane domain, close to the fourth transmembrane domain, and Tyr139, in the fourth transmembrane domain [7] (Fig. 2). However, conflicting topology models with three or four transmembrane segments have been proposed for human VKOR [7], [10], [11] and biochemical and structural consequences of the mutation detected in resistant wild rats still remain to be elucidated.
In the present study, the rat VKORC1 and its main spontaneous mutants observed all over Europe were expressed as membrane-bound proteins using the Pichia pastoris expression system, in order to study the consequences of such mutations on the VKOR activity. Comparison between kinetic parameters obtained using recombinant P. pastoris or rat liver microsomes were performed to validate the expression system. Finally, the link between the different mutations and the efficiency of the different AVKs available for the rodent control was discussed.
Section snippets
Chemicals
Vitamin K1 (Phylloquinone) was converted to vitamin K > O according to Tishler et al. [12]. Purity was estimated by LC/MS and was higher than 99%. Sodium warfarin, difenacoum and brodifacoum were purchased from Sigma–Aldrich (Saint Quentin Fallavier, France). Chlorophacinone, bromadiolone and difethialone were supplied by Liphatech (Pont de Casse, France). Methanol HPLC grade, and acetic acid (analysis grade) were obtained from Merck (Germany).
Plasmids construction and mutagenesis
The coding sequence corresponding to the rat VKORC1
Engineering of an efficient heterologous expression system allowing the production of a catalytically active form of rat VKORC1
To produce the rat VKORC1 for kinetic analysis, we expressed rVKORC1-WT as a c-myc fused protein in P. pastoris. This recombinant protein was intracellularly expressed as a membrane-bound protein. Immunoblot analysis of membrane fractions obtained from recombinant yeast cells expressing the wild type VKORC1 using c-myc antibodies revealed the presence of a single immunoreactive protein migrating at ∼20 kDa, consistent with the theoretical molecular mass of the rat VKORC1 fused with a c-myc tag
Discussion
Mutations in the VKORC1 gene [2], [3] in humans and rodents have been largely associated with resistance phenotypes to AVKs drugs. Despite this relationship, there is an evident lack of information about the direct consequences of the mutations while the improvement of drug efficacy requires a better understanding of the molecular mechanism of AVKs resistance. This lack of insight stems from the difficulty of obtaining VKORC1 enzyme, a 18-kDa transmembrane protein with four [7], [9]
Acknowledgments
This work was supported by Grants from Agence Nationale pour la Recherche (RODENT 2009-CESA-008-03) and by DGER.
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These authors contributed equally to this work.