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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 17, 2001 as doi:10.1096/fj.01-0337fje. |
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Departments of Internal Medicine and
* Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA
2Correspondence: Rheumatology, Department of Internal Medicine, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157, USA. E-mail: rwallin{at}wfubmc.edu
SPECIFIC AIMS
The anticoagulant warfarin targets the vitamin K cycle and prevents production of competent vitamin K-dependent proteins. To understand the molecular mechanism of genetic resistance to the drug, the targeted enzyme of the cycle, vitamin K 2,3-epoxide reductase (VKOR), was investigated in a colony of laboratory rats that are genetically resistant to the drug.
PRINCIPAL FINDINGS
1. Identification of warfarin binding proteins by photoaffinity labeling with 4'-azido-warfarin- 3H-alcohol
The unsuccessful attempts by several research groups to purify VKOR from the ER membrane led us to believe that affinity labeling of the enzyme in a partially purified preparation was necessary in order to identify the enzyme. The 3H-alcohol derivative of 4'azido-warfarin was found to be a potent inhibitor of VKOR and was selected as the labeling reagent. After labeling, proteins were separated by 2D-SDS-PAGE, stained with Coomassie blue, and the radioactively labeled proteins in the gel were identified. Three proteins were shown to have significant radioactivity over background. The proteins were excised from the gel and tryptic peptides were analyzed by microcapillary reverse-phase HPLC nano electrospray tandem mass spectrometry. The ion pattern of peptides conclusively identified the most heavily labeled protein as the ER resident protein calumenin. The second protein was identified as cytochrome b5, but the third protein could not be identified. Since heme has been shown not to be involved in vitamin K epoxide reduction by VKOR, we focused on calumenin as the putative warfarin receptor.
2. The primary structure of calumenin in normal and warfarin-resistant rats
PCR was used to amplify the entire coding region of calumenin in normal Sprague Dawley rats and warfarin-resistant rats. The nucleotide sequence of the rat protein encodes a 315 residue protein. The sequence was found to be 100% identical in both rat strains and was 91% identical to human calumenin. The signal peptide has the only two-cysteine residues in the calumenin precursor. Calumenin is a water-soluble acidic protein and would be expected to be present in the ER lumen.
3. Calumenin is overexpressed in livers of warfarin-resistant rats
Northern blots of calumenin in the lung, heart, and liver from normal Sprague Dawley rats and warfarin-resistant rats are shown in Fig. 1
A. The results from normal rats are consistent with published data showing low expression of calumenin mRNA in liver (Fig. 1
A). On the other hand, calumenin mRNA expression in livers from warfarin-resistant rats was found to be equivalent to its expression in heart and lung. Analysis of other tissues, including spleen, testis, skeletal muscle, and brain, also showed no difference in calumenin expression between normal and warfarin-resistant rats (data not shown). Western blotting of calumenin was carried out to confirm that overexpression of calumenin could be demonstrated at the protein level. A strong immune reaction was seen with a 47 kDa protein present in microsomes from resistant rat liver (Fig. 1
, lane Resist.) whereas this protein was hardly detectable in microsomes from normal rats (Fig. 1
, lane Normal.).
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A Southern blot of PstI cut liver DNA isolated from normal and resistant rats is shown in Fig. 1C
. This restriction enzyme produced DNA fragments of different sizes from the two rat strains. Digestion with HindIII, EcoRI, and MspI gave identical fragmentation patterns (not shown). Since the coding sequences of calumenin in normal and resistant rats are identical, the PstI cleavage site that is different in normal and warfarin-resistant rats must be outside the coding region.
4. Partial purification of VKOR from livers of warfarin-resistant rats
In agreement with previous reports, we found that liver microsomes from warfarin-resistant rats have less VKOR activity than liver microsomes from normal rats. However, when VKOR was purified from warfarin-resistant rat livers according to a standard protocol developed by our laboratory, the enzyme preparations from normal and warfarin-resistant rats had the same specific activity (18.3 nmol/mg/30 min). The kinetic constant Km (5.2 µM) was also found to be the same, and VKOR in both preparations showed the same sensitivity to warfarin inhibition. The last finding is in sharp contrast to warfarin inhibition of VKOR in microsomes from normal and warfarin-resistant rats, where a significant difference in inhibition is noted. Quantitative dot blot analysis showed that the calumenin concentration in the partially purified VKOR preparations from normal and warfarin-resistant rats was the same within the experimental error (0.5 µg/mg of protein±0.1). Thus, the excess of calumenin protein present in liver microsomes from warfarin-resistant rats (see Fig. 1B
) had been removed from VKOR by subjecting VKOR to purification according our protocol. Based on these results, we hypothesized that calumenin is responsible for suppression of VKOR activity in warfarin-resistant rat liver.
5. The effect of calumenin on VKOR activity
To test our hypothesis that calumenin inhibits VKOR activity, we designed a reconstitution test system where calumenin was added to preparations of VKOR when excess calumenin had been removed by extraction or chromatography. The calumenin used in the reconstitution experiments (r-calumenin) was expressed in Escherichia coli and purified by a nondenaturing procedure to electrophoretic homogeneity as a 47 kDa protein. When added to the reconstitution system, r-calumenin was found to inhibit VKOR activity in a dose-dependent manner. Heat denatured r-calumenin and the ER chaperone GRP78 (BiP) had no effect on VKOR activity. Furthermore, increasing r-calumenin concentrations in the test system was found to increasingly protect the enzyme from inhibition by warfarin.
6. Overexpression of calumenin in COS-1 cells
Transient transfection of COS-1 cells resulted in overexpression of r-calumenin. Figure 2
A shows a Western blot of control cells (lane Co.) and transfected cells from four different culture dishes, respectively (lanes 1–4). Control cells and transfected cells were prepared for enzyme assays to test for VKOR activity and the ability of VKOR to support the vitamin K-dependent 
-carboxylation system. As shown in Fig. 2B
and consistent with the reconstitution data, VKOR activity was reduced 40% in transfected cells compared with the controls. Also consistent with the reconstitution data, VKOR activity in transfected cells became more resistant to warfarin inhibition (Fig. 2B
). Warfarin (5 µM) added to control cells inhibited the activity 80% whereas this concentration of warfarin added to transfected cell inhibited the activity by 52%. The effect of calumenin on VKOR-supported -carboxylase activity could also be demonstrated in transfected cells. As shown in Fig. 2C
, this activity was reduced 46% in transfected cells (transf.) vs. control cells (cont.). These results add strong support to the conclusion that calumenin affects the vitamin K-dependent -carboxylation system.
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CONCLUSIONS AND SIGNIFICANCE
This paper provides a molecular model for one form of genetic warfarin resistance in the rat and may apply to warfarin resistance in certain patients on anticoagulant therapy. Our results suggest that the concentration of calumenin in liver will control resistance to the drug as well as the efficiency of the vitamin K-dependent -carboxylation system to postribosomally modify vitamin K-dependent proteins.
Our hypothesis that calumenin interacts with VKOR and confers resistance to warfarin is strengthened by earlier localization of the warfarin resistance gene in mice to chromosome 7, the chromosome carrying the calumenin gene.
Calumenin belongs to the CREC family of Ca2+ binding proteins found in the secretory pathway of mammalian cells. All members of the CREC family have been shown to have chaperone functions. So far, calumenin has been shown to interact only with serum amyloid P component. Our finding that calumenin interacts with proteins of the -carboxylation system provides new information about the physiological functions of this protein.
Earlier work on VKOR in normal and warfarin-resistant rats has suggested that a mutant and less functional enzyme resides in the ER membrane of these rats. Our results raise questions about these earlier studies of crude liver microsomes and propose a novel molecular model for warfarin resistance in the rat. The model shown in Fig. 3
proposes that calumenin affects the vitamin K-dependent -carboxylation system in liver by binding to VKOR as a chaperone. The interaction between calumenin and VKOR is responsible for reduced VKOR activity, which results in a less effective, vitamin K-dependent -carboxylation system. Since calumenin is not a warfarin binding protein, we hypothesize that calumenin will bind to VKOR in such a way that the drug is prevented from reaching its binding site on VKOR (see Fig. 3
).
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0337fje; to cite this article, use FASEB J. (September 17, 2001) 10.1096/fj.01-0337fje 
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