Vitamin K epoxide reductase significantly improves carboxylation in a cell line overexpressing factor X.
Previously we reported that we could increase the fraction of carboxylated factor X by reducing the affinity of the propeptide for its binding site on human gamma glutamyl carboxylase. We attributed this to an increased turnover rate. However, even with the reduced affinity propeptide, when sufficient overproduction of factor X is achieved, there is still a significant fraction of uncarboxylated recombinant factor X. We report here that the factor X of such a cell line was only 52% carboxylated but that the fraction of carboxylated factor X could be increased to 92% by coexpressing the recently identified gene for vitamin K epoxide reductase. Because vitamin K is in excess in both the untransfected and vitamin K epoxide reductase (VKOR)-transfected cells, the simplest explanation for this result is that VKOR catalyzes both the reduction of vitamin K epoxide to vitamin K and the conversion of vitamin K to vitamin K hydroquinone. In addition to its mechanistic relevance, this observation has practical implications for overproducing recombinant vitamin K-dependent proteins for therapeutic use.
Sun
Yan-Mei
YM
Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA.
Jin
Da-Yun
DY
Camire
Rodney M
RM
Stafford
Darrel W
DW
D019735:Carbon-Carbon Ligases / Q000378:metabolism*; D002460:Cell Line; D005170:Factor X / Q000378:metabolism*; D006801:Humans; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D014162:Transfection; D014812:Vitamin K / Q000378:metabolism*; D064417:Vitamin K Epoxide Reductases
D012333:RNA, Messenger; D011994:Recombinant Proteins; D014812:Vitamin K; D005170:Factor X; D006899:Mixed Function Oxygenases; D064417:Vitamin K Epoxide Reductases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase