r-VKORC1 Expression in Factor IX BHK Cells Increases the Extent of Factor IX Carboxylation but Is Limited by Saturation of Another Carboxylation Component or by a Shift in the Rate-Limiting Step

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Abstract
Carboxylation of vitamin K-dependent (VKD) proteins is required for their activity and depends on reduced vitamin K generated by vitamin K oxidoreductase (VKOR) and a redox protein that regenerates VKOR activity. VKD protein carboxylation is inefficient in mammalian cells, and to understand why carboxylation becomes saturated, we developed an approach that directly measures the extent of intracellular VKD protein carboxylation. Analysis of factor IX (fIX)-expressing BHK cells indicated that slow egress of fIX from the endoplasmic reticulum and preferential secretion of the carboxylated form contribute to secreted fIX being more fully carboxylated. The analysis also revealed the first reported in vivo VKD protein turnover, which was 14-fold faster than that which occurs in vitro, suggesting facilitation of this process in vivo. r-VKORC1 expression increased the rate of fIX carboxylation and the extent of secreted carboxylated fIX ∼2-fold, which shows that carboxylation is the rate-limiting step in fIX turnover and which was surprising because turnover in vitro is limited by release of carboxylated fIX. Interestingly, the increases were significantly smaller than the amount of VKOR overexpression (15-fold). However, when cell extracts were tested in single-turnover experiments in vitro, where redox protein is functionally substituted with dithiothreitol, VKOR overexpression increased the fIX carboxylation rate 14-fold, showing r-VKORC1 is functional for supporting fIX carboxylation. These data indicate that the effect of VKOR overexpression is limited in vivo, possibly because a carboxylation component like the redox protein becomes saturated or because another step is now rate-limiting. The studies illustrate the complexity of carboxylation and potential importance of component stoichiometry to overall efficiency.