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

Abstract

Carboxylation of vitamin K-dependent (VKD) proteins is required for their activity and depends upon 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 intracellular VKD protein carboxylation. Analysis of factor IX (fIX)-expressing BHK cells indicated that slow fIX egress 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 occurs in vitro, suggesting facilitation of this process in vivo. r-VKORC1 expression increased the rate of fIX carboxylation and extent of 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 less 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 by 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.

Gla-rich Protein (GRP), A New Vitamin K-dependent Protein Identified from Sturgeon Cartilage and Highly Conserved in Vertebrates

“…Indeed, several protein factors are involved in the synthesis of functional VKD proteins: the ␥-glutamyl carboxylase (36), the vitamin K oxidoreductase, a redox protein that regenerates vitamin K activity, a vitamin K co-factor, and a propeptide processing enzyme (36). Studies performed with Gla-containing coagulation proteins indicate that overall efficiency is dependent on the stoichiometry of all the components involved in this complex process (37). Our results show that, in sturgeon, GRP mRNA only associates with cartilaginous tissues, being expressed in chordoblasts as well as immature and mature chondrocytes.…”

The Vitamin K Oxidoreductase Is a Multimer That Efficiently Reduces Vitamin K Epoxide to Hydroquinone to Allow Vitamin K-dependent Protein Carboxylation

“…In each case, ϳ20 clones were screened by Western analysis using an anti-VKORC1 antibody (21) to identify a cell line with the desired level of expression. Recombinant and endogenous VKORC1 were distinguishable on SDS-PAGE, and quantitation of each form on a LICOR Odyssey scanner was used to determine the level of VKORC1 overexpression.…”

“…This test was possible because VKORC1 is rate-limiting for carboxylation (21,32) and because of a previous development that improved the sensitivity of the carboxylation assay (27). Endogenous levels of VKORC1 in 293 cells are low, and this assay (described below) therefore enabled analysis of VKORC1 activity.…”

Novel Insight into the Mechanism of the Vitamin K Oxidoreductase (VKOR)

“…Generation and Expression of VKOR Variants-Wild type human VKOR was generated by PCR using r-VKORC1/ ZEM229 as the template and the primers VKRC1S and VKRC1AS1, all of which have previously been described (22). The PCR product was digested with BglII and then gel isolated and subcloned into the BamHI site of BacPak8 to generate pBacPak8-VKOR.…”