Carboxylase Overexpression Effects Full Carboxylation but Poor Release and Secretion of Factor IX:  Implications for the Release of Vitamin K-Dependent Proteins

Abstract

Vitamin K-dependent (VKD) proteins are modified by the VKD carboxylase as they transit through the endoplasmic reticulum. In a reaction required for their activity, clusters of Glu's are converted to Gla's, and fully carboxylated VKD proteins are normally secreted. In mammalian cell lines expressing high levels of r-VKD proteins, however, under- and uncarboxylated VKD forms are observed. Overexpression of r-carboxylase does not improve carboxylation, but the lack of effect is not understood, and the intracellular events that occur during VKD protein carboxylation have not been investigated. We analyzed carboxylation in 293- and BHK cell lines expressing r-factor IX (fIX) and endogenous carboxylase or overexpressed r-carboxylase. The fIX secreted from the four cell lines was highly carboxylated, indicating fIX-carboxylase engagement during intracellular trafficking. The r-carboxylase was functional for carboxylation: overexpression resulted in a proportional increase in fIX-carboxylase complexes that yielded full fIX carboxylation. Interestingly, the carboxylated fIX product was not efficiently released from the carboxylase in r-fIX/r-carboxylase cells, resulting in decreased fIX secretion. r-Carboxylase overexpression changed the ratios of intracellular fIX to carboxylase, and we therefore developed an in vitro assay to test whether fIX levels affect release. FIX-carboxylase complexes were in vitro carboxylated with or without excess VKD substrate or propeptide. These analyses are the first to dissect the rates of release versus carboxylation and showed that release was much slower than carboxylation. In the absence of excess VKD substrate/propeptide, fIX in the fIX-carboxylase complex was fully carboxylated by 10 min, but 95% was still complexed with carboxylase after 30 min. The presence of excess VKD substrate/propeptide, however, led to a significant increase in VKD product release, possibly through a second propeptide binding site in the carboxylase. The intracellular analyses also showed that the fIX carboxylation rate was slow in vivo and was similar in r-fIX versus r-fIX/r-carboxylase cells, despite the large differences in carboxylase levels. The results suggest that the vitamin K cofactor may be limiting for carboxylation in the cell lines.

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

“…3A), similar to results previously obtained with this cell line (16). That pulse-chase study also included a 22 hr time point, which showed that 100% of the fIX is ultimately secreted into media (16).…”

“…This response presents a major impediment for producing recombinant VKD proteins, several of which have therapeutic potential, for example in hemophilia and sepsis (13,14). Coexpression of r-carboxylase with VKD proteins does not improve carboxylation even though overexpression of carboxylase protein results in a proportional increase in activity (15,16), and the change in VKD protein/carboxylase stoichiometry due to carboxylase overexpression actually results in decreased secretion of the VKD protein (i.e. fIX) (16).…”

“…fIX) (16). Intracellular analysis of fIX BHK and fIX 293 cells that contain endogenous carboxylase or that also express rcarboxylase suggested that the availability of KH 2 cofactor may limit carboxylation (16). We therefore initiated studies to directly analyze the intracellular events in fIX carboxylation in mammalian cells.…”

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

“…7, a and b). The cells contain endogenous carboxylase, whose ability to recycle KH 2 to KO was unknown because the carboxylase requires activation by VKD proteins (29,30), which were present in only trace amounts in these cells (31). Therefore, the experiment was repeated in cells pretreated with carboxylase siRNA prior to the addition of KO.…”

Binding of the Factor IX γ-Carboxyglutamic Acid Domain to the Vitamin K-dependent γ-Glutamyl Carboxylase Active Site Induces an Allosteric Effect That May Ensure Processive Carboxylation and Regulate the Release of Carboxylated Product

“…Hallgren et al (32) proposed an active release mechanism based upon their observation that, in a chase experiment, the amount of carboxylated factor IX-carboxylase complex remains unchanged unless a competing propeptide is present. They concluded that another propeptide binds to a second propeptide-binding site, facilitating the release of the carboxylated proFIX.…”