The γ-carboxylation recognition site is sufficient to direct vitamin K- dependent carboxylation on an adjacent glutamate-rich region of thrombin in a propeptide-thrombin chimera

Abstract

The propeptides of the vitamin K-dependent proteins contain a γ- carboxylation recognition site that is required for γ-glutamyl carboxylation. To determine whether the propeptide is sufficient to direct carboxylation, two mutant prothrombin species were expressed and characterized with regard to posttranslational γ-carboxylation. A double point mutant, in which serine substituted for cysteines 17 and 22 disrupted a conserved loop formed by a disulfide bond, was fully carboxylated when expressed in Chinese hamster ovary cells. A propeptide/thrombin chimeric protein, constructed by deleting the Gla, aromatic amine acid stack, and kringle domains of prothrombin, has the signal peptide and propeptide juxtaposed to a glutamate-rich COOH-terminal region of prothrombin, residues 249-530. Of the 8 glutamic acid residues contained within the first 40 residues of the NH2 terminus adjacent to the propeptide, at least seven were fully carboxylated as demonstrated by direct γ-carboxyglutamic acid analysis of the alkaline hydrolysate and by NH2-terminal sequence analysis. These results indicate that the γ-carboxylation recognition site within the prothrombin propeptide in a prothrombin propeptide-thrombin chimeric protein is sufficient to direct γ-carboxylase-catalyzed carboxylation of adjacent glutamic acid residues in a glutamate-rich region of thrombin that is not normally γ-carboxylated. Furthermore, the disulfide loop in the Gla domain of prothrombin is not required for complete carboxylation.