GGCX mutants that impair hemostasis reveal the importance of processivity and full carboxylation to VKD protein function

Abstract

γ-Glutamyl carboxylase (GGCX) generates multiple carboxylated Glus (Glas) in vitamin K–dependent (VKD) proteins that are required for their functions. GGCX is processive, remaining bound to VKD proteins throughout multiple Glu carboxylations, and this study reveals the essentiality of processivity to VKD protein function. GGCX mutants (V255M and S300F) whose combined heterozygosity in a patient causes defective clotting and calcification were studied using a novel assay that mimics in vivo carboxylation. Complexes between variant carboxylases and VKD proteins important to hemostasis (factor IX [FIX]) or calcification (matrix Gla protein [MGP]) were reacted in the presence of a challenge VKD protein that could potentially interfere with carboxylation of the VKD protein in the complex. The VKD protein in the complex with wild-type carboxylase was carboxylated before challenge protein carboxylation occurred and became fully carboxylated. In contrast, the V255M mutant carboxylated both forms at the same time and did not completely carboxylate FIX in the complex. S300F carboxylation was poor with both FIX and MGP. Additional studies analyzed FIX- and MGP-derived peptides containing the Gla domain linked to sequences that mediate carboxylase binding. The total amount of carboxylated peptide generated by the V255M mutant was higher than that of wild-type GGCX; however, the individual peptides were partially carboxylated. Analysis of the V255M mutant in FIX HEK293 cells lacking endogenous GGCX revealed poor FIX clotting activity. This study shows that disrupted processivity causes disease and explains the defect in the patient. Kinetic analyses also suggest that disrupted processivity may occur in wild-type carboxylase under some conditions (eg, warfarin therapy or vitamin K deficiency).

Association among VKORC1 rs9923231, CYP4F2 rs2108622, GGCX rs11676382 polymorphisms and acute ischemic stroke

“…[10] GGCX encodes the gamma-glutamyl carboxylase enzyme, which is required for the posttranslational modification of vitamin K-dependent proteins, including blood coagulation factors and bone matrix proteins. [11] Variants of these genes have been associated with a range of clinical outcomes, including bleeding disorders, bone metabolism disorders, and cardiovascular diseases. In particular, SNPs in the VKORC1 gene have been shown to affect the efficacy and safety of warfarin therapy, [9] while variants of CYP4F2 and GGCX genes have been implicated in the pathogenesis of hypertension, thrombosis, and osteoporosis.…”

“…In particular, SNPs in the VKORC1 gene have been shown to affect the efficacy and safety of warfarin therapy, [9] while variants of CYP4F2 and GGCX genes have been implicated in the pathogenesis of hypertension, thrombosis, and osteoporosis. [11][12][13][14] Owing to the critical role of the key enzymes encoded by these genes, SNPs impacting their function lead to alterations in the metabolic pathways of vitamin K, which may become symptomatic in certain contexts, like in the case of the administration of various drugs such as oral anticoagulants. Numerous studies have shown the need to adjust drug doses in patients harboring such variants.…”