Vitamin K-dependent (VKD) protein activities require carboxylated Glus (Glas) generated by the gamma-glutamyl carboxylase. Some carboxylase mutations cause severe bleeding, while others cause pseudoxanthoma elasticum (PXE)-like associated with excessive calcification. How carboxylase mutations cause PXE-like was unknown. We analyzed two mutants (V255M and S300F) whose compound heterozygosity causes PXE-like. Substrates derived from VKD proteins important to calcification (MGP) or clotting (factor IX) were studied, which contained the Gla domain and exosite-binding domain that mediates carboxylase binding. Surprisingly, the V255M mutant was more active (4-5 fold) than wild type carboxylase, while S300F activity was low. The V255M results suggested faster substrate release, which could impact carboxylase processivity, where the carboxylase remains bound to VKD proteins throughout multiple Glu to Gla conversions. To assess mutant processivity, we performed a novel challenge assay in which MGP-carboxylase and factor IX-carboxylase complexes were reacted in the presence of excess challenge VKD peptide. Tight complexes between VKD proteins and wild type carboxylase excluded access of the challenge peptide during the carboxylation of VKD protein in the complex. In contrast, VKD protein complexes with V255M or S300F allowed promiscuous access of challenge peptide. Both mutants therefore impair processivity. Most of the V255M product was carboxylated challenge peptide, which could explain mild PXE-like observed in the mother and aunt of the proband. Both have wild type and V255M carboxylase alleles; however, higher V255M production of a potentially defective MGP product could account for their phenotype. The results are an important advance in understanding why carboxylase mutations cause the PXE-like disease.

Compound heterozygosity of the gamma-glutamyl carboxylase mutants V255M and S300F causes pseudoxanthoma elasticum-like disease through impaired processivity