Multiple carboxylated Glus (Glas) is produced by γ-Glutamyl carboxylase (GGCX) in vitamin K-dependent (VKD) proteins, which are necessary for their activities. For a study, researchers demonstrated the significance of processivity to the function of VKD proteins by demonstrating how GGCX is processive and remains attached to VKD proteins during repeated Glu carboxylations.
A new assay replicating in vivo carboxylation was used to study GGCX mutants (V255M and S300F), whose combined heterozygosity in a patient produces impaired coagulation and calcification. In the presence of a challenge VKD protein that would possibly interfere with carboxylation of the VKD protein in the complex, complexes between variant carboxylases and VKD proteins crucial to hemostasis (factor IX [FIX]) or calcification (matrix Gla protein [MGP]) responded. Before the challenge protein’s carboxylation occurred, the VKD protein was partially carboxylated in combination with wild-type carboxylase. The V255M mutant, however, carboxylated both forms simultaneously while only partially carboxylating FIX in the complex. Both FIX and MGP had subpar S300F carboxylation.
Additional research examined peptides with the Gla domain connected to sequences that facilitate carboxylase binding obtained from FIX and MGP. The V255M mutant produced a greater quantity of total carboxylated peptide than wild-type GGCX, but each individual peptide was only partly carboxylated. Poor FIX clotting activity was discovered by analyzing the V255M mutant in FIX HEK293 cells deficient in native GGCX.
According to the study, sickness was brought on by interrupted processivity, which also explained the patient’s problem. Additionally, kinetic investigations revealed that under some circumstances, wild-type carboxylase may have disturbed processivity (e.g., warfarin therapy or vitamin K deficiency).
