Cellular and biochemical approaches to define GGCX carboxylation of vitamin K-dependent proteins.

The gamma-glutamyl carboxylase (GGCX) generates clusters of carboxylated glutamic acid residues in vitamin K-dependent (VKD) proteins, which is required for their diverse functions including hemostasis and regulation of calcification. The GGCX modifies a VKD protein using several substrates and cofactors, and has regulatory mechanisms like processivity that ensures full carboxylation of VKD proteins. The GGCX mechanism is incompletely understood. GGCX mutations cause disease: vitamin K clotting factor deficiency (VKCFD1) associated with severe bleeding and pseudoxanthoma elasticum (PXE)-like, where bleeding is mild but calcification is excessive. Why mutations cause disease has only been revealed for a few GGCX mutants. The chapter describes biochemical and cellular assays developed to define GGCX mechanism and determine why GGCX mutations cause disease. Multiple components are important to VKD protein carboxylation. GGCX requires reduced vitamin K generated by the vitamin K epoxide reductase (VKORC1), which is activated by redox protein(s). GGCX activity is also supported by the reductases VKORC1-like and ferroptosis suppressor protein-1 (FSP1). Carboxylation occurs in the endoplasmic reticulum during VKD protein secretion and is impacted by quality control mechanisms. This chapter emphasizes the importance in maintaining an appropriate stoichiometry of the components when studying recombinant (r-) r-GGCX function in cells. Also emphasized is the essentiality in using both cellular and biochemical approaches to study r-GGCX function, as cellular analysis can detect altered VKD protein carboxylation by r-GGCX mutants but cannot explain the r-GGCX defects. This point is illustrated by studies on a GGCX mutant that causes the PXE-like disease, where biochemical analysis was necessary for revealing mutant dysfunction, i.e. impaired processivity.