Assessment of gamma-glutamyl carboxylase activity in its native milieu.

Gamma-glutamyl carboxylase (GGCX), a polytopic membrane protein found in the endoplasmic reticulum, catalyzes the posttranslational modification of a variety of vitamin K-dependent (VKD) proteins to their functional forms. GGCX uses the free energy from the oxygenation of reduced vitamin K to remove the proton from the glutamate residue to drive VKD carboxylation. During the process of carboxylation, reduced vitamin K is oxidized to vitamin K epoxide. Therefore, GGCX is a dual-function enzyme that possesses both glutamate carboxylation and vitamin K epoxidation activities. Genetic variations in GGCX are mainly associated with bleeding disorders referred to as combined VKD coagulation factors deficiency. Comorbid non-bleeding phenotypes are also observed in patients carrying GGCX mutations. Our current knowledge concerning GGCX's function has been obtained mainly from in vitro experimentation under artificial conditions, which limits its use in interpreting the clinical phenotypes associated with GGCX genotypes. In this chapter, we describe the background, establishment, and application of mammalian cell-based assays for both the carboxylation and epoxidation activities of GGCX. We provide detailed procedures for making the reporter cell lines, creating CRISPR-Cas9-mediated gene-knockout reporter cell lines, and using these cell lines for functional studies of GGCX and its naturally occurring mutations. Combined with different reporter proteins, this cell-based strategy has been successfully used for the functional study of vitamin K-related enzymes, high-throughput screening of VKD carboxylation inhibitors, and genome-wide CRISPR-Cas9 knockout library screening of the unknown enzymes associated with vitamin K reduction.