γ-Glutamyl carboxylase mutations differentially affect the biological function of vitamin K–dependent proteins

Abstract

Gamma-glutamyl carboxylase (GGCX) is an integral membrane protein that catalyzes posttranslational carboxylation of a number of vitamin K-dependent (VKD) proteins involved in a wide variety of physiological processes, including blood coagulation, vascular calcification, and bone metabolism. Naturally occurring GGCX mutations are associated with multiple distinct clinical phenotypes. However, the genotype-phenotype correlation of GGCX remains elusive. Here, we systematically examined the effect of all naturally occurring GGCX mutations on the carboxylation of three structure-function distinct VKD proteins in a cellular environment. GGCX mutations were transiently introduced into GGCX-deficient human embryonic kidney 293 cells stably expressing chimeric coagulation factor, matrix Gla protein (MGP), or osteocalcin as VKD reporter-proteins, then the carboxylation efficiency of these reporter-proteins were evaluated. Our results show that GGCX mutations differentially affect the carboxylation of these reporter-proteins and the efficiency of using vitamin K as a cofactor. Carboxylation of these reporter-proteins by a C-terminal truncation mutation (R704X) implies that GGCX's C-terminus plays a critical role in the binding of osteocalcin, but not in the binding of coagulation factors and MGP. This has been confirmed by probing the protein-protein interaction between GGCX and its protein substrates in live cells using bimolecular fluorescence complementation and chemical cross-linking assays. Additionally, using a minigene splicing assay, we demonstrated that several GGCX missense mutations affect GGCX's pre-mRNA splicing rather than altering the corresponding amino acid residues. Results from this study interpreted the correlation of GGCX's genotype and its clinical phenotypes, and clarified why vitamin K administration rectified bleeding disorders but not non-bleeding disorders.

GGCX mutations show different responses to vitamin K thereby determining the severity of the hemorrhagic phenotype in VKCFD1 patients

“…Another study has previously shown that GGCX mutations can also affect the nonhemostatic VKD proteins MGP and BGLAP differentially. 43 The selective deficiency to γ-carboxylate for example FX or FII for some mutations are new, important findings, which will help to treat respective patients with clotting factor concentrates more efficiently.…”

“…Low‐responding mutations also affect protein stability as do mutations GGCX:p.(R83P/W) and GGCX:p.(M174R). The loss‐of‐function mutation GGCX:p.(F299S) and the low responding mutation GGCX:p.(S300F) potentially affect vitamin K epoxidation 43 . Patients harboring a low responding mutation in homozygous state or in compound heterozygous state with a loss‐of‐function (GGCX:p.(F299S), GGCX:p.(M174R)), or a nonsense mutation are difficult to treat with vitamin K and result in the most severe hemostatic phenotypes.…”

Splicing Outcomes of 5′ Splice Site GT>GC Variants That Generate Wild-Type Transcripts Differ Significantly Between Full-Length and Minigene Splicing Assays

“…RNA analysis using either patient blood cells or immortalized lymphoblastoid cells represents an alternative option, providing that the gene of interest is normally expressed in these cells (Wai et al, 2020). In case of the non-feasibility of both approaches, a cell culture-based minigene splicing assay has often been devised (for some most recent examples, see Damasio et al, 2021;Hao et al, 2021;Kim et al, 2021;Kortum et al, 2021;Le Tertre et al, 2021;Morbidoni et al, 2021;Qian et al, 2021;Saint-Martin et al, 2021;Torrado et al, 2021).…”

GGCX variants leading to biallelic deficiency to γ‐carboxylate GRP cause skin laxity in VKCFD1 patients

“…Two pathogenic GGCX variants directly affecting this binding site (GGCX:p.(Phe299Ser) and GGCX:p.(Ser300Phe)) resulted in loss‐of‐function for γ‐carboxylation of all analyzed VKD proteins. Both GGCX variants were also found in another study to cause loss‐of‐function or diminished ability to γ‐carboxylate VKD clotting factors (Hao et al, 2021). Hao et al also showed that these two residues show reduced vitamin k epoxidation when mutated.…”