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

• Published in: Blood Vol. 137; no. 4; pp. 533 - 543
• Main Authors: Hao, Zhenyu, Jin, Da-Yun, Chen, Xuejie, Schurgers, Leon J., Stafford, Darrel W., Tie, Jian-Ke
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28.01.2021; American Society of Hematology
• Subjects: Amino Acid Sequence; Base Sequence; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - metabolism; Carbon-Carbon Ligases - chemistry; Carbon-Carbon Ligases - genetics; Carboxy-Lyases - chemistry; Carboxy-Lyases - genetics; Extracellular Matrix Proteins - genetics; Extracellular Matrix Proteins - metabolism; Genes, Reporter; Genetic Association Studies; Genetic Pleiotropy; HEK293 Cells; Hemorrhagic Disorders - drug therapy; Hemorrhagic Disorders - genetics; Humans; Matrix Gla Protein; Mutation; Mutation, Missense; Osteocalcin - genetics; Osteocalcin - metabolism; Protein C - genetics; Protein C - metabolism; Protein Domains; Protein Interaction Mapping; Protein Isoforms - genetics; Protein Isoforms - metabolism; Protein Processing, Post-Translational - genetics; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; RNA Precursors - metabolism; RNA Splicing; Structure-Activity Relationship; Thrombosis and Hemostasis; Vitamin K - physiology; Vitamin K - therapeutic use
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.2020006329

γ-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 physiologic 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 3 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, and then the carboxylation efficiency of these reporter proteins was 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–messenger RNA 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 nonbleeding disorders. •GGCX mutations differentially affect the carboxylation of vitamin K–dependent proteins associated with distinct clinical phenotypes.•Some GGCX missense mutations affect the pre–messenger RNA splicing rather than altering the corresponding amino acid residues. [Display omitted]