γ-Glutamyl carboxylase mutations differentially affect the biological function of vitamin K dependent proteins
γ-Glutamyl carboxylase mutations differentially affect the biological function of vitamin K dependent proteins
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DOI：
10.1182/blood.2020006329
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发表时间：
2021-01-28
期刊：
影响因子：
20.3
通讯作者：
Tie, Jian-Ke
中科院分区：
文献类型：
--
作者：
Hao, Zhenyu;Jin, Da-Yun;Tie, Jian-Ke
γ-谷氨酰羧化酶（GGCX）是一种整合的膜蛋白，其催化多种维生素K依赖性（VKD）蛋白的翻译后羧化，所述蛋白参与多种生理过程，包括血液凝固、血管钙化和骨代谢。自然发生的GGCX突变与多种不同的临床表型相关。然而，GGCX的基因型-表型相关性仍然难以捉摸。在这里，我们系统地研究了所有天然存在的GGCX突变对细胞环境中3种结构-功能不同的VKD蛋白羧化的影响。将GGCX突变瞬时导入稳定表达嵌合凝血因子、基质Gla蛋白（MGP）或骨钙素作为VKD报告蛋白的GGCX缺陷型人胚肾293细胞中，然后评价这些报告蛋白的羧化效率。我们的研究结果表明，GGCX突变差异影响这些报告蛋白的羧化和使用维生素K作为辅因子的效率。这些报告蛋白的羧基化的C-末端截短突变（R704 X）意味着GGCX的C末端骨钙素的结合中起着关键作用，但不是在凝血因子和MGP的结合。这已经通过使用双分子荧光互补和化学交联测定来探测活细胞中GGCX与其蛋白质底物之间的蛋白质-蛋白质相互作用而得到证实。此外，使用小基因剪接测定，我们证明了几个GGCX错义突变影响GGCX的前信使RNA剪接，而不是改变相应的氨基酸残基。这项研究的结果解释了GGCX基因型与其临床表型的相关性，并阐明了为什么维生素K治疗可以纠正出血性疾病，而不能纠正非出血性疾病。
gamma-Glutamyl carboxylase (GGCX) is an integral membrane protein that catalyzes post-translational 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.