N-Glycan-calnexin interactions in human factor VII secretion and deficiency
N-Glycan-calnexin interactions in human factor VII secretion and deficiency
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N-聚糖-钙连接蛋白在人凝血因子 VII 分泌和缺乏中的相互作用
DOI：
10.1016/j.biocel.2019.05.017
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发表时间：
2019
影响因子：
4
通讯作者：
Wu Qingyu
中科院分区：
文献类型：
--
作者：
Wang Hao;Wang Lina;Li Shuo;Dong Ningzheng;Wu Qingyu
凝血因子VII（FVII）是凝血过程中的关键丝氨酸蛋白酶。已显示FVII中的N-糖基化对于蛋白质分泌至关重要。然而，到目前为止，潜在的生化机制仍不清楚。最近，我们发现跨膜丝氨酸蛋白酶corin中的N-聚糖对于钙连接蛋白辅助蛋白折叠和细胞外表达至关重要。在这项研究中，我们测试了FVII蛋白酶结构域中的N-聚糖介导钙连接蛋白辅助蛋白折叠以及自然发生的F7突变消除N-糖基化损害FVII分泌的假设。我们在存在或不存在葡萄糖苷酶抑制剂的情况下，在HEK 293和HepG 2细胞中表达人FVII野生型（WT）和缺乏一个或两个N-糖基化位点的突变蛋白。通过免疫染色、免疫共沉淀、Western印迹和ELISA检测FVII的表达、分泌和与内质网伴侣的结合。我们发现，人FVII蛋白酶结构域中N360的N-糖基化，而不是前肽结构域中N183的N-糖基化是蛋白分泌所必需的。N360处N-糖基化的消除损害了钙连接蛋白辅助的FVII折叠和分泌。当通过葡萄糖苷酶抑制阻断N-聚糖-钙连接蛋白相互作用时，在WT FVII中观察到类似的结果。在HEK 293和HepG 2细胞中，自然发生的F7突变消除了N360处的N-糖基化，减少了FVII的分泌。这些结果表明FVII蛋白酶结构域中的N-聚糖介导钙连接蛋白辅助的蛋白折叠和随后的细胞外表达。自然发生的F7突变消除FVII中的N-糖基化可能会损害这一机制，从而降低患者的FVII水平。
Factor VII (FVII) is a key serine protease in blood coagulation. N-glycosylation in FVII has been shown to be critical for protein secretion. To date, however, the underlying biochemical mechanism remains unclear. Recently, we found thatN-glycans in the transmembrane serine protease corin are critical for calnexin-assisted protein folding and extracellular expression. In this study, we tested the hypothesis thatN-glycans in the FVII protease domain mediate calnexin-assisted protein folding and that naturally occurringF7mutations abolishingN-glycosylation impair FVII secretion. We expressed human FVII wild-type (WT) and mutant proteins lacking one or bothN-glycosylation sites in HEK293 and HepG2 cells in the presence or absence of a glucosidase inhibitor. FVII expression, secretion and binding to endoplasmic reticulum chaperones were examined by immune staining, co-immunoprecipitation, Western blotting, and ELISA. We found thatN-glycosylation at N360 in the protease domain, but not N183 in the pro-peptide domain, of human FVII is required for protein secretion. Elimination ofN-glycosylation at N360 impaired calnexin-assisted FVII folding and secretion. Similar results were observed in WT FVII whenN-glycan-calnexin interaction was blocked by glucosidase inhibition. Naturally occurringF7mutations abolishingN-glycosylation at N360 reduced FVII secretion in HEK293 and HepG2 cells. These results indicate thatN-glycans in the FVII protease domain mediate calnexin-assisted protein folding and subsequent extracellular expression. Naturally occurringF7mutations abolishingN-glycosylation in FVII may impair this mechanism, thereby reducing FVII levels in patients.