N-glycan in the scavenger receptor cysteine-rich domain of hepsin promotes intracellular trafficking and cell surface expression
N-glycan in the scavenger receptor cysteine-rich domain of hepsin promotes intracellular trafficking and cell surface expression
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Hepsin 清道夫受体富含半胱氨酸结构域中的 N-聚糖促进细胞内运输和细胞表面表达
DOI：
10.1016/j.ijbiomac.2020.06.109
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发表时间：
2020
影响因子：
8.2
通讯作者：
Dong Ningzheng
中科院分区：
文献类型：
--
作者：
Sun Shijin;Wang Lina;Zhang Shengnan;Zhang Ce;Chen Yue;Wu Qingyu;Dong Ningzheng
A组清道夫受体富含半胱氨酸（SRCR）结构域是存在于参与多种生物过程的许多蛋白质中的保守模块。Hepsin是一种与许多癌症有关的肝蛋白酶，由胞质尾区、跨膜结构域和具有A组SRCR结构域和丝氨酸蛋白酶结构域的胞外区组成。与许多含SRCR的蛋白质一样，hepsin中的SRCR结构域具有N-糖基化位点，但其功能意义尚不清楚。在这项研究中，我们证实了N-糖基化的Asn 112在肝蛋白酶的糖苷酶消化和定点突变在人肝癌细胞。在Western印迹、荧光底物分析、流式细胞术和蛋白追踪实验中，我们发现Asn 112突变为Gln（N112 Q）抑制了hepsin的细胞内运输、细胞表面表达和酶原激活。通过免疫荧光染色，我们发现N112 Q突变体比野生型hepsin在内质网（ER）中更丰富。进一步的免疫共沉淀研究表明N112 Q突变体与钙连接蛋白和结合免疫球蛋白（BiP）（两种ER伴侣）的结合增加。我们的研究结果表明，N-聚糖的SRCR结构域的hepsin促进细胞内运输和细胞表面的表达，可能是通过钙连接蛋白依赖的机制，促进ER退出。
The group A scavenger receptor cysteine-rich (SRCR) domain is a conserved module present in numerous proteins involved in diverse biological processes. Hepsin, a hepatic protease implicated in many cancers, consists of a cytoplasmic tail, a transmembrane domain and an extracellular regions with a group A SRCR domain and a serine protease domain. Like in many SRCR-containing proteins, the SRCR domain in hepsin has an N-glycosylation site, but its functional significance is unknown. In this study, we confirmed N-glycosylation at Asn112 in hepsin by glycosidase digestion and site-directed mutagenesis in human hepatoma cells. In Western blotting, fluorogenic substrate assay, flow cytometry, and protein-chase experiments, we found that Asn112 to Gln (N112Q) mutation inhibited hepsin intracellular trafficking, cell surface expression, and zymogen activation. By immunofluorescent staining, we found that the N112Q mutant was more abundant than wild-type hepsin in the endoplasmic reticulum (ER). Further co-immunoprecipitation studies indicated increased binding of the N112Q mutant to calnexin and binding-immunoglobulin protein (BiP), two ER chaperones. Our results indicate that the N-glycan in the SRCR domain of hepsin promotes intracellular trafficking and cell surface expression, possibly by a calnexin-dependent mechanism in facilitating ER exiting.