Autoactivation and calpain-1-mediated shedding of hepsin in human hepatoma cells
Autoactivation and calpain-1-mediated shedding of hepsin in human hepatoma cells
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人肝癌细胞中 hepsin 的自激活和 calpain-1 介导的脱落
DOI：
10.1042/bcj20190375
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发表时间：
2019
影响因子：
4.1
通讯作者：
Wu Qingyu
中科院分区：
文献类型：
--
作者：
Wang Lina;Zhang Ce;Sun Shijin;Chen Yue;Hu Yae;Wang Hao;Liu Meng;Dong Ningzheng;Wu Qingyu
Hepsin是一种跨膜丝氨酸蛋白酶，参与许多生物过程，包括肝细胞生长、尿蛋白分泌、听神经发育和癌症转移。酶原活化对肝素的功能至关重要。迄今为止，hepsin如何在细胞中被激活和调节仍然是一个谜。在这项研究中，我们对最初发现hepsin的人肝癌HepG2细胞和SMMC-7721细胞进行了定点诱变、细胞表达、质膜蛋白标记、胰蛋白酶消化、Western blotting和流式细胞术实验。我们的研究结果表明，hepsin是由细胞表面的自催化而不是细胞内的自催化激活的。此外，我们发现肝素经历了外畴脱落。在HepG2和SMMC-7721细胞的条件培养基中，我们检测到几乎包含整个hepsin细胞外区域的可溶性片段。通过测试蛋白酶抑制剂、基因敲低和定点突变，我们发现calpain-1是一种主要的蛋白酶，它在细胞外作用，在肝蛋白酶的近膜空间切割Tyr52。这些结果为研究调节hepsin表达和活性的生化和细胞机制提供了新的见解。
Hepsin is a transmembrane serine protease implicated in many biological processes, including hepatocyte growth, urinary protein secretion, auditory nerve development, and cancer metastasis. Zymogen activation is critical for hepsin function. To date, how hepsin is activated and regulated in cells remains an enigma. In this study, we conducted site-directed mutagenesis, cell expression, plasma membrane protein labeling, trypsin digestion, Western blotting, and flow cytometry experiments in human hepatoma HepG2 cells, where hepsin was originally discovered, and SMMC-7721 cells. Our results show that hepsin is activated by autocatalysis on the cell surface but not intracellularly. Moreover, we show that hepsin undergoes ectodomain shedding. In the conditioned medium from HepG2 and SMMC-7721 cells, we detected a soluble fragment comprising nearly the entire extracellular region of hepsin. By testing protease inhibitors, gene knockdown, and site-directed mutagenesis, we identified calpain-1 as a primary protease that acted extracellularly to cleave Tyr52 in the juxtamembrane space of hepsin. These results provide new insights into the biochemical and cellular mechanisms that regulate hepsin expression and activity.