N-Glycosylation Is Required for Matriptase-2 Autoactivation and Ectodomain Shedding
N-Glycosylation Is Required for Matriptase-2 Autoactivation and Ectodomain Shedding
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Matriptase-2 自动激活和胞外域脱落需要 N-糖基化
DOI：
10.1074/jbc.m114.555110
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发表时间：
2014-07-11
影响因子：
4.8
通讯作者：
He, Yang
中科院分区：
文献类型：
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作者：
Jiang, Jiang;Yang, Jianfeng;He, Yang
间质蛋白酶-2是一种调节铁稳态的肝膜丝氨酸蛋白酶。间质蛋白酶-2的缺陷导致缺铁性贫血。在细胞中，间质蛋白酶-2作为酶原合成。迄今为止，间质蛋白酶-2的表达和活化是如何调节的仍然知之甚少。在此，我们在HEK 293和肝BEL-7402、SMMC 7721和QGY-7703细胞中表达人间质蛋白酶-2。通过标记细胞表面蛋白和Western分析，我们研究了matriptase-2细胞表面表达，酶原激活和胞外域脱落。我们的研究结果表明，matriptase-2被激活的细胞表面，但不是胞内。活化的间质蛋白酶-2经历胞外域脱落，在条件培养基中产生可溶性片段。通过测试失活突变体R576 A和S762 A，我们发现间质蛋白酶-2的活化和脱落是由其自身的催化活性介导的，并且间质蛋白酶-2的单链形式在胞外域脱落中几乎没有活性。我们制备了另外的间质蛋白酶-2突变体N136 Q、N184 Q、N216 Q、N338 Q、N433 Q、N453 Q和N518 Q，其中预测的N-糖基化位点中的每一个被突变。所有这些突变体都在细胞表面表达。然而，突变体N216 Q、N453 Q和N518 Q，而不是其它突变体，具有受损的酶原活化和胞外域脱落。我们的结果表明，特定位点的N-聚糖对于matriptase-2的激活至关重要。总之，这些数据为matriptase-2的细胞表面表达、酶原激活和胞外域脱落提供了新的见解。
Matriptase-2 is a hepatic membrane serine protease that regulates iron homeostasis. Defects in matriptase-2 cause iron deficiency anemia. In cells, matriptase-2 is synthesized as a zymogen. To date, how matriptase-2 expression and activation are regulated remains poorly understood. Here we expressed human matriptase-2 in HEK293 and hepatic BEL-7402, SMMC7721, and QGY-7703 cells. By labeling cell surface proteins and Western analysis, we examined matriptase-2 cell surface expression, zymogen activation, and ectodomain shedding. Our results show that matriptase-2 was activated on the cell surface but not intracellularly. Activated matriptase-2 underwent ectodomain shedding, producing soluble fragments in the conditioned medium. By testing inactive mutants, R576A and S762A, we found that matriptase-2 activation and shedding were mediated by its own catalytic activity and that the one-chain form of matriptase-2 had little activity in ectodomain shedding. We made additional matriptase-2 mutants, N136Q, N184Q, N216Q, N338Q, N433Q, N453Q, and N518Q, in which each of the predicted N-glycosylation sites was mutated. All of these mutants were expressed on the cell surface. However, mutants N216Q, N453Q, and N518Q, but not the other mutants, had impaired zymogen activation and ectodomain shedding. Our results indicate that N-glycans at specific sites are critical for matriptase-2 activation. Together, these data provide new insights into the cell surface expression, zymogen activation, and ectodomain shedding of matriptase-2.