Activation of epithelial sodium channels by mouse channel activating proteases (mCAP) expressed in Xenopus oocytes requires catalytic activity of mCAP3 and mCAP2 but not mCAP1
Activation of epithelial sodium channels by mouse channel activating proteases (mCAP) expressed in Xenopus oocytes requires catalytic activity of mCAP3 and mCAP2 but not mCAP1
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DOI：
10.1681/asn.2005060637
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发表时间：
2006-04-01
影响因子：
13.6
通讯作者：
Rossier, Bernard C.
中科院分区：
文献类型：
--
作者：
Andreasen, Ditte;Vuagniaux, Gregoire;Rossier, Bernard C.
小鼠通道激活蛋白1、2和3(mCAP1、mCAP2和mCAP3)最近被描述为上皮性钠通道(ENaC)的调节因子。MCap是一种膜结合的丝氨酸蛋白酶，是以失活的酶原合成的。为了成熟为活性的蛋白水解酶，它们经历了自催化和/或异催化过程的分子内切割。通过Western印迹分析，制备针对每种MCap的特异性抗体，以区分酶原和活性蛋白酶。在MCap的催化区或蛋白-蛋白质相互作用区引入各种点突变，野生型和突变型酶在非洲爪哇卵母细胞表达系统中表达，以测试激活ENaC的能力。在mCAP3中，一个完整的催化三联体是激活ENaC所必需的，但不是蛋白酶分子内切割所必需的。这表明了一种异催化机制。突变mCAP2的催化三联体不仅完全取消了ENaC的激活，而且还阻止了该酶的切割。因此，mCAP2的加工似乎是自催化的。此外，位于两个蛋白质相互作用区域的mCAP2保守残基的突变显著调节了ENaC的激活。令人惊讶的是，mCAP1催化失活的突变体仍然能够完全激活ENaC，并且没有看到mCAP1分子内切割的证据。然而，需要存在完整的糖基磷脂酰肌醇锚定。结论：在非洲爪哇卵母细胞表达系统中，自体和异源催化的要求是针对每个CAP的，内源性伙伴是MCAP激活ENaC的必要条件。
Mouse channel activating proteases 1, 2, and 3 (mCAP1, mCAP2, and mCAP3) were described recently as regulators of the epithelial sodium channel (ENaC). The mCAP are membrane-bound serine proteases that are synthesized as inactive proenzymes. To mature into active proteases, they undergo intramolecular cleavage by auto- and/or heterocatalytic processing. Specific antibodies against each mCAP were developed to distinguish between proenzyme and active protease by Western blot analysis. Various point mutations were introduced in the catalytic or protein-protein interacting domains of mCAP and wild-type and mutant enzymes were expressed in the Xenopus oocyte expression system to test for ability to activate ENaC. In mCAP3, an intact catalytic triad was necessary for activation of ENaC but not for intramolecular cleavage of the protease. This suggests a heterocatalytic mechanism. Mutating the catalytic triad of mCAP2 not only abolished ENaC activation completely but also impeded cleavage of the protease. Processing of mCAP2 therefore seems to be autocatalytic. Furthermore, mutations in conserved residues of mCAP2 located in two protein-protein interacting domains significantly modulated ENaC activation. Surprisingly, mCAP1 catalytically inactive mutants were still able to fully activate ENaC, and no evidence of mCAP1 intramolecular cleavage was seen. The presence of an intact glycosylphosphatidylinositol anchor, however, was required. It is concluded that auto- and heterocatalytic requirements are specific for each CAP and that endogenous partners are a necessity for activation of ENaC by mCAP in the Xenopus oocyte expression system.