The murine goblet cell protein mCLCA3 is a zinc-dependent metalloprotease with autoproteolytic activity
The murine goblet cell protein mCLCA3 is a zinc-dependent metalloprotease with autoproteolytic activity
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DOI：
10.1007/s10059-011-0158-8
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发表时间：
2011-12-01
影响因子：
3.8
通讯作者：
Gruber, Achim D.
中科院分区：
文献类型：
--
作者：
Bothe, Melanie K.;Mundhenk, Lars;Gruber, Achim D.
CLCA 蛋白家族的几个成员最初被称为钙激活的氯离子通道，已被证明可以通过未知的机制调节各种细胞类型中的氯离子电导。此外，人类 (h) hCLCA1 被认为可以调节哮喘和囊性纤维化 (CF) 患者的疾病严重程度。所有 CLCA 蛋白均在翻译后裂解为两个亚基，最近，已鉴定出保守的 HEXXH 锌结合氨基酸基序，表明 CLCA 蛋白作为金属蛋白酶的作用。在这里，我们表征了鼠模型蛋白 mCLCA3 的切割和自蛋白水解活性，该蛋白代表人 hCLCA1 的鼠直系同源物。使用来自转染的 HEK293 细胞的粗膜部分，我们证明 mCLCA3 裂解是锌依赖性的，并且完全被阳离子螯合金属蛋白酶抑制剂抑制。由于在 mCLCA3 的 HEXXH 基序中插入 E157Q 突变而导致裂解缺陷，细胞转运和分泌并未受到影响。 hCLCA1 和 mCLCA3 的猪 (p) 直系同源物 pCLCA1 进一步证实了这些关键结果的种间保守性。重要的是，mCLCA3E157Q 突变体在 HEK293 细胞中与野生型 mCLCA3 共转染后被裂解，表明发生了分子间自蛋白水解事件。蛋白质片段的 Edman 降解和 MALDI-TOF-MS 鉴定出 mCLCA3 中氨基酸 695 和 696 之间的单个切割位点。数据强烈表明，分泌的 CLCA 蛋白质具有锌依赖性自蛋白水解活性，并且它们可能切割其他蛋白质。
Several members of the CLCA family of proteins, originally named chloride channels, calcium-activated, have been shown to modulate chloride conductance in various cell types via an unknown mechanism. Moreover, the human (h) hCLCA1 is thought to modulate the severity of disease in asthma and cystic fibrosis (CF) patients. All CLCA proteins are post-translationally cleaved into two subunits, and recently, a conserved HEXXH zinc-binding amino acid motif has been identified, suggesting a role for CLCA proteins as metalloproteases. Here, we have characterized the cleavage and autoproteolytic activity of the murine model protein mCLCA3, which represents the murine orthologue of human hCLCA1. Using crude membrane fractions from transfected HEK293 cells, we demonstrate that mCLCA3 cleavage is zinc-dependent and exclusively inhibited by cation-chelating metalloprotease inhibitors. Cellular transport and secretion were not affected in response to a cleavage defect that was introduced by the insertion of an E157Q mutation within the HEXXH motif of mCLCA3. Interspecies conservation of these key results was further confirmed with the porcine (p) orthologue of hCLCA1 and mCLCA3, pCLCA1. Importantly, the mCLCA3E157Q mutant was cleaved after co-transfection with the wild-type mCLCA3 in HEK293 cells, suggesting that an intermolecular autoproteolytic event takes place. Edman degradation and MALDI-TOF-MS of the protein fragments identified a single cleavage site in mCLCA3 between amino acids 695 and 696. The data strongly suggest that secreted CLCA proteins have zinc-dependent autoproteolytic activity and that they may cleave additional proteins.