Impaired Autoproteolytic Cleavage of mCLCA6, a Murine Integral Membrane Protein Expressed in Enterocytes, Leads to Cleavage at the Plasma Membrane Instead of the Endoplasmic Reticulum
Impaired Autoproteolytic Cleavage of mCLCA6, a Murine Integral Membrane Protein Expressed in Enterocytes, Leads to Cleavage at the Plasma Membrane Instead of the Endoplasmic Reticulum
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DOI：
10.1007/s10059-012-2217-1
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发表时间：
2012-03-01
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（钙激活氯离子通道调节剂）与分泌失调相关的疾病有关，包括囊性纤维化（CF）和哮喘。已显示它们可能通过充当金属蛋白酶来调节内源性氯离子电导。基于翻译后切割后亚基的差异加工，可以区分CLCA蛋白的两个亚组。在一个亚组中，两个亚基都被分泌，在另一组中，羧基末端亚基具有跨膜区段，导致仅氨基末端亚基脱落。CLCA的翻译后切割和蛋白水解活性的最新数据仅限于分泌型CLCA。在这项研究中，我们的特点是切割的mCLCA6，小鼠CLCA具有跨膜段。对于分泌型CLCA，在mCLCA 6的HEXXH基序中具有E157Q突变的蛋白质未观察到内质网中的切割，表明该突变蛋白质和分泌型CLCA家族成员共享相似的自体蛋白水解切割机制。与具有E157Q突变的分泌型CLCA蛋白相反，mCLCA6E157Q突变体的未切割前体到达质膜，在质膜处其被切割并且氨基末端亚基脱落到上清液中。使用粗膜馏分，我们表明，裂解的mCLCA6E157Q蛋白是锌依赖性和敏感的金属蛋白酶抑制剂，这表明二次裂解的金属蛋白酶。有趣的是，mCLCA6E157Q锚定在质膜上对于其次级切割不是必需的，因为mCLCA6TM E157Q突变体仍然经历切割。我们的数据表明，CLCA蛋白的加工比以前认识到的更复杂。
CLCA proteins (calcium-activated chloride channel regulators) have been linked to diseases involving secretory disorders, including cystic fibrosis (CF) and asthma. They have been shown to modulate endogenous chloride conductance, possibly by acting as metalloproteases. Based on the differential processing of the subunits after post-translational cleavage, two subgroups of CLCA proteins can be distinguished. In one subgroup, both subunits are secreted, in the other group, the carboxy-terminal subunit possesses a transmembrane segment, resulting in shedding of only the amino-terminal subunit. Recent data on the post-translational cleavage and proteolytic activity of CLCA are limited to secreted CLCA. In this study, we characterized the cleavage of mCLCA6, a murine CLCA possessing a transmembrane segment. As for secreted CLCA, the cleavage in the endoplasmic reticulum was not observed for a protein with the E157Q mutation in the HEXXH motif of mCLCA6, suggesting that this mutant protein and secreted CLCA family members share a similar autoproteolytic cleavage mechanism. In contrast to secreted CLCA proteins with the E157Q mutation, the uncleaved precursor of the mCLCA6E157Q mutant reached the plasma membrane, where it was cleaved and the amino-terminal subunit was shed into the supernatant. Using crude membrane fractions, we showed that cleavage of the mCLCA6E157Q protein is zinc-dependent and sensitive to metalloprotease inhibitors, suggesting secondary cleavage by a metalloprotease. Interestingly, anchorage of mCLCA6E157Q to the plasma membrane is not essential for its secondary cleavage, because the mCLCA6.TM E157Q mutant still underwent cleavage. Our data suggest that the processing of CLCA proteins is more complex than previously recognized.