Stomach-specific calpain, nCL-2/calpain 8, is active without calpain regulatory subunit and oligomerizes through C2-like domains
Stomach-specific calpain, nCL-2/calpain 8, is active without calpain regulatory subunit and oligomerizes through C2-like domains
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DOI：
10.1074/jbc.m703168200
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发表时间：
2007-09-21
影响因子：
4.8
通讯作者：
Sorimachi, Hiroyuki
中科院分区：
文献类型：
--
作者：
Hata, Shoji;Doi, Naoko;Sorimachi, Hiroyuki
钙蛋白酶是细胞内Ca2+调节的半胱氨酸蛋白酶家族，在多种细胞功能的调节中不可或缺。钙蛋白酶的不当激活会导致致死性或各种疾病，如肌肉萎缩症和肿瘤形成。nCL-2/ calpain8主要在胃中表达，它似乎参与胃表面粘液细胞（坑细胞）的膜运输。虽然nCL-2的初级结构与普遍存在的m-calpain大亚基非常相似，但nCL-2的酶学性质尚未报道。为了表征nCL-2，利用大肠杆菌表达系统制备了重组蛋白，并进行了纯化。nCL-2作为一种可溶性活性酶稳定地产生，没有常规的钙蛋白酶调节亚基（30K）。纯化后的nCL-2表现出Ca2+依赖性活性，在约0.3 mM Ca2+时活性达到半峰，与m-calpain相似，但其最适pH和温度较低。免疫沉淀分析显示，nCL-2以单体和同源低聚形式存在，但不以30K或30K-2的异源二聚体形式存在，并且低聚化发生在5EF-hand结构域IV以外的结构域，最有可能通过结构域III，这表明nCL-2存在一种新的调控系统。
Calpains constitute a family of intracellular Ca2+-regulated cysteine proteases that are indispensable in the regulation of a wide variety of cellular functions. The improper activation of calpain causes lethality or various disorders, such as muscular dystrophies and tumor formation. nCL-2/calpain 8 is predominantly expressed in the stomach, where it appears to be involved in membrane trafficking in the gastric surface mucus cells (pit cells). Although the primary structure of nCL-2 is quite similar to that of the ubiquitous m-calpain large subunit, the enzymatic properties of nCL-2 have never been reported. Here, to characterize nCL-2, the recombinant protein was prepared using an Escherichia coli expression system and purified to homogeneity. nCL-2 was stably produced as a soluble and active enzyme without the conventional calpain regulatory subunit (30K). Purified nCL-2 showed Ca2+-dependent activity, with half-maximal activity at about 0.3 mM Ca2+, similar to that of m-calpain, whereas its optimal pH and temperature were comparatively low. Immunoprecipitation analysis revealed that nCL-2 exists in both monomeric and homo-oligomeric forms, but not as a heterodimer with 30K or 30K-2, and that the oligomerization occurs through domains other than the 5EF-hand domain IV, most probably through domain III, suggesting a novel regulatory system for nCL-2.