Calpain 8/nCL-2 and calpain 9/nCL-4 constitute an active protease complex, G-calpain, involved in gastric mucosal defense.
Calpain 8/nCL-2 and calpain 9/nCL-4 constitute an active protease complex, G-calpain, involved in gastric mucosal defense.
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DOI：
10.1371/journal.pgen.1001040
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发表时间：
2010-07-29
期刊：
影响因子：
4.5
通讯作者：
Sorimachi H
中科院分区：
文献类型：
--
作者：
Hata S;Abe M;Suzuki H;Kitamura F;Toyama-Sorimachi N;Abe K;Sakimura K;Sorimachi H
钙蛋白酶是一类钙离子依赖性的半胱氨酸蛋白酶，是细胞内各种生理过程中不可缺少的蛋白。在15种哺乳动物钙蛋白酶中，钙蛋白酶8/nCL-2和钙蛋白酶9/nCL-4主要在胃肠道中表达，并且仅限于胃中的胃表面粘液（小凹）细胞。钙蛋白酶8可能的功能报道在囊泡之间的ER和高尔基体的运输，和钙蛋白酶9有牵连，在抑制肿瘤的发生。这些突出表明，钙蛋白酶8和9的调节彼此不同，并从传统的钙蛋白酶，因此，在胃肠道中具有潜在的重要的，特定的功能。然而，没有直接证据表明钙蛋白酶8或9与人类疾病有关，其性质和生理功能目前尚不清楚。为了解决它们的生理作用，我们分析了这些钙蛋白酶Capn 8和Capn 9基因突变的小鼠。Capn 8 −/−和Capn 9 −/−小鼠具有生育能力，其胃粘膜表现正常。然而，这两种小鼠均对乙醇给药诱导的胃粘膜损伤敏感。此外，Capn 8 −/−胃中的钙蛋白酶9和8均显着降低，Capn 9 −/−也是如此。与这一发现相一致的是，在野生型胃中，钙蛋白酶8和9形成了一种我们称为“G-钙蛋白酶”的复合物，其中两者都是活性所必需的。这是第一个“杂交”钙蛋白酶复合体的例子。为了解决钙蛋白酶8蛋白水解活性的生理相关性，我们产生了钙蛋白酶8：C105 S“敲入”（Capn 8 CS/CS）小鼠，其表达蛋白水解无活性但结构完整的钙蛋白酶8。尽管与Capn 8 −/−胃不同，Capn 8 CS/CS小鼠的胃表达稳定且活跃的钙蛋白酶9，但小鼠对乙醇诱导的胃损伤敏感。这些结果提供了第一个证据，这两个胃肠道特异性钙蛋白酶是必不可少的胃粘膜防御，他们指出G-钙蛋白酶作为一个潜在的目标，由外部压力引起的胃病。持续或不当摄入刺激物，包括酒精、非甾体抗炎药（NSAID）和幽门螺杆菌，通常会导致严重的胃病，影响广泛的人群。复杂的胃防御系统有助于抵御这些威胁，例如分泌粘液。在这里，我们报告，两个胃肠道特异性钙蛋白酶，钙蛋白酶8/nCL-2和钙蛋白酶9/nCL-4，参与粘膜防御应激诱导的胃病。钙蛋白酶是一种钙离子依赖性的细胞溶质蛋白酶，在各种细胞过程中不可或缺。钙蛋白酶活性不适当可导致死亡或严重疾病，如肌营养不良症和无脑畸形，尽管没有关于钙蛋白酶在胃肠道疾病中的作用的报道。在这里，我们表明，小鼠的基因突变的钙蛋白酶8和9是易受酒精诱导的胃损伤。此外，这些钙蛋白酶形成稳定的复合物，其中两种分子都是活性所必需的。因此，人类钙蛋白酶8和9可能有助于胃对酒精等刺激物引起的应激的敏感性。事实上，预测这些钙蛋白酶中的一些报道的人类单核苷酸多态性（SNP）会损害它们的蛋白水解活性。我们的突变小鼠提供了独特的动物模型，潜在的人类胃病所造成的这样的SNP。
Calpains constitute a superfamily of Ca2+-dependent cysteine proteases, indispensable for various cellular processes. Among the 15 mammalian calpains, calpain 8/nCL-2 and calpain 9/nCL-4 are predominantly expressed in the gastrointestinal tract and are restricted to the gastric surface mucus (pit) cells in the stomach. Possible functions reported for calpain 8 are in vesicle trafficking between ER and Golgi, and calpain 9 are implicated in suppressing tumorigenesis. These highlight that calpains 8 and 9 are regulated differently from each other and from conventional calpains and, thus, have potentially important, specific functions in the gastrointestinal tract. However, there is no direct evidence implicating calpain 8 or 9 in human disease, and their properties and physiological functions are currently unknown. To address their physiological roles, we analyzed mice with mutations in the genes for these calpains, Capn8 and Capn9. Capn8−/− and Capn9−/− mice were fertile, and their gastric mucosae appeared normal. However, both mice were susceptible to gastric mucosal injury induced by ethanol administration. Moreover, the Capn8−/− stomach showed significant decreases in both calpains 9 and 8, and the same was true for Capn9−/−. Consistent with this finding, in the wild-type stomach, calpains 8 and 9 formed a complex we termed “G-calpain,” in which both were essential for activity. This is the first example of a “hybrid” calpain complex. To address the physiological relevance of the calpain 8 proteolytic activity, we generated calpain 8:C105S “knock-in” (Capn8CS/CS) mice, which expressed a proteolytically inactive, but structurally intact, calpain 8. Although, unlike the Capn8−/− stomach, that of the Capn8CS/CS mice expressed a stable and active calpain 9, the mice were susceptible to ethanol-induced gastric injury. These results provide the first evidence that both of the gastrointestinal-tract-specific calpains are essential for gastric mucosal defense, and they point to G-calpain as a potential target for gastropathies caused by external stresses. The continuous or improper ingestion of irritants, including alcohol, nonsteroidal anti-inflammatory drugs (NSAIDs), and Helicobacter pylori, often leads to serious gastropathies, affecting a wide range of people. A complex gastric defense system helps protect against these threats, for example by secreting mucus. Here we report that two gastrointestinal-tract-specific calpains, calpain 8/nCL-2 and calpain 9/nCL-4, are involved in the mucosal defense against stress-induced gastropathies. Calpains are Ca2+-dependent cytosolic proteases that are indispensable for various cellular processes. Improper calpain activities can result in death or serious disorders, such as muscular dystrophies and lissencephaly, although no role for calpains in gastrointestinal diseases has been reported. Here we show that mice with mutations in the genes for calpains 8 and 9 are susceptible to alcohol-induced gastric injury. Moreover, these calpains form a stable complex, in which both molecules are essential for activity. Thus, human calpains 8 and 9 may contribute to the stomach's susceptibility to stress caused by irritants such as alcohol. Indeed, some reported human single nucleotide polymorphisms (SNPs) in these calpains are predicted to compromise their proteolytic activity. Our mutant mice provide unique animal models for potential human gastropathies caused by such SNPs.
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