Ma H, Fukiage C, Kim Y H, Duncan M K, Reed N A, Shih M, Azuma M, Shearer T R
Department of Oral Molecular Biology, Oregon Health & Science University, Portland, Oregon 97201, USA.
J Biol Chem. 2001 Jul 27;276(30):28525-31. doi: 10.1074/jbc.M100603200. Epub 2001 May 25.
Calpains are calcium-dependent intracellular nonlysosomal proteases that are believed to hydrolyze specific substrates important in calcium-regulated signaling pathways. Recently, an atypical member of the calpain family, calpain 10, was described, and genetic variation in this gene was associated with an increased risk of type II diabetes mellitus in humans. In the present report, a polyclonal antibody directed against rat calpain 10 was developed. This antibody was used to monitor the expression of calpain 10 protein in tissues from rats, mice, and humans. Calpain 10 protein was found to be present in all tissues examined by Western blotting including the lens, retina, brain, heart, and skeletal muscle. Although some calpain 10 was detectable in the water-soluble protein fraction of these tissues, it was preferentially found in the water-insoluble fraction. In the lens, immunohistochemistry revealed that calpain 10 was predominately located in the cytoplasm of epithelial and newly differentiating lens fibers at the transition zone. However, calpain 10 was found to be associated with the plasma membrane of differentiated lens fiber cells and the sarcolemma of skeletal muscle. In the lens epithelium-derived cell line, alphaTN4-1, the calpain 10 protein was found in a punctate distribution in the cell nucleus as well as the cytoplasm. After the elevation of intracellular calcium levels with ionomycin, calpain 10 protein levels in the nucleus of alphaTN4-1 cells increased markedly, whereas those in the cytoplasm decreased. In the lens, the elevation of intracellular calcium levels after selenite administration resulted in increased levels of calpain 10 RNA within 1 day and a loss of calpain 10 protein from the lens nucleus coincident with the onset of selenite cataract. In conclusion, calpain 10 seems to be a ubiquitous calpain, the expression level and subcellular distribution of which are dynamically influenced by calcium.
钙蛋白酶是一种依赖钙的细胞内非溶酶体蛋白酶，据信可水解钙调节信号通路中重要的特定底物。最近，有人描述了钙蛋白酶家族的一个非典型成员——钙蛋白酶10，该基因的遗传变异与人类患II型糖尿病的风险增加有关。在本报告中，制备了一种针对大鼠钙蛋白酶10的多克隆抗体。该抗体用于监测大鼠、小鼠和人类组织中钙蛋白酶10蛋白的表达。通过蛋白质印迹法发现在所有检测的组织中都存在钙蛋白酶10蛋白，包括晶状体、视网膜、脑、心脏和骨骼肌。虽然在这些组织的水溶性蛋白部分可检测到一些钙蛋白酶10，但它主要存在于水不溶性部分。在晶状体中，免疫组织化学显示钙蛋白酶10主要位于过渡区上皮细胞和新分化的晶状体纤维的细胞质中。然而，发现钙蛋白酶10与分化的晶状体纤维细胞的质膜和骨骼肌的肌膜有关。在晶状体上皮衍生的细胞系alphaTN4-1中，发现钙蛋白酶10蛋白在细胞核和细胞质中呈点状分布。用离子霉素提高细胞内钙水平后，alphaTN4-1细胞核中的钙蛋白酶10蛋白水平显著增加，而细胞质中的水平则下降。在晶状体中，亚硒酸钠给药后细胞内钙水平升高导致1天内钙蛋白酶10 RNA水平增加，并且随着亚硒酸钠性白内障的发生，晶状体核中的钙蛋白酶10蛋白丢失。总之，钙蛋白酶10似乎是一种普遍存在的钙蛋白酶，其表达水平和亚细胞分布受到钙的动态影响。
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