David L L, Azuma M, Shearer T R
Department of Oral Molecular Biology, School of Dentistry, Oregon Health Sciences University, Portland 97201.
Invest Ophthalmol Vis Sci. 1994 Mar;35(3):785-93.
To determine if limited proteolysis of beta-crystallins is associated with insolubilization of proteins in rats lens during maturation and to test if the protease, calpain II, is involved.
Soluble and insoluble lens proteins from 4-day-old to 4-month-old rat lens cortexes and nuclei were separated by two-dimensional electrophoresis. The insoluble proteins from 4-month-old nuclei were electroblotted and the NH2 termini of proteins sequenced. Cleavage sites appearing at 4 months of age were compared to cleavage sites produced by purified calpain II and to cleavage sites appearing in cataracts induced by selenite in vivo or in lenses cultured with calcium ionophore A23187 or diamide.
In solubilization of more than 50% of proteins occurred in the nucleus of the transparent rat lens by 4 months of age. The insoluble protein that formed contained an abundance of partially degraded beta-crystallin polypeptides missing portions of their NH2 terminal extensions. In contrast, these truncated beta-crystallins were largely absent from both the cortex and soluble fraction of the nucleus. The cleavage sites in the insoluble beta-crystallins appearing during maturation in the lens nucleus were similar to cleavage sites produced by purified calpain II and also similar to cleavage sites appearing in the insoluble protein of cataractous lenses.
These results suggest that proteolysis of beta-crystallins by the protease calpain II contributes to protein insolubilization during lens maturation and that acceleration of this insolubilization process is associated with cataract formation in rodent lenses.
确定β-晶状体蛋白的有限蛋白水解是否与大鼠晶状体成熟过程中蛋白质的不溶性有关，并测试蛋白酶钙蛋白酶II是否参与其中。
通过二维电泳分离4日龄至4月龄大鼠晶状体皮质和核中的可溶性和不溶性晶状体蛋白。对4月龄核中的不溶性蛋白进行电印迹，并对蛋白质的NH2末端进行测序。将4月龄时出现的切割位点与纯化的钙蛋白酶II产生的切割位点以及体内亚硒酸盐诱导的白内障或用钙离子载体A23187或二酰胺培养的晶状体中出现的切割位点进行比较。
到4月龄时，透明大鼠晶状体核中超过50%的蛋白质发生了溶解。形成的不溶性蛋白质含有大量部分降解的β-晶状体蛋白多肽，这些多肽缺失了其NH2末端延伸部分。相比之下，这些截短的β-晶状体蛋白在皮质和核的可溶部分中基本不存在。晶状体核成熟过程中出现的不溶性β-晶状体蛋白中的切割位点与纯化的钙蛋白酶II产生的切割位点相似，也与白内障晶状体不溶性蛋白中出现的切割位点相似。
这些结果表明，蛋白酶钙蛋白酶II对β-晶状体蛋白的蛋白水解有助于晶状体成熟过程中的蛋白质不溶性，并且这种不溶性过程的加速与啮齿动物晶状体中的白内障形成有关。
