Ma H, Hata I, Shih M, Fukiage C, Nakamura Y, Azuma M, Shearer T R
Departments of Oral Molecular Biology, Biochemistry and Molecular Biology, and Ophthalmology, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Exp Eye Res. 1999 Apr;68(4):447-56. doi: 10.1006/exer.1998.0625.
The purpose of this study was to characterize Lp82 calpain in normal mouse. Lp82 is a lens-specific, calcium-activated isozyme from the calpain super family of cysteine proteases (EC 34.22.17). RT-PCR and molecular cloning were performed on total RNA from 12 day-old mice. Lp82 and m-calpain protein levels and proteolytic activities in lenses were measured by casein zymography, immunoblotting, and ELISA after partial purification by DEAE-HPLC. The 2334-bp cDNA encoding for mouse Lp82 contained a single large open reading frame encoding a protein of 709 amino acid residues with a calculated molecular weight of 82.2 kDa and a predicted pI of 5.8. The amino acid sequence of mouse lens Lp82 was 99% homologous to rat lens Lp82. As in rat, mouse lens Lp82 showed a unique N -terminus and deletion of the IS1 and IS2 regions. In contrast to rat, Lp82 was the dominant calpain in young mouse lens. Lp82 was lens-specific, and the lens nucleus contained the highest specific activity of Lp82 and very little m-calpain. Endogenous Lp82 in lens soluble proteins was activated by addition of calcium and caused limited proteolysis of crystallins even in the presence of large amounts of recombinant domain I from the natural calpain inhibitor calpastatin. Loss of Lp82 protein accompanied aging of mouse lens. Lp82 may be responsible for a major portion of crystallin proteolysis occurring during normal lens development and maturation, or during cataract formation in young mice.
本研究的目的是对正常小鼠中的Lp82钙蛋白酶进行表征。Lp82是一种来自半胱氨酸蛋白酶钙蛋白酶超家族（EC 34.22.17）的晶状体特异性钙激活同工酶。对12日龄小鼠的总RNA进行了逆转录聚合酶链反应（RT-PCR）和分子克隆。通过酪蛋白酶谱法、免疫印迹法以及用二乙氨基乙基纤维素（DEAE）-高效液相色谱法（HPLC）进行部分纯化后的酶联免疫吸附测定（ELISA），测定了晶状体中Lp82和m-钙蛋白酶的蛋白质水平及蛋白水解活性。编码小鼠Lp82的2334碱基对的cDNA包含一个单一的大开放阅读框，编码一个由709个氨基酸残基组成的蛋白质，计算分子量为82.2 kDa，预测的等电点为5.8。小鼠晶状体Lp82的氨基酸序列与大鼠晶状体Lp82的同源性为99%。与大鼠一样，小鼠晶状体Lp82显示出独特的N端以及IS1和IS2区域的缺失。与大鼠不同的是，Lp82是幼龄小鼠晶状体中的主要钙蛋白酶。Lp82是晶状体特异性的，晶状体核中Lp82的比活性最高，而m-钙蛋白酶含量极少。即使在存在大量来自天然钙蛋白酶抑制剂钙蛋白酶抑制蛋白的重组结构域I的情况下，向晶状体可溶性蛋白中添加钙也可激活内源性Lp82，并导致晶状体蛋白发生有限的蛋白水解。Lp82蛋白的缺失伴随着小鼠晶状体的老化。Lp82可能是正常晶状体发育和成熟过程中，或幼龄小鼠白内障形成过程中发生的大部分晶状体蛋白水解的原因。
