Ma H, Shih M, Hata I, Fukiage C, Azuma M, Shearer T R
Departments of Oral Molecular Biology, Biochemistry and Molecular Biology, and Ophthalmology, Oregon Health Sciences University, Portland, OR 97201, USA.
Curr Eye Res. 2000 Mar;20(3):183-9.
To clone and sequence the cDNA for Lp85 calpain from young rat lens, and to test for Lp85 protein expression and proteolytic activity.
RT-PCR and molecular cloning were performed on total RNA from 12 day-old rats. Lp85 protein expression was visualized by immunoblotting using a specific antibody developed to the unique peptide sequence in Lp85. Proteolytic activity was assessed by casein zymography. Transient expression of Lp85 and previously characterized lens-specific calpain Lp82 were separately performed in mammalian COS-7 cells.
The 2410-bp cDNA for rat lens Lp85 encoded a protein of 737 amino acid residues with a calculated molecular weight of 85.0 kDa and a predicted pI of 5.67. The amino acid sequence of Lp85 was identical to Lp82 except for an insert region of 28 amino acids in domain IV of the calcium-binding region. mRNA and protein for Lp85 were present only in rat and mouse lenses and not in other tissues or species. Lp85 protein concentrations were highest in the nuclear region, most concentrated in the insoluble fraction, disappeared with lens maturation, and Lp85 exhibited migration similar to Lp82 on native PAGE gels. Lp85 was enzymatically active when expressed in COS-7 cells.
Lp85 is a newly classified, lens- and rodent-specific, enzymatically active, member of the AX1 (alternative exon 1) subclass of calpains. In conjunction with Lp82 and m-calpain in lens, Lp85 may be responsible for proteolysis during normal lens development and maturation or during cataract formation in young rodents.
克隆并测序幼鼠晶状体中Lp85钙蛋白酶的cDNA，并检测Lp85蛋白的表达及蛋白水解活性。
对12日龄大鼠的总RNA进行逆转录聚合酶链反应（RT-PCR）和分子克隆。使用针对Lp85中独特肽序列开发的特异性抗体，通过免疫印迹法观察Lp85蛋白的表达。通过酪蛋白酶谱法评估蛋白水解活性。分别在哺乳动物COS-7细胞中瞬时表达Lp85和先前鉴定的晶状体特异性钙蛋白酶Lp82。
大鼠晶状体Lp85的2410个碱基对的cDNA编码一个由737个氨基酸残基组成的蛋白质，计算分子量为85.0 kDa，预测等电点为5.67。Lp85的氨基酸序列与Lp82相同，除了钙结合区域结构域IV中有一个28个氨基酸的插入区域。Lp85的mRNA和蛋白仅存在于大鼠和小鼠的晶状体中，而不存在于其他组织或物种中。Lp85蛋白浓度在核区域最高，最集中于不溶性部分，随着晶状体成熟而消失，并且Lp85在天然聚丙烯酰胺凝胶电泳（PAGE）上的迁移与Lp82相似。当在COS-7细胞中表达时，Lp85具有酶活性。
Lp85是钙蛋白酶AX1（替代外显子1）亚类中一个新分类的、晶状体和啮齿动物特异性的、具有酶活性的成员。与晶状体中的Lp82和m-钙蛋白酶一起，Lp85可能在正常晶状体发育和成熟过程中或幼龄啮齿动物白内障形成过程中负责蛋白水解。
