Ma H, Shih M, Fukiage C, Azuma M, Duncan M K, Reed N A, Richard I, Beckmann J S, Shearer T R
Departments of Oral Molecular Biology, Oregon Health Sciences University, Portland, Oregon, USA.
Invest Ophthalmol Vis Sci. 2000 Dec;41(13):4232-9.
To determine the influence of specific regions within Lp82 calpain on protein stability, enzymatic activity, and localization within lens and to test the influence of an Lp82 knockout mouse on normal maturational proteolysis in lens.
DNA constructs for Lp82 and Lp82-related proteins were subcloned into the pcDNA 3.1 vector. The constructs contained a substitution of the novel sequence (NS) region from p94 for the AX1 N-terminal region of Lp82 and insertions of the p94 IS1 and IS2 regions into Lp82. Transient expression of these Lp82-related proteins was performed in COS-7 mammalian cells. Immunoblotting and casein zymography were used to measure protein stability and enzymatic activity of the expressed proteins. Homologous recombination was used to knock out p94 gene expression and p94 splice variants such as Lp82 and Lp85 in the lenses of 10-day-old mice. Confocal microscopy revealed the immunohistochemical localization Lp82 and Lp85 within lens.
Insertion of IS1 into Lp82 resulted in a lack of stable protein and loss of enzymatic activity. In contrast, substitution of the NS region for AX1 and insertion of IS2 into Lp82 had no effect on the stability of the Lp82-related proteins. p94 knockout mice at 10 days of age exhibited a total absence of Lp82 activity in the lens but normal activity for the separate mu- and m-calpain gene products. Calcium-induced in vitro proteolysis was retarded in these Lp82/p94 knockout lenses. Lp82 and Lp85 immunostaining was intense throughout the cytoplasm of the cortical and nuclear fibers of newborn mouse lenses with little staining in the epithelium. In contrast, immunostaining for the ubiquitous m-calpain was highest in the epithelium and bow region, with much lower levels in the nucleus. The naturally occurring IS3 insert in Lp85 also promoted the association of Lp85 with the perinuclear region of the nucleated lens fibers.
The lack of the IS1 region in Lp82 accounts for the stability and abundance of enzymatically active Lp82 protein in rodent lenses. Conversely, the presence of the IS1 region is responsible for the lability of p94 and Rt88 calpains in muscle and retina, respectively. The insert in Lp85 may promote membrane association. A consequence of the specific loss of Lp82 in the lens may be to retard normal maturational proteolysis.
确定Lp82钙蛋白酶特定区域对蛋白质稳定性、酶活性及在晶状体中定位的影响，并测试Lp82基因敲除小鼠对晶状体正常成熟蛋白水解的影响。
将Lp82及Lp82相关蛋白的DNA构建体亚克隆到pcDNA 3.1载体中。构建体包含用p94的新序列（NS）区域替换Lp82的AX1 N端区域，以及将p94的IS1和IS2区域插入Lp82。在COS-7哺乳动物细胞中进行这些Lp82相关蛋白的瞬时表达。采用免疫印迹法和酪蛋白酶谱法测量所表达蛋白的稳定性和酶活性。利用同源重组敲除10日龄小鼠晶状体中的p94基因表达及p94剪接变体，如Lp82和Lp85。共聚焦显微镜检查揭示了Lp82和Lp85在晶状体中的免疫组织化学定位。
将IS1插入Lp82导致蛋白不稳定且酶活性丧失。相反，用NS区域替换AX1以及将IS2插入Lp82对Lp82相关蛋白的稳定性没有影响。10日龄的p94基因敲除小鼠晶状体中Lp82活性完全缺失，但μ-钙蛋白酶和m-钙蛋白酶基因产物的活性正常。在这些Lp82/p94基因敲除的晶状体中，钙诱导的体外蛋白水解受到抑制。Lp82和Lp85免疫染色在新生小鼠晶状体皮质和核纤维的整个细胞质中强烈，而上皮细胞中染色较少。相比之下，普遍存在的m-钙蛋白酶的免疫染色在上皮细胞和弓形区域最高，在细胞核中水平低得多。Lp85中天然存在的IS3插入也促进了Lp85与有核晶状体纤维核周区域的结合。
Lp82中缺乏IS1区域导致啮齿动物晶状体中具有酶活性的Lp82蛋白的稳定性和丰度。相反，IS1区域的存在分别导致肌肉和视网膜中p94和Rt88钙蛋白酶的不稳定性。Lp85中的插入可能促进膜结合。晶状体中Lp82特异性缺失的一个后果可能是延缓正常的成熟蛋白水解。
