Conformational and functional differences between recombinant human lens alpha A- and alpha B-crystallin
Conformational and functional differences between recombinant human lens alpha A- and alpha B-crystallin
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DOI：
10.1074/jbc.272.10.6220
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发表时间：
1997-03-07
影响因子：
4.8
通讯作者：
Liang, JJN
中科院分区：
文献类型：
--
作者：
Sun, TX;Das, BK;Liang, JJN
人类和其他哺乳动物的透镜蛋白由三种主要的晶状体蛋白组成：α-晶状体蛋白、β-晶状体蛋白和γ-晶状体蛋白。α-晶体蛋白在超分子组装中起着突出的作用，需要60保持透镜的透明度。随着年龄的增长，晶状体蛋白，特别是α-晶状体蛋白，经历翻译后修饰，这可能破坏超分子组装，并且透镜变得对导致白内障形成的其他应力敏感。由于这些修饰甚至发生在相对年轻的年龄，因此难以获得用于体外实验的纯的、未修饰的晶体蛋白。α-晶体蛋白由两个亚基α A和α B组成。在应用重组DNA技术之前，这两个α-晶状体蛋白亚基在变性状态下从小牛透镜中分离，并通过去除变性剂而重构，但它们没有正确地重折叠。在本研究中，我们应用重组DNA技术制备天然的、未修饰的α A-和α B-晶体蛋白用于构象和功能研究。从大肠杆菌表达的蛋白质是在天然状态，可以直接研究。首先，从人透镜上皮细胞cDNA文库中分离α A和α B cDNA。将cDNA克隆到pAED 4表达载体中，在E. coli菌株BL 21（DE 3）。通过凝胶过滤和DEAE液相色谱纯化后获得纯的重组α A-和α B-晶体蛋白。他们进行了构象研究，涉及各种光谱测量和评估分子伴侣样活性。α A-和α B-晶体蛋白不仅具有不同的二级结构，而且具有不同的三级结构。1-苯胺基-8-萘磺酸盐荧光表明α B-晶状体蛋白比α A-晶状体蛋白更疏水。如通过保护胰岛素聚集的能力所测量的，α B-晶状体蛋白的分子伴侣样活性比α A-晶状体蛋白大约4倍。所得数据为进一步研究人透镜α-晶状体蛋白提供了基线。
Human and other mammalian lens proteins are composed of three major crystallins: alpha-, beta-, and gamma-crystallin. alpha-Crystallin plays a prominent role in the supramolecular assembly required 60 maintain lens transparency. With age, the crystallins, especially alpha-crystallin, undergo posttranslational modifications that may disrupt the supramolecular assembly, and the lens becomes susceptible to other stresses resulting in cataract formation. Because these modifications occur even at a relatively young age, it is difficult to obtain pure, unmodified crystallins for in vitro experiments. alpha-Crystallin is composed of two subunits, alpha A and alpha B. Before the application of recombinant DNA technology, these two alpha-crystallin subunits were separated from calf lens in the denatured state and reconstituted by the removal of the denaturant, but they were not refolded properly. In the present studies, we applied the recombinant DNA technology to prepare native, unmodified alpha A- and alpha B-crystallins for conformational and functional studies. The expressed proteins from Escherichia coli are in the native state and can be studied directly. First, alpha A and alpha B cDNAs were isolated from a human lens epithelial cell cDNA library. The cDNAs were cloned into a pAED4 expression vector and then expressed in E. coli strain BL21(DE3). Pure recombinant alpha A- and alpha B-crystallins were obtained after purification by gel filtration and DEAE liquid chromatography. They were subjected to conformational studies involving various spectroscopic measurements and an assessment of chaperone-like activity. alpha A- and alpha B-crystallins have not only different secondary structure, but also tertiary structure. 1-Anilino-8-naphthalene sulfonate fluorescence indicates that alpha B-crystallin is more hydrophobic than alpha A-crystallin. The chaperone-like activity, as measured by the ability to protect insulin aggregation, is about 4 times greater for alpha B- than for alpha A-crystallin. The resulting data provide a base line for further studies of human lens alpha-crystallin.