Sun T X, Das B K, Liang J J
Center for Ophthalmic Research, Brigham and Women's Hospital, and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1997 Mar 7;272(10):6220-5. doi: 10.1074/jbc.272.10.6220.
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 to 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, alphaA and alphaB. 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 alphaA- and alphaB-crystallins for conformational and functional studies. The expressed proteins from Escherichia coli are in the native state and can be studied directly. First, alphaA and alphaB 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 alphaA- and alphaB-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. alphaA- and alphaB-crystallins have not only different secondary structure, but also tertiary structure. 1-Anilino-8-naphthalene sulfonate fluorescence indicates that alphaB-crystallin is more hydrophobic than alphaA-crystallin. The chaperone-like activity, as measured by the ability to protect insulin aggregation, is about 4 times greater for alphaB- than for alphaA-crystallin. The resulting data provide a base line for further studies of human lens alpha-crystallin.
α-、β-和γ-晶状体蛋白。α-晶状体蛋白在维持晶状体透明度所需的超分子组装中起重要作用。随着年龄的增长，晶状体蛋白，尤其是α-晶状体蛋白，会发生翻译后修饰，这可能会破坏超分子组装，晶状体变得易受其他应激影响，从而导致白内障形成。由于这些修饰甚至在相对年轻时就会发生，因此很难获得用于体外实验的纯的、未修饰的晶状体蛋白。α-晶状体蛋白由两个亚基αA和αB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晶状体中分离出来，并通过去除变性剂进行重组，但它们没有正确复性。在本研究中，我们应用重组DNA技术制备天然的、未修饰的αA和αB晶状体蛋白，用于构象和功能研究。从大肠杆菌中表达的蛋白质处于天然状态，可以直接进行研究。首先，从人晶状体上皮细胞cDNA文库中分离出αA和αB cDNA。将这些cDNA克隆到pAED4表达载体中，然后在大肠杆菌BL21(DE3)菌株中表达。通过凝胶过滤和DEAE液相色谱纯化后，获得了纯的重组αA和αB晶状体蛋白。对它们进行了涉及各种光谱测量的构象研究以及伴侣样活性评估。αA和αB晶状体蛋白不仅具有不同的二级结构，还具有不同的三级结构。用1-苯胺基-8-萘磺酸盐荧光法检测表明，αB晶状体蛋白比αA晶状体蛋白更疏水。通过保护胰岛素聚集的能力来衡量，αB晶状体蛋白的伴侣样活性比αA晶状体蛋白大约高4倍。所得数据为进一步研究人晶状体α-晶状体蛋白提供了基线。
