Conformational and functional differences between recombinant human lens alpha A- and alpha B-crystallin

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.