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

Abstract Human and other mammalian lens proteins are composed of three major crystallins: α-, β-, and γ-crystallin. α-Crystallin plays a prominent role in the supramolecular assembly required to maintain lens transparency. With age, the crystallins, especially α-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. α-Crystallin is composed of two subunits, αA and αB. Before the application of recombinant DNA technology, these two α-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 αA- and αB-crystallins for conformational and functional studies. The expressed proteins from Escherichia coli are in the native state and can be studied directly. First, αA and αB 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 αA- and αB-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. αA- and αB-crystallins have not only different secondary structure, but also tertiary structure. 1-Anilino-8-naphthalene sulfonate fluorescence indicates that αB-crystallin is more hydrophobic than αA-crystallin. The chaperone-like activity, as measured by the ability to protect insulin aggregation, is about 4 times greater for αB- than for αA-crystallin. The resulting data provide a base line for further studies of human lens α-crystallin.