CONFORMATIONAL AND FUNCTIONAL DIFFERENCES BETWEEN RECOMBINANT HUMAN LENS ALPHA-A-CRYSTALLIN AND ALPHA-B-CRYSTALLIN

Human and other mammalian lens proteins are composed of three major cr ystallins: 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-crystal lin, undergo posttranslational modifications that may disrupt the supr amolecular assembly, and the lens becomes susceptible to other stresse s resulting in cataract formation. Because these modifications occur e ven at a relatively young age, it is difficult to obtain pure, unmodif ied crystallins for in vitro experiments. alpha-Crystallin is composed of two subunits, alpha A and alpha B. Before the application of recom binant DNA technology, these two alpha-crystallin subunits were separa ted from calf lens in the denatured state and reconstituted by the rem oval of the denaturant, but they were not refolded properly. In the pr esent studies, we applied the recombinant DNA technology to prepare na tive, unmodified alpha A- and alpha B-crystallins for conformational a nd functional studies. The expressed proteins from Escherichia coil ar e 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 lib rary. The cDNAs were cloned into a pAED4 expression vector and then ex pressed in E. coli strain BL21(DE3). Pure recombinant alpha A- and alp ha B-crystallins were obtained after purification by gel filtration an d DEAE liquid chromatography. They were subjected to conformational st udies involving various spectroscopic measurements and an assessment o f chaperone-like activity. alpha A- and alpha B-crystallins have not o nly different secondary structure, but also tertiary structure. 1-Anil ino-8-naphthalene sulfonate fluorescence indicates that alpha B-crysta llin is more hydrophobic than alpha A-crystallin. The chaperone-like a ctivity, as measured by the ability to protect insulin aggregation, is about 4 times greater for alpha B- than for alpha A-crystallin. The r esulting data provide a base line for further studies of human lens al pha-crystallin.