Conformational study of N-epsilon-(carboxymethyl)lysine adducts of recombinant alpha-crystallins

Purpose. Lens proteins underwent nonenzymatic glycation, and the advanced g lycation end products (AGEs) were detected by immunological assays. One of the major AGE structures is N epsilon-(carboxymethyl)lysine (CML). Since th e involvement of AGEs in the pathogenesis of diabetic complications is spec ulated, the effects of CML formation on proteins were studied. Methods. CML adducts were generated in recombinant alpha A- and alpha B-cry stallins by incubation with glyoxylic acid and NaBH3CN. SDS-PAGE and size e xclusion chromatography were used to detect subunit degradation and high-mo lecular-weight (HMW) aggregation. Conformational change was determined by f luorescence and circular dichroism (CD) measurements. The chaperone functio n was studied by DTT-induced aggregation of insulin. Results. Lysine modification was estimated to be 60-90% depending on the co nditions of incubation. No subunit degradation or HMW aggregation was obser ved. Fluorescence and CD measurements detected a conformational change in C ML adducts. Measurements of chaperone-like activity, however, indicated tha t the formation of CML increased the protein's ability to protect insulin a gainst DTT-induced aggregation. Conclusions. Although CML adducts of alpha A- and alpha B-crystallins, the major AGE structures formed in vitro, changed protein conformation, no subu nit degradation and HMW aggregation were observed. Moreover, the CML adduct s increased chaperone-like activity of both alpha A- and alpha B-crystallin s. The results suggest that CML formation alone may not play a major role i n protein aggregation and lens opacity.