Conformational study of N?-(carboxymethyl)lysine adducts of recombinant a-crystallins

Abstract

Purpose. Lens proteins underwent nonenzymatic glycation, and the advanced glycation end products (AGEs) were detected by immunological assays. One of the major AGE structures is N?-(carboxymethyl)lysine (CML). Since the involvement of AGEs in the pathogenesis of diabetic complications is speculated, the effects of CML formation on proteins were studied. Methods. CML adducts were generated in recombinant aA-and aB-crystallins by incubation with glyoxylic acid and NaBH 3 CN. SDS-PAGE and size exclusion chromatography were used to detect subunit degradation and high-molecular-weight (HMW) aggregation. Conformational change was determined by fluorescence and circular dichroism (CD) measurements. The chaperone function was studied by DTT-induced aggregation of insulin. Results. Lysine modification was estimated to be 60–90% depending on the conditions of incubation. No subunit degradation or HMW aggregation was observed. Fluorescence and CD measurements detected a conformational change in CML adducts. Measurements of chaperone-like activity, however, indicated that the formation of CML increased the protein's ability to protect insulin against DTT-induced aggregation. Conclusions. Although CML adducts of aA- and aB-crystallins, the major AGE structures formed in vitro, changed protein conformation, no subunit degradation and HMW aggregation were observed. Moreover, the CML adducts increased chaperone-like activity of both aA- and aB-crystallins. The results suggest that CML formation alone may not play a major role in protein aggregation and lens opacity.