Conformational study of N(epsilon)-(carboxymethyl)lysine adducts of recombinant alpha-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(epsilon)-(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 alphaA- and alphaB-crystallins by incubation with glyoxylic acid and NaBH3CN. 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 alphaA- and alphaB-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 alphaA- and alphaB-crystallins. The results suggest that CML formation alone may not play a major role in protein aggregation and lens opacity.