title: Conformational study of N(epsilon)-(carboxymethyl)lysine adducts of recombinant alpha-crystallins.
abstract: OBJECTIVE
  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.
authors.affiliation: Center for Ophthalmic Research, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115-5822, USA.
authors.forename: N J
authors.initials: NJ
authors.lastname: Akhtar
authors.forename: T X
authors.initials: TX
authors.lastname: Sun
authors.forename: J J
authors.initials: JJ
authors.lastname: Liang
mesh_terms: D002850:Chromatography, Gel / Q000379:methods; D002942:Circular Dichroism; D003459:Crystallins / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005453:Fluorescence; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D018832:Molecular Chaperones / Q000502:physiology; D008968:Molecular Conformation; D011994:Recombinant Proteins
chemical_list: D003459:Crystallins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; C067242:N(epsilon)-(carboxymethyl)hydroxylysine; D008239:Lysine