title: Oxidation of human lens recombinant alphaA-crystallin and cysteine-deficient mutants.
abstract: Disulfide cross-linking, one of the results of oxidative stress, has been thought to play an important role in cataractogenesis. High molecular mass (HMM) protein aggregation also contributes to cataract development, and a prevailing speculation is that disulfide cross-linking induces HMM aggregation. However, there is no direct evidence to support this speculation. Dimerization is an effect of disulfide cross-linking but cannot explain the size of HMM aggregates observed in the lens. alphaA-crystallin has two cysteine residues (Cys131 and Cys142) and we have prepared three Cys-deficient mutants, two single mutants (C131I and C142I) and one double mutant (C131I/C142I). They were subjected to H202 oxidation in an ascorbate-FeCl(3)-EDTA-H202 system. The effects of oxidation on the mutants, including changes in aggregate size and conformation, were compared with those of the wild-type alphaA-crystallin by FPLC gel filtration, absorption, fluorescence, and circular dichroism measurements. The results indicated that other amino acid residues besides Cys, such as Trp and Tyr, were also oxidized by H202. Disulfide dimerization alone seems to play a less important role in HMM aggregation than does the secondary conformational change resulting from the combined effect of the oxidation of Trp and Tyr as well as Cys.
authors.affiliation: Center for Ophthalmic Research, Brigham and Women's Hospital, Boston, MA 02115, USA.
authors.forename: S J
authors.initials: SJ
authors.lastname: Chen
authors.forename: T X
authors.initials: TX
authors.lastname: Sun
authors.forename: N J
authors.initials: NJ
authors.lastname: Akhtar
authors.forename: J J
authors.initials: JJ
authors.lastname: Liang
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003459:Crystallins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D007908:Lens, Crystalline / Q000737:chemistry*; D009154:Mutation / Q000235:genetics*; D016877:Oxidants / Q000378:metabolism / Q000494:pharmacology*; D010084:Oxidation-Reduction / Q000187:drug effects; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000378:metabolism
chemical_list: D003459:Crystallins; D016877:Oxidants; D011994:Recombinant Proteins; D014364:Tryptophan; D006861:Hydrogen Peroxide; D003545:Cysteine