Effect of oxidized betaB3-crystallin peptide on lens betaL-crystallin: interaction with betaB2-crystallin.
OBJECTIVE
To investigate the interaction of oxidized betaB3-crystallin peptide (residues 152-166) with betaL-crystallin and to identify peptide-interaction sites.


METHODS
Peptides were oxidized by using CuSO4 and H2O2. Aggregation and light-scattering assays of bovine betaL-crystallin were conducted at 55 degrees C and 37 degrees C, respectively. Assays were performed in the presence of oxidized and nonoxidized betaB3-crystallin peptides and in the presence of alpha-crystallin. Peptide-induced change in hydrophobicity was determined by bis-ANS (4,4'-dianilino-1,1' binaphthyl-5,5' disulfonic acid) binding study. Oxidized betaB3-peptide binding sites were identified by sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-{p-azidobenzamido}-hexanoamido] ethyl-1-3 dithiopropionate) labeling and mass spectrometric analysis.


RESULTS
Aggregation and relative light-scattering of betaL-crystallin was higher in the presence of oxidized betaB3-crystallin peptide than with betaL-crystallin, without oxidized peptide and with nonoxidized peptide. Enhanced aggregation was observed despite the presence of alpha-crystallin in the assay. Furthermore, a significant increase in aggregation and light-scattering was observed in the presence of oxidized betaB3-peptide at 37 degrees C. Bis-ANS binding to betaL-crystallin treated with oxidized betaB3-peptide was two to three times higher than in the controls at 37 degrees C. The oxidized betaB3-peptide preferentially interacted with betaB2-crystallin. The data were confirmed by mass spectrometric analysis.


CONCLUSIONS
Oxidized betaB3-peptide interacts with betaB2-crystallin and enhances its aggregation and precipitation. Peptide-induced aggregation and increased hydrophobicity of the lens crystallin at 37 degrees C are relevant to crystallin aggregation in the aging lenses.
Department of Ophthalmology, University of Missouri, 65212, USA.
E G Padmanabha
EG
Udupa
K Krishna
KK
Sharma
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D019327:Copper Sulfate / Q000494:pharmacology; D003459:Crystallins / Q000494:pharmacology*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007908:Lens, Crystalline / Q000737:chemistry*; D008027:Light; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D012542:Scattering, Radiation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D038206:beta-Crystallin B Chain / Q000378:metabolism*; D038204:beta-Crystallins / Q000378:metabolism*
C103908:CRYBB3 protein, human; D003459:Crystallins; D010446:Peptide Fragments; D038206:beta-Crystallin B Chain; D038204:beta-Crystallins; C113634:beta-crystallin B2; D006861:Hydrogen Peroxide; D019327:Copper Sulfate