Udupa E G Padmanabha, Sharma K Krishna
Department of Ophthalmology, University of Missouri, 65212, USA.
Invest Ophthalmol Vis Sci. 2005 Jul;46(7):2514-21. doi: 10.1167/iovs.05-0031.
To investigate the interaction of oxidized betaB3-crystallin peptide (residues 152-166) with betaL-crystallin and to identify peptide-interaction sites.
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.
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.
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.
研究氧化型βB3-晶状体蛋白肽（第152 - 166位氨基酸残基）与βL-晶状体蛋白的相互作用，并确定肽相互作用位点。
使用硫酸铜和过氧化氢氧化肽。分别在55℃和37℃对牛βL-晶状体蛋白进行聚集和光散射测定。在氧化型和非氧化型βB3-晶状体蛋白肽存在以及α-晶状体蛋白存在的情况下进行测定。通过双-ANS（4,4'-二苯胺基-1,1'-联萘-5,5'-二磺酸）结合研究确定肽诱导的疏水性变化。通过磺基-SBED（磺基琥珀酰亚胺基-2-[6-(生物素酰胺基)-2-{对叠氮苯甲酰胺基}-己酰胺基]乙基-1,3-二硫代丙酸酯）标记和质谱分析鉴定氧化型βB3-肽结合位点。
与βL-晶状体蛋白、无氧化肽和非氧化肽存在时相比，在氧化型βB3-晶状体蛋白肽存在下，βL-晶状体蛋白的聚集和相对光散射更高。尽管测定中存在α-晶状体蛋白，但仍观察到聚集增强。此外，在37℃氧化型βB3-肽存在下，聚集和光散射显著增加。在37℃，双-ANS与经氧化型βB3-肽处理的βL-晶状体蛋白的结合比对照高两到三倍。氧化型βB3-肽优先与βB2-晶状体蛋白相互作用。质谱分析证实了该数据。
氧化型βB3-肽与βB2-晶状体蛋白相互作用并增强其聚集和沉淀。肽在37℃诱导的晶状体蛋白聚集和疏水性增加与老化晶状体中的晶状体蛋白聚集有关。
