Srivastava Om P, Srivastava Kiran
Department of Physiological Optics, School of Optometry, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Mol Vis. 2003 Dec 8;9:644-56.
[corrected] The aims of this study were to determine in vitro crosslinking of a 9 kDa gammaD-crystallin fragment alone and with alpha-, beta-, or gamma-crystallins, the existence of covalent multimers of the polypeptide in vivo, and posttranslational modifications in the three isoforms of the polypeptide.
A mixture of crystallin fragments (3-14 kDa), a 9 kDa gammaD-crystallin polypeptide or the polypeptide and individual alpha-, beta-, or gamma-crystallins, were incubated at 37 degrees C for a desired length of time and the crosslinked species were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), size exclusion Agarose A 1.5 gel chromatography, and western blot analysis. In addition, the existence of covalent multimers of the 9 kDa polypeptide in human lens water soluble (WS) and water insoluble (WI) protein fractions of normal and cataractous human lenses was determined by western blot analyses. The posttranslationally modified amino acids of three isofroms of the polypeptide were identified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) and ES-MS/MS mass spectrometric analyses.
Following incubation of a mixture of the crystallin fragments or the 9 kDa polypeptide, covalently crosslinked species held via non-disulfide bonding were seen on SDS-PAGE analysis. The polypeptide also exhibited crosslinking with individual alpha-, beta-, and gamma-crystallins. After western blot analysis with site specific anti-9 kDa antibodies, both WS and WI protein fractions from normal and cataractous lenses showed immunoreactive 27 and 45 kDa multimers. The mass spectrometric analysis of the three isoforms of the polypeptide (with identical molecular weight but different charges) showed oxidized methionine and tryptophan residues, with the latter residue containing two oxygens.
The data suggest that a 9 kDa gammaD-crystallin fragment demonstrated crosslinking properties, which might be due to oxidation of its methionine and tryptophan residues.
[已修正] 本研究的目的是确定单独的9 kDa γD-晶状体蛋白片段以及与α-、β-或γ-晶状体蛋白一起时的体外交联情况、该多肽在体内共价多聚体的存在情况以及该多肽三种同工型的翻译后修饰。
将晶状体蛋白片段混合物（3 - 14 kDa）、9 kDa γD-晶状体蛋白多肽或该多肽与单个α-、β-或γ-晶状体蛋白在37℃孵育所需时长，然后通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、尺寸排阻琼脂糖A 1.5凝胶色谱和蛋白质印迹分析对交联产物进行分析。此外，通过蛋白质印迹分析确定正常和白内障患者晶状体的水溶性（WS）和水不溶性（WI）蛋白组分中9 kDa多肽共价多聚体的存在情况。通过基质辅助激光解吸电离飞行时间（MALDI-TOF）和电喷雾串联质谱（ES-MS/MS）分析鉴定该多肽三种同工型的翻译后修饰氨基酸。
在孵育晶状体蛋白片段混合物或9 kDa多肽后，SDS-PAGE分析显示存在通过非二硫键结合的共价交联产物。该多肽还与单个α-、β-和γ-晶状体蛋白发生交联。用位点特异性抗9 kDa抗体进行蛋白质印迹分析后，正常和白内障晶状体的WS和WI蛋白组分均显示出免疫反应性的27 kDa和45 kDa多聚体。对该多肽的三种同工型（分子量相同但电荷不同）进行质谱分析显示存在甲硫氨酸和色氨酸残基氧化，后者残基含有两个氧原子。
数据表明9 kDa γD-晶状体蛋白片段具有交联特性，这可能是由于其甲硫氨酸和色氨酸残基的氧化所致。
