Purpose: [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.
Methods: 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.
Results: 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.
Conclusions: 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.