Isolation and Characterization of Non-Disulfide in vivo Cross-Linked Gamma S-Crystallin from Cataractous Lenses

Abstract

Abstract: : Purpose: Cataractogenesis is characterized by aggregation and insolubilization of lens crystallins. Non-disulfide cross-linking is among the major reactions leading to formation of high molecular weight aggregates. Due to the variety of crystallins involved in cross-linking and the complicity of the reactions, molecular analysis of the non-disulfide cross-links has been difficult. The goal of this research was to isolate from cataractous lenses in vivo cross-linked dimers of gamma S-crystallin suitable for detailed structural analyses. Methods: The cross-linked gamma S-crystallin dimers were purified from cataractous lenses using several steps of size exclusion chromatography, ion-exchange and reversed phase HPLC. MALDI and electrospray ionization mass spectrometry were used to analyze the intact proteins and peptides formed by enzymatic digestion and CNBr cleavage. Results: A procedure for isolation/purification of in vivo cross-linked dimeric gamma S-crystallin (molecular mass of approximately 42 kDa) from cataractous lenses was developed. Peptide mapping indicated that the protein consists of full-length gamma S-crystallin and the region including residues 19-35 is involved in the non-disulfide cross-linking. The mass spectra of the intact protein showed the presence of several major components with different cross-linkers with molecular weights ranging from 150 to 240 Da. Characterization of peptides containing the cross-links will be presented. Conclusion: The region including residues 19-35 of gamma S-crystallin is involved in the in vivo formation of non-disulfide cross-links in cataractous lenses.

Keywords: 525 protein modifications-post translational • 526 protein purification and characterization • 338 cataract