Existence of deamidated αB-crystallin fragments in normaland cataractous human lenses

[摘要] Purpose: The aims of this study were to characterize lens crystallinfragments having a molecular mass of <10 kDa, isolated bysolubilization in trichloroacetic acid, in order to identify cleavagesites in the parent crystallins for their origin and determinepost-translational modifications in the fragments.Methods: The water-soluble (WS) and water-insoluble (WI) proteinfractions were isolated from normal human lenses of 60 to 80 year olddonors and from age-matched cataractous lenses. Both WS and WI proteinfractions were treated with TCA at 60 °C for 2 h and the TCA-solublefractions were recovered following centrifugation. The preparations weredialyzed against H2O to remove TCA, concentrated by lyophilizationand subjected to two dimensional gel electrophoresis (2D-GE). The spotsfrom 2D-gels were analyzed by western blot analysis, partial N-terminalsequencing, or excised for mass spectrometric analysis.Results: SDS-PAGE analysis showed that TCA solubilized polypeptideshaving a molecular mass of <10 kDa from both WS and WI proteinfractions of normal and cataractous lenses. Following 2D-GE ofTCA-solubilized species from normal lenses, 8 and 5 polypeptides fromthe WS and WI protein fractions, respectively, were observed. Usingsimilar 2D-GE analysis of TCA solubilized species from cataractouslenses, 9 and 5 polypeptides from WS and WI protein fractions,respectively, were seen. Partial N-terminal sequence analysis showedthat the majority of the polypeptides from both WS and WI proteinfractions of normal and cataractous lenses were derived from αB-crystallin following cleavage at the D129-P130 bond.Western blot and partial N-terminal sequence analyses identified threeadditional 4-kDa αA-crystallin fragments with cleavage at theD136-G137 bond in the WS proteins from normal lenses.MALDI-TOF mass spectrometric analysis showed that all TCA solublepolypeptides from cataractous lenses, except one from normal lenses,contained residue number 130 to 175 from αB-crystallin. No furthertruncation occurred at the C-terminal region of the αB-crystallinpolypeptides. Following comparison of the isotopic distribution inMALDI-TOF profiles of a tryptic fragment having a mass of 2,014 amongthe αB-crystallin polypeptides, a gain of one single Dalton wasobserved. This suggested deamidation of the N146 residue in αB-crystallin fragments.Conclusions: The results show that the N146 residue in humanαB-crystallin undergoes in vivo deamidation and several fragmentscontaining this modification exist in both WS and WI protein fractionsof normal and cataractous human lenses.

[学科分类] 生物化学/生物物理