Existence of deamidated alphaB-crystallin fragments in normal and cataractous human lenses.
OBJECTIVE
The aims of this study were to characterize lens crystallin fragments having a molecular mass of <10 kDa, isolated by solubilization in trichloroacetic acid, in order to identify cleavage sites in the parent crystallins for their origin and determine post-translational modifications in the fragments.


METHODS
The water-soluble (WS) and water-insoluble (WI) protein fractions were isolated from normal human lenses of 60 to 80 year old donors and from age-matched cataractous lenses. Both WS and WI protein fractions were treated with TCA at 60 degrees C for 2 h and the TCA-soluble fractions were recovered following centrifugation. The preparations were dialyzed against H2O to remove TCA, concentrated by lyophilization and subjected to two dimensional gel electrophoresis (2D-GE). The spots from 2D-gels were analyzed by western blot analysis, partial N-terminal sequencing, or excised for mass spectrometric analysis.


RESULTS
SDS-PAGE analysis showed that TCA solubilized polypeptides having a molecular mass of <10 kDa from both WS and WI protein fractions of normal and cataractous lenses. Following 2D-GE of TCA-solubilized species from normal lenses, 8 and 5 polypeptides from the WS and WI protein fractions, respectively, were observed. Using similar 2D-GE analysis of TCA solubilized species from cataractous lenses, 9 and 5 polypeptides from WS and WI protein fractions, respectively, were seen. Partial N-terminal sequence analysis showed that the majority of the polypeptides from both WS and WI protein fractions of normal and cataractous lenses were derived from alphaB-crystallin following cleavage at the D129-P130 bond. Western blot and partial N-terminal sequence analyses identified three additional 4-kDa alphaA-crystallin fragments with cleavage at the D136-G137 bond in the WS proteins from normal lenses. MALDI-TOF mass spectrometric analysis showed that all TCA soluble polypeptides from cataractous lenses, except one from normal lenses, contained residue number 130 to 175 from alphaB-crystallin. No further truncation occurred at the C-terminal region of the alphaB-crystallin polypeptides. Following comparison of the isotopic distribution in MALDI-TOF profiles of a tryptic fragment having a mass of 2,014 among the alphaB-crystallin polypeptides, a gain of one single Dalton was observed. This suggested deamidation of the N146 residue in alphaB-crystallin fragments.


CONCLUSIONS
The results show that the N146 residue in human alphaB-crystallin undergoes in vivo deamidation and several fragments containing this modification exist in both WS and WI protein fractions of normal and cataractous human lenses.
Srivastava
Om P
OP
Department of Physiological Optics, School of Optometry, University of Alabama at Birmingham, Birmingham, AL 35294, USA. Srivastava@icare.opt.uab.edu
Srivastava
Kiran
K
D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism; D015153:Blotting, Western; D002386:Cataract / Q000378:metabolism*; D003641:Deamination; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism*; D008875:Middle Aged; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D038203:alpha-Crystallin B Chain / Q000378:metabolism*
D010446:Peptide Fragments; D010455:Peptides; D038203:alpha-Crystallin B Chain; D001216:Asparagine