Abstract
The accumulation of molecular damage by ascorbylation products impairs molecules of critical importance for the maintenance of homeostatic functions in the lens epithelium. The purpose of this study was to compare the protein composition of the B-3 line of transformed human lens epithelial cells (HLE B-3) exposed to the stress produced from dehydroascorbic acid (DHA) to that of normal control cells, and to identify protein targets of ascorbylation stress in HLE B-3 cells treated with DHA. Using two-dimensional SDS-polyacrylamide gel electrophoresis (2DE) with 7 cm mini gels and electrospray ion trap mass spectrometry with nano spray (ESI-MS), we could compared four two dimensional (2D) gel protein maps at the same time.
As a result, in the identified proteins, mass spectral data revealed that there were specifically modified proteins by DHA treatment in senescent cells. These newly observed modifications are methionine oxidation (alfa-enolase, vimentin, and mutant beta-actin), tryptophane oxidation (calreticulin), deamidation (alfa-enolase and vimentin), C-terminal carbamidomethylation (alfa-enolase, vimentin and mutant beta-actin), and N-terminal carbamylation (mutant beta-actin).
Mass-mapping profile also showed the remarkably increment of several beta-B2crystallins in the same cells. These results suggest that epithelial cell senescence is associated with a breakdown of anti-ascorbylation mechanisms.
