Abstract
Purpose: :
Senile cataracts are associated with progressive oxidation, fragmentation, cross-linking, insolubilization, and yellow pigmentation of lens crystallins, a phenomenon that was reproduced in the hSVCT2 transgenic mouse.(PNAS,2006). At 12 mos of age all AGEs attributable to ASA (pentosidine, CML, K2P, vesperlysine A and total fluorescence) were highly significantly increased, and the lenses were yellow like those from a 70 yrs old human lens. We now further determined other modification and crystallin aggregation both in vitro and in vivo by ascorbic acid oxidation.
Methods: :
Mouse lens protein extract and recombinant mouse gamma D crystallin were incubated with ASA (3 mM) or dehydroascorbic acid (100uM) under anaerobic condition and analyzed by either LC/MS/MS tandem mass sequencing or LC/MS of protease digest for protein modifications. A 1:1 ratio of C12 and universal labeled C13 labeled ASA were used for unknown modification identification. The protein turbidity was measured spectrophotometrically at 550nm. For in vivo study, young and old age hSVCT2 transgenic mouse lenses protein were extracted. The modifications were quantitively determined by LC/MS. Protein aggregation was determined by light scattering measurement.
Results: :
NΔ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1), the most abundant AGEs in human lens was identified in mouse lens protein extract following incubation with ASA. R10, R95, R147 of gamma D crystallin; R95, R163, R180 of gamma F crystallin and R186 of alpha A crystallin formed methylglyoxal hydroimidazolones (MG-H1). MG-H1 was also found significantly elevated in transgenic hSVCT2 mouse lens both with LC/MS and western-blot using MG-H1 antibody. 3-deoxythreosone arginine adduct was also found in gamma D, F and alpha A crystallin by in vitro incubation. ASA caused gamma D crystallin aggregation during in vitro incubation in less than 36 hours. Aggregation could be significantly blocked by co-incubation with either DTT or GSH, indicating that disulfide formation may play an important role in gamma D crystallin precipitation or aggregation. QLS analysis of transgenic mouse lens protein extract showed that proteins eluting in the HMW fraction of the Superose column form aggregates about 4x larger in size than those normally present in WT mouse, providing support to our hypothesis that protein ascorbylation enhances aggregation in vivo.
Conclusions: :
These results show that ASA oxidation and modification unequivocally proceed in the aging lens, resulting in crosslinking and aggregation.
Keywords: crystallins • oxidation/oxidative or free radical damage • aging