Effect of a single AGE modification on the structure and chaperone activity of human alphaB-crystallin.

During aging, human lens proteins undergo several post-translational modifications, one of which is glycation. This process leads to the formation of advanced glycation end products (AGEs) which accumulate with time possibly leading to the formation of cataract. alphaB-Crystallin, a predominant protein in the lens, is a member of the small heat shock proteins (sHSPs) which are a ubiquitous class of molecular chaperones that interact with partially denatured proteins to prevent aggregation. This chaperone function is considered to be vital for the maintenance of lens transparency and in the prevention of cataract. In the present study, we introduced an analog of the advanced glycation end product, OP-lysine, at the 90th position of a mutated human alphaB-crystallin (K90C) by covalent modification of the cysteine residue with N-(2-bromoethyl)-3-oxidopyridinium hydrobromide. The AGE-modified K90C-alphaB-crystallin is termed as K90C-OP. We compared the structural and functional properties of K90C-OP with the original K90C mutant, with K90C chemically modified back to a lysine analog (K90C-AE), and with wild-type human alphaB-crystallin. Modified K90C-OP showed decreased intrinsic tryptophan fluorescence and bis-ANS binding without significant alterations in either the secondary, tertiary, or quaternary structure. K90C-OP, however, exhibited a reduced efficiency in the chaperoning ability with alcohol dehydrogenase, insulin, and citrate synthase as substrates compared to the other alpha-crystallin proteins. Therefore, introduction of a single AGE near the chaperone site of human alphaB-crystallin can alter the chaperoning ability of the protein with only minor changes in the local environment of the protein.

Influence of glutathione fructosylation on its properties.

Incubation of fructose and glutathione leads to the formation of N-2-deoxy-glucos-2-yl glutathione as the major glycation product, with characteristic positive ion at 470 Th in LC-MS spectra. Glutathione disulfide and fructose generate two compounds: N-2-deoxy-glucos-2-yl glutathione disulfide (m/z=775 Th) and bis di-N,N'-2-deoxy-glucos-2-yl glutathione disulfide (m/z=937 Th). N-2-deoxy-glucos-2-yl glutathione is 2.5-fold less effective than glutathione in reducing dehydroascorbic acid. Glutathione peroxidase and glutahione-S-transferase exhibit marginal activity toward N-2-deoxy-glucos-2-yl glutathione, while glyoxalase I shows 44.9% of the enzyme's specific activity. Glutathione reductase demonstrates 6.9% of the enzyme's specific activity with bis di-N,N'-2-deoxy-glucos-2-yl glutathione, while with mono-N-glucosyl glutathione disulfide retained 5 6.1% of the original activity. Glutathione reductase could not reduce N-2-deoxy-glucos-2-yl glutathione in mixed disulfide with gammaS-crystallin, but reduced glutathione in mixed disulfide with gammaS-crystallin by 90%. The presence of N-2-deoxy-glucos-2-yl glutathione in mixed disulfide with gammaS-crystallin makes this molecule more susceptible to unfolding than native gammaS-crystallin.

Glucose-derived Amadori compounds of glutathione.

Under the chromatographic conditions used in these studies we observed time- and concentration-dependent formation of N-1-Deoxy-fructos-1-yl glutathione as the major glycation product formed in the mixtures of GSH with glucose. N-1-Deoxy-fructos-1-yl glutathione had a characteristic positively charged ion with m/z=470 Th in its LC-MS spectra. Mixtures of glutathione disulfide and glucose generated two compounds: N-1-Deoxy-fructos-1-yl GSSG (m/z=775 Th) as major adduct and bis di-N, N'-1-Deoxy-fructos-1-yl GSSG (m/z=937 Th) as the minor one. All three compounds showed a resonance signal at 55.2 ppm in the 13C-NMR spectra as C1 methylene group of deoxyfructosyl, which represents direct evidence that they are Amadori compounds. All three compounds purified from GSSG/Glc or GSH/Glc mixtures also showed LC-MS/MS fragmentation patterns identical to those of the synthetically synthesized N-1-Deoxy-fructos-1-yl glutathione, N-1-Deoxy-fructos-1-yl GSSG and bis di-N, N'-1-Deoxy-fructos-1-yl GSSG. N-1-Deoxy-fructos-1-yl glutathione was shown to be a poor substrate for glutathione peroxidase (6.7% of the enzyme's original specific activity) and glutathione-S-transferase (25.7% of the original enzyme's specific activity). Glutathione reductase failed to recycle the disulfide bond within the structure of di-substituted bis di-N, N'-1-Deoxy-fructos-1-yl GSSG. It showed only 1% of the original enzyme's specific activity, but retained its ability to reduce the disulfide bond within the structure of N-1-Deoxy-fructos-1-yl GSSG by 57% of its original specific activity. Since the GSH concentration in diabetic lens is significantly decreased and the glucose concentration can increase 10-fold and higher, the formation of Amadori products of the different forms of glutathione with this monosaccharide may be favored under these conditions and could contribute to a lowering of glutathione levels and an increase of oxidative stress observed in diabetic lens.