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V.M. Monnier, Z. Dai, X. Wu; Mapping of Enzymatic Deglycation Sites in Amidated Glycated RNAse A Using Ion Trap Mass Spectrometry . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2352.
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Purpose: Lens crystallins are modified by glycation with age and acquire yellow chromophores and cross-links that predispose them toward aggregation. Examples include carboxymethyllysine, pentosidine and argpyrimidine. Recently, glucosepane has been identified as a major Amadori-derived crosslink in crystallins from old human lenses (Biemel et al., JBC 2002). In order to reverse protein glycation, we are developing fungal Amadoriase enzymes. Although these enzymes are inactive against native glycated proteins, we found activity could be obtained against glycated histones, glycated amided lens crystallins and other proteins. Using glycated amidated RNAse A as a model protein of known crystal structure, we have now mapped its enzymatic deglycation sites in relationship to the geographic distribution of its negative charges on the molecule. Methods: WT RNAse A was amidated, and glycated in 1M glucose : 13C6-glucose (1:1) for 7 days. RNAse A mutants D38N, E86Q, D38N/E86Q were generated and glycated. The glycated proteins were incubated with recombinant Amadoriase I for 120 min. at 37 °C, dialyzed, and digested with trypsin or chymotrypsin. Peptide mapping by HPLC was performed with an LCQ ESI ion-trap instrument in the positive mode. Results: All 10 lysine residues of RNAseA were glycated. Glycated peptides t1-t11 were typically recognized as [Mi +162] with double peaks (Δm/z = 6u). Six chymotryptic peptides (c1-c6) were obtained. Quantitation of individual peptides following deglycation showed a decrease in t2, t3, and t4 suggesting deglycation of K1, K7 and K91. Peptides c2 and c6 were also decreased confirming K1, K7 and K91 as major deglycation sites, although no clear distinction between K1 and K7 could be made. Surface area calculations for solvent exposure of K residues (EMBL Heidelberg website) showed K1 and K91 were the most exposed lysines in ribonuclease. Surprisingly, mutagenesis of adjacent negative charges at sites 38, 86, and both, into the amides N and Q respectively, did not result in deglycation of native glycated protein. Conclusions: Fungal amadoriase enzymes are able to deglycate highly positively charged proteins, such as histones, but not lower pI proteins. Amidation of negative charges on the substrate helps overcome electrostatic inhibition and leads to deglycation of the most exposed K residues. However, mutation of negative into neutral charges at the vicinity of these residues is not sufficient for deglycation to occur, suggesting presence of complex allosteric effects of negative charges on protein-enzyme interaction. CR : None Support: NEI/NIH EY 07099 and P30EY11373
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