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VM Monnier, X Wu; Glycated Proteins Become Substrates of Amadoriase Enzyme Upon Amidation of Carboxylate: Role of Charge on the Substrate Specificity of Amadoriase . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2384.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Glycation of lens crystallins increase their susceptibility toward oxidation and crosslinking during aging. Glycated crystallins also have impaired chaperone activity. When combined these processes may greatly increase the predisposition of the lens toward cataractogenesis. To counter the effects of excessive glycation, we are developing genetic and pharmacological strategies, among which amadoriase enzymes that can reverse the glycation process. Amadoriase is a deglycation enzyme that oxidizes glycated substrate and generates glucosone, H2O2 and free amine group. However, amadoriase so far only recognizes small molecules as substrates and cannot deglycate large protein such as glycated BSA. We report the unexpected finding that glycated proteins and lens crystallins can become substrates of amadoriase upon amidation of the carboxylate residues. Method: Glycated BSA as model protein and bovine α-crystallins were amidated by reacting the protein with NH4Cl using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as coupling reagent. Deglycation activity was measured in terms of H2O2 release using amplex red and horseradish peroxidase. Result: Incubation of the substrate with recombinant Amadoriase I from Aspergillus sp. led to progressive formation of H2O2 and release of glucosone with no detectable activity against non-amidated glycated BSA or crystallins. The extent of deglycation of lysine residues (furosine method) confirmed that around 10% of the glycation sites on the protein were deglycated. Further studies with affinity purified albumin from diabetic patient serum revealed in vivo glycated proteins can also become substrates of amadoriase as a function of degree of modification. CD spectra of BSA, glycated BSA and amidated glycated BSA showed no major conformational changes after amidation. Negative ions such as PO4-3 and SO4-2 also inhibited the activity, while esterifying glycated BSA, which also removes negative charges, restored the activity. The sites of deglycation on albumin and crystallins are under investigation by mass spectrometry. Conclusion: These results suggest not only steric hindrance, but electrostatic repulsion between the negative charges of the enzyme-protein substrate complex can selectively affect substrate accessibility. This discovery provides clues for the future engineering of amadoriases with the new activity towards glycated protein. By extrapolation, these results suggest the age-related deamidation of lens crystallins may alter the behavior of lens crystallins as substrate toward certain enzymes.
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