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Jianye Zhang, Benlian Wang, Xingjun Fan, Xiaoqin Liu, Vincent M. Monnier; Identification Of The Glucose-derived Modification And Cross-link Sites In Alpha A Crystallin. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4746.
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Aging lens proteins accumulate two kinds of glucose-derived cross-links forming between lysine and arginine residues in lens crystallins, glucosepane and 3-deoxyglucosone-derived imidazolium cross-link (DODIC). These cross-links may impair the chaperone functions of alpha crystallins and thus favor aggregation of lens proteins and cataract formation. Identification of the cross-link sites in crystallins should help understand the interaction among lens proteins and the process of diabetic cataracto-genesis.
Recombinant αA crystallin was incubated in high concentration of glucose (500mM) under metal free condition for 4weeks, then digested by trypsin and analyzed by MALDI-TOF and LC-MS/MS. The resulting data were analyzed by Mascot and xQuest.
: all the seven lysine (K11, K70, K78, K88, K99, K145, K163) residues in αA crystallin were observed to be modified by glucose to form the Amadori products (Δm/z = 162). The intra-peptide cross-links of both glucosepane and DODIC were observed at K166-R163. The inter-peptides cross-link of glucosepane was observed at K166-R103 and K166-R117, while DODIC was only observed at K166-R103. Considering the structure of the heat shock proteins, these inter-peptides cross-links maybe also represent inter-molecular cross-links. K166 was also found to be modified by methylglyoxal(MGO) to form carboxyethyl lysine (CEL), and by 3-deoxyglucosone (Δm/z = 144) the precursor of DODIC. Meanwhile, methylglyoxal derived hydro-imidazolone (MGH1) was observed at R12, R103, R157, R163, and 3-deoxyglucosone-derived hydroimidazolone (3-DGH) was observed at R12, R65, R103, R112, R157, R163. No Glyoxal derived products could be observed.
The K166 is the most reactive lysine in αA crystallin, which probably plays an important role in the αA crystallin oligomer structure. The glycation at K166 facilitates the cross-linking and aggregation of αA crystallin. Under the metal free condition, the degradation of glucose favors the formation of MGO rather than that of glyoxal.
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