April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Identification Of The Glucose-derived Modification And Cross-link Sites In Alpha A Crystallin
Author Affiliations & Notes
  • Jianye Zhang
    Pathology,
    Case Western Reserve University, cleveland, Ohio
  • Benlian Wang
    Center for proteomics and Bioinformatics,
    Case Western Reserve University, cleveland, Ohio
  • Xingjun Fan
    Pathology, Case Western Reserve Univ, Cleveland, Ohio
  • Xiaoqin Liu
    Pathology,
    Case Western Reserve University, cleveland, Ohio
  • Vincent M. Monnier
    Pathology,
    Case Western Reserve University, cleveland, Ohio
  • Footnotes
    Commercial Relationships  Jianye Zhang, None; Benlian Wang, None; Xingjun Fan, None; Xiaoqin Liu, None; Vincent M. Monnier, None
  • Footnotes
    Support  EY07099, VSCR P30EY-11373
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 4746. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : 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.

Methods: : 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.

Results: : : 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.

Conclusions: : 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.

Keywords: protein modifications-post translational • diabetes • proteomics 
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