June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Molecular Basis for Lens Crystallin Precipitation by Vitamin C Glycation and Oxidation Products in Age-Related Cataracts
Author Affiliations & Notes
  • Grant Hom
    Biology, Fairview High School, Cleveland, OH
    Pathology, Case Western Reserve University, Cleveland, OH
  • Xingjun Fan
    Pathology, Case Western Reserve University, Cleveland, OH
  • Benlian Wang
    Center for Proteomics, Case Western Reserve University, Cleveland, OH
  • Vincent M Monnier
    Pathology, Case Western Reserve University, Cleveland, OH
    Biochemistry, Case Western Reserve University, Cleveland, OH
  • Footnotes
    Commercial Relationships Grant Hom, None; Xingjun Fan, None; Benlian Wang, None; Vincent Monnier, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 5882. doi:
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      Grant Hom, Xingjun Fan, Benlian Wang, Vincent M Monnier; Molecular Basis for Lens Crystallin Precipitation by Vitamin C Glycation and Oxidation Products in Age-Related Cataracts. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5882.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Research by Ortwerth and colleagues (1988) showed that incubation of bovine lens protein fractions with vitamin C, or dehydroascorbic acid itself, was associated with advanced glycation (AGE)/ascorbylation product formation, protein aggregation, crosslinking, precipitation/insolubilization and opacification as observed in age-related human cataracts. The goal of this research was to test the hypothesis that the reported protein precipitation was associated with selective modification of aminoacid residues in precipitated but not soluble proteins.

Methods: To test this hypothesis, calf lens crystallin homogenate was separated into α-, βH-,βL- and γ-crystallin rich fractions by Sephadex G-200 gel filtration chromatography, and fractions were incubated with 20 mM ascorbic acid until precipitation occurred (3-7 days). Proteomic analysis with mass spectrometry was used to reveal and identify protein modification sites that were selectively present in precipitated but not soluble proteins, focusing on the AGE hydroimidazolones from xylosone (XYH), methylglyoxal (MG-H1), CML, CEL, and tryptophan oxidation to N-formyl kynurenine (NFK) and kynurenine(KYN).

Results: Formation of AGEs and oxidation was observed in both precipitated and soluble crystallins. However, modification of selective lysine (K) and arginine (R) residues, and oxidation of embedded tryptophan (W) residues occurred in precipitated but not soluble fractions rich in CRYBB1, CRYBB2, CRYBA4 and CRYBA1 crystallins: Major sites include CRYBB1 (K61CML, K119CML, R93XYH, W102NFK), CRYBB2(K68CML, R98XYH, K101CML, K168CML) CRYBA1 (K15CML, K114CML, R78XYH, R194XYH,W79NFK, W181NFK) and CRYBA4 (W31, R39, R40, R59XYH, K62CML). Surprisingly, gamma crystallins (CRYGS,B,C, and Z ) were in both soluble and precipitated form and partially oxidized at W46 and W137 (CRYGS), but no AGE formation sites were detected and no relationship with precipitation was found.

Conclusions: This study is the first to provide evidence of specific modification sites by glycation and oxidation in relationship to protein precipitation, whereby beta-crystallins emerge as major targets and constituents in the precipitate. Molecular modeling studies are in progress to clarify whether ascorbylation favors protein unfolding and exposure of the protected tryptophan residues

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