May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
Glycation–Induced Aggregation of Lens Proteins by the Oxidation Products of Ascorbic Acid
Author Affiliations & Notes
  • B.J. Ortwerth
    Mason Eye Inst East, University of Missouri, Columbia, MO
  • R. Cheng
    Mason Eye Inst East, University of Missouri, Columbia, MO
  • B. Lin
    Mason Eye Inst East, University of Missouri, Columbia, MO
  • Q. Feng
    Mason Eye Inst East, University of Missouri, Columbia, MO
  • Footnotes
    Commercial Relationships  B.J. Ortwerth, None; R. Cheng, None; B. Lin, None; Q. Feng, None.
  • Footnotes
    Support  NIH Grant EY07070, RPB
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 2887. doi:
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    • Get Citation

      B.J. Ortwerth, R. Cheng, B. Lin, Q. Feng; Glycation–Induced Aggregation of Lens Proteins by the Oxidation Products of Ascorbic Acid . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2887.

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

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Abstract

Abstract: : Purpose: To determine whether the glycation of calf lens proteins in vitro by the oxidation products of ascorbic acid is able to cause protein aggregation. Methods: Dialyzed water–soluble (WS) proteins from fetal calf lenses were incubated with 20 mM ascorbic acid under air for 4 weeks with aliquots removed at weekly intervals. Each aliquot was analyzed by Superose–6 gel filtration chromatography, monitoring for protein at A280nm and for advanced glycation endproducts (AGEs) at A330nm. Similar experiments were carried out with 1.0 mM ascorbate in the presence and absence of 100 mM cyanoborohydride, 2–aminoguanidine or semicarbizide, all of which inhibit AGE formation. The incorporation of ascorbate into aggregated proteins was also determined following the incubation of 10 mg/mL calf lens proteins with 20 mM[U–14C]ascorbate (0.24 mCi/mmole). These data were compared to the Superose–6 chromatographic profile of the WS proteins from aged human lenses. Results: The aggregation of calf lens proteins was observed after only one week of incubation with 20 mM ascorbate in air. Aggregate peaks eluted prior to α–crystallin (∼1–2 x 106 Da), which may represent chaperone complexes, and in the void volume of the Superose–6 column as a high molecular weight (HMW) peak of 4 x 107 Da or larger. Both aggregate peaks increased proportionately over the next 3 weeks with high levels of yellow compounds associated with each peak. No aggregation or yellowing was seen in protein samples incubated without ascorbate. Incubations with 1.0 mM ascorbate also showed extensive protein aggregation. A similar HMW peak was seen when the WS proteins from aged human lens were separated. Glycation was implicated by the incorporation of radioactive ascorbate into each of the aggregate peaks. Further, the addition of cyanoborohydride or other inhibitors of AGE formation blocked protein aggregation. Absorption spectra of the glycated proteins showed a correlation between the extent of yellowing and aggregate formation. Conclusions: Protein aggregation increases during nuclear cataract formation until the aggregate size is large enough to scatter light (cataract). Increasing evidence argues that AGE formation due to the oxidation products of ascorbic acid is a prominent feature in aged and cataractous lenses. We show here that ascorbate glycation alone can perturb the structure of fetal calf lens proteins sufficiently to cause them to aggregate into very high molecular weight complexes similar to those seen in aged human lenses.

Keywords: protein modifications-post translational • cataract • protein structure/function 
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