September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Identification of novel amino acid modifications in human lens proteins: mechanisms and relevance.
Author Affiliations & Notes
  • Stefan Rakete
    Ophthalmology, UC Denver, Aurora, Colorado, United States
  • Ram H Nagaraj
    Ophthalmology, UC Denver, Aurora, Colorado, United States
  • Footnotes
    Commercial Relationships   Stefan Rakete, None; Ram Nagaraj, None
  • Footnotes
    Support  NH Grant EY022061 & EY023286
Investigative Ophthalmology & Visual Science September 2016, Vol.57, No Pagination Specified. doi:
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      Stefan Rakete, Ram H Nagaraj; Identification of novel amino acid modifications in human lens proteins: mechanisms and relevance.. Invest. Ophthalmol. Vis. Sci. 201657(12):.

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

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Abstract

Purpose : The formation of cataracts is accelerated by post-translational modifications of amino acids in lens proteins. We showed that erythrulose and kynoxazine were able to modify and crosslink proteins. Those compounds are directly related to the degradation of ascorbate in the human lens. The investigation of the underlying pathways in vivo may help to understand how ascorbate degradation influences protein modification and therefore cataract formation.

Methods : The 3-deoxythreosone-arginine-derived hydroimidazolinone and the respective lysine-arginine-hydroimidazolinone-crosslink were isolated from erythrulose/boc-lysine/boc-arginine incubations and characterized by NMR and mass spectrometry. Purified recombinant αB-crystallin was incubated under different conditions and analyzed by SDS-PAGE. Lens proteins were hydrolyzed by enzymatic digestion and analyzed by UPLC-MS2. α–Dicarbonyls were trapped as stable quinoxalines and analyzed by UPLC-MS2.

Results : αB-Crystallin was modified and crosslinked by kynoxazine and erythrulose. The crosslinking of αB-crystallin could be prevented by the addition of aminoguanidine, a α–dicarbonyl quencher. Furthermore, blockage of all lysine residues in αB-crystallin also prevented crosslinking. We found that kynoxazine and erythrulose generate substantial amounts of 3-deoxythreosone at physiological conditions. Based on that, a 3-deoxythreosone-derived arginine modification and a 3-deoxythreosone-derived arginine-lysine crosslink were isolated, characterized and used for UPLC-MS2 analysis of enzymatically digested lens proteins. We were able to detect both modifications in lens proteins. The levels of the 3-deoxythreosone-derived arginine modification correlated positively with the age of the lenses.

Conclusions : αB-Crystallin is modified and crosslinked by kynoxazine and erythrulose via 3-deoxythreosone, which reacted with arginine and lysine residues. Those 3-deoxythreosone-derived amino acid modifications were identified in human lens proteins for the first time. They are likely to alter the solubility of lens proteins, cause protein aggregation and therefore promote cataract formation.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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