June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
THE RELATIONSHIP BETWEEN OXIDATION, DISULFIDE FORMATION AND AGGREGATION OF HUMAN GAMMA D CRYSTALLIN
Author Affiliations & Notes
  • Ramkumar Srinivasagan
    Pathology, Case Western Reserve University, Cleveland, Ohio, United States
  • Benlian Wang
    Center for Proteiomics and Bioinformatics, Case Western Reserve University, Cleveland, Ohio, United States
  • Xingjun Fan
    Pathology, Case Western Reserve University, Cleveland, Ohio, United States
  • Vincent M Monnier
    Pathology, Case Western Reserve University, Cleveland, Ohio, United States
  • Footnotes
    Commercial Relationships   Ramkumar Srinivasagan, None; Benlian Wang, None; Xingjun Fan, None; Vincent Monnier, None
  • Footnotes
    Support  NONE
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 5309. doi:
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      Ramkumar Srinivasagan, Benlian Wang, Xingjun Fan, Vincent M Monnier; THE RELATIONSHIP BETWEEN OXIDATION, DISULFIDE FORMATION AND AGGREGATION OF HUMAN GAMMA D CRYSTALLIN. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5309.

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

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Abstract

Purpose : Gamma crystallins are highly conserved sulfhydryl rich proteins which play a critical structural role in the lens nucleus where they are present as the single major protein. Recent work from our laboratory lead to discovery of dimers and multimers involving gamma C, D, S with themselves and other cystallins. Major disulfide formation sites were identified at C23, 42, 79/80, 109 and 154 in γC and C19, 33 and 42 in γD. In order to understand the role of oxidation on the stability of γ- crystallins we oxidized recombinant γD and determined molecular changes

Methods : Recombinant wild type (WT) and cysteine mutant of γD-crystallin were oxidized with diamide or Cu2+ and molecular changes were determined using Western blotting and LC-MS. The structural stability of cysteine mutants were determined by near- and far UV CD, Trp fluorescence, solubility by turbidity assay and unfolding by GnHCl

Results : Oxidation with the cysteine specific diamide showed WT protein formed dimers at C19-111, C109-111 and C111-111. Only C111A mutant but no other mutant (C19, 33, 42, 72) suppressed dimerization by diamide. Co-incubation with αB crystallin did not protect WT C111 oxidation. No changes in structural and stability properties (Far- and near UV CD, Trp fluorescence, unfolding) of the C111 mutant vs WT were noted. Similarly to Quintanar et al., (Quintanar et al (ACS Chem Biol.; 11: 263, 2016) we found Cu2+ induced 80% WT protein precipitation and dimerization. However C111A mutant suppressed precipitation. Iodoacetamide treated WT also suppressed oxidation and precipitation.

Conclusions : Cysteine 111 plays a major role in dictating the propensity of Human γD-crystallin to dimerize and precipitates. Once C111 is mutated, Cu2+ oxidation induces other cysteines to form disulfides but precipitation is subdued. More research is needed to understand why C111 is not oxidized in vivo and why the in vivo sites 19, 33, and 42 were not detected in vitro under mild oxidation.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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