Abstract
Purpose: :
Based on the evidence of oxidative stress and oxidative protein modifications during glaucomatous neurodegeneration, this study aimed to analyze the glaucomatous patient sera for protein oxidation and immunoreactivity to oxidized proteins.
Methods: :
Serum samples were collected from 22 patients with glaucoma and 18 age-matched control subjects without glaucoma. Oxidized proteins containing reactive carbonyl groups were measured in serum samples by a specific ELISA. In addition, 2D-PAGE and oxyblot analyses followed by LC-MS/MS were performed using the patient sera to validate and identify oxidized proteins. Since circulating antibodies reflect a molecular imprint of disease-related antigens, the second part of the study aimed to determine serum immunoreactivity to oxidized proteins. Therefore, in vitro experiments were conducted using primary cultures of retina and optic nerve head cells incubated in the presence and absence of oxidants to compare the immunoreactivity of patient sera against oxidized versus control proteins. In vitro data were then correlated with immunoreactivities of the same serum samples against human retinal proteins obtained from glaucomatous and non-glaucomatous donors, as well as with oxidized proteins revealed by the oxyblot analysis of human retinal proteins.
Results: :
Comparison of serum samples from glaucomatous and non-glaucomatous subjects exhibited a significant increase (p<0.01) in protein carbonyls in glaucoma, which reflect oxidatively modified proteins. Findings of in vitro experiments provided supportive evidence that the immunoreactivity of the glaucomatous patient sera against oxidatively stressed retinal cell culture proteins was over two-fold greater than the immunoreactivity of the same sera to control proteins. Proteomic analyses validated that many of the immunoreactivies detected in glaucomatous serum samples against glaucomatous retinal proteins (as opposed to control proteins) corresponded to oxidized proteins identified in the glaucomatous retina and serum samples.
Conclusions: :
Importance of these findings is two-fold. First, oxidized proteins exhibiting elevated titers in the glaucomatous patient sera may represent a subset of (stressed) tissue-specific antigens in glaucoma and serve as disease markers in glaucoma patients. Second, by changing the antigenic features of proteins, oxidative modifications during glaucomatous neurodegeneration may be an immunostimulatory signal that induces increased autoantibody production and contributes to dysregulation of immune response.
Keywords: oxidation/oxidative or free radical damage • immunomodulation/immunoregulation • proteomics