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Yasir J. Sepah, Ronald Copeland, Roomasa Channa, Junfeng Ma, Afsheen Khwaja, Elham Hatef, Mohamed Ibrahim, Diana V. Do, Gerald W. Hart, Quan D. Nguyen; The Potential Role of O-GlcNAcylation as Biomarker in Diabetic Retinopathy and Diabetic Macular Edema. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4457.
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O-linked b-N-acetylglucosamine (O-GlcNAc) is a post-translational modification consisting of addition of a single GlcNAc moiety to serine and/or threonine residues of numerous nuclear and cytoplasmic proteins. Recent data supports the hypothesis that a hyperglycemia-induced increase in O-GlcNAcylation underlies insulin resistance and glucose toxicity. O-GlcNAc has not been established on secreted proteins. Presence of such proteins in the ocular fluid (OF) of eyes with DR/DME has also not been investigated. We aim to establish the presence of protein O-GlcNAcylation in OF of patients with diabetic retinopathy (DR) and diabetic macular edema (DME).
Anterior chamber (AC) fluid was collected from one eye of each of 25 subjects with DME and non-proliferative or inactive proliferative DR who are participating in a clinical trial for DME. The eyes have not been treated with anti-VEGF/steroid therapies for at least 3 months prior to sample collection. AC fluids samples were stored at -80C°. Proteins contained in AC fluid (approx. 10 µl) was subjected to SDS-PAGE analysis and transferred onto polyvinylidenefluroride (PVDF) membranes. Membranes were blocked in Tris-buffer saline with Tween (0.1%, [v/v] Tween 20) containing 3% BSA followed by overnight blotting with O-GlcNAc specific antibody. To test for specificity, the O-GlcNAc antibody was preincubated with free GlcNAc for 1h prior incubation with PVDF membrane. Detection was performed by enhanced chemiluminescence.
Immunoblotting demonstrated that at least 4 protein bands were reactive to an O-GlcNAc specific antibody in all 25 samples. Immunoreactivity of the antibody could be competed away with GlcNAc, suggesting that proteins contained within the AC of eyes with DR and DME are O-GlcNAcylated. The identity of O-GlcNAcylated proteins is being confirmed using mass spectrometry.
The index study has shown the presence of O-GlcNAcylated proteins in AC fluid of eyes with DR and DME, which has not been illustrated previously. Results from our novel study suggest that O-GlcNAcylation may be a "sensor" that could be employed to predict the various clinical stages and therapeutic responses of DR and DME.
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