Abstract
Purpose: :
To investigate whether tyrosine sulfation, a post-translational modification that improves bioactivity and ability of proteins to interact with other proteins, is altered by high glucose (HG) in retinal vascular cells. Basement membrane proteins such as fibronectin, and collagen IV, and lysyl oxidase, a cross-linking enzyme, are sulfated proteins known to exhibit altered expression and activity in diabetes. However, how HG compromises protein functionality is currently not well understood. In this study, we examined if HG modulates tyrosine sulfation levels in protein isolated from retinal endothelial cells and pericytes.
Methods: :
Rat retinal endothelial cells (RRECs) or bovine retinal pericytes (BRPs) were grown in normal (N; 5mM) or HG medium (30 mM) for 7 days. Total protein isolated from the cells was analyzed for tyrosine sulfation using Western blot (WB) analysis performed with PSG2 antibody, which recognizes sulfated tyrosines.
Results: :
WB analysis indicated that tyrosine sulfation level was significantly altered in proteins isolated from RRECs or BRPs grown in HG medium compared to those grown in N medium. RRECs grown in HG medium exhibited mostly increased tyrosine sulfation ranging from 1.1 to 13.0 fold in nine distinct protein bands (~23 kD to 250 kD). In BRPs, nine protein bands that appear similar to those with altered sulfation in the endothelial cells were detected. Interestingly, tyrosine sulfation level was reduced (2-4 fold) in the BRP protein bands grown in HG medium compared to those grown in N medium.
Conclusions: :
Findings from this study indicate that HG exposure alters either the sulfation pattern or steady state levels of specific proteins in retinal vascular cells. Importantly, the observed differential effects of HG on sulfation appear cell specific. HG-induced altered tyrosine sulfation may compromise protein interactions and/or function and contribute to the pathogenesis of diabetic retinopathy.
Keywords: diabetic retinopathy • extracellular matrix • protein modifications-post translational