Changes in MHC-II/peptide binding affinity following protein glycation, as observed in Diabetes

Abstract

Abstract Increased oxidized ribose, glucose, glyoxal and methylglyoxal concentrations are observed in several metabolic diseases including diabetes. These highly reactive moieties bind to the cellular proteome to form advanced glycation end-products (AGEs), which can alter the structure and function of targeted proteins. To address whether the AGE-mediated modification of immunologically relevant proteins could have functional consequences we incubated soluble recombinant HLA- DR1 (HLA-DRA*01:01/DRB1*01:01) in presence of glucose at concentration commonly observed in diabetic subjects (15–20 mM). Analysis of the AGE adducts was achieved by using bottom-up proteomics and tandem mass spectrometry analysis. Major observed modifications were glyoxal-derived hydroimiadazolone, argpyrimidine, dihydroxy and methylglyoxal adducts on arginine residues, pyralline and glyceraldehyde-derived pyridinium compound adducts of lysine and carboxy ethylation and methylation of both arginine and lysine residues. These AGE-related modifications were comparable to what observed on MHCII, immunoprecipitated from the PBMC of Diabetic patients. AGE PTMs mapped to both the binding groove and HLA-DM binding domains. AGE-modified and unmodified HLA-DR1 were then assessed for their binding affinity of several relevant peptides in a fluorescence polarization assay in the presence or absence of HLA-DM. Changes in binding affinity for the selected peptides was observed in AGE-HLA-DR1 as compared to the unmodified HLA-DR1. Altogether our data indicate that protein PTM, as commonly observed in conditions of metabolic and oxidative stress can change HLA-DR1/peptide binding affinity thus compromising MHC-II restricted immune responses.