Extracellular molecular interactions in celiac disease
- This dissertation is aimed to explore two critical extracellular interactions in the pathogenesis of celiac disease: thioredoxin mediated extracellular tissue transglutaminase (TG2) activation and binding interaction between gluten peptides and HLA-DQ2. The mechanism of activation of TG2 in the extracellular matrix remained a fundamental mystery in biology as well as in celiac disease pathogenesis. Earlier investigations have highlighted the role of a disulfide bond formed by vicinal Cys residues in maintaining calcium-bound TG2 in an inactive state. We characterized the redox potential of this disulfide bond to be approximately -190 mV, which is very oxidative for a disulfide bond in proteins. We then identified a physiological factor human thioredoxin (Trx) for the activation of oxidized TG2. With a kcat/KM of 1.6 [mu]m-1min-1. Trx was a highly specific activator of oxidized human TG2. Both recombinant Trx and interferon-[gamma] stimulated Trx released from monocytes can activate extracellular TG2 around the fibroblasts. In addition to explaining how TG2 can be activated by dietary gluten in the small intestinal mucosa of celiac sprue patients, our findings reveal a new strategy for inhibiting the undesirable consequences of TG2 activity in this widespread and lifelong disease. Inspired by the finding of Trx mediated TG2 activation, we then sought to develop small molecule disulfide inhibitors targeting extracellular Trx. We performed a careful structural-activity relationship study based on an imidazolyl isobutyl disulfide compound. Two rounds of inhibitor screening suggested that aromatic ring part mainly contributes to ligand binding, and a new lead compound was identified with ~26 fold increased inhibition selectivity. Potential inhibitor binding site was also predicted near Cys73 by docking model and Trx mutant results. Our findings provide novel tools to study Trx-TG2 interaction in celiac disease as well as a promising therapeutic candidate. Understanding the binding interaction between HLA-DQ2 and antigenic peptides lies at the center of antigen presentation in celiac disease. We use capillary electrophoresis-fluorescence detection to investigate competition and displacement of endogenous peptides by an immunogenic gluten peptide (the 20-mer) for binding to HLA-DQ2. Remarkably, this immunogenic peptide could displace CLIP peptides from DQ2 binding site under neutral but not acidic pH condition. This unique property of gluten peptide supports a direct loading mechanism of antigen presentation in the extracellular environment.
|Type of resource
|electronic; electronic resource; remote
|1 online resource.
|Jin, Xi, active 2012
|Stanford University, Department of Chemistry
|Khosla, Chaitan, 1964-
|Khosla, Chaitan, 1964-
|Statement of responsibility
|Submitted to the Department of Chemistry.
|Thesis (Ph.D.)--Stanford University, 2012.
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