Celiac disease (CD) is a severe pathological condition of the small intestine caused by CD4+ T cell-mediated inflammatory response to dietary gluten. CD is strongly associated with HLA DQ2/DQ8 genetic background and can affect 1% of the European and North American population. Gluten proteins are complex aggregates of two subunits called gliadins and glutenins. Among the gluten proteins, those which are involved in eliciting the inflammatory response are important candidates for studying the pathogenic mechanism, but the identity and complete profile of disease causing antigens is not available.
Therefore, the aims of my thesis were the identification, cloning, expression, biochemical and immunological characterization of CD-specific wheat antigens. We therefore, developed a combined proteomic and immunological approach using CD patient sera for identifying wheat antigens involved in CD. We identified [gamma]-gliadins with a molecular mass of 37 kDa to be highly CD-specific and the identity of the 37 kDa [gamma]-gliadin protein was deduced by mass spectrometry. The [gamma]-gliadin 1 (GG1) gene was cloned, expressed and purified as a recombinant antigen in Escherichia coli. Biochemical characterization was performed with the recombinant [gamma]-gliadin 1 (rGG1), which revealed that rGG1 was insoluble in physiological solutions, unfolded and formed high molecular weight aggregates. Immunological characterization was performed by studying the IgA reactivity of CD and control patients to rGG1. The rGG1 protein was a recognized by 73% of CD patients, with a CD-specificity of up to 93%. Thus rGG1 was a major CD-specific IgA-reactive wheat antigen. The protein was also specifically recognized by the majority of Dermatitis herpetiformis (DH) patients and therefore could serve as a candidate marker for DH diagnosis. ^The specificity of rGG1 with CD condition was further demonstrated by showing that treated CD patients lost their reactivity to rGG1 when they were put on a gluten-free diet (GFD). Thus rGG1 could be used for monitoring the adherence of CD patients to GFD. Further, we used synthetic overlapping peptides of rGG1 and identified that the N-terminal proline/glutamine-rich region was highly IgA reactive, among which two peptides were the major immunodominant epitopes. We also demonstrated that this proline/glutamine-rich region was resistant to enzymatic digestion, a feature important for disease eliciting antigens.
Current results should be useful for understanding the disease mechanism and for developing strategies for treating and diagnosing CD.