Abstract
This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells.
Highlights
This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions
The human monoclonal antibodies (hmAbs) were incubated with size-separated fractions (Supplementary Figure 1) of transglutaminase 2 (TG2)-gliadin and hmAb-peptide complexes were isolated with magnetic protein G beads
Of the thirteen gliadin-reactive hmAbs tested, nine derived from IgA+ plasma cells isolated by flow cytometry with two different synthetic gliadin peptides used for staining (biotin-GSGSGS-PLQPEQPFP, PLQPEQPFP for short; biotin-(PEG)-LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF, deamidated 33mer for short) and four hmAbs derived from in vitro cultured plasma cells secreting IgA reactive to complex deamidated gliadin (Table 1)
Summary
This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. Many gluten-derived peptides are excellent substrates for the enzyme transglutaminase 2 (TG2), which can deamidate glutamine residues in certain sequence contexts and thereby convert them into glutamic acid Both the T-cell and B-cell response in celiac disease seem to be directed toward gluten peptides that have been deamidated by TG210–12. We report epitope mapping of gliadin-reactive hmAbs by antibody pull-down of fragments from complex proteolytic digests of gluten followed by sequencing of the isolated peptides by mass spectrometry
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