Gliadin Antibodies of CD Patients Recognize Deamidated Gliadin

Although the gliadin-deamidating activity of tTG toward gliadin was known since 1998 [79-82], attention was focused on the deamidation effect on binding of modified gliadin peptides to HLA molecules. This was due to the fact that the typical mucosal lesion in CD is basically regarded as a result of T-cell activation after presentation of gliadin peptides. Antibodies in CD were considered of relatively minor importance in disease pathogenesis and were generally viewed as side products of the immune response of some usefulness in diagnosis. Several attempts were made to screen different gliadin amino acid sequences for epitopes of antibodies of CD patients [154-157]. Unfortunately, these studies used sequences from native unmodified gliadin.

To identify the gliadin epitope, we screened libraries of phage-displayed peptides for binding to antibodies from CD patients [157]. The technique of screening of phage-displayed peptides (phage display) is based on application of a large pool of filamentous coli phage that express a vast number of different short peptides fused to phage coat proteins (combinatorial library of random peptides) on their surface [158-160]. Each single phage expresses only peptides of one common sequence. Due to the size of the phage pool, the number of different expressed peptides is tremendous and can be tested for binding ligands of interest. If antibodies are applied as ligands, those phage with peptides on their surface mimicking the antibody epitopes will be bound and can be amplified in further rounds of selection. Finally, the phage DNA can be sequenced and the peptide sequence responsible for binding can be deduced from the DNA sequence. We screened a phage-displayed dodeca-peptide library with human IgA from sera positive for anti-endomysium antibodies and with high concentration of gliadin antibodies [157]. As a result, 200 different antibody-binding dodecapeptide sequences were identified. These were synthesized onto cellulose membranes and subjected to further binding studies with human sera. Only four of the dodecapeptides showed a significantly stronger binding of IgA of sera with high gliadin antibody concentrations compared to sera low in gliadin antibodies. All of them contained the tripeptide motif PEQ. Contrary to the phage display experiments, the PEQ sequence could not be found when short peptides (synthesized onto cellulose membranes) covering in overlapping manner the complete native sequence of a- and 7-type gliadins were investigated for binding to gliadin antibodies (pepscan). But if the third amino acid residue in one of the main peptides detected in the pepscan investigations, QPQQPF, was substituted by glutamic acid yielding a PEQ motif, binding of IgA antibodies from sera of CD patients increased considerably. From these findings, together with knowledge about the deamidating activity of tTG, we concluded that gliadin antibodies of CD patients selectively recognized deamidated gliadin.

Later, other investigators [161] reported that gliadin peptide sequences QQLPQPQQPQQSFPQQQRPF, amino acid positions 134-153 of a 7-type gliadin [80] and QLQPFPQPQLPYPQPQS, amino acid positions 56-75 of a a-type gliadin [162] were specifically deamidated by tTG. This study tested binding of IgA and IgG from sera of CD patients to the native peptide and the glutamine-glutamic acid substituted peptides. These authors found that selective deamidation specifically increased recognition by gliadin antibodies from CD patients.

These results demonstrated that the epitope repertoire of gliadin antibodies of CD patients was restricted to a small number of similar amino acid sequences. In this regard it is worth mentioning that human antibodies of the IgE class, which can be found in wheat allergic patients, are also commonly directed against the (nondeamidated) QQPFP and PQQPF motifs [163, 164]. Furthermore, the native gliadin peptide QQPFP is also the main epitope of monoclonal antibodies raised against gliadin or secalin in mice [23]. The restricted number of epitope motifs in gliadin was promising for the development of antibody assays based on a small number of defined gliadin-analogous peptides.

The analysis of the epitope repertoire of antibodies against tTG in CD patients was not, however, conclusive enough to allow the construction of peptide-based tests for estimation of autoantibodies [165-168].

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