Epitope mapping of anti-amelogenin IgA in coeliac disease

Previously we detected increased levels of IgA to the enamel matrix protein amelogenin in children with untreated coeliac disease (CeD). The biological impact of autoantibodies to amelogenin may depend on which part of the amelogenin (epitopes) they bind. Sera or blood samples from 146 untreated children with CeD from two different cohorts and 25 non-CeD control children were tested for IgA anti-amelogenin (Emdogain) reactivity. The 32 CeD children and six controls with the highest reactivity were selected for detailed IgA anti-amelogenin (AMELX) epitope mapping using 31 overlapping, 10-22mer peptides in ELISA. The dominating reactivity were to six peptides in a 75-amino-acid (aa)-long central segment (aa 75-150) containing enzymatic cleavage sites for matrix metalloproteinase 20 (MMP-20) and kallikrein-related peptidase 4 (KLK-4) and to two N-terminal peptides (aa 13-41) included in the tyrosine-rich amelogenin peptide fragment, which is important for self-assembly. Only two of the six control children reacted to single, but different peptides. Solid-phase extraction with gliadin- and Emdogain-coated Sepharose revealed a gliadin cross-reactive central region and a putative conformation-dependent, apparently non-cross-reactive N-terminal region. High IgA anti-amelogenin reactivity may interfere with normal amelogenesis by inhibiting amelogenin self-assembly, amelogenin-hydroxyapatite interaction, and/or enzymatic degradation.