Epitope mapping of anti‐amelogenin IgA in coeliac disease

Abstract

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.

Epitope mapping of anti‐amelogenin IgG in untreated celiac children

“…Patient cohorts: Some of the patients (n = 23) studied here were included in a previous study in which the IgA anti‐AMELX epitopes were mapped (12). The present cohort 1 included blood samples collected for diagnostic purposes were obtained from 73 children (median age 8 years; age range 1–16 years) with biopsy‐proven, but untreated, CeD and from 25 children (median age 8 years; age range, 1–17 years) proven by biopsy not to have CeD.…”

“…Diagnosis was based on the level of anti‐TG2 IgA, HLA‐DQ genotype, clinical examination, and histopathological and immunohistochemical examination of jejunal biopsy samples. Blood sampling was conducted as described in detail previously (12). Cohort 2 comprised 73 children with untreated CeD: all had sera containing anti‐TG2 IgA titers >10 times the upper normal limit that had been sent to Oslo University Hospital, Ullevaal, Norway, to be examined anti‐endomysium IgA reactivity.…”

“…Sera from all patients (146 with CeD and 34 non‐CeD controls) were tested for IgG reactivity to amelogenin, using porcine amelogenin (Emdogain; Straumann) as antigen in ELISA, as detailed elsewhere (11). The following samples (n = 42) from patients with CeD were selected for further epitope mapping, specifically: samples from patients in cohorts 1 (n = 14) and 2 (n = 22) with anti‐amelogenin IgG immune reactivity higher than the median value; samples from patients with enamel defects (cohort 1, n = 3); and samples from three patients who had participated in the anti‐AMELX IgA study (12) (these patients were included despite having anti‐amelogenin IgG levels lower than the median). The control consisted of the five sera with the highest levels anti‐amelogenin IgG and the five blood samples with the highest levels of anti‐amelogenin IgG, from 10 controls (cohort 1).…”

“…The severity of DEDs caused by CeD ranges (according to the Aine classification) from hypomineralized, discolored spots (Grade 1) to severe substance defects (Grade 4) (6). The etiology of CeD‐DEDs is unclear and is presumably multifactorial as malnutrition (7), genetics (8,9), CeD‐associated immune activity and autoimmunity may be contributory factors (10–12).…”

“…Although it remains to be clarified whether anti‐amelogenin IgG is involved in the etiology of CeD‐DEDs, the functional implication of the cross‐reactivity would depend on the amelogenin regions to which the IgG reacts. We previously reported the epitope mapping of anti‐AMELX IgA in children with untreated CeD (12). The current paper is an extended epitope‐mapping of serum anti‐amelogenin IgG in children with newly diagnosed CeD and disease controls selected from the same two cohorts.…”