048 Characterization of a conformational epitope on the EC1 domain of desmoglein 1 recognized by IgG4 autoantibodies from Fogo Selvagem

Fogo Selvagem (FS) is mediated by pathogenic IgG4 restricted autoantibodies against Desmoglein-1 (Dsg1), which do not react with Dsg3 or Dsg4. Double purified FS anti-Dsg1 IgG4 (affinity purified on llama anti-human IgG4-Agarose and a human Dsg1-ectodomain-Agarose affinity media) yield highly pathogenic IgG4 in the mouse model. Hybrid Dsg1 molecules bearing the EC1 domain of Dsg4 no longer bind FS IgG4. Moreover, Dsg4 bearing the EC1 domain of Dsg1 gain reactivity with FS IgG4 and are able to affinity-purify FS IgG4 autoantibodies that are also pathogenic in the mouse model, further suggesting that pathogenic epitope(s) are clustered on the EC1 domain of Dsg1. We showed previously that highly purified FS IgG4 from 19/20 FS sera bind a single peptide on the EC1 domain of Dsg1 using MALDI/MS Epitope Mapping. A 16 amino acid peptide (ALNSMGQDLERPLELR) that overlaps RAL motif of Dsg1 was recognized by 19 IgG4 fractions. Mutations of M133 and Q135 of this peptide reduced the binding (∼50%) of IgG4 autoantibodies to Dsg1 by ELISA. Using molecular modeling we identified two additional residues, distant from the previous peptide, but within the EC1 domain, that are complementary components of the epitope bound by FS IgG4 autoantibodies. Mutations of these additional residues completely abolish the binding of FS IgG4 from 19 FS sera. These results suggest that highly restricted and pathogenic IgG4 autoantibodies from FS patients are exquisitely specific for a conformational epitope on the EC1 domain of Dsg1. This strongly suggests that binding of FS IgG4 autoantibodies to this conformational epitope, that partially overlaps the adhesive site of Dsg1, may directly interfere with the adhesive function of this molecule and lead to acantholysis.