Mapping of B cell epitopes on desmoglein 3 in pemphigus vulgaris patients by the use of overlapping peptides

BackgroundPemphigus vulgaris (PV) is a severe autoimmune blistering disease associated with autoantibodies to desmoglein 3 (Dsg 3), a transmembrane glycoprotein of the cadherin family. Previous studies mainly focused on the mapping of conformational epitopes of Dsg 3 using recombinant fragments of Dsg 3 and competition ELISA.ObjectiveHere, we performed a mapping of linear B cell epitopes on Dsg 3 in PV patients by the use of overlapping synthetic peptides.MethodsA set of 254 overlapping synthetic peptides of 14 amino acids length covering the entire Dsg 3 extracellular domain was generated. Sera of patients with active PV (n = 10) and healthy volunteers (n = 10) were tested for IgG reactivity with the 254 peptides by ELISA. Testing each peptide separately, 7 major antigenic sites were identified. In order to validate these reactivities, 7 corresponding peptides of 13–33 amino acids in length were generated and employed by ELISA. Additional sera of active PV patients (n = 17) and healthy volunteers (n = 20) were tested and the most reactive peptide was used to specifically purify anti-Dsg 3 antibodies from PV sera (n = 3).ResultsThe major autoantibody reactivity in PV sera was mapped to amino acids 333–356 within the EC3 domain. Purifying patients IgG using the identified peptide, however, failed to induce acantholysis in keratinocyte dissociation assay.ConclusionWe conclude that linear epitopes do not play a major pathogenic role in human PV.