Hyperglucosylated adhesin-derived peptides as antigenic probes in multiple sclerosis: Structure optimization and immunological evaluation

Peptides mimicking antigenic epitopes targeted by antibodies can be powerful tools to be used as antigen surrogates for the specific diagnosis and treatment of autoimmune diseases. Obtaining structural insights about the nature of peptide-antibody interaction in complex mixtures such as sera is a critical goal. In multiple sclerosis (MS), we previously demonstrated that theN-linked beta-d-glucopyranosyl moieties (N-Glc) containing epitopes in nontypeableHaemophilus influenzaeadhesin C-terminal portion HMW1(1205-1526) were essential for high-affinity antibody binding in a subpopulation of MS patients. With the aim of developing peptide probes and assessing their binding properties to antibodies from sera of representative patients, we performed the systematic analysis of synthetic peptides based on HMW1(1347-1354) fragment bearing one or twoN-Glc respectively on Asn-1349 and/or Asn-1352. TheN-glucosylated nonapeptides efficiently bind to IgG antibodies, displaying IC(50)in the range 10(-8)-10(-10)M by competitive indirect enzyme-linked immunosorbent assay (ELISA) in three representative MS patient sera. We selected the di-N-glucosylated adhesin peptide Ac-KAN (Glc)VTLN (Glc)TT-NH(2)as the shortest sequence able to inhibit high-avidity interaction withN-Glc targeting IgM antibodies. Nuclear magnetic resonance (NMR)- and circular dichroism (CD)-based characterization showed that the binding properties of these antigens could not be ascribed to structural differences induced by the presence of up to twoN-glucosyl moieties. Therefore, the antibody binding is not easily correlated to the position of the sugar or to a determined conformation in water.