Selective Capture of Anti-N-glucosylated NTHi Adhesin Peptide Antibodies by a Multivalent Dextran Conjugate.

Tentacle-like polymers decorated with several copies of peptide antigens can be interesting tools for increasing the ability to capture circulating antibodies in patient sera, using cooperative effects for stronger avidity. We previously showed that antibodies from multiple sclerosis (MS) patient sera preferentially recognize hyperglucosylated adhesin protein HMW1ct of non-typeable Haemophilus influenzae (NTHi). We selected the C-terminal HMW1ct(1347-1354) minimal epitope and prepared the diglucosylated analogue Ac-KAN(Glc)VTLN(Glc)TTG-K(N3 )-NH2 to graft a 40 kDa dextran scaffold modified with glycidyl-propargyl moieties to perform a copper catalyzed alkyne-azide coupling reaction (CuAAC). Quantitative NMR measurements allowed the characterization of the peptide loading (19.5 %) on the multivalent dextran conjugate. This novel polymeric structure displayed optimal capturing properties of both IgG and, more interestingly, IgM antibodies in MS sera. Specific antibodies from a representative MS serum, were successfully depleted using a Sepharose resin bearing the new glucosylated multivalent conjugate, as confirmed by ELISA. These results may offer a promising proof-of-concept for the selective purification of high affinity autoantibodies from sera of autoimmune patients, in general, and of specific high affinity antibodies against a minimally glcosylated epitope Asn(Glc) from sera of multiple sclerosis (MS) patients, in particular.

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 the N-linked ß-d-glucopyranosyl moieties (N-Glc) containing epitopes in nontypeable Haemophilus influenzae adhesin 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 two N-Glc respectively on Asn-1349 and/or Asn-1352. The N-glucosylated nonapeptides efficiently bind to IgG antibodies, displaying IC50 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-NH2 as the shortest sequence able to inhibit high-avidity interaction with N-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 two N-glucosyl moieties. Therefore, the antibody binding is not easily correlated to the position of the sugar or to a determined conformation in water.

Glycoreplica peptides to investigate molecular mechanisms of immune-mediated physiological versus pathological conditions.

Investigation of the role of saccharides and glycoconjugates in mechanisms of immune-mediated physiological and pathological conditions is a hot topic. In fact, in many autoimmune diseases cross-reactivity between sugar moieties exposed on exogenous pathogens and self-molecules has long been hinted. Several peptides have been reported as mimetics of glycans specifically interacting with sugar-binding antibodies. The seek for these glycoreplica peptides is instrumental in characterizing antigen mimicry pathways and their involvement in triggering autoimmunity. Therefore, peptides mimicking glycan-protein interactions are valuable molecular tools to overcome the difficulties of oligosaccharide preparations. The clinical impact of peptide-based probes for autoimmune diseases diagnosis and follow-up is emerging only recently as just the tip of the iceberg of an overlooked potential. Here we provide a brief overview of the relevance of the structural and functional aspects of peptide probes and their mimicry effect in autoimmunity mechanisms for promising applications in diagnostics and therapeutics.

Individual Human Metabolic Phenotype Analyzed by (1)H NMR of Saliva Samples.

Saliva is an important physiological fluid that contains a complex mixture of analytes that may produce a characteristic individual signature. In recent years, it has been demonstrated that urine possesses a clear signature of the individual metabolic phenotype. Here NMR-based metabolomics was employed to analyze saliva from 23 healthy volunteers. About six saliva samples were collected daily from each individual for 10 consecutive days: 7 days in a real-life situation (days 1-7, Phase I) and 3 days (days 8-10, Phase II) under a standardized diet plus a physical exercise program at day 10. The result is the first demonstration of the existence of an individual metabolic phenotype in saliva. A systematic comparative analysis with urine samples from the same collection scheme demonstrates that the individual phenotype in saliva is slightly weaker than that in urine but less influenced by diet.