Antibody Recognition in multiple sclerosis and rett syndrome using a collection of linear and cyclic N‐glucosylated antigenic probes

Abstract

Antibody detection in autoimmune disorders, such as multiple sclerosis (MS) and Rett syndrome (RTT) can be achieved more efficiently using synthetic peptides. The previously developed synthetic antigenic probe CSF114(Glc), a type I' β-turn N-glucosylated peptide structure, is able to recognize antibodies in MS and RTT patients' sera as a sign of immune system derangement. We report herein the design, synthesis, conformational analysis, and immunological evaluation of a collection of glycopeptide analogs of CSF114(Glc) to characterize the specific role of secondary structures in MS and RTT antibody recognition. Therefore, we synthesized a series of linear and cyclic short glucosylated sequences, mimicking different β-turn conformations, which were evaluated in inhibition enzyme-linked immunosorbent assays (ELISA). Calculated IC50 ranking analysis allowed the selection of the candidate octapeptide containing two (S)-2-amino-4-pentynoic acid (L-Pra) residues Ac-Pra-RRN(Glc)GHT-Pra-NH2 , with an IC50 in the nanomolar range. This peptide was adequately modified for solid-phase ELISA (SP-ELISA) and surface plasmon resonance (SPR) experiments. Pra-RRN(Glc)GHT-Pra-NH2 peptide was modified with an alkyl chain linked to the N-terminus, favoring immobilization on solid phase in SP-ELISA and differentiating IgG antibody recognition between patients and healthy blood donors with a high specificity. However, this peptide displayed a loss in IgM specificity and sensitivity. Moreover, an analog was obtained after modification of the octapeptide candidate Ac-Pra-RRN(Glc)GHT-Pra-NH2 to favor immobilization on SPR sensor chips. SPR technology allowed us to determine its affinity (KD = 16.4 nM), 2.3 times lower than the affinity of the original glucopeptide CSF114(Glc) (KD = 7.1 nM).

Structure–Activity Relationship Studies, SPR Affinity Characterization, and Conformational Analysis of Peptides That Mimic the HNK‐1 Carbohydrate Epitope

“…Hence, the negatively charged groups of the two molecules cannot be superimposed efficiently; therefore, the increased affinity observed with peptide 5 cannot be explained in terms of correct mimicry of the relative positions of the two negatively charged groups of the peptide, as compared with the sugar. On the other hand, previously reported conformational studies on parent peptide 1 revealed the presence of a β‐turn around the Ser 2 ‐Glu 3 ‐Thr 4 ‐Thr 5 sequence . In our peptide 5 (and 3 ) a Glu residue replaces Thr 5 , and a stable hydrogen bond between the side chains of residues 2 and 5 (not reported for peptide 1 ) is observed.…”

“…Indeed, despite the presence of the N‐terminal PEG extension, when peptide 13 was tested in solution on the immobilized antibody, it showed a K D value of 6.32×10 −7 m , corresponding to a marginal loss of affinity (six‐fold), as compared with the original sequence. We previously observed similar behavior, that is, a modest decrease in affinity due to N‐terminal modification of antigenic peptides, possibly through the introduction of steric hindrance, which, however, did not hamper the use of these peptides in ELISA . Accordingly, using SP‐ELISA, we tested the peptide Ttds‐LSETETdL ( 13 ), which can be efficiently adsorbed on PVC plates.…”

Surface plasmon resonance-based methodology for anti-adalimumab antibody identification and kinetic characterization

“…Experiments were conducted following the previously described SPR protocol [29] properly optimized for the present study. ADA was immobilized on the sensor chip CM5-type surface.…”

“…Kinetic experiments were conducted following the previously described SPR protocol [29] optimizing the dissociation phase times and regeneration solutions for the present study. Diluted samples were injected in duplicate over immobilized ADA for 120 s at a flow rate of 30 μL/min.…”

Modeling interaction between gp120 HIV protein and CCR5 receptor

“…Surface plasmon resonance experiments with peptides in solution were performed using a Biacore T100 instrument from GE Healthcare (Uppsala, Sweden). Experiments were conducted following the previously described SPR protocol, properly optimized for the present study . The V3 loop peptide was covalently immobilized on a CM5‐type sensor chip surface, according to the amine coupling strategy.…”