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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(N 3 )-NH 2 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., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The role of pathologic auto-antibodies against myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis is a highly controversial matter. As the use of animal models may enable to unravel the molecular mechanisms of the human disorder, numerous studies on multiple sclerosis are carried out using experimental autoimmune encephalomyelitis (EAE). In particular, the most extensively used EAE model is obtained by immunizing C57BL/6 mice with the immunodominant peptide MOG(35-55). In this scenario, we analyzed the anti-MOG antibody response in this model using the recombinant refolded extracellular domain of the protein, MOG(1-117). To assess the presence of a B-cell intramolecular epitope spreading mechanism, we tested also five synthetic peptides mapping the 1-117 sequence of MOG, including MOG(35-55). For this purpose, we cloned, expressed in Escherichia coli and on-column refolded MOG(1-117), and we applied an optimized microwave-assisted solid-phase synthetic strategy to obtain the designed peptide sequences. Subsequently, we set up a solid-phase immunoenzymatic assay testing both naïve and EAE mice sera and using MOG protein and peptides as antigenic probes. The results obtained disclose an intense IgG antibody response against both the recombinant protein and the immunizing peptide, while no response was observed against the other synthetic fragments, thus excluding the presence of an intramolecular epitope spreading mechanism. Furthermore, as the properly refolded recombinant probe is able to bind antibodies with greater efficiency compared with MOG(35-55), we hypothesize the presence of both linear and conformational epitopes on MOG(35-55) sequence., (Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.)

Sophisticated approaches have recently led to the identification of novel autoantigens associated with Multiple Sclerosis (MuS), e.g. neurofascin, contactin, CNPase, and other T-cell receptor membrane anchored proteins. These putative antigens, although differing from the conventional myelin derivatives, are conceptually based on an animal model of experimental autoimmune encephalomyelitis. In this report we describe the identification of putative antigens based on their recognition by autoantibodies isolated from MuS patient serum. In a previous work from this laboratory we have shown that a peptide probe, named CSF114(Glc), specifically identifies serum autoantibodies in a subset of MuS patients, representing ∼30% of the patient population. The autoantibodies, purified from MuS patients' sera (six), through CSF114(Glc) affinity chromatography, detected three immunoreactive protein bands present in the rat brain. Proteomic analysis of the immunoreactive bands, involving MALDI and MS/MS techniques, revealed the presence of four proteins distinguishable by their mass: alpha fodrin, alpha actinin 1, creatine kinase, and CNPase. The immunoreactive profile of these rat brain proteins was compared with that of commercially available standard proteins by challenging against either CSF114(Glc) purified MuS autoantibodies, or monoclonal antibodies. Further discrimination among the rat brain proteins was provided by the following procedure: whereas monoclonal antibodies recognized all rat brain proteins, isolated MuS specific antibodies recognize only alpha actinin 1 as a putative antigen. In fact, alpha actinin 1 displayed a robust immunoreactive response against all MuS patients' sera examined, whereas the other three bands were not consistently detectable. Thus, alpha actinin 1, a cytoskeleton protein implicated in inflammatory/degenerative autoimmune diseases (lupus nephritis and autoimmune hepatitis) might be regarded as a novel MuS autoantigen, perhaps a prototypic biomarker for the inflammatory/degenerative process typical of the disease.

We previously reported that CSF114(Glc) detects diagnostic autoantibodies in multiple sclerosis sera. We report herein a bioinformatic analysis of myelin proteins and CSF114(Glc), which led to the identification of five sequences. These glucopeptides were synthesized and tested in enzymatic assays, showing a common minimal epitope. Starting from that, we designed an optimized sequence, SP077, showing a higher homology with both CSF114(Glc) and the five sequences selected using the bioinformatic approach. SP077 was synthesized and tested on 50 multiple sclerosis patients' sera, and was able to detect higher antibody titers as compared to CSF114(Glc). Finally, the conformational properties of SP077 were studied by NMR spectroscopy and structure calculations. Thus, the immunological activity of SP077 in the recognition of specific autoantibodies in multiple sclerosis patients' sera may be ascribed to both the optimized design of its epitopic region and the superior surface interacting properties of its C-terminal region.

Synthetic N-glycosylated CSF114(Glc) and related peptides were proved to be able to recognize specific and high-affinity autoantibodies circulating in blood of relapsing-remitting multiple sclerosis (MS) patients and correlating with disease activity. The effect of these peptides has been linked to the β-turn structure around the minimal epitope Asn(Glc). In this work we performed Hamiltonian replica exchange molecular dynamics simulations on the central heptapeptide fragment of a CSF114(Glc)-derived peptide in water and in a water/hexafluoroacetone mixture, confirming a significant incidence of β-turn structures in both solvents. The structural similarity of the glycosylated and unglycosylated forms in all environments proves that the conformation of the heptapeptide is only marginally affected by the presence of the sugar. Moreover, the presence of a significant amount of bioactive hairpin-like conformations in the water environment suggests a possible use not only in the diagnosis but also in the treatment of MS.

In multiple sclerosis (MS) the gold standard for the diagnosis and prognosis is, up to now, the use of magnetic resonance imaging markers. No alternative simpler assays proven of use, except for cerebrospinal fluid analysis, have been provided in MS diagnosis. Therefore, there is a need to develop non-invasive, sensitive, simple new techniques for the clinical routine. Herein we present the evaluation of the feasibility of a glycopeptide-based biosensor to detect MS specific antibodies in sera using the surface plasmon resonance technology. The previously described glycopeptide antigen CSF114(Glc) has been immobilized on a gold sensor chip and the method has been optimized for real-time specific autoantibody detection directly in sera. A population of 60 healthy blood donors and 61 multiple sclerosis patients has been screened. The receiver operating characteristic (ROC)-based analysis has established the optimal diagnostic cut-off value for the method obtaining a sensitivity of 36% and a specificity of 95%. Sample sera have been also screened with a previously validated ELISA.

Circulating autoantibodies have been recognized as disease biomarkers of autoimmune diseases. We have previously disclosed a synthetic glycopeptide that is able to detect specific autoantibodies in sera of patients who are affected by multiple sclerosis (MS). This glycopeptide is characterized by a type I' beta-turn around the minimal epitope Asn(Glc) that allows an efficient exposure of this moiety to antibody interactions in the context of a solid-phase immunoenzymatic assay. With the aim of optimizing the glycopeptide-antibody interactions, we analyze a series of new glycopeptides based on different turn structures. Our results confirm the role of conformation in the recognition and binding of synthetic antigenic probes to MS autoantibodies. Glycopeptide 2, which is characterized by a type I beta-turn around the minimal epitope Asn(Glc), shows the highest antibody affinity (IC50 = 11.8 nM), and thus it appears to be a promising tool for the detection of specific autoantibodies as MS biomarker in patients' sera.

Glycopeptide analogues of CSF114(Glc), modified at N-terminus with new ferrocenyl carboxylic acid and a new ferrocenyl-thiphosphino amino acid, were used to implement a new electrochemical biosensor for autoantibody detection in multiple sclerosis. The ferrocenyl moiety of these "electrochemical probes" did not affect autoantibody recognition both in SP-ELISA and in inhibition experiments. By electrochemical monitoring the interactions of the modified peptides Fc-CSF114(Glc) and 4-FcPhP(S)Abu-CSF114(Glc) with the autoantibodies, we demonstrated that autoantibodies could be detected with a sensitivity comparable to ELISA method. The new electrochemical probes can be proposed to characterize autoantibodies as biomarkers of multiple sclerosis by a simple, rapid, and reproducible cyclic voltammetry-based diagnostic methodology., (Copyright (c) 2008 Wiley Periodicals, Inc.)

Sera from patients suffering from autoimmune disorders often contain multiple types of autoantibodies, some of which can be exclusive of a disease and thus used as biomarkers for diagnosis. Identification of these autoantibodies, as disease biomarkers, should be achieved using native antigens in simple biological assays. However, posttranslational modifications, such as glycosylation, may play a fundamental role for specific autoantibody recognition. In line with these observations, we described synthetic glycopeptides able to detect high autoantibody titers in sera of patients affected by multiple sclerosis, an inflammatory, demyelinating disease of the central nervous system. We describe here the conformation-activity relationship of a focused library of glycopeptides based on structural diversity, with the aim of defining the structural requirements for the interaction of these glycopeptide antigens with specific autoantibodies. The final goal is the optimization of an antigenic probe for multiple sclerosis, to be used in the development of a simple diagnostic test based on an immunoenzymatic assay. The reported results clearly indicate that glycopeptides able to reveal high antibody titers in multiple sclerosis sera are characterized by a type I' beta-turn around the minimal epitope Asn(Glc), which allows an efficient exposure of this moiety to antibodies interactions, in the context of a solid-phase immunoenzymatic assay.

Multiple sclerosis (MS) is a complex disease that seems to depend on several pathophysiological processes. Because of its varied clinical presentation, natural history, and response to therapeutic interventions, MS can be considered to be a group of diseases that have not been yet characterized, thus resulting in difficult evaluation of prognosis. In the last few years, the role of autoAbs in MS has been reevaluated, and, therefore, their identification as specific biomarkers became a relevant target. In this paper, we demonstrate that an aberrant N-glucosylation is a fundamental determinant of autoAb recognition in MS. Thus, we developed CSF114(Glc), an antigenic probe accurately measuring IgM autoAbs in the sera of a patient population, as disease biomarker. The relevance of CSF114(Glc) is demonstrated by its clinical application and correlation with disease activity and prognosis. In fact, CSF114(Glc), a structure-based designed glycopeptide, is able to recognize, by ELISA, the presence of specific IgM autoAbs in the sera of a MS patient population but not in blood donors and other autoimmune conditions. AutoAbs specific for CSF114(Glc) isolated from MS patients recognized myelin and oligodendrocyte antigens by immunohistochemistry but not other nonrelevant tissues. We demonstrate that CSF114(Glc) is a reliable, specific probe in a longitudinal study of untreated MS patients. Development of IgG/IgM anti-CSF114(Glc) Abs paralleled clinical activity and brain lesions positive to MRI. Therefore, a CSF114(Glc)-based immunoassay on sera may have important prognostic value in monitoring MS disease progression guiding optimal therapeutic treatment.

Multiple sclerosis (MS) is an inflammatory and autoimmune disorder, in which an antibody‐mediated demyelination mechanism plays a critical role. We prepared two glucosylated peptides derived from the human myelin proteins, that is, oligodendrocyte‐myelin glycoprotein (OMGp) and reticulon‐4 receptor (RTN4R), selected by a bioinformatic approach for their conformational homology with CSF114(Glc), a designed β‐turn antigenic probe derived from myelin oligodendrocyte glycoprotein (MOG), a glycoprotein present in the CNS. This synthetic antigen is specifically recognized by antibodies in sera of MS patients. We report herein the antigenic properties of these peptides, showing, on the one hand, that MS patient antibodies recognize the two glucosylated peptides and, on the other hand, that these antibodies cross‐react with CSF114(Glc) and with the previously described hyperglucosylated nontypeable Haemophilus influenzae bacterial adhesin protein HMW1ct(Glc). These observations point to an immunological association between human and bacterial protein antigens, underpinning the hypothesis that molecular mimicry triggers the breakdown of self‐tolerance in MS and suggesting that RTN4R and OMGp can be considered as autoantigens. [ABSTRACT FROM AUTHOR]

The interaction of lipid environments with the type I' β-turn peptide structure called CSF114 and its N-glucosylated form CSF114(Glc), previously developed as a synthetic antigenic probe recognizing specific autoantibodies in a subpopulation of multiple sclerosis patients' serum, was investigated by fluorescence spectroscopy and electrochemical experiments using large unilamellar vesicles, mercury supported lipid selfassembled monolayers (SAMs) and tethered bilayer lipid membranes (tBLMs). The synthetic antigenic probe N-glucosylated peptide CSF114(Glc) and its unglucosylated form interact with the polar heads of lipid SAMs of dioleoylphosphatidylcholine at nonzero transmembrane potentials, probably establishing a dual electrostatic interaction of the trimethylammonium and phosphate groups of the phosphatidylcholine polar head with the Glu5 and His9 residues on the opposite ends of the CSF114(Glc) β-turn encompassing residues 6-9. His9 protonation at pH 7 eliminates this dual interaction. CSF114(Glc) is adsorbed on top of SAMs of mixtures of dioleoylphosphatidylcholine with sphingomyelin, an important component of myelin, whose proteins are hypothesized to undergo an aberrant N-glucosylation triggering the autoimmune response. Incorporation of the type I' β-turn peptide structure CSF114 into lipid SAMs by potential scans of electrochemical impedance spectroscopy induces defects causing a slight permeabilization toward cadmium ions. The N-glucopeptide CSF114(Glc) does not affect tBLMs to a detectable extent. [ABSTRACT FROM AUTHOR]

Rett syndrome (RTT) is a devastating neurodevelopmental disease, previously included into the autistic spectrum disorders, affecting almost exclusively females (frequency 1:10,000). RTT leads to intellective deficit, purposeful hands use loss and late major motor impairment besides featuring breathing disorders, epilepsy and increased risk of sudden death. The condition is caused in up to 95% of the cases by mutations in the X-linked methyl-CpG binding protein 2 ( MECP2 ) gene. Our group has shown a number of previously unrecognized features, such as systemic redox imbalance, chronic inflammatory status, respiratory bronchiolitis-associated interstitial lung disease-like lung disease, and erythrocyte morphology changes. While evidence on an intimate involvement of MeCP2 in the immune response is cumulating, we have recently shown a cytokine dysregulation in RTT. Increasing evidence on the relationship between MeCP2 and an immune dysfunction is reported, with, apparently, a link between MECP2 gene polymorphisms and autoimmune diseases, including primary Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, and systemic sclerosis. Antineuronal (i.e., brain proteins) antibodies have been shown in RTT. Recently, high levels of anti-N-glucosylation (N-Glc) IgM serum autoantibodies [i.e., anti-CSF114(N-Glc) IgMs] have been detected by our group in a statistically significant number of RTT patients. In the current review, the Authors explore the current evidence, either in favor or against, the presence of an autoimmune component in RTT. [ABSTRACT FROM AUTHOR]

Abstract: Antibodies to MOG in serum have a dubious prognostic value in multiple sclerosis. The MOG recombinant protein conformational properties relevant to the antigenic activity are unknown. We employed a solid-phase ELISA based on a product (rMOGED(His)6) expressed in E. coli after subcloning the cDNA of the extracellular domain of rat MOG, performing a refolding procedure on column and affinity purification. The far-UV Circular Dichroism (CD) spectra of rMOGED(His)6 showed a β-sheet, a characteristic feature of the Ig-fold. However, in MS sera and controls we failed to detected IgM or IgG antibodies. [Copyright &y& Elsevier]