A Promising Compound for Green Multiresponsive Materials Based on Acyl Carrier Protein.

A gel that exhibits intrinsically multiple-responsive behavior was prepared from an oligopeptide and studied. ACP(65-74) is an active decapeptide fragment of acyl carrier protein. We investigated 3% w/v ACP(65-74)-NH2 self-healing physical gels in water, glycerol carbonate (GC), and their mixtures. The morphology was investigated by optical, birefringence, and confocal laser scanning microscopy, circular dichroism, Fourier transform infrared, and fluorescence spectroscopy experiments. We found that all samples possess pH responsiveness with fully reversible sol-to-gel transitions. The rheological properties depend on the temperature and solvent composition. The temperature dependence of the gels in water shows a peculiar behavior that is similar to that of thermoresponsive polymer solutions. The results reveal the presence of several ß-sheet structures and amyloid aggregates, offering valuable insights into the fibrillation mechanism of amyloids in different solvent media.

Design, synthesis, conformational analysis, and biological activity of Cα1-to-Cα6 1,4- and 4,1-disubstituted 1H-[1,2,3]triazol-1-yl-bridged oxytocin analogues.

Oxytocin (OT) is a neurohypophyseal peptide hormone containing a disulphide-bridged pseudocyclic conformation. The biomedical use of OT peptides is limited amongst others by disadvantageous pharmacokinetic parameters. To increase the stability of OT by replacing the disulphide bridge with the stable and more rigid [1,2,3]triazol-1-yl moiety, we employed the Cu2+-catalysed side chain-to-side chain azide-alkyne 1,3-cycloaddition. Here we report the design, synthesis, conformational analysis, and in vitro pharmacological activity of a homologous series of Cα1-to-Cα6 side chain-to-side chain [1,2,3]triazol-1-yl-containing OT analogues differing in the length of the bridge, location, and orientation of the linking moiety. Exploiting this macrocyclisation approach, it was possible to generate a systematic series of compounds providing interesting insight into the structure-conformation-function relationship of OT. Most analogues were able to adopt similar conformation to endogenous OT in water, namely, a type I ß-turn. This approach may in the future generate stabilised pharmacological peptide tools to advance understanding of OT physiology.

Glucopeptides derived from myelin-relevant proteins and hyperglucosylated nontypeable Haemophilus influenzae bacterial adhesin cross-react with multiple sclerosis specific antibodies: A step forward in the identification of native autoantigens in multiple sclerosis.

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 ß-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.

Accelerated solid-phase synthesis of glycopeptides containing multiple N-glycosylated sites.

Peptide fragments of glycoproteins containing multiple N-glycosylated sites are essential biochemical tools not only to investigate protein-protein interactions but also to develop glycopeptide-based diagnostics and immunotherapy. However, solid-phase synthesis of glycopeptides containing multiple N-glycosylated sites is hampered by difficult couplings, which results in a substantial drop in yield. To increase the final yield, large amounts of reagents but also time-consuming steps are required. Therefore, we propose herein to utilize heating and stirring in combination with low-loading solid supports to set up an accelerated route to obtain, by an efficient High-Temperature Fast Stirring Peptide Synthesis (HTFS-PS), glycopeptides containing multiple N-glycosylated sites using equimolar excess of the precious glycosylated building blocks.

Corrections to "An Optimized Safe Process from Bench to Pilot cGMP Production of API Eptifibatide Using a Multigram-Scale Microwave-Assisted Solid-Phase Peptide Synthesizer".

[This corrects the article DOI: 10.1021/acs.oprd.1c00368.].

Role of Helical Structure in MBP Immunodominant Peptides for Efficient IgM Antibody Recognition in Multiple Sclerosis.

The involvement of Myelin Basic Protein (MBP) in Multiple Sclerosis (MS) has been widely discussed in the literature. This intrinsically disordered protein has an interesting α-helix motif, which can be considered as a conformational epitope. In this work we investigate the importance of the helical structure in antibody recognition by MBP peptides of different lengths. Firstly, we synthesized the peptide MBP (81-106) (1) and observed that its elongation at both N- and C-termini, to obtain the peptide MBP (76-116) (2) improves IgM antibody recognition in SP-ELISA, but destabilizes the helical structure. Conversely, in competitive ELISA, MBP (81-106) (1) is recognized more efficiently by IgM antibodies than MBP (76-116) (2), possibly thanks to its more stable helical structure observed in CD and NMR conformational experiments. These results are discussed in terms of different performances of peptide antigens in the two ELISA formats tested.

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.

ELISA based on peptide antigens reproducing cross-reactive viral epitopes to detect antibodies in latent autoimmune diabetes in adults vs. type 1 diabetes.

Diagnosis of Latent Autoimmune Diabetes in Adults (LADA) is based on the adult-age, anti-islet autoantibodies, and temporary insulin-independence. As in Type-1-Diabetes (T1DM), autoimmunity may trigger LADA and enteroviruses-infections can play a role. Anti-human Glutamic-Acid-Decarboxylase (hGAD) autoantibodies are accepted clinical biomarkers, but do not discriminate LADA vs. T1DM. The hypothesis is that protein antigens detecting anti-hGAD antibodies do not expose epitopes specific for different disease forms. We investigated the diagnostic value of autoantibodies in LADA vs. T1DM to peptides of hGAD65/67 isoforms, and Enterovirus-Coxsackie-B4 (CVB4), as antigens sharing the epitope PEVKXK (X: E/T) included in CD8 T-cell CVB4 epitope restricted by diabetes-associated HLA-A2.1. Statistically significant differences of IgM and/or IgG in LADA and T1DM vs. controls were identified. In LADA IgMs to GAD65/67 peptides are diagnostics, IgGs to GAD65/67 peptides correlate with anti-CVB4 peptide antibodies. IgM and/or IgG to all tested peptides can predict LADA, monitoring CVB4 infected patients, improving LADA vs. T1DM stratification.•A customized SP-ELISA based on synthetic peptides Ac-hGAD65(250-273)-NH2 (1), Ac-hGAD67(258-281)-NH2 (2), and Ac-CVB4P2C(28-50)-NH2 (3) is described.•The method was designed to detect specific IgM and/or IgG in LADA, T1DM, vs. controls•Final aim is improvement of LADA vs. T1DM patient stratification.

Cross-reactive peptide epitopes of Enterovirus Coxsackie B4 and human glutamic acid decarboxylase detecting antibodies in latent autoimmune diabetes in adults versus type 1 diabetes.

BACKGROUND: Diagnosis of latent autoimmune diabetes in adults (LADA) is usually based on the adult age, anti-pancreatic islet cell antibodies detection, and insulin independence. This study investigates the diagnostic value of antibodies against human glutamic acid decarboxylase (hGAD) peptides in LADA and type 1 diabetes mellitus (T1DM) patients, and their cross-reactivity with an Enterovirus Coxsackie B4 (CVB4) shared epitope. METHODS: Sera from 27 LADA patients, 23 T1DM patients, and 24 controls were tested in ELISA for antibodies against hGAD peptides and a selected sequence of P2C protein of CVB4 (CVB4P2C). Diagnostic power of peptides was analyzed by ROC-curve analysis and cross-reactivity among peptides evaluated. RESULTS: IgM and IgG antibodies showed significant differences between LADA and T1DM versus controls for all peptides. Antibody responses present high agreement among peptides for IgM and IgG-isotypes in T1DM, which is not reproduced in LADA. IgM antibodies showed high predicting diagnostic power particularly in LADA (sensitivity > 85%, specificity 95.8%). CONCLUSIONS: Our study highlights the usefulness of peptides as diagnostic antigens in T1DM and LADA, and extends previous findings by comparing IgM and IgG-isotype antibodies in the same population. Additionally, results highlight the role of the entourage in the shared sequon PEVKXK in GAD and CVB4P2C particularly in IgMs identification.

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.

A Multiple N-Glucosylated Peptide Epitope Efficiently Detecting Antibodies in Multiple Sclerosis.

Diagnostics of Multiple Sclerosis (MS) are essentially based on the gold standard magnetic resonance imaging. Few alternative simple assays are available to follow up disease activity. Considering that the disease can remain elusive for years, identification of antibodies fluctuating in biological fluids as relevant biomarkers of immune response is a challenge. In previous studies, we reported that anti-N-glucosylated (N-Glc) peptide antibodies that can be easily detected in Solid-Phase Enzyme-Linked ImmunoSorbent Assays (SP-ELISA) on MS patients' sera preferentially recognize hyperglucosylated adhesin of non-typeable Haemophilus Influenzae. Since multivalency can be useful for diagnostic purposes to allow an efficient coating in ELISA, we report herein the development of a collection of Multiple N-glucosylated Peptide Epitopes (N-Glc MEPs) to detect anti-N-Glc antibodies in MS. To this aim, a series of N-Glc peptide antigens to be represented in the N-GlcMEPs were tested in competitive ELISA. We confirmed that the epitope recognized by antibodies shall contain at least 5-mer sequences including the fundamental N-Glc moiety. Using a 4-branched dendrimeric lysine scaffold, we selected the N-Glc MEP 24, carrying the minimal epitope Asn(Glc) anchored to a polyethylene glycol-based spacer (PEG) containing a 19-atoms chain, as an efficient multivalent probe to reveal specific and high affinity anti-N-Glc antibodies in MS.

An Optimised Di-Boronate-ChemMatrix Affinity Chromatography to Trap Deoxyfructosylated Peptides as Biomarkers of Glycation.

We report herein a novel ChemMatrix® Rink resin functionalised with two phenylboronate (PhB) moieties linked on the N-α and N-ε amino functions of a lysine residue to specifically capture deoxyfructosylated peptides, compared to differently glycosylated peptides in complex mixtures. The new PhB-Lys(PhB)-ChemMatrix® Rink resin allows for exploitation of the previously demonstrated ability of cis diols to form phenylboronic esters. The optimised capturing and cleavage procedure from the novel functionalised resin showed that only the peptides containing deoxyfructosyl-lysine moieties can be efficiently and specifically detected by HR-MS and MS/MS experiments. We also investigated the high-selective affinity to deoxyfructosylated peptides in an ad hoc mixture containing unique synthetic non-modified peptides and in the hydrolysates of human and bovine serum albumin as complex peptide mixtures. We demonstrated that the deoxyfructopyranosyl moiety on lysine residues is crucial in the capturing reaction. Therefore, the novel specifically-designed PhB-Lys(PhB)-ChemMatrix® Rink resin, which has the highest affinity to deoxyfructosylated peptides, is a candidate to quantitatively separate early glycation peptides from complex mixtures to investigate their role in diabetes complications in the clinics.

A Photochromic Azobenzene Peptidomimetic of a ß-Turn Model Peptide Structure as a Conformational Switch.

The insertion of azobenzene moiety in complex molecular protein or peptide systems can lead to molecular switches to be used to determine kinetics of folding/unfolding properties of secondary structures, such as α-helix, ß-turn, or ß-hairpin. In fact, in azobenzene, absorption of light induces a reversible trans ↔ cis isomerization, which in turns generates a strain or a structure relaxation in the chain that causes peptide folding/unfolding. In particular azobenzene may permit reversible conformational control of hairpin formation. In the present work a synthetic photochromic azobenzene amino acid derivative was incorporated as a turn element to modify the synthetic peptide [Pro7,Asn8,Thr10]CSF114 previously designed to fold as a type I ß-turn structure in biomimetic HFA/water solution. In particular, the P-N-H fragment at positions 7-9, involved in a ß-hairpin, was replaced by an azobenzene amino acid derivative (synthesized ad hoc) to investigate if the electronic properties of the novel peptidomimetic analog could induce variations in the isomerization process. The absorption spectra of the azopeptidomimetic analog of the type I ß-turn structure and of the azobenzene amino acid as control were measured as a function of the irradiation time exciting into the respective first ππ* and nπ* transition bands. Isomerization of the azopeptidomimetic results strongly favored by exciting into the ππ* transition. Moreover, conformational changes induced by the cis↔ trans azopeptidomimetic switch were investigated by NMR in different solvents.

Antibodies to post-translationally modified mitochondrial peptide PDC-E2(167-184) in type 1 diabetes.

BACKGROUND: Mitochondria play a role in type 1 diabetes (T1D) particularly in the treatment and prevention of disorder consequences. Due to their demonstrated role in diabetes pathology, mitochondrial proteins can be an interesting starting point to study candidate antigens in T1D. We investigated the role of relevant post-translational modifications (PTM) on a synthetic mitochondrial peptide as putative antigen. METHODS: The antibody response in T1D was evaluated by solid phase-ELISA using a collection of synthetic peptides bearing different PTMs. We investigated the role of lipoylation, phosphorylation, and glycosylation. The PTMs were introduced at position 173 of the mitochondrial pyruvate dehydrogenase E2 complex peptide PDC-E2(167-184) and at position 7 of a structure-based designed ß-turn peptide as an irrelevant sequence to investigate the role of the specific PDC-E2 peptide sequence. RESULTS: IgM titres in 31 T1D patients were higher than IgGs to all the synthetic PTM peptides. Results demonstrated the crucial role of lysine lipoamide, serine O-phosphorylation, and O-glycosylation into the PDC-E2(167-184) peptide sequence for IgM antibody recognition. CONCLUSIONS: Results highlight the importance of immune dysregulation in T1D, furthermore, if confirmed in a large number of patients, they will contribute to add novel diagnostic markers for the understanding the physiopathology of the disease.

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

The design of molecules that mimic biologically relevant glycans is a significant goal for understanding important biological processes and may lead to new therapeutic and diagnostic agents. In this study we focused our attention on the trisaccharide human natural killer cell-1 (HNK-1), considered the antigenic determinant of myelin-associated glycoprotein and the target of clinically relevant auto-antibodies in autoimmune neurological disorders such as IgM monoclonal gammopathy and demyelinating polyneuropathy. We describe a structure-activity relationship study based on surface plasmon resonance binding affinities aimed at the optimization of a peptide that mimics the HNK-1 minimal epitope. We developed a cyclic heptapeptide that shows an affinity of 1.09×10-7 m for a commercial anti-HNK1 mouse monoclonal antibody. Detailed conformational analysis gave possible explanations for the good affinity displayed by this novel analogue, which was subsequently used as an immunological probe. However, preliminary screening indicates that patients' sera do not specifically recognize this peptide, showing that murine monoclonal antibodies cannot be used as a guide to select immunological probes for the detection of clinically relevant human auto-antibodies.

Antibodies from multiple sclerosis patients preferentially recognize hyperglucosylated adhesin of non-typeable Haemophilus influenzae.

In autoimmune diseases, there have been proposals that exogenous "molecular triggers", i.e., specific 'non-self antigens' accompanying infectious agents, might disrupt control of the adaptive immune system resulting in serious pathologies. The etiology of multiple sclerosis (MS) remains unclear. However, epidemiologic data suggest that exposure to infectious agents may be associated with increased MS risk and progression may be linked to exogenous, bacterially-derived, antigenic molecules, mimicking mammalian cell surface glycoconjugates triggering autoimmune responses. Previously, antibodies specific to a gluco-asparagine (N-Glc) glycopeptide, CSF114(N-Glc), were identified in sera of an MS patient subpopulation. Since the human glycoproteome repertoire lacks this uniquely modified amino acid, we turned our attention to bacteria, i.e., Haemophilus influenzae, expressing cell-surface adhesins including N-Glc, to establish a connection between H. influenzae infection and MS. We exploited the biosynthetic machinery from the opportunistic pathogen H. influenzae (and the homologous enzymes from A. pleuropneumoniae) to produce a unique set of defined glucosylated adhesin proteins. Interestingly we revealed that a hyperglucosylated protein domain, based on the cell-surface adhesin HMW1A, is preferentially recognized by antibodies from sera of an MS patient subpopulation. In conclusion the hyperglucosylated adhesin is the first example of an N-glucosylated native antigen that can be considered a relevant candidate for triggering pathogenic antibodies in MS.

Serpin†A1 C-Terminal Peptides as Collagen Turnover Modulators.

The modulation of collagen turnover can be a relevant pharmacological target in the context of treating either pathological or pathophysiological conditions, such as collagen-related diseases and skin aging. Our recent work has focused on the search for short-chain peptides as lead compounds for further development of compounds that enhance the production of type I collagen. In this study we selected and synthesized overlapping peptides of the C-terminal portion of serpin A1 (residues 393-418), the impact of which on collagen production has been reported previously, in order to identify shorter and still active fragments and to provide insight on the mechanisms involved. The biological activity of each fragment was evaluated with cultured normal human dermal fibroblasts, and changes in the amounts of collagen were monitored in collected culture media by a sandwich ELISA technique developed in house. Interestingly, we identified a decapeptide, termed SA1-III (Ac-MGKVVNPTQK-NH2 ), as a promising candidate for our purposes; it is able to induce a significant increase in type I collagen levels in the culture medium of treated cells at micromolar concentrations.

Epitope mapping of anti-myelin oligodendrocyte glycoprotein (MOG) antibodies in a mouse model of multiple sclerosis: microwave-assisted synthesis of the peptide antigens and ELISA screening.

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.

Interaction Study of Phospholipid Membranes with an N-Glucosylated ß-Turn Peptide Structure Detecting Autoantibodies Biomarkers of Multiple Sclerosis.

The interaction of lipid environments with the type I' ß-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 self-assembled 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 His8 residues on the opposite ends of the CSF114(Glc) ß-turn encompassing residues 6-9. His8 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' ß-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.