Synthesis of lipopeptides of the immunodominant epitope hMBP(83–99) containing amide or C-C bond linked hydrophobic chains for the study of T cell response

Summary We previously demonstrated that the lipopeptide of the myelin basic protein (MBP) immunodominant epitope in Lewis rat Palm-GpMBP(74-85) (Gp: guinea pig), which induced experimental autoimmune encephalomyelitisin vivo strongly increased the T cell proliferative responsein vitro. We extended this study to the human immunodominant epitope hMBP(83-99), synthesizing different lipophilic peptides bearing a hydrophobic chain linked through an amide or a C-C bond. To this aim, we developed a synthetic pathway for (±)-N-Fmoc-Ahd-OH (Ahd: 2-aminohexadecanoic acid) which was used to synthesize diastereomeric peptides which were successfully separated by reverse-phase high-performance liquid chromatography. MBP-specific T cell lines recognizing the immunodominant epitope hMBP(83-99) have been generated from patients affected by multiple sclerosis. Their proliferative response to the native peptide and to some lipoderivatives has been investigated. In contrast to the animal model, none of the investigated lipopeptides exhibited ‘superagonist’ activity. Prof. L. Amaducci passed away on 11 January 1998. His memory will hearten those pursuing this research.     

Synthesis of lipopeptides of the immunodominant epitope hMBP(83–99) containing amide or C-C bond linked hydrophobic chains for the study of T cell response

We previously demonstrated that the lipopeptide of the myelin basic protein (MBP) immunodominant epitope in Lewis rat Palm-GpMBP(74–85) (Gp: guinea pig), which induced experimental autoimmune encephalomyelitis in vivo strongly increased the T cell proliferative response in vitro. We extended this study to the human immunodominant epitope hMBP(83–99), synthesizing different lipophilic peptides bearing a hydrophobic chain linked through an amide or a C-C bond. To this aim, we developed a synthetic pathway for (±)-N-Fmoc-Ahd-OH (Ahd: 2-aminohexadecanoic acid) which was used to synthesize diastereomeric peptides which were successfully separated by reverse-phase high-performance liquid chromatography. MBP-specific T cell lines recognizing the immunodominant epitope hMBP(83–99) have been generated from patients affected by multiple sclerosis. Their proliferative response to the native peptide and to some lipoderivatives has been investigated. In contrast to the animal model, none of the investigated lipopeptides exhibited superagonist activity.     

Novel conformational peptide antigen which simulates an immunodominant epitope of the 2nd extracellular loop of β1-adrenoreceptor: Computer simulation, synthesis, spatial structure

By the means of computer simulation, the polypeptide antigen conformational structure that initiates the immunodominant epitope of the 2nd extracellular loop of β₁-adrenoreceptor has been built. A linear 25-member peptide corresponding to the calculated sequence was synthesized by means of the solid-phase method using Fmoc-technology, then by means of the closure of disulfide bridges, the original bicyclic polypeptide was obtained corresponding to the proposed structure of the conformational antigen. With the help of high-resolution NMR spectroscopy, the 3D structure of synthetic conformational antigen was investigated. It was shown, that the structure of bicyclic polypeptide is similar to that of the computer model. Bicyclic conformational antigen was suitable for the detection of autoantibodies in the blood serum of patients with rhythm and conductivity violation without evidence of organic disease of the cardiovascular system.     

NMR structural elucidation of myelin basic protein epitope 83–99 implicated in multiple sclerosis

Myelin basic protein peptide 83–99 (MBP83–99) is the most immunodominant epitope playing a significant role in the multiple sclerosis (MS), an autoimmune disease of the central nervous system. Many peptide analogues, linear or cyclic have been designed and synthesized based on this segment in order to inhibit the experimental autoimmune encephalomyelitis, the best well-known animal model of MS. In this study, the solution structural motif of MBP_83–99 has been performed using 2D ¹H-NMR spectroscopy in dimethyl sulfoxide. A rather extended conformation, along with the formation of a well defined α-helix spanning residues Val⁸⁷–Phe⁹⁰ is proposed, as no long-range NOE are presented. Moreover, the residues of MBP peptide that are important for T-cell receptor recognition are solvent exposed. The spatial arrangement of the side chain all over the sequence of our NMR based model exhibits great similarity with the solid state model, while both TCR contacts occupy the same region in space.     

Competitive ELISA for the identification of 35–55 myelin oligodendrocyte glycoprotein immunodominant epitope conjugated with mannan

Analogs of immunodominant myelin peptides involved in multiple sclerosis (MS: the most common autoimmune disease) have been extensively used to modify the immune response over the progression of the disease. The immunodominant 35–55 epitope of myelin oligodendrocyte glycoprotein (MOG_35–55) is an autoantigen appearing in MS and stimulates the encephalitogenic T cells, whereas mannan polysaccharide (Saccharomyces cerevisiae) is a carrier toward the mannose receptor of dendritic cells and macrophages. The conjugate of mannan-MOG_35–55 has been extensively studied for the inhibition of chronic experimental autoimmune encephalomyelitis (EAE: an animal model of MS) by inducing antigen-specific immune tolerance against the clinical symptoms of EAE in mice. Moreover, it presents a promising approach for the immunotherapy of MS under clinical investigation. In this study, a competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the MOG_35–55 peptide that is conjugated to mannan. Intra- and inter-day assay experiments proved that the proposed ELISA methodology is accurate and reliable and could be used in the following applications: (i) to identify the peptide (antigen) while it is conjugated to mannan and (ii) to adequately address the alterations that the MOG_35–55 peptide may undergo when it is bound to mannan during production and stability studies.     

Radioactive synthesis and biodistribution study of β-elemene–99mTc(CO)3 conjugates

β-Elemene, (5S,7R,10S)-(−)-(1-methyl-1-vinyl-2,4-diisopropenylcyclohexane), is an anticancer agent from traditional Chinese herbal medicine. Three novel ^99mTc(CO)₃–β-elemene conjugates were synthesized successfully, and compared with β-elemene exhibited improved water solubility. A biodistribution and micro single photon emission computed tomography image study showed there is a visible accumulation in Lewis lung cancer tumors. Y. Sun and Y. Ren contributed equally to this paper.     

Design and synthesis of novel hybrid benzamide–peptide histone deacetylase inhibitors

We designed and synthesized a series of novel hybrid histone deacetylase inhibitors based on conjugation of benzamide-type inhibitors with either linear or cyclic peptides. Linear tetrapeptides (compounds 13 and 14), cyclic tetrapeptides (compounds 1 and 11), and heptanediamide–peptide conjugates (compounds 10, 12, 15 and 16) were synthesized through on-resin solid-phase peptide synthesis (SPPS). All compounds were found to be moderate HDAC1 and HDAC3 inhibitors, with IC₅₀ values ranging from 1.3 μM to 532 μM. Interestingly, compound 15 showed 19-fold selectivity for HDAC3 versus HDAC1.     

A novel convergent method for the synthesis of α-acyl-γ-hydroxylactams and its application in the total synthesis of PI-090 and 091

A novel convergent method for the synthesis of α-acyl-γ-hydroxylactams utilizing the aldol reaction of N-Boc-protected γ-methoxylactams was developed. As the first application of this method for the synthesis of biologically active natural products, the total synthesis of platelet aggregation inhibitors PI-090 and PI-091 were also investigated and successfully achieved.     

Cyclic peptide analogs of 558–565 epitope of A2 subunit of Factor VIII prolong aPTT. Toward a novel synthesis of anticoagulants

Novel anticoagulant therapies target specific clotting factors in blood coagulation cascade. Inhibition of the blood coagulation through Factor VIII–Factor IX interaction represents an attractive approach for the treatment and prevention of diseases caused by thrombosis. Our research efforts are continued by the synthesis and biological evaluation of cyclic, head to tail peptides, analogs of the 558–565 sequence of the A2 subunit of FVIII, aiming at the efficient inhibition of Factor VIIIa–Factor IXa interaction. The analogs were synthesized on solid phase using the acid labile 2-chlorotrityl chloride resin, while their anticoagulant activities were examined in vitro by monitoring activated partial thromboplastin time and the inhibition of Factor VIII activity. The results reveal that these peptides provide bases for the development of new anticoagulant agents.     

Solution conformations of the pituitary opioid peptide dynorphin-(1–13)

Circular dichroism spectra have been measured for dynorphin-(1–13) in water and in solutions of sodium dodecyl sulfate and L-α-lysophosphatidylcholine (palmitoyl). Spectra in water have the features expected for a peptide containing little, if any, order. Small changes are brought about by L-α-lysophosphatidylcholine (palmitoyl), but the resulting spectrum retains the characteristics expected for a random coil. In contrast, sodium dodecyl sulfate produces significant changes which are those expected for induction of α helical content. Quantitative analysis of the circular dichroism spectra suggests the conformation changes from about 5% helix in water to 17% helix in sodium dodecyl sulfate. These results from experiment are in excellent agreement with those obtained from our formulation of the configuration partition function. This formulation predicts a change in helical content from 1% to 19%. The ordering influence is felt most strongly by those residues immediately following the enkephalin sequence.     

Identification and immunogenicity of immunodominant mimotopes of outer membrane protein U (OmpU) of Vibrio mimicus from phage display peptide library

Vibrio mimicus (V. mimicus) is the causative agent of ascites disease in aquatic animals. Outer membrane protein U (OmpU) is an important antigen of V. mimicus, but its protective epitopes are still unclear. A random 12-mer phage-displayed peptide library was used to screen and identify immunodominant mimotopes of the OmpU protein in V. mimicus by panning against purified OmpU-specific polyclonal antibody. Then the immunogenicity and immunoprotection in fish of these mimotopes was evaluated. Nine positive phage clones presented seven different 12- peptide sequences and more than 50% of them carried a consensus core motif of DSSK–P. These positive clones reacted with the target antibody and this interaction could be blocked, in a dose-dependent manner, by OmpU protein. Intraperitoneal injection of seven positive phage clones into fish induced a speci?c antibody response to OmpU protein. The fish immunized respectively with the positive phage clones C17, C24, C60 and C66 obtained 100% immunoprotective effect against experimental V. mimicus challenge. Taken together, these mimotopes presented by clone C17, C24, C60 and C66 were immunodominant mimotopes of the OmpU protein and exhibited a more appropriate candidate as epitope-based vaccine against V. mimicus infection in aquatic animals.     

Solution structure of the human CD4 (403–419) receptor peptide

The cytoplasmic part of CD4 is known to be essential for the interaction with the human immunodeficiency virus type 1 proteins Vpu and Nef. The 17 amino acid synthetic peptide CD4 (403–419) with the amino acid sequence of the membrane proximal part of the cytoplasmic domain of the human CD4 receptor was structurally investigated by circular dichroism and nuclear magnetic resonance spectroscopy. The average -helical content of the peptide could be estimated to be around 25%. Chemical shift index analysis and the connectivity pattern in nuclear Overhauser enhancement spectra located the -helical part of the peptide from Gln403 to Arg412. It may be speculated that this amphipathic -helix is the contact region with the Vpu and Nef proteins.The authors thank Prof. F.X. Schmid for help with the CD spectra.     

Cell surface proteoglycan-mediated uptake and accumulation of the Alzheimer's disease peptide Aβ(1–42)

Proteoglycans (PGs) have been found in Alzheimer's disease amyloid-β (Aβ) plaques and their glycosaminoglycan chains reportedly influence Aβ aggregation, neurotoxicity and intracellular accumulation in cell and animal models, but their exact pathophysiological role(s) remain unclear. We have studied the cellular uptake of fluorescently labelled Aβ(1–42) and Aβ(1–40) peptides in normal CHO cells (K1) and the mutant cell line (pgsA-745) which lacks all protein-attached heparan and chondroitin sulfate chains. After 24 h of incubation, CHO-K1 accumulates more Aβ(1–42) and Aβ(1–40) compared with CHO-pgsA-745, consistent with the suggested role of PGs in Aβ uptake. However, after short incubation times (≤3 h) there was no difference; moreover, the time evolution of Aβ(1–42) accumulation in CHO-K1 followed an unusual sigmoidal-like trend, indicating a possible involvement of PG-mediated peptide aggregation in Aβ endocytosis. Neither Aβ(1–42) nor Aβ(1–40) could stimulate uptake of a 10 kDa dextran (a general endocytosis marker) suggesting that Aβ-induced upregulation of endocytosis does not occur. CHO-K1 cells contained a higher number of Aβ(1–42)-positive vesicles, but the intensity difference per vesicle was only marginal suggesting that the superior accumulation of Aβ(1–42) stems from a higher number of endocytic events. FRET imaging support that intracellular Aβ(1–42) is aggregated in both cell types. We also report that CHO-pgsA-745 cells perform less endocytosis than CHO-K1 and, albeit this does not explain their difference in Aβ internalisation, we discuss a general method for data compensation. Altogether, this study contributes new insights into the mechanisms of PG-mediated Aβ uptake that may be relevant for our understanding of their role in AD pathology.     

pH-dependence of the specific binding of Cu(II) and Zn(II) ions to the amyloid-β peptide

Metal ions like Cu(II) and Zn(II) are accumulated in Alzheimer's disease amyloid plaques. The amyloid-β (Aβ) peptide involved in the disease interacts with these metal ions at neutral pH via ligands provided by the N-terminal histidines and the N-terminus. The present study uses high-resolution NMR spectroscopy to monitor the residue-specific interactions of Cu(II) and Zn(II) with (15)N- and (13)C,(15)N-labeled Aβ(1-40) peptides at varying pH levels. At pH 7.4 both ions bind to the specific ligands, competing with one another. At pH 5.5 Cu(II) retains its specific histidine ligands, while Zn(II) seems to lack residue-specific interactions. The low pH mimics acidosis which is linked to inflammatory processes in vivo. The results suggest that the cell toxic effects of redox active Cu(II) binding to Aβ may be reversed by the protective activity of non-redox active Zn(II) binding to the same major binding site under non-acidic conditions. Under acidic conditions, the protective effect of Zn(II) may be decreased or changed, since Zn(II) is less able to compete with Cu(II) for the specific binding site on the Aβ peptide under these conditions.     

Microwave-assisted solid-phase peptide synthesis of the 60–110 domain of human pleiotrophin on 2-chlorotrityl resin

A fast and efficient microwave-assisted solid phase peptide synthesis (MW-SPPS) of a 51mer peptide, the main heparin-binding site (60–110) of human pleiotrophin (hPTN), using 2-chlorotrityl chloride resin (CLTR-Cl) following the 9-fluorenylmethyloxycarbonyl/tert-butyl (Fmoc/tBu) methodology and with the standard N,N′-diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt) coupling reagents, is described. An MW-SPPS protocol was for the first time successfully applied to the acid labile CLTR-Cl for the faster synthesis of long peptides (51mer peptide) and with an enhanced purity in comparison to conventional SPPS protocols. The synthesis of such long peptides is not trivial and it is generally achieved by recombinant techniques. The desired linear peptide was obtained in only 30 h of total processing time and in 51% crude yield, in which 60% was the purified product obtained with 99.4% purity. The synthesized peptide was purified by reversed phase high performance liquid chromatography (RP-HPLC) and identified by electrospray ionization mass spectrometry (ESI-MS). Then, the regioselective formation of the two disulfide bridges of hPTN 60–110 was successfully achieved by a two-step procedure, involving an oxidative folding step in dimethylsulfoxide (DMSO) to form the Cys⁷⁷–Cys¹⁰⁹ bond, followed by iodine oxidation to form the Cys⁶⁷–Cys⁹⁹ bond.     

Facile synthesis of peptide–porphyrin conjugates: Towards artificial catalase

A facile synthetic method for peptide–porphyrin conjugates containing four peptide units on one porphyrin was developed using chemoselective reactions. The key building blocks, 5,10,15,20-tetrakis(3-azidophenyl)porphyrin 1 and 5,10,15,20-tetrakis(5-azido-3-pyridyl)porphyrin 2, were efficiently synthesized and used as substrates for two well-known chemoselective reactions, traceless Staudinger ligation and copper-catalyzed azide alkyne cycloaddition (so-called click chemistry). Both reactions gave the desired compounds, and click chemistry was superior for our purpose. To confirm the value of the established methodology, nine peptide–porphyrin conjugates were synthesized, and their catalase- and peroxidase-like activity in water was evaluated. Our synthetic strategy is expected to be valuable for the preparation of artificial heme protein models.     

Functional structure of the intermediate and ventral hippocampo–prefrontal pathway in the prefrontal convergent system

The hippocampo–prefrontal pathway is a unique projection that connects distant ends of the cerebral cortex. The direct hippocampo–prefrontal projection arises from the ventral to intermediate third of the hippocampus, but not from the dorsal third. It forms a funnel-shaped structure that collects information from the large hippocampal area and projects it to the prefrontal cortex. The anatomical regional differentiation of the projection has not been described. The hippocampal region is differentiated into structural and behavioural roles. For example, it has been shown that the ventral, but not the dorsal, hippocampus reciprocally connects with the amygdala and influences emotional behaviours. These data imply that hippocampal variation along the dorso–ventral axis is contained within the hippocampo–prefrontal pathway. Here, we present electrophysiological studies that demonstrate regional differences in short- but not long-term plasticity in the intermediate/posterior-dorsal and ventral routes of the hippocampo–prefrontal pathway. Furthermore, behavioural studies revealed that each route appears to play a different role in working memory. These results suggest that hippocampal regional information is processed through different routes, with the integration of individual regulatory functions in the prefrontal convergent system.     

Influence of the metal center and linker on the intracellular distribution and biological activity of organometal–peptide conjugates

Organometallic complexes conjugated to cell-penetrating peptides (CPPs) are promising systems for diagnostic imaging and therapeutic applications in human medicine. Recently, we reported on the synthesis of cymantrene(CpMn(CO)₃)–CPP conjugates with biological activity on different cancer cell lines. However, the precise mechanism of cytotoxicity remained elusive in these studies. To investigate the role of the metal center and the linker between the CpM(CO)₃ moiety and the peptide, a number of derivatives with manganese replaced by rhenium and the keto linker originally used substituted by a methylene group were prepared and fully characterized by ¹H NMR spectroscopy, infrared spectroscopy, electrospray ionization mass spectrometry, and elemental analysis as well as X-ray structure determination. The organometal–peptide conjugates as well as carboxyfluorescein-labeled derivatives thereof were prepared by solid-phase peptide synthesis, purified by high-performance liquid chromatography, and analyzed by mass spectrometry. Fluorescence microscopy studies of MCF-7 human breast cancer cells revealed an efficient cellular uptake and pronounced nuclear localization of the bioconjugates with the methylene linker compared with systems with the keto group. In addition, the latter also showed a higher cytotoxicity. In contrast, the variation of the metal center from manganese to rhenium had a negligible effect. The structure–activity relationships determined in the present work will aid in the further tuning of the biological activity of organometal–peptide conjugates.