Peptide binding motifs and specificities for HLA-DQ molecules

 HLA-DQ molecules have been associated with susceptibility to a number of autoimmune and other diseases, possibly through the peptide repertoire that can be presented by different allelic products. It is thus of importance to understand which peptides can be bound by different HLA-DQ allelic products. Recently, a model for HLA-DQ has been described and used to derive peptide positional environments for HLA-DQ allelic products. By combining the peptide positional environments with known HLA-DQ peptide binding motifs, a set of predictions of likely anchor motifs for many of the products of HLA-DQ allelic variants are made and presented in a table referred to as a roadmap for HLA-DQ peptide binding specificities. Received: 3 March 1999 / Revised: 20 May 1999     

The use of norbornene derivatives in the synthesis of conformationally constrained peptides and pseudo-peptides

Summary The desymmetrisation ofendo-norborn-5-ene-2,3-dicarboxylic anhydride by proline esters has been used to prepare conformationally constrained pseudo-peptides with two peptide chains parallel to one another. A Curtius rearrangement on the desymmetrication adduct produced the corresponding isocyanate which was used to prepare both a peptide incorporating anendo-2-amino-3-carboxy-norborn-5-ene unit, and a pseudo-peptide with two peptide chains parallel to one another but offset by the presence of a urea unit. The conformational analysis of the resulting peptides was carried out, and the norbornene unit was found to induce the formation of β-turns and parallel β-sheets.     

Identification of Brucella cell wall antigenic determinants by immunoscreening of a random peptide library expressed at the surface of filamentous phage

Summary Antibodies against the 89 kDa Brucella abortus outer membrane protein (OMP) are detected in 68% of B. abortus infected cows. A monoclonal antibody, specifically directed against Brucella OMP89, has been used to screen a phage-displayed peptide library. We describe here results obtained from affinity selection of phages with mAb A7617E3C3 in three rounds of biopanning using a cysteine-constrained peptide library expressed on the surface of filamentous bacteriophages pVIII major coat protein. Deduced amino acid sequences of the peptide region of clones positively detected by colony immunoblotting experiments indicate the presence of a consensus sequence. Peptides corresponding to the most frequently represented sequence have been synthesized. Monoclonal antibody binding to selected phages is inhibited by corresponding cyclic peptides, but not by linear peptides. This confirms the specificity of the peptide sequence for its paratope but also the importance of a certain conformation for binding.     

Peptide purification by affinity chromatography based on α‐ketoacyl group chemistry

Significant advances have been achieved in the fields of peptide/protein synthesis, permitting the preparation of large, complex molecules. Shortcomings, however, continue to exist in the area of peptide purification. This paper details some studies we undertook to develop a new strategy for peptide purification based on a reactivity of α‐ketoacyl groups in peptides. The α‐ketoacyl peptide was generated from N^ε‐acyl‐lysyl‐peptide in the solid phase via a transamination reaction using glyoxylic acid and nickel(II) ion. Cleavage of the α‐ketoacyl group with o‐phenylenediamine gave the target peptide in an acceptable yield and purity. We first carried out a careful step‐by‐step optimization of the purification conditions using a model peptide. The strategy was then used in the purification of a transmembrane peptide that could not be effectively purified using a conventional RP‐HPLC system due to the strong hydrophobicity of the peptide and its high tendency to aggregate. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

The use of inulinase pre-pro leader peptide for secretion of heterologous proteins in Saccharomyces cerevisiae

Summary The pre-pro and pre regions of inulinase leader peptide have been used to secrete the heterologous proteins such as human lipocortin-1 and human interleukin-2 (IL2) in Saccharomyces cerevisiae. The inulinase leader peptide consisting of both pre and pro regions directed an efficient secretion of such heterologous proteins in S. cerevisiae, while deletion of pro region prevents them from being secreted. Both pre and pro regions of inulinase leader peptide appears to be necessary to direct secretion of heterologous proteins in S. cerevisiae.     

Enhancement of the inhibitory effect of an IL‐15 antagonist peptide by alanine scanning

IL‐15 is a proinflammatory cytokine that acts early in the inflammatory response and has been associated with several autoimmune diseases including rheumatoid arthritis, where it had been proposed as a therapeutic target. We recently reported an IL‐15 antagonist peptide corresponding to sequence 36–45 of IL‐15 (KVTAMKCFLL) named P8, which specifically binds to IL‐15Rα and inhibits IL‐15 biological activity with a half maximal inhibitory concentration (IC50) of 130 µ m in CTLL‐2 proliferation assay. In order to improve binding of peptide P8 to the receptor IL‐15Rα, we used an Ala scan strategy to study contribution of each individual amino acid to the peptide's antagonist effect. Here, we found that Phe and Cys are important for peptide binding to IL‐15Rα. We also investigated other single site mutations and replaced the second Lys in the sequence by the polar non‐charged amino acid threonine. The resulting peptide [K6T]P8 exhibited a higher activity than P8 with an IC50 of 24 µm . We also found that this peptide was more active than peptide P8 in the inhibition of TNFα secretion by synovial cells from rheumatoid arthritis patients. The peptide [K6T]P8 described in this work is a new type of IL‐15 antagonist and constitutes a potential therapeutic agent for rheumatoid arthritis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Design and Synthesis of De Novo Peptide for Manganese Binding

The de novo peptide with 63-residues (MHB) has been synthesized biochemically and used for the binding of manganese (II) ions. In designed peptide, the leucine of the peptide dA1 (prototype) was replaced by His27 and Asp41 for binding the manganese (II) ions. The different chromatography studies and mass determination showed that new peptide folds into a monomeric, highly helical with a active site structure similar to the native Mn–SOD in an aqueous solution. Electron paramagnetic resonance (EPR) study suggested that the peptide binds single manganese (II) ion per molecule loosely with K _D value of about 36 μM. The circular dichroism (CD) studies demonstrated that the helical contents of the peptide did not change significantly even after binding the metal ions. The SOD activity study of the Mn–peptide complex showed that the IC₅₀ values is 8.08 μM.     

Characterization of a new maleimido functionalization of gold for surface plasmon resonance spectroscopy

Para‐maleimidophenyl (p‐MP) modified gold surfaces have been prepared by one‐step electrochemical deposition and used in surface plasmon resonance (SPR) studies. Therefore, a FITC mimotope peptide (MP1, 12 aa), a human mucin 1 epitope peptide (MUC, 9 aa) and a protein with their specific antibodies were used as model systems. The peptides were modified with an N‐terminal cysteine for covalent and directed coupling to the maleimido functionalized surface by means of Michael addition. The coupling yield of the peptide, the binding characteristics of antibody and the unspecific adsorption of the analytes were investigated. The results expand the spectrum of biosensors usable with p‐MP by widely used SPR and support its potential to be versatile for several electrochemical and optical biosensors. This allows the combination of an electrochemical and optical read‐out for a broad variety of biomolecular interactions on the same chip. Copyright © 2014 John Wiley & Sons, Ltd.     

Functional roles of peptide cotransmitters at neuromuscular synapses inAplysia

Neuromuscular synapses inAplysia have been used as model systems to study peptidergic cotransmission. Here we describe neuromuscular preparations in which it has been possible to investigate the physiological consequences of peptide transmitter release in detail. In the first preparation, the release of peptide cotransmitters from identified motor neuron B15 has been shown to be sensitive to the pattern of stimulation. High frequencies and long burst durations evoke peptide release that modulates muscle contractions in a manner similar to that produced by exogenous cotransmitter. By contrast, the release of the same peptide transmitters from motor neuron B1 show little dependence on pattern. We conclude that there are no stimulation patterns that are prerequisites for peptide release. Peptide cotransmitter release from motor neuron B47 has also been studied. B47, depending on the stimulation pattern, uses either ACh, which acts as a conventional inhibitory transmitter, or Ach plus neuropeptides, which act as excitatory modulatory cotransmitters. Thus, neuropeptide cotransmitters have the capability to greatly increase synaptic plasticity at neuromuscular synapses.     

Identification and Cloning of Peptide Synthetase Genes of Thermostable Bacilli Using the Polymerase Chain Reaction

Fourteen thermophilic and thermostable strains of the genus Bacillus were studied. Total DNA was isolated from these strains and used as a template to identify and clone peptide synthetase genes by means of polymerase chain reaction. Amplified DNA fragments were cloned into a phasmid vector, and nucleotide sequences of cloned fragments were determined. Stringent thermophilic strains were shown to lack genetic systems, which are responsible for the synthesis of secondary metabolites and homologous to the known peptide synthetase genes. On the contrary, thermostable strains had peptide synthetases and produced antimicrobial secondary metabolites. Analysis of nucleotide sequences and deduced amino acid sequences of cloned PCR fragments from B. licheniformis strains VK2, VK21, and VK2101 showed that they are absolutely identical. The cloned DNA fragment was found to be a portion of the open reading frame, which we termed ORF1. Data from analysis of a partial nucleotide sequence of the peptide synthetase gene of strain VK21 indicated that a 9.5-kb region of chromosomal DNA contains sequences of two genes homologous to the B. subtilis peptide synthetase genesdhbB and dhbF. Strains VK2, VK21, and VK2101 were shown to synthesize siderophores. A method for screening bacteria with peptide synthetase genes has been developed.     

Identification of preferred substrate sequences of microbial transglutaminase from Streptomyces mobaraensis using a phage-displayed peptide library

Microbial transglutaminase (TGase) from Streptomyces mobaraensis (MTG) has been used in many industrial applications because it effectively catalyzes the formation of covalent cross-linking between glutamine residues in various substrate proteins and lysine residues or primary amines. To better understand the sequence preference around the reactive glutamine residue by this enzymatic reaction, we screened preferred peptide sequences using a phage-displayed random peptide library. Most of the peptides identified contained a consensus sequence, which was different from those previously found for mammalian TGases. Of these, most sequences had a specific reactivity toward MTG when produced as a fusion protein with glutathione-S-transferase. Furthermore, the representative sequence was found to be reactive even in the peptide form. The amino acid residues in the sequence critical for the reactivity were further analyzed, and the possible interaction with the enzyme has been discussed in this paper.     

Development of a novel T cell-oriented vaccine using CTL/Th-hybrid epitope long peptide and biodegradable microparticles,against an intracellular bacterium

Antigen-specific CD8+ T-lymphocytes (cytotoxic T-lymphocytes: CTL), as well as CD4+ T-lymphocytes (helper T-lymphocytes: Th), simultaneously play an important role in the elimination of intracellular bacteria such as Mycobacterium tuberculosis and Listeria monocytogenes. Administration of T-cell epitope short peptide needs large numbers of peptides for effective vaccination due to its easily degradable nature in vivo. In this respect, biocompatible and biodegradable microparticles combined with CTL/Th-hybrid epitope long peptide (long peptide) have been used to diminish the degradation of loaded peptide. The aim of this study is to develop a novel T cell-oriented vaccine against intracellular bacteria that is composed of long peptide and poly (lactic-co-glycolic acid) (PLGA) microparticles. Mouse bone marrow-derived dendritic cells (BMDCs) were loaded with L. monocytogenes listeriolysin O (LLO)-derived or ovalbumin (OVA)-derived long peptide/PLGA or other comparative antigens. The antigen-loaded BMDCs were injected subcutaneously into the flank of mice twice, and then, the spleens were collected and lymphocyte proliferation and interferon-γ production were evaluated. The median diameter of the PLGA spheres was 1.38 μm. Both LLO- and OVA-long peptide/PLGA showed significantly more robust CTL and Th proliferations with higher interferon-γ production than the long peptide alone or CTL and Th short peptides/PLGA vaccination. Furthermore, the LLO-long peptide/PLGA vaccination showed a significantly lower bacterial burden in spleens compared with the long peptide alone or the CTL and Th short peptides/PLGA vaccination after the challenge of lethal amounts of L. monocytogenes. These results suggest that the novel vaccine taking advantages of CTL/Th-hybrid epitope long peptide and PLGA microparticle is effective for protection against intracellular bacteria.     

Standard dimensions forcis N-methyl peptide unit and flexibility ofcis peptide units

The mean dimensions of thecis N-methyl peptide unit have been arrived at by analysing the crystal structure data on compounds containing such units. These dimensions can be used as standard in conformational studies on cyclic peptides. While the bonds meeting at C are almost coplanar, those meeting at N show a slight pyramidal disposition. A comparison of the dimensions of the normal and N-methylatedcis peptide units show that there are perceptible differences in the parameters connected with N. In addition, the flexibility of thecis peptide unit has been analysed by studying the distribution of the parameters in different classes of compounds such as cyclic di, tri and higher peptides. The salient features are: (i) The angle C^αCN in cyclic dipeptide and the angle C^δNC^α in higher peptides tend to be lower, when the peptide unit is associated with a prolyl residue; (ii) in cyclic tripeptides the internal anglesviz., C^αCN and CNC^α are significantly larger thereby increasing the intra-annular space; (iii) the bond C^α-C is distinctly shorter when it occurs in cyclic dipeptides. The results lead to the conclusion that thecis peptide unit takes up aneed-based flexibility in its dimension.     

Functional expression,purification, and antimicrobial activity of a novel antimicrobial peptide MLH in Escherichia coli

In this study, a novel heterozygous antimicrobial peptide MLH was synthesized, expressed, purified, and characterized. The peptide Md-cec-LL-37_Hp (MLH) was selected through bioinformatic analysis using musca domestica antimicrobial peptide (Cec-Med), human antimicrobial peptide LL-37, and helicobacter pylori antimicrobial peptide (Hp) as parent peptides. The target gene was synthesized by overlap extension PCR (SOE-PCR) and connected to the expression vector pET-32a (+), and the recombinant plasmid pET-32a-MLH was transformed to Escherichia coli for constructing pET-32a-MLH/BL21 (DE3). Isopropyl β-D-thiogalactoside (IPTG) was used to induce protein expression, and SDS-PAGE and western blot were adopted to test the target protein. And fermentation condition was optimized to get the mass expression of the fusion protein. The Ni²⁺ affinity chromatographic column was used to purify. Active heterozygous peptide was obtained after renaturation. Finally, the activity of the heterozygous antimicrobial peptide was identified. The fusion peptide showed significant antimicrobial effect on both E. coli and Staphylococcus aureus.     

Blockade of a mitochondrial cationic channel by an addressing peptide: An electrophysiological study

Summary A voltage-dependent cationic channel of large conductance is observed in phospholipid bilayers formed at the tip of microelectrodes from proteoliposomes derived from mitochondrial membranes. This channel was blocked by a 13-residue peptide with the sequence of the amino terminal extremity of the nuclear-coded subunit IV of cytochromec oxidase. The blockade was reversible, voltage- and dose-dependent. The peptide did not affect the activity of aTorpedo chloride channel observed under the same conditions. From experiments with phospholipid monolayers, it is unlikely that the peptide inserts into bilayers under the experimental conditions used. The blockade was observed from both sides of the membrane, being characterized by more frequent transitions to the lower conductance states, and a maximum effect was observed around 0 mV. Channels, the gating mechanism of which had been eliminated by exposure to trypsin, were also blocked by the peptide. For trypsinized channels, the duration of the closure decreased and the blockade saturated at potentials below –30 mV. These observations are consistent with a translocation of the peptide through the channel. Dynorphin B, which has the same length and charge as the peptide, had some blocking activity. Introduction of negative charges in the peptide by succinylation suppressed the activity.     

Recombinant peptide fusion construction for protein-templated catalytic palladium nanoparticles

Although peptide-enabled synthesis of nanostructures has garnered considerable interest for use in catalytic applications, it has so far been achieved mostly via Fmoc based solid phase peptide synthesis. Consequently, the potential of longer peptides in nanoparticle synthesis have not been explored largely due to the complexities and economic constraints of this chemical synthesis route. This study examines the potential of a 45-amino acid long peptide expressed as fusion to green fluorescence protein (GFPuv) in Escherichia coli for use in palladium nanoparticle synthesis. Fed-batch fermentation with E. coli harboring an arabinose-inducible plasmid produced a product containing three copies of Pd4 peptide fused to N-terminus of GFPuv ((Pd4)₃-GFPuv). Using the intrinsic fluorescence of GFPuv, expression and enrichment of the fusion product was easily monitored. Crude lysate, desalted lysate, and an ion-exchange enriched fraction containing (Pd4)₃-GFPuv were used to test the hypothesis that high purity of the biologic material used as the nanoparticle synthesis template may not be necessary. Nanoparticles were characterized using a variety of material science techniques and used to catalyze a model Suzuki–Miyaura coupling reaction. Results demonstrated that palladium nanoparticles can be synthesized using the soluble cell extract containing (Pd4)₃-GFPuv without extensive purification or cleavage steps, and as a catalyst the crude mixture is functional.     

Development of a bicistronic expression system in the branchiopod crustacean Daphnia magna

The viral 2A peptides have recently been used for bicistronic expression in various organisms. In this system, a single mRNA that codes for two proteins flanking the 2A peptide can be translated simultaneously into each protein by ribosomal skipping at this peptide sequence. Here, we tested the function of the Thosea asigna insect virus 2A (T2A) peptide in the branchiopod crustacean Daphnia magna—an emerging model of evolutionary developmental biology. First, we used transgenic Daphnia that expresses a potential bicistronic RNA containing mCherry and histone H2B‐ green fluorescent protein (GFP) open reading frames upstream and downstream of the T2A sequence, respectively. Microscopic observation revealed difference of localization of the two proteins in the cell, homogenous distribution of mCherry and nuclear localization of H2B‐GFP. Second, we changed localization of mCherry from cytoplasm to plasma membrane by attachment of a consensus myristoylation motif in the bicistronic reporter. RNA that codes for this new bicistronic reporter was injected into eggs. At gastrulation stage, we found spectrally distinct fluorescence with enough intensity and resolution to detect membrane localized mCherry and nuclear GFP. These results indicate that the T2A peptide functions in D. magna and T2A‐mediated bicistronic expression would be a promising tool for evo‐devo studies of this species.     

Cis-trans isomerism of the peptide bond

The molecular orbital method PCILO is applied to eight. N-monsubstituted amides. Experimentally known geometric properties are reasonably predicted by minimization of total energy with respect to molecular geometry. The same procedure shows that molecular deformations during rotation around the peptide bond significantly lower calculated barriers. Experimental heats of activation and the free-energy changes associated with cis–trans isomerism are in good agreement with those calculated, which include qualitative estimates of configurational entropy contributions to the isomerism energies. Both the calculations and revised infrared data indicate that N-phenylurethane, which has been used as a model for the cis peptide bond, should be predominantly trans. However the variations in rotational barriers and cis–trans isomerism energies among the N-monosubstituted amides provide no reason to suppose that the cis peptide bond should be excluded from stable protein conformations.     

Using a neural network to identify potential HLA-DR1 binding sites within proteins

The presentation by antigen-presenting cells of immunodominant peptide segments in association with major histocompatibility complex (MHC) encoded proteins is fundamental to the efficacy of a specific immune response. One approach used to identify immunodominant segments within proteins has involved the development of predictive algorithms which utilize amino acid sequence data to identify structural characteristics or motifs associated with in vivo antigenicity. The parallel-computing technique termed ‘neural networking’ has recently been shown to be remarkably efficient at addressing the problem of pattern recognition and can be applied to predict protein secondary structure attributes directly from amino acid sequence data. In order to examine the potential of a neural network to generalize peptide structural feature related to binding within class II MHC-encoded proteins, we have trained a neural network to determine whether or not any given amino acid of a protein is part of a peptide segment capable of binding to HLA-DR1. We report that a neural network trained on a data base consisting of peptide segments known to bind to HLA-DR1 is able to generalize features relating to HLA-DR1-binding capacity (r = 0.17 and p = 0.0001).     

A large‐scale proteogenomics study of apicomplexan pathogens—Toxoplasma gondii and Neospora caninum

Proteomics data can supplement genome annotation efforts, for example being used to confirm gene models or correct gene annotation errors. Here, we present a large‐scale proteogenomics study of two important apicomplexan pathogens: Toxoplasma gondii and Neospora caninum. We queried proteomics data against a panel of official and alternate gene models generated directly from RNASeq data, using several newly generated and some previously published MS datasets for this meta‐analysis. We identified a total of 201 996 and 39 953 peptide‐spectrum matches for T. gondii and N. caninum, respectively, at a 1% peptide FDR threshold. This equated to the identification of 30 494 distinct peptide sequences and 2921 proteins (matches to official gene models) for T. gondii, and 8911 peptides/1273 proteins for N. caninum following stringent protein‐level thresholding. We have also identified 289 and 140 loci for T. gondii and N. caninum, respectively, which mapped to RNA‐Seq‐derived gene models used in our analysis and apparently absent from the official annotation (release 10 from EuPathDB) of these species. We present several examples in our study where the RNA‐Seq evidence can help in correction of the current gene model and can help in discovery of potential new genes. The findings of this study have been integrated into the EuPathDB. The data have been deposited to the ProteomeXchange with identifiers PXD000297and PXD000298.