European Peptide Society--28th Symposium and Third International Peptide Symposium. Peptides--bridges between disciplines. 5-10 September 2004, Prague, Czech Republic.
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Animals↗
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[Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP)].
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Atrial Natriuretic Factor↗
[Peptide syntheses in the aqueous phase. Synthesis of some peptides and a mixture of erratic sequence peptide by the n-carboxyanhydride method (author's transl)].
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Amino Acid Sequence↗
[Pattern of cleavage, by peptide hydrolases, of mixtures of erratic sequence peptides obtained by peptide syntheses in the aqueous phase (author's transl)].
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Amino Acid Sequence↗
New dendrimer-peptide host-guest complexes: towards dendrimers as peptide carriers.
Adamantyl urea and adamantyl thiourea modified poly(propylene imine) dendrimers act as hosts for N-terminal tert-butoxycarbonyl (Boc)-protected peptides and form chloroform-soluble complexes. Investigations with NMR spectroscopy show that the peptide is bound to the dendrimer by ionic interactions between the dendrimer outer shell tertiary amines and the C-terminal carboxylic acid of the peptide, and also through host-urea to peptide-amide hydrogen bonding. The hydrogen-bonding nature of the peptide-dendrimer interactions was further confirmed by using Fourier transform IR spectroscopy, for which the NH- and CO-stretch signals of the peptide amide moieties shift towards lower wavenumbers upon complexation with the dendrimer. Spatial analysis of the complexes with NOESY spectroscopy generally shows close proximity of the N-terminal Boc group of the peptide to the peripheral adamantyl groups on the dendrimer host. The influence of side-chain motif on interactions with the host is analyzed by using seven different N-Boc-protected tripeptides as guests for the dendrimer. Downfield shifts of up to 1.3 ppm were observed for the guest amide NH-proton signals. These shifts decreased with increasing 'bulkiness' of the amino acid side chains. Despite this, the dendrimer was capable of making multiple peptide-dendrimer complexes when presented with a library of seven peptides. The different peptides were all present in the host, which did not show specific preferences, and could be released under mild acidic conditions. These results show the general nature of the peptide-dendrimer interactions in the formation of either single- or multiple-peptide-dendrimer complexes.
Amines↗
Fine manipulation of antibody affinity for synthetic epitopes by altering peptide structure: antibody binding to looped peptides*.
Linear peptides weakly imitate antibody binding sites on globular proteins when the peptides are shown to be effective at all. As a step toward enhancing the ability of peptides to mimic epitopes, we have examined the effects of various alterations in peptide structure on antibody binding. Synthetic peptides containing the core amino acid sequence of residues 41 to 48 from horse cytochrome c were examined for their ability to bind antibodies elicited against the 41-48 peptide coupled to bovine serum albumin (BSA). Since residues 41-48 in native cytochrome c are part of an omega loop, in some peptides cysteines were incorporated for intrachain disulfide bonding to stabilize loop structure. In additional cases, glycine was incorporated as a spacer between the natural sequence and the cysteine residues with the intent of relaxing loop structure slightly. Eleven analogues containing the 41-48 sequence were tested. These included native cytochrome c and the 1-80 and 1-65 cyanogen bromide-cleaved fragments. The native protein did not bind the anti-41-48 antibodies. The other analogues differed by over three orders of magnitude in their binding. The affinity of binding was inversely related to the extent of predicted loop structure indicating that the antibodies were elicited against the 41-48 sequence in a more unfolded conformation despite the Pro Gly sequence at positions 44 and 45 that generally favors a beta turn. Surprisingly, the immunizing peptide, containing residues 41-48 only, was the poorest binding peptide. The relative impotence of 41-48 was shown to be largely due to differences at the amino terminus between the free and BSA-coupled peptides as the antibodies were elicited against the latter. The distinctions among the synthetic peptides containing the 41-48 sequence show the exquisite sensitivity of antibody binding to amino acid changes that may occur outside of an epitope and suggest modifications in peptide structure at the periphery of an epitope that can lead to desired changes in antibody affinity.
Animals↗
Presentation of a horse cytochrome c peptide by multiple H-2b class I major histocompatibility complex (MHC) molecules to C57BL/6- and bm1-derived cytotoxic T lymphocytes: presence of a single MHC anchor residue may confer efficient peptide-specific CTL recognition.
In this study the immunogenic tryptic fragment from a horse cytochrome c (cyt c) digest recognized by cytotoxic T lymphocytes (CTL) induced by in vitro peptide stimulation from C57BL/6 (B6) and mutant B6.C-H-2bm1 (bm1) mice is identified. An identical sequence, p40-53, is recognized by CTL from both B6 and bm1 mice. In addition, both B6 bm1 cloned CTL lines display unusual major histocompatibility complex (MHC) class I-restricted recognition of this peptide in that they respond to it in the context of H-2Kb, H-2Db, and H-2Kbm1 class I molecules, although the sequence lacks the usual structural Kb and Db peptide-binding motifs. Truncated analogues which resemble the lengths of naturally processed MHC class I-presented peptides, confer reactivity for B6 and bm1 CTL against EL4 (H-2b) targets as well as the L cell transfectants, L+Kb, L+Db, and L+Kbm1. The antigenic peptide with the greatest potency is p41-49, which appears to be generated by angiotensin converting enzyme cleavage of the full-length p40-53 tryptic peptide. The minimum antigenic peptide recognized by both B6 and bm1 CTL, and which targets lysis on each of the transfectants, is the hexamer p43-48 peptide from horse cyt c. Residues Pro44 and Thr47, which occupy polymorphic positions with respect to other species-variant cyt c molecules, influence recognition of these peptides differently for the B6 and bm1 CTL. The ability of H-2Kb, H-2Db, and mutant H-2Kbm1 class I molecules to present the same peptide to a single cloned CTL is discussed in the context of current knowledge of peptide anchor residues and side chain-specific binding pockets in the MHC class I peptide-binding site.
Amino Acid Sequence↗
Invariant chain peptides enhancing or inhibiting the presentation of antigenic peptides by major histocompatibility complex class II molecules.
Two soluble invariant chain (Ii) peptides with overlapping sequences had contrasting effects on the presentation of antigenic peptides by murine Ad, Ak, Ed, and Ek major histocompatibility complex (MHC) class II molecules. Naturally produced class II-associated invariant chain peptides human (h)Ii81-104/murine (m)Ii80-103 inhibited antigen presentation on these MHC class II alleles in a manner consistent with competitive inhibition. The Ii-4 peptides hIi77-92/mIi76-91 enhanced presentation of antigenic peptides on I-E class II alleles by promoting the exchange of peptides at the cell surface. Treatment of antigen-presenting cells (APC) with Ii-4 before the addition of antigenic peptide greatly enhanced subsequent T cell responses, while treatment of APC with Ii-4 after antigenic peptide binding decreased subsequent T cell responses. The hIi81-104 and mIi80-103 peptides inhibited T cell responses in both types of assays. The binding of biotinylated antigenic peptide to MHC class II-transfected L cells, as measured by flow cytometry, was inhibited by mIi80-103 and enhanced by mIi-4. Segments of Ii fragments remaining associated with MHC class II, or released Ii peptides, appear to regulate the formation of stable antigenic peptide/MHC class II complexes either positively or negatively through interactions at or near the antigenic peptide binding site. These findings open a pathway for the design of novel therapeutics based on the structure and function of natural and rationally designed fragments of Ii.
Amino Acid Sequence↗
Selective solid-phase isolation of methionine-containing peptides and subsequent matrix-assisted laser desorption/ionisation mass spectrometric detection of methionine- and of methionine-sulfoxide-containing peptides.
Methionine residues and the oxidised forms in proteins are becoming more and more important in view of their biological function. In particular, methionine sulfoxide seems to have a regulatory function. This paper presents a fast strategy for simultaneous determination of methionine- and methionine-sulfoxide-containing peptides, involving application of methionine-specific solid-phase reagent chemistry combined with matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). In the first step, methionine-containing peptides are covalently bound as sulfonium salts to glass beads, whereas methionine-sulfoxide-containing peptides and other methionine-free peptides are not bound and are washed out. The wash solution is used for MALDI-MS analysis to determine the molecular masses of these peptides and to perform, if necessary, seamless post-source decay (PSD) fragment ion analysis. Methionine-sulfoxide-containing peptides can be identified due to the characteristic metastable loss of methanesulfenic acid from the protonated molecules. In the second step, the bound peptides are cleaved from the matrix of the beads by addition of 2-mercaptoethanol at pH 8.5-8.8. The resulting peptides, mainly methionine-containing peptides, are analysed in a straightforward manner by MALDI-MS and seamless PSD. The strategy allows the fast identification of methionine- and methionine-sulfoxide-containing peptides even in complex tryptic digests, as demonstrated here for the glycoprotein antithrombin. These results show that sometimes methionine-containing tryptic peptides are not detected due to steric restrictions (e.g. glycosylation near the methionine residue) on the binding reaction, and that, on the other hand, some methionine-free peptides can be quite strongly bound non-covalently to the matrix of the beads. The latter observation indicates the necessity of seamless PSD fragment ion analysis for unambiguous identification. Furthermore, there are indications that oxidation of some methionine residues occurred to a minor extent during the solid-phase isolation steps.
Humans↗
Phospholipid structure determines the effects of peptides on membranes. Differential scanning calorimetry studies with pentagastrin-related peptides.
The effect of phospholipid structure on the interaction between small peptides and phospholipid membranes has been studied by high-sensitivity differential scanning calorimetry. The peptides used, N-Boc-beta-Ala-Trp-Met-Arg-Phe-NH2 and N-Boc-beta-Ala-Trp-Met-Lys-Phe-NH2, are basic analogs of the hormone pentagastrin. These peptides split the gel-to-liquid crystalline phase transition of synthetic phosphatidylcholines into two components. For dimyristoyl (DMPC), dipalmitoyl (DPPC) and 1-stearoyl-2-oleoyl (SOPC) phosphatidylcholines, one component remains at the temperature corresponding to that of pure lipid and the other one is shifted towards higher temperatures. With increasing peptide concentration there is a gradual increase in the enthalpy of the high-temperature component at the expense of the low-temperature one, and there is also an increase in the total enthalpy of the transition. A mixture of the peptide with distearoylphosphatidylcholine (DSPC) behaves differently, with the transition occurring at a temperature below that of the pure lipid increasing with peptide concentration. The susceptibility of various phosphatidylcholines to perturbation by the peptides increases in the order DMPC greater than SOPC greater than DPPC greater than DSPC. The effect of these peptides on the phase transitions of acidic phosphatidylglycerols is generally greater than with the corresponding phosphatidylcholines, but the dependence on the length of lipid hydrocarbon chains is similar. Perturbation of the thermotropic phase transition is strongest for dimyristoylphosphatidylglycerol, followed by the dipalmitoyl and the distearoyl analogs. The effect of the peptides on the phase transition of dimyristoylphosphatidylserine is significantly smaller compared to that observed with dimyristoylphosphatidylglycerol and it is further reduced for dimyristoylphosphatidic acid. The phase transition of this latter lipid remains virtually unchanged, even in the presence of high concentrations of the peptide. Similar resistance to the perturbation of the phase transitions by the peptides is observed for synthetic phosphatidylethanolamine. The different susceptibility of various phospholipids to perturbation by the peptides is suggested to be related to different degrees of intermolecular interaction between phospholipid molecules, and particularly to different abilities of phospholipids to form intermolecular hydrogen bonding.
Calorimetry, Differential Scanning↗
Antibodies raised against peptide fragments of bovine alpha s1-casein cross-react with the intact protein only when the peptides contain both B and T cell determinants.
In order to compare the immune response to peptides with that to the native protein, we used bovine alpha s1-casein (alpha s1-CN), a major milk protein whose conformation seems to be in a highly disordered state in solution, as the model antigen. Firstly, the T and B cell determinants on this protein were localized by a T cell proliferation assay and an enzyme-linked immunosorbent assay (ELISA), using 13 synthetic overlapping peptides encompassing the entire sequence of alpha s1-CN. T cells from alpha s1-CN-primed C3H/He mice, a high responder to alpha s1-CN, strongly proliferated in the presence of peptides 91-110, 61-80 and 46-65. In addition, peptides 151-170, 136-155 and 106-125 were also found to contain T cell determinants. On the other hand, peptides 181-199, 46-65, 76-95 and 106-125 were generally found to be immunodominant B cell determinants, while peptides 121-140, 136-155, 91-110 and 151-170 also had antibody-binding activity. The peptides were then tested for their ability to elicit a specific antibody. This revealed that only peptides 91-110, 106-125, 136-155 and 46-65 were able to produce specific antibodies that bound to the native protein as well as the peptides themselves. These peptides contained both B and T cell determinants on the intact protein. Thus, we confirmed that a peptide corresponding to both T and B cell determinants on alpha s1-CN was capable of eliciting a specific antibody that reacted with the protein as well as the peptide itself.
Allergens↗
Both major and minor peptide-binding pockets in HLA-A2 influence the presentation of influenza virus matrix peptide to cytotoxic T lymphocytes.
Most of the polymorphic residues in class I MHC molecules are concentrated in the alpha 1- and alpha 2-domains with their side chains pointing towards the antigen peptide site. Previous crystal structure analysis revealed six pockets inside the peptide-binding groove and the "extra" electron density in some of the pockets indicated that the pockets are involved in direct peptide binding. In order to investigate the functional role of individual positions from each pocket in antigen presentation, 37 HLA-A2 variants with single amino acid substitution in the peptide-binding groove were generated and used to analyse the specificity of influenza A virus matrix peptide-specific, HLA-A2-restricted CTL. The ability to present peptide by each variant was studied in detail by peptide titration, cold target inhibition, time course and limiting dilution analysis. The direct effect on peptide binding by these substitutions was determined by cell surface class I MHC molecule reconstitution analysis. The results demonstrated that each of the six peptide binding pockets plays a role in T cell recognition. Substitutions introduced into pocket F had less effect on CTL recognition than substitutions introduced in other pockets. With the exception of Tyr substitution for Phe9, single amino acid substitutions in the peptide-binding groove had only minor effects on peptide binding. Therefore, the impact of the substitutions in altering the epitopes recognized by CTL seems to be mediated through an alteration in the conformation of the bound peptide.
Antigen Presentation↗
The N-terminal 12 residue long peptide of HIV gp41 is the minimal peptide sufficient to induce significant T-cell-like membrane destabilization in vitro.
Here, we predicted the minimal N-terminal fragment of gp41 required to induce significant membrane destabilization using IMPALA. This algorithm is dedicated to predict peptide interaction with a membrane. We based our prediction of the minimal fusion peptide on the tilted peptide theory. This theory proposes that some protein fragments having a peculiar distribution of hydrophobicity adopt a tilted orientation at a hydrophobic/hydrophilic interface. As a result of this orientation, tilted peptides should disrupt the interface. We analysed in silico the membrane-interacting properties of gp41 N-terminal peptides of different length derived from the isolate BRU and from an alignment of 710 HIV strains available on the Los Alamos National Laboratory. Molecular modelling results indicated that the 12 residue long peptide should be the minimal fusion peptide. We then assayed lipid-mixing and leakage of T-cell-like liposomes with N-terminal peptides of different length as first challenge of our predictions. Experimental results confirmed that the 12 residue long peptide is necessary and sufficient to induce membrane destabilization to the same extent as the 23 residue long fusion peptide. In silico analysis of some fusion-incompetent mutants presented in the literature further revealed that they cannot insert into a modelled membrane correctly tilted. According to this work, the tilted peptide model appears to explain at least partly the membrane destabilization properties of HIV fusion peptide.
Algorithms↗
Application of a peptide bank from porcine brain in isolation of regulatory peptides.
Over the past years, the introduction of biological assay systems, random peptide sequencing and orphan receptor screening has led to the isolation and identification of new regulatory peptides with potential clinical impact. We have developed a method for separating peptides into about 300 fractions from large amounts of porcine brain tissue. The preparation of this peptide bank consists of three steps including ultrafiltration followed by cation-exchange separation and reversed-phase chromatography. These fractions represent the peptide bank with desalted and lyophilized peptides from brain tissue. Molecular masses of the peptides in the fractions are determined by matrix-assisted laser desorption ionization MS and a mass data bank is subsequently generated. For systematic analysis of the peptides, a subsequent two-step purification procedure is followed by Edman sequencing resulting in the identification of different peptides. A survival assay with a neuronal cell line revealing the stimulatory and inhibitory activities is applied as a model to test the 300 fractions. This primary screen indicates that the biological activities of the extracted peptides are easily characterized and, moreover, can be related to the biochemical entities. We conclude that the established peptide bank is an efficient and useful tool for the isolation of regulatory brain peptides applying different purification strategies.
Animals↗
Mimicry of a 21.5 kDa myelin basic protein peptide by a Maedi Visna virus polymerase peptide does not contribute to the pathogenesis of encephalitis in sheep.
Epitope mimicry is the theory that an infectious agent such as a virus causes pathological effects via mimicry of host proteins and thus elicits a cross-reactive immune response to host tissues. Weise and Carnegie (1988) found a region of sequence similarity between the pol gene of the Maedi Visna virus (MVV), which induces demyelinating encephalitis in sheep, and myelin basic protein (MBP), which is known to induce experimental allergic encephalitis (EAE) in laboratory animals. In this study, cross-reactions between sera raised in sheep against synthetic peptides of MVV (TGKIPWILLPGR) and 21.5 kDa MBP (SGKVPWLKPGR) were demonstrated using enzyme-linked immunosorbant assay (ELISA) and thin layer chromatography (TLC) immunoprobing. The antibody responses of MVV-infected sheep were investigated using ELISA against the peptides, and MBP protein, immunoprobing of the peptides on TLC plates and Western blotting against MBP. Slight significant reactions to the 21.5 kDa MBP peptide (P < 0.001) and to a lesser extent sheep MBP (P < 0.004) were detected in ELISA. The MBP peptide evoked stronger responses from more sera than the MVV peptide on immunoprobed TLC plates. On the Western blots, eight of the 23 sheep with Visna had serum reactivity to MBP. This slight reaction to MBP in MVV-infected sheep is of interest because of the immune responses to MBP evident in multiple sclerosis and EAE, but its relevance in Visna is limited since no correlation with disease severity was observed. The cell-mediated immune responses of MVV-infected sheep against similar peptides was assessed. The peptides did not stimulate proliferation of peripheral blood lymphocytes of MVV-infected sheep. Since the MVV peptide was not recognised by antibodies or T lymphocytes from MVV-infected and encephalic sheep, it was concluded that epitope mimicry of this 21.5 kDa MBP peptide by the similar MVV pol peptide was not contributing to the immunopathogensis of Visna. The slight antibody response to MBP and the MBP peptide can be attributed to by-stander effects of the immunopathology of MVV-induced encephalitis.
Animals↗
Construction of peptides with nucleobase amino acids: design and synthesis of the nucleobase-conjugated peptides derived from HIV-1 Rev and their binding properties to HIV-1 RRE RNA.
In order to develop a novel molecule that recognizes a specific structure of RNA, we have attempted to design peptides having L-alpha-amino acids with a nucleobase at the side chain (nucleobase amino acid (NBA)), expecting that the function of a nucleobase which can specifically recognize a base in RNA is regulated in a peptide conformation. In this study, to demonstrate the applicability of the NBA units in the peptide to RNA recognition, we designed and synthesized a variety of NBA-conjugated peptides, derived from HIV-1 Rev. Circular dichroism study revealed that the conjugation of the Rev peptide with an NBA unit did not disturb the peptide conformation. RNA-binding affinities of the designed peptides with RRE IIB RNA were dependent on the structure of the nucleobase moieties in the peptides. The peptide having the cytosine NBA at the position of the Asn40 site in the Rev showed a higher binding ability for RRE IIB RNA, despite the diminishing the Asn40 function. Furthermore, the peptide having the guanine NBA at the position of the Arg44 site, which is the most important residue for the RNA binding in the Rev, bound to RRE IIB RNA in an ability similar to Rev34-50 with native sequence. These results demonstrate that an appropriate NBA unit in the peptide plays an important role in the RNA binding with a specific contact such as hydrogen bonding, and the interaction between the nucleobase in the peptide and the base in the RNA can enhance the RNA-binding affinity and specificity.
Amino Acids↗
The class II MHC protein HLA-DR1 in complex with an endogenous peptide: implications for the structural basis of the specificity of peptide binding.
BACKGROUND: Class II major histocompatibility complex (MHC) proteins are cell surface glycoproteins that bind peptides and present them to T cells as part of the mechanism for detecting and responding to foreign material in the body. The peptide-binding activity exhibits allele-specific preferences for particular sidechains at some positions, although the structural basis of these preferences is not understood in detail. We have determined the 2.45 A crystal structure of the human class II MHC protein HLA-DR1 in complex with the tight binding endogenous peptide A2 (103-117) in order to discover peptide-MHC interactions that are important in determining the binding motif and to investigate conformational constraints on the bound peptide. RESULTS: The bound peptide adopts a polyproline II-like conformation and places several sidechains within pockets in the binding site. Bound water molecules mediate MHC-peptide contacts at several sites. A tryptophan residue from the beta 2 'lower' domain of HLA-DR1 was found to project into a pocket underneath the peptide-binding domain and may be important in modulating interdomain interactions in MHC proteins. CONCLUSIONS: The peptide-binding motif of HLA-DR1 includes an aromatic residue at position +1, an arginine residue at position +2, and a small residue at position +6 (where the numbering refers to the normal MHC class II convention); these preferences can be understood in light of interactions observed in the peptide-MHC complex. Comparison of the structure with that of another MHC-peptide complex shows that completely different peptide sequences bind in essentially the same conformation and are accommodated with only minimal rearrangement of HLA-DR1 residues. Small conformational differences that are observed appear to be important in interactions with other proteins.
Amino Acid Sequence↗
Determination of substrate specificity for peptide deformylase through the screening of a combinatorial peptide library.
Peptide deformylase is an essential Fe2+ metalloenzyme that catalyzes the removal of the N-terminal formyl group from nascent polypeptides in eubacteria. In vivo, the deformylase is capable of deformylating most of the polypeptides in a bacterial cell, which contain diverse N-terminal sequences. In this work, we have developed a combinatorial method to systematically examine the sequence specificity of peptide deformylase. A peptide library that contains all possible N-terminally formylated tetrapeptides was constructed on TentaGel resin, with a unique peptide sequence on each resin bead. Limited treatment with the Escherichia coli deformylase resulted in the deformylation of those peptides that are the most potent substrates of the enzyme. By using an enzyme-linked assay, the beads containing the deformylated peptides were identified and isolated. Peptide sequence analysis using matrix-assisted laser desorption ionization mass spectrometry revealed a consensus sequence, formyl-Met-X-Z-Tyr (X = any amino acid except for aspartate and glutamate; Z = lysine, arginine, tyrosine, or phenylalanine), for the E. coli enzyme. The deformylase is also capable of efficient deformylation of formyl-Phe-Tyr-(Phe/Tyr) peptides. These results demonstrate that, despite being a broad-specificity enzyme, the peptide deformylase deformylates different peptides at drastically different rates. In addition, the selectivity of peptide deformylase for the N-formyl over the N-acetyl group has been studied with N-alpha-fluoroacetyl peptides, and the results suggest that both electronic and steric factors are responsible for the observed specificity. The deformylase was also shown to exhibit esterase activity. These results will facilitate the design of specific deformylase inhibitors as potential antibacterial agents. This combinatorial method should be generally applicable to the study of the substrate specificity of other acylases and peptidases.
Amidohydrolases↗