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PubMed2002

Liquid-phase peptide synthesis on polyethylene glycol (PEG) supports using strategies based on the 9-fluorenylmethoxycarbonyl amino protecting group: application of PEGylated peptides in biochemical assays.

Stepwise synthetic assembly of polypeptide chains reversibly linked to polyethylene glycol represents a hybrid between traditional solution and solid-phase chemistries and combines the inherent advantages of both approaches. The technical simplicity and scalability of the liquid-phase peptide synthesis method renders it particularly attractive for multiple parallel syntheses, combinatorial approaches and the large-scale preparation of peptides. The versatile protection strategy based on the N alpha-fluorenylmethoxycarbonyl group commonly used in solid-phase peptide synthesis was adapted to the liquid-phase approach. Fluoride ions were used rather than the conventional organic base piperidine for the repetitive amino-deprotection step. Using a range of acid- and base-labile linkers between the polymer and the peptide, it was shown that free and fully side-chain protected peptides can be obtained using our version of the liquid-phase peptide synthesis method. Protocols for simultaneous multiple syntheses requiring a minimum of equipment are presented and the use of polyethylene glycol-bound peptides in biochemical binding and functional assay systems is demonstrated.

Amino Acids↗
PubMed2005

Normalization of relative peptide ratios derived from in-gel digests: applications to protein variant analysis at the peptide level.

The ability to detect protein variants and post-translational modifications by mass spectrometry has become increasingly important. Unfortunately, the ability to detect variants in large intact proteins (>80,000 Da) is limited. Even in the analysis of smaller proteins, algorithms are required to determine the presence of a 2 Da mass shift in an intact 13 kDa protein because the isotopic distribution of the multiply charged ions of the variant overlaps the wild-type distribution. Fortunately, most modern instruments are capable of detecting variants in tryptic peptides derived from intact proteins. If a single common variant protein is known, the presence of a variant tryptic peptide can be easily demonstrated. A more difficult issue is the case where a multiplicity of peptides with multiple amino acid substitutions can be associated with pathology. In these cases a decrease in the relative amount of a variant peptide relative to other internal tryptic fragments would be diagnostic. However, the variability associated with the analysis of in-gel or solution digests of proteins, related to efficiencies in digestion, extraction and ionization, confounds variant analysis at the peptide level. A strategy was developed to normalize for this variability by utilizing multiple isotopically labeled internal standards for multiple peptides derived from the same protein. Erythrocyte spectrin from 36 normal and 25 abnormal osmotic fragility samples was analyzed as a test case. Three isotopically labeled target peptides comprising the alpha/beta-spectrin self-association sites were added to purified digested alpha-spectrin. The utilization of multiple internal standards demonstrates the capability to normalize for sample variability due to ionization efficiency, solvent effects, digestion and extraction efficiency.

Algorithms↗
PubMed1997

Conformational preferences of a peptide corresponding to the major antigenic determinant of foot-and-mouth disease virus: implications for peptide-vaccine approaches.

The conformational preferences in solution of a peptide corresponding to the GH loop of the VP1 capsid protein from the foot-and-mouth disease virus were examined by proton nuclear magnetic resonance and circular dichroism. The GH loop is the major antigenic determinant of the virus and participates in cell attachment through an integrin-like Arg-Gly-Asp sequence. The synthetic peptide, corresponding to residues Gly132 to Ser162 of the VP1 capsid protein of the serotype O, is largely disordered in aqueous solution as shown by the absence of long- and medium-range NOE contacts and by random-like chemical shifts values. Helical contents in aqueous solution were estimated to be less than 10%, as determined by extrapolation of trifluoroethanol titration from CD measurements, in good agreement with estimations from NMR experiments. In the presence of 40% trifluoroethanol an alpha-helix, flanked by two proline residues between Asn12 (Asn143 in the intact protein) and Leu28 (159), is induced. This contrasts with the 3(10) helix observed between residues Leu148 and Val155 in the crystal structure of the dithiothreitol-reduced virus, indicating that the cosolvent does not stabilize a residual, low-populated structure, similar to that in the intact virus. Several algorithms also fail to predict the structure found in the intact virus because these are based mainly on local sequence information. The lack of structure of the peptide in aqueous solution strongly suggests that the conformational determinants sufficient for the structure stabilization of this highly immunogenic antigen are mostly dictated by interactions of the loop with other regions of the virus structure, and do not arise from local amino acid sequence information. The ability of designed GH-VP1 peptides to neutralize anti-virus antibodies is likely to arise from antibody-induced conformation of the peptide and its application as peptide vaccines is not straightforward. Similarly, insertion of these peptides in carriers or macromolecular assemblies as vaccine vectors would depend on the conformation adopted at the insertion site and its success cannot be predicted.

Algorithms↗
PubMed1993

Recombinant technology as an alternative to chemical peptide synthesis: expression and characterization of HIV-1 Rev recombinant peptides.

Recombinant DNA technology has been widely used for the production of proteins in recent years. In this paper, we describe the expression and the purification of two specific peptides corresponding to parts of the human immunodeficiency virus Rev protein. The strategy of this method relies on the chemical synthesis of a pair of two complementary oligodeoxynucleotides corresponding to the coding region of the peptide of interest and the subsequent cloning into a prokaryotic expression vector. Transformation of Escherichia coli with these synthetic gene constructs yielded high production levels of recombinant protein in the bacteria. The recombinant protein was composed of two moieties, one corresponding to an "affinity handle" and the second corresponding to the peptide. Chemical cleavage of the fused protein followed by a combination of affinity chromatography and rp-HPLC led to rapid and convenient peptide purification. Peptide fused to the affinity handle as well as cleaved peptide were fully characterized by N-terminal microsequencing and mass spectrometry. The data presented demonstrate that although the major recombinant products had the expected amino acid composition, we detected unexpected processing such as alternative cleavage within the signal peptide, modified cysteines, and deamidations. These results emphasize the importance of the complete characterization of recombinant products by efficient analytical tools such as N-terminal microsequencing and mass spectrometry.

Amino Acid Sequence↗
PubMed1997

The three-dimensional structures of a polysaccharide binding antibody to Cryptococcus neoformans and its complex with a peptide from a phage display library: implications for the identification of peptide mimotopes.

The three-dimensional structure of 2H1, a protective monoclonal antibody to Cryptococcus neoformans, has been solved at 2.4 A resolution, in both its unbound form and in complex with the 12 amino acid residue peptide PA1 (GLQYTPSWMLVG). PA1 was previously identified as a potential mimotope of the cryptococcal capsular polysaccharide by screening of a phage display peptide library. Peptide binding is associated with only minor rearrangements of some side-chains and a small shift in the H2 loop of the antibody. The peptide assumes a tightly coiled conformation consisting of one inverse gamma-turn and one type II beta-turn that serves to place the entire peptide motif, consisting of ThrP5, ProP6, TrpP8, MetP9 and LeuP10, into a depression in the antibody combining site. A small number of H-bonds between peptide and antibody contribute to the affinity and specificity. Poor steric complementarity between PA1 and the antibody heavy chain along with the fact that the majority of the interactions between 2H1 and PA1 involve van der Waals interactions with the light chain may explain why this peptide acts as only a partial mimotope of the capsular polysaccharide epitope.

Amino Acid Sequence↗
PubMed2000

[Antimicrobial peptides and peptide antibiotics].

Antimicrobial peptides are naturally occurring antibiotics. As part of the innate immune system of vertebrates they have direct antimicrobial function. Further, they can act as mediators of inflammation. Their antimicrobial spectrum covers gram-positive and -negative bacteria as well as fungi and certain viruses. Based on their structure, antimicrobial peptides can be divided into several families. Peptides of the defensin, cathelicidin, and histatin families have been isolated from humans, where they have been found in defense cells, such as macrophages or neutrophils, as well as in epithelial cells. Decreased production of antimicrobial peptides is associated with immune deficiencies. Further, lung disease in cystic fibrosis may be linked to the dysfunction of antimicrobial peptides. Based on naturally occurring antimicrobial peptides, derivates of these molecules were developed as innovative antibiotic drugs. The present review focuses on the biology of antimicrobial peptides as well as their potential as drugs.

Anti-Bacterial Agents↗
PubMed2004

WT1 peptide vaccination combined with BCG-CWS is more efficient for tumor eradication than WT1 peptide vaccination alone.

A Wilms' tumor gene WT1 is expressed at high levels not only in most types of leukemia but also in various types of solid tumors, including lung and breast cancer. WT1 protein has been reported to serve as a target antigen for tumor-specific immunotherapy both in vitro in human systems and in vivo in murine models. We have shown that mice immunized with WT1 peptide or WT1 cDNA could reject a challenge from WT1-expressing tumor cells (a "prophylactic" model). However, it was not examined whether WT1 peptide vaccination had the potency to reject tumor cells in a "therapeutic" setting. In the present study, we demonstrated for the first time that WT1 peptide vaccination combined with Mycobacterium bovis bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) was more effective for eradication of WT1-expressing tumor cells that had been implanted into mice before vaccination (a "therapeutic" model) compared with WT1 peptide vaccination alone. An intradermal injection of BCG-CWS into mice, followed by that of WT1 peptide at the same site on the next day, generated WT1-specific cytotoxic T lymphocytes (CTLs) and led to rejection of WT1-expressing leukemia or lung cancer cells. These results showed that BCG-CWS, which was well known to enhance innate immunity, could enhance WT1-specific immune responses (acquired immunity) in combination with WT1 peptide vaccination. Therefore, WT1 peptide vaccination combined with BCG-CWS may be applied to cancer immunotherapy in clinical settings.

Animals↗
PubMed1984

Peptide mapping by polyacrylamide gel electrophoresis after cleavage at aspartyl-prolyl peptide bonds in sodium dodecyl sulfate-containing buffers.

Protein samples prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis are preferentially cleaved at aspartyl-prolyl peptide bonds upon heating at 110 degrees C. The presence of aspartyl-prolyl peptide bonds in a protein can therefore be detected by gel electrophoresis of heated samples and the resulting peptides mapped. The method of heat cleavage also works well with proteins in bands cut from electrophoresed gels using modified stacking conditions in the second electrophoresis. An immunoblotting procedure for peptide mapping of nanogram quantities of specific proteins in complex mixtures is demonstrated. Peptide maps produced by aspartyl-prolyl peptide bond cleavage of fructose-1,6-bisphosphatases from different sources show the effectiveness of the above techniques and suggest a conservation of aspartyl-prolyl peptide bonds in pig kidney and mouse and rat liver fructose-1,6-bisphosphatases.

Animals↗
PubMed1992

Reciprocal effects between opioid peptides and human polymorphonuclear leukocytes--II. Enhancement of phorbol myristate acetate-induced respiratory burst in human polymorphonuclear leukocyte by opioid peptides previously exposed to activated oxygen species.

Activated oxygen species (AOS) have often been shown to promote strong modifications in peptide structures and thus in their biological functions. In the present study, the immunomodulatory effects of Leu-enkephalin, beta-endorphin, dynorphin and some fragments are evaluated, before and after exposure of peptides to AOS, by studying their influence on human polymorphonuclear leukocyte (PMN) respiratory burst. None of the tested opioid peptides (modified or not) were shown to affect resting oxidative metabolism in the PMNs. The effects of peptides on phorbol myristate acetate (PMA)-stimulated production of AOS were measured in a lucigenin-enhanced chemiluminescence assay. Before AOS exposure, the opioid peptides suppressed the PMA-stimulated respiratory burst in human PMNs and a U-shaped dose-response relationship was observed. Conversely, after AOS exposure the opioid peptides enhanced the PMA-stimulated respiratory burst in human PMNs and an inverted U-shaped dose-response relationship was observed. In both cases, the maximal effect was reached at peptide concentrations of 10(-10)M-10(-12) M.

Binding Sites↗
PubMed1995

Use of a constrain phage displayed-peptide library for the isolation of peptides binding to HIV-1 nucleocapsid protein (NCp7).

It has been shown that peptide libraries are powerful tools for the identification of peptides showing new binding specificity. This technology was applied to the isolation of peptides binding to HIV-1 nucleocapsid protein (NCp7). Three different prolin reach peptide sequences, interacting with NCp7, were isolated, from a constrained phage displayed-peptide library of 10(8) independent clones. The three peptide sequences, isolated from the peptide library, were shown to bind NCp7 in the region 30-52. Moreover, two of them share the PP-(D/E)R consensus sequence.

Amino Acid Sequence↗
PubMed1990

Sequence analysis of homogeneous peptides of shark immunoglobulin light chains by tandem mass spectrometry: correlation with gene sequence and homologies among variable and constant region peptides of sharks and mammals.

Morphologically, sharks are living fossils that are remarkably similar to their Devonian ancestors of ca. 400 million years ago. If a parallel conservation in biochemical properties characterizes shark evolution, knowledge of the properties of shark immunoglobulins should provide information on the structure of primordial immunoglobulins and their genes. The problem of polyclonality of shark immunoglobulins has precluded detailed analysis of shark immunoglobulin light polypeptide chains. Here, we approach the problem of obtaining direct sequence information on polyclonal light chains of shark immunoglobulins by isolating homogeneous peptides from tryptic digests of shark light chains and sequencing these by tandem mass spectrometry. To confirm the location of the peptides, we isolated a complementary DNA (cDNA) clone from a sandbar shark cDNA library in the expression vector lambda gt11, identifying the clone by its ability to produce a peptide serologically detectable using rabbit antibody to purified shark light chain. The correspondence between peptide sequence and that derived from gene sequence provided direct proof that the gene studied was that of a major expressed serum light chain. Using this combined approach, we isolated homogeneous peptides from both constant and variable regions. The variable region peptides showed homology to corresponding sequences of mammalian V lambda and V kappa sequences. The constant region gene sequence we obtained was homologous to mammalian C lambda sequence. The four constant region tryptic peptides we sequenced corresponded exactly to stretches of the C lambda sequence derived from the DNA sequence. The combined approach described here shows that shark light chains exhibit heterogeneity at both the protein and gene level, but that the constant regions of these chains can be identified as homologs of mammalian lambda chains and that evolutionary conservation has occurred in V region sequences ranging from elasmobranchs to man.

Amino Acid Sequence↗
PubMed1994

Lipophilic multiple antigen peptide system for peptide immunogen and synthetic vaccine.

We describe the development and structural requirements of a new lipophilic multiple antigen peptide (lipoMAP) system for immunogens that contains a built-in lipophilic adjuvant and has the ability to elicit cytotoxic T-lymphocytes (CTLs). In addition to the peptide antigens of choice at the amino terminus, the basic lipoMAP design consists of three components: a tetravalent symmetrical core matrix containing two levels of branching beta-alanyl-lysine as a building unit, a hydrophilic Ser-Ser dipeptide linker, and at the carboxyl terminus, palmitoyl lysines (PL) with alternating chirality. An 18-residue peptide from the third variable region in the gp120 of HIV-1 was used as antigen in eight models for a structure-function study. Alternating palmitoyl lysine (PL) was introduced as the lipid anchor and built-in adjuvant because D and L Lys (Pal) was found via molecular modeling to best mimic phosphatidylcholine and thus provide the most stable peptide antigens on the ordered lipid membranes. The requirements of the palmitoyl lysines and the L-Ser-L-Ser linker were crucial, since replacement with palmitoyl serines or L-Ser-D-Ser linkers led to a marked decrease in immune response. The stoichimetric ratio of PL vs MAP was also important. Multiple antigen peptide (MAP) constructs without the lipophilic PLs, those that were underlipidated and contained one PL, or those that were overlipidated containing four PLs, were ineffective. LipoMAPs containing three palmitic acids elicited significant humoral responses in oil-based emulsion and liposomes, but not in water or alum formulations. LipoMAP containing only two PLs was found best to be incorporated in liposomes and elicited a significant immune response and cytotoxic T-lymphocytes (CTLs). These models were compared favorably with a preparation using tripalmitoyl-S-glyceryl cysteine (P3C) as the lipid anchor. We also developed a modular synthesis of MAP-P3C that incorporated P3C as a premade unit containing a thiopyridine, which simplified the overall scheme and minimized oxidation during stepwise peptide synthesis. This lipoMAP model is a new addition to the design of our macromolecular assemblage approach mimicking peptide antigens on the surface of micro-organisms. It may be a potentially useful approach to the design of a synthetic vaccine for humans.

Amino Acid Sequence↗
PubMed1994

HLA-B27 binding peptides derived from the 57 kD heat shock protein of Chlamydia trachomatis: novel insights into the peptide binding rules.

In this study we investigate the 57 kD heat shock protein of Chlamydia trachomatis for potential HLA-B27 restricted T cell epitopes. This protein is known to elicit T cell immunity, as judged by delayed type hypersensitivity. We synthesized 24 peptides containing the B27 anchor amino acid arginine at position 2, according to the rules previously described for peptide binding to MHC class I molecules. The nonamer peptides were tested in an in vitro assembly assay; six out of the 24 peptides bind to HLA-B27 although their sequences only partially match the HLA-B27 binding motif. Two of these six peptides carry negatively charged amino acids which apparently fit into the P1 pocket and in three out of the six a positively charged amino acid fits into the P3 pocket. In addition, two octamer peptides stabilized the HLA-B27 molecule without containing an appropriate amino or carboxy terminus. Therefore our data suggest that current binding rules will need to be refined before they can be used to accurately predict potential T cell epitopes. Furthermore our HLA-B27-binding peptides should prove useful probes for the study of the processing and presentation of this bacterial antigen, and of changes in the T cell repertoire induced by this form of infection.

Amino Acid Sequence↗
PubMed1994

Experimental allergic encephalomyelitis induced by the peptide encoded by exon 2 of the MBP gene, a peptide implicated in remyelination.

The discovery of T lymphocytes reactive to the peptide encoded by exon 2 of the myelin basic protein (MBP) gene in multiple sclerosis (MS) patients has drawn attention to MBP isoforms harboring that peptide as candidate autoantigens. Previously, immunological studies in MS had almost exclusively used the more abundant 18.5 kDa isoform of MBP, which does not contain the exon 2 peptide. Investigations of experimental allergic encephalomyelitis (EAE) have also focussed on the 18.5 kDa MBP isoform and its peptides. Since EAE is an animal model widely used to study MS, we examined the encephalitogenic potential of exon 2 peptide in the SJL/J mouse. Evidence for increased expression of exon 2-containing isoforms during remyelination in mouse CNS suggested that exon 2-sensitized T cells, with encephalitogenic capacity, might be important in the perpetuation of relapsing EAE (rEAE). Our experiments have demonstrated that exon 2 peptide is inherently immunogenic in SJL mice and that EAE could be induced by the adoptive transfer of exon 2-sensitized lymphocytes. Furthermore, the disease could be accentuated by the transfer of short-term exon 2-reactive lines or by a combination of adoptive transfer and antigenic challenge with exon 2 peptide. The immunodominant epitope(s) appeared to localize to the segment bordered by amino acids 59-85.

Amino Acid Sequence↗
PubMed1993

Biological distribution of 99mTc-labeled YIGSR and IKVAV laminin peptides in rodents: 99mTc-IKVAV peptide localizes to the lung.

Two laminin-derived peptides containing either YIGSR or IKVAV (single amino acid code) sequences were radiolabeled with 99mTc and their biological distribution evaluated in rodents. Both 99mTc-peptides cleared rapidly from the circulation though the kidney, and to a lesser extent, through the liver. 99mTc-YIGSR peptide did not accumulate in any organ examined in normal, tumored, and emphysemic mice. The 99mTc-IKVAV peptide localized within 10 min to the lung of normal animals, resulting in lung-to-blood ratios of approximately 23:1. The 99mTc-IKVAV peptide localized to lung after submicron filtration and after intraperitoneal injection, suggesting that particulates do not major role in localization. Pre-incubation of 99mTc-IKVAV peptide in whole blood decreased lung localization, suggesting that margination of radiolabeled cells does not play a major role in the lung localization. When 99mTc-IKVAV was injected into mice with tumored lungs (melanoma), the lung uptake was markedly increased (up to 20% injected dose higher than control lungs) at all time points examined (10, 30, and 120 min). When 99mTc-IKVAV was injected into mice with genetic emphysema, the lung uptake was markedly decreased at all time points. The localization of the 99mTc-IKVAV-containing peptide to the lung is consistent with a receptor-based mechanism.

Amino Acid Sequence↗
PubMed1994

Differences in peptide presentation between B27 subtypes: the importance of the P1 side chain in maintaining high affinity peptide binding to B*2703.

Susceptibility to spondyloarthropathies is strongly associated with the MHC class I molecule HLA-B27, and is hypothesized to result from the presentation of arthritogenic peptides. Subtypes of B27 that differ structurally but are disease-associated ought to be capable of presenting such peptides, while nondisease-associated subtypes would not. We demonstrate that B*2703, the predominant West African B27 subtype that may not predispose to disease, is not recognized by most B*2705-alloreactive CTL, and does not efficiently present a known B*2705-restricted influenza A nucleoprotein (NP) peptide. We show inefficient presentation is due to a reduced binding affinity of B*2703 for the NP peptide. Furthermore, substituting Arg for the naturally occurring Ser at P1 of the NP peptide, restores high affinity binding and efficient presentation by B*2703. Our results suggest that B*2703 will bind and present efficiently only a subset of the peptides that bind to B*2705, in particular those with Arg or Lys at P1. The apparent lack of disease in individuals with B*2703 may be due to an inability to bind and present putative arthritogenic peptides.

Alleles↗
PubMed2006

Activities of four frog skin-derived antimicrobial peptides (temporin-1DRa, temporin-1Va and the melittin-related peptides AR-23 and RV-23) against anaerobic bacteria.

The activities of two antimicrobial peptides belonging to the temporin family (temporin-1DRa from Rana draytonii and temporin-1Va from Rana virgatipes) and two peptides with structural similarity to the bee venom peptide melittin (AR-23 from Rana tagoi and RV-23 from R. draytonii) were evaluated against a range of reference strains and clinical isolates of anaerobic bacteria. These peptides were selected because they show broad-spectrum growth inhibitory activity against reference strains of several medically important aerobic microorganisms and against clinical isolates of methicillin-resistant Staphylococcus aureus. All peptides showed relatively high potency (minimum inhibitory concentration (MIC) </=25 microM) against the Gram-positive bacilli Propionibacterium acnes and Clostridium tertium and the Gram-positive cocci Peptostreptococcus anaerobius. Activity was lower and more variable against Clostridium septicum, Clostridium perfringens and Peptostreptococcus asaccharolyticus. Growth of the Gram-negative bacilli Bacteroides fragilis and Fusobacterium spp. was poorly inhibited, but all the peptides were active (MIC</=25 microM) against Prevotella melaninogenica. The clinical utility of the melittin-related peptides is limited by their toxicities, but temporin-1DRa and temporin-1Va have relatively low haemolytic activity against human erythrocytes and so represent candidates for drug development, particularly for topical therapy of infected surface lesions.

Amino Acid Sequence↗
PubMed2003

The role of acidic residues and of sodium ion adduction on the gas-phase H/D exchange of peptides and peptide dimers.

Gas-phase H/D exchange is widely used for characterizing the structure of ions. However, many structural parameters that affect the rate of H/D exchange are poorly understood, which complicates the interpretation of experimental data. Here, the effects of sodium ion adduction on the rate of H/D exchange with D2O for a series of peptides and peptide dimers with varying numbers of acidic residues are described. The maximum number of sodium ion adducts that can be accommodated by the peptides and peptide dimers in this study is N + 1, where N is the number of free carboxylic acid groups. The formation of methyl-esters at all carboxylic acid groups, or the replacement of all the acidic hydrogens with sodium ions, effectively shuts down H/D exchange with D2O. In contrast, both the rate and the extent of H/D exchange with D2O are increased for most of the peptides and peptide dimers by the adduction of an intermediate number of sodium ions. These results are consistent with the H/D exchange occurring via a salt-bridge mechanism and show that the presence of two carboxylic acid groups is much better than one. The results with peptide dimers also indicate that surface accessibility may not be a dominant factor in the extent of H/D exchange for these ions.

Acids↗