Use Of Peptides Research Articles

Solid‐phase synthesis and purification of a set of uniformly 13C, 15N labelled de novo designed membrane fusogenic peptides

The transmembrane segments of soluble N-ethylmaleimide-sensitive factor (SNARE) proteins or viral envelope proteins drive membrane fusion, which suggests that simple synthetic biology constructs for fusion exist and can be evaluated. We describe the high-yield synthesis of a set of de novo designed fusogenic peptides for use in functional investigations, which are highly enriched in 13C and 15N using three equivalents of labelled amino acids and optimized reaction conditions minimizing aggregation. The biomimetic peptides have a high purity >90% and show reproducible and fusogenic activity that correlates well with the intended functional design characteristics, from strongly fusogenic to almost non-fusogenic.

A novel and easy method for the production of recombinant peptides for use in the generation of monospecific antisera against Lama glama IgG2b and IgG2c subclasses

We describe an easy method for the production of small recombinant peptides of 8 amino acid residues expressed as a fusion peptide with glutathione S-transferase (GST) and employed in an immunisation schedule to obtain polyclonal antibodies. The chosen peptides corresponded to specific fragments of the hinge regions of llama ( Lama glama) IgG2 subisotypes b (2bH) and c (2cH). The DNA sequences encoding each peptide were ligated individually into pGEX-5X-2, which encodes GST. Once purified from a bacterial lysate by glutathione affinity chromatography, GST-2bH and GST-2cH were used to immunize rabbits. In parallel, polyclonal antibodies were generated against specific synthetic fragments of the hinge regions of llama IgG2a (2aH) and IgG3 (3H) coupled to keyhole limpet haemocyanin (KLH). Polyclonal antibodies raised against GST-peptides and KLH-peptides were detected by enzyme-linked immunosorbent assay (ELISA), Western blot (WB) and indirect immunofluorescence (IIF). The results obtained by ELISA demonstrated that monospecific anti-IgG2 and anti-IgG3 antisera were obtained using KLH-2aH, GST-2bH, GST-2cH and KLH-3H as antigens. All antisera showed reactivity with their specific IgG isotype by WB and IIF. This simple and novel recombinant DNA methodology for the generation of two monospecific anti-isotype antisera using small peptides expressed as fusion peptides with GST offers the possibility of large scale peptide production as an alternative to chemical peptide synthesis.

Peptide-based vaccination: where do we stand?

Allergen-specific immunotherapy represents the only causative approach towards allergy treatment. Specific immunotherapy can, however, include allergic reactions and occasionally life-threatening anaphylaxis. Peptides have been evaluated as a potential therapeutic approach in atopic allergic disease because they have the potential to inhibit T-cell function but not induce anaphylaxis. Data from early clinical trials of peptide vaccination revealed that therapy was associated with a modest improvement in allergic disease, and was accompanied by a high frequency of adverse reactions. More recent studies have demonstrated improved clinical outcomes, improved safety, and have defined the mechanisms of adverse events observed in earlier studies. Mechanisms of peptide vaccination include the hyporesponsiveness of allergen-specific responses and the induction of regulatory T cells and cytokines. Novel peptide design has allowed the generation of fragments that contain T-cell stimulatory epitopes, lack B cell epitopes, and can induce protective IgG responses in both mice and humans. Other approaches have focused on hypoallergenic B-cell epitopes that induce inhibitory IgG antibodies. Peptides that specifically induce regulatory cytokine production would also enhance peptide vaccines. Several recent studies have described immunodominant epitopes from major allergens that may form candidate peptides for use in peptide vaccination. The manipulation of peptide epitopes may provide a strategy for the rational design of peptide allergy vaccines further improving safety and efficacy.

Dendrimers and Antivirals: A Review

In response to the need for antiviral agents, dendrimers, hyper-branched, well-defined, and chemically versatile molecules, have been found to have a number of potential uses. How they are used is based on knowledge of 1) how a virus interacts with its target cells, 2) how it replicates, and 3) which viral components are recognized by the immune response of the host. Many viral-host cell interactions are initiated by viral proteins binding to specific cell surface carbohydrates. Dendrimers offer an efficient means of presenting multiple ligands, or sites of contact, on a single molecule. Derivatized with carbohydrate residues, the multivalent ligands have been shown to inhibit viral binding. Dendrimers derivatized with peptides or anionic groups have also been found to inhibit infection. The availability of a number of different types of dendrimers permits synthesis of potential inhibitors of viral binding to be tailored to meet the dimensions needed for optimum adherence by the virus. Future directions should see increased studies of the use of dendrimers as carriers of 1) multiple indicators on a viral probe to increase diagnostic sensitivity, 2) multiple peptides for use as immunogens or as inhibitors of viral binding, and 3) inhibitors of viral enzymes. While the field of dendrimer chemistry is relatively young, promising results indicate that dendrimers may provide the scaffolding needed for development of effective antivirals.

Crystal Structure of an Anti-meningococcal Subtype P1.4 PorA Antibody Provides Basis for Peptide–Vaccine Design

In various western countries, subtype P1.4 of Neisseria meningitidis serogroup B causes the greatest incidence of meningococcal disease. To investigate the molecular recognition of this subtype, we crystallised a peptide (P1HVVVNNKVATH(P11)), corresponding to the subtype P1.4 epitope sequence of outer membrane protein PorA, in complex with a Fab fragment of the bactericidal antibody MN20B9.34 directed against this epitope. Structure determination at 1.95 A resolution revealed a unique complex of one P1.4 antigen peptide bound to two identical Fab fragments. One Fab recognises the putative epitope residues in a 2:2 type I beta-turn at residues P5NNKV(P8), whereas the other Fab binds the C-terminal residues of the peptide that we consider a crystallisation artefact. Interestingly, recognition of the P1.4 epitope peptide is mediated almost exclusively through the complementarity-determining regions of the heavy chain. We exploited the observed turn conformation for designing conformationally restricted cyclic peptides for use as a peptide vaccine. The conformational stability of the two peptide designs was assessed by molecular dynamics simulations. Unlike the linear peptide, both cyclic peptides, conjugated to tetanus toxoid as a carrier protein, elicited antibody responses in mice that recognised meningococci of subtype P1.7-2,4. Serum bactericidal assays showed that some, but not all, of the sera induced with the cyclic peptide conjugates could activate the complement system with titres that were very high compared to the titres induced by complete PorA protein in its native conformation administered in outer membrane vesicles.

Selecting peptides for use in nanoscale materials using phage-displayed combinatorial peptide libraries

Materials that combine inorganic components and biological molecules provide a new paradigm for synthesizing nanoscale and larger structures with tailored physical properties. These synthesis techniques utilize the molecular recognition properties of many biological molecules to nucleate and control growth of the nanoscale structure. Phage-displayed peptide libraries are a powerful tool to identify peptides that selectively recognize and bind to a variety of inorganic surfaces that are utilized in electronic and photonic devices. These libraries have been used extensively to study the peptide-mediated nucleation and growth of some metallic and semiconducting materials, and the application to designed nanostructures has been demonstrated.

An efficient synthesis of 5-aminolaevulinic acid (ALA)-containing peptides for use in photodynamic therapy

An efficient and cost-effective procedure has been devised for the preparation of urethane-protected 5-aminolaevulinic acid (5-ALA) dipeptide ester derivatives which avoids problems associated with the instability of 5-ALA under basic conditions. The procedure is also applicable to the direct synthesis of N-(α)-acetyl amino acid-ALA dipeptides in high enantiomeric purity as potential novel prodrugs for photodynamic therapy (PDT).

In vitro intestinal transport and antihypertensive activity of ACE inhibitory pea and whey digests

Angiotensin I converting enzyme (ACE) inhibitory peptides cause an antihypertensive effect if they reach the systemic circulation. This was investigated for the high ACE inhibitory activity present in peas and whey in vitro gastrointestinal digests. The samples retained high ACE inhibitory activity when incubated in Caco-2 homogenates or rat intestinal acetone powder, both sources of small intestine peptidases. Only little ACE inhibitory activity was transported through Caco-2 cell monolayers in 1 h. As the Caco-2 model is tighter than intestinal mammalian tissue, sufficient absorption of these peptides might occur in vivo. After intravenous administration of 50 mg protein kg−1 BW in spontaneously hypertensive rats (SHR), pea digest exerted a transient, but strong antihypertensive effect of 44.4 mmHg. Whey digest exerted no effect at this dose. These results suggest that pea digest could be a promising source of ACE inhibitory peptides for use in the prevention and treatment of hypertension.

Selection of active ScFv to G-protein-coupled receptor CCR5 using surface antigen-mimicking peptides.

This study describes the use of cyclic peptides for use in the selection of single-chain (ScFv) antibodies specific for the HIV-1 coreceptor CCR5, a representative G-protein-coupled receptor (GPCR). A tandem ligation strategy was developed for preparing biotinylated cyclic peptides, first through an orthogonal end-to-end ligation and then a chemoselective ligation with functionalized biotin. Cyclic peptides mimicking the extracellular loops of CCR5 and their unconstrained counterparts were then used for solution-phase selection of ScFv antibodies from a phage display antibody library. Antibodies reactive with CCR5 on cells were detected using a homogeneous high throughput assay. Of 19 isolated ScFv antibodies that bound to CCR5+ cells, three inhibited CCR5-mediated but not CXCR4-mediated HIV infection. Only ScFvs selected by binding to cyclic constrained peptides exhibited inhibitory activity. Our results demonstrate that surface-antigen mimetics of a GPCR are effective tools for selecting active, site-specific ScFv antibodies that hold promise as immunological reagents and therapeutics.

High-yield expression of isotopically labeled peptides for use in NMR studies.

Fusion protein constructs of the 56 amino acid globular protein GB-1 with various peptide sequences, coupled with the incorporation of a histidine tag for affinity purification, have generated high-yield fusion protein constructs. Methionine residues were inserted into the constructs to generate pure peptides following CNBr cleavage, yielding a system that is efficient and cost effective for isotopic labeling of peptides for NMR studies and other disciplines such as mass spectroscopy. Six peptides of varying sequences and hydrophobicities were expressed using this GB-1 fusion protein technique and produced soluble fusion protein constructs in all cases. The ability to easily express and purify recombinant peptides in high yields is applicable for biomedical research and has medicinal and pharmaceutical applications.

Photocrosslinkable polyvinyl alcohol hydrogels that can be modified with cell adhesion peptides for use in tissue engineering

Photoactive polyvinyl alcohol hydrogels (PVA) have been investigated for use as tissue engineering scaffolds. These materials allow in situ polymerization for minimally invasive implantation methods. The mechanical properties of these materials can be tailored for a variety of soft tissue applications. The Young's modulus and ultimate tensile strength of PVA hydrogels are increased with increasing polymer concentration, and highly elastic hydrogels can be formed by altering the number of crosslinkable groups per chain. Fibroblasts homogeneously seeded within 3 mm thick PVA hydrogels remained viable throughout 2 weeks in culture, with no differences in viability across the thickness of the hydrogel. Cells seeded within the PVA hydrogels also produce extracellular matrix proteins, as indicated by the production of hydroxyproline during culture. Intrinsically cell non-adhesive, these PVA hydrogels were functionalized with the cell-adhesive peptide RGDS and found to support the attachment and spreading of fibroblasts in a dose-dependent manner. These results suggest that photopolymerizable PVA hydrogels are promising for tissue engineering applications.

A panel of MHC class I restricted viral peptides for use as a quality control for vaccine trial ELISPOT assays

Vaccines in general and HIV vaccines in particular are focusing ever more on the induction of cellular immunity specifically the generation of cytotoxic T cells (CTL). As progress is made towards developing a safe and effective HIV vaccine, there is a need for a robust, sensitive and reproducible assay to evaluate vaccine-induced cellular immunogenicity in Phase II/III trials. The enzyme-linked immunospot (ELISPOT) assay fits these criteria and is a technology that is readily transferable and amenable to high-through-put screening. There is a need for reagents that can be used as positive controls and for optimizing and standardizing the assay. We selected a panel of 23 8–11 mer Influenza virus (Flu), Cytomegalovirus (CMV) and Epstein Barr virus (EBV) epitopes recognized by CD8 positive T cells and presented by 11 class I HLA-A and HLA-B alleles whose cumulative frequencies represent >100% of Caucasian individuals. We examined interferon-gamma (IFN-γ) secretion in peripheral blood mononuclear cells (PBMC) incubated with the peptides using a modified ELISPOT assay. IFN-γ secretion was detected in 15/17 (88%) HIV-1 seronegative and 14/20 (70%) HIV-1 seropositive individuals. Release of IFN-γ in response to the pool of peptides was CD8+ T cell mediated and HLA restricted. In vitro stimulation of PBMC with individual peptides or the pool of peptides led to the expansion of T cells capable of killing target cells expressing the appropriate viral antigen in a CTL assay. The size, shape and appearance of the spots produced using this peptide panel provided a standard for the establishment of acceptance criteria of spots for the evaluation of ELISPOT plates using an automated reader system.

The intracellular function of extracellular signaling peptides

A novel class of extracellular signaling peptides has been identified in Gram-positive bacteria that are actively transported into the cell to interact with intracellular receptors. The defining members of this novel class of signaling peptides are the Phr peptides of Bacillus subtilis and the mating pheromones of Enterococcus faecalis. These peptides are small and unmodified, gene encoded, and secreted by the bacterium. Most of these peptides diffuse into the extracellular medium, and when their concentration is sufficiently high, they are then actively transported into the cell by an oligopeptide permease (Opp). Once inside the cell, these peptides interact with an array of intracellular receptors. In B. subtilis, the Phr peptides regulate development of environmentally resistant spores and genetically competent cells (i.e. the natural ability to take up exogenous DNA). In E. faecalis, the mating pheromones regulate cell-cell transfer of plasmids, many of which encode antibiotic resistance or virulence factors. At least one component of the signaling pathway for these peptides is conserved in many bacteria, Opp. Opp is a non-specific transporter that transports peptides for use as carbon and nitrogen sources. The possibility that other bacteria could posses similar intracellularly functioning signaling peptides is discussed.

Peptide chemical ligation inside living cells: in vivo generation of a circular protein domain

Here we describe the first example of a peptide chemical ligation reaction performed inside a living cell. A cell-based native chemical ligation approach was developed and used to generate a circular version of the N-terminal Src homology 3 (SH3) domain from the murine c-Crk adapter protein inside Escherichia coli cells. The in vivo cyclization reaction was extremely efficient and the resulting circular protein domain was fully biologically active and able to adopt the native SH3 folded structure. This work represents an important step towards the in vivo generation of small backbone cyclic peptides for use in basic biological research.

A comparative study of the adsorption of amino acids on to calcium minerals found in renal calculi

To assess the binding of individual amino acids to the principal calcium minerals found in human kidney stones, the adsorption of 20 amino acids on to calcium oxalate monohydrate, CaHPO4*2H2O, Ca3(PO4)2 and Ca5(PO4)3OH crystals was determined over the physiological urinary pH range (pH 5-8) in aqueous solutions. All amino acids adsorbed most strongly at pH 5, and this decreased in all cases as the pH was increased. The amino acids which adsorbed most strongly were aspartic acid, glutamic acid and gamma-carboxyglutamic acid, with the last displaying the strongest affinity. All amino acids bound more avidly to calcium oxalate monohydrate than to any of the phosphate minerals. Adsorption on to CaHPO4*2H2O was generally higher than for Ca3(PO4)2 and Ca5(PO4)3OH, for which all amino acids, with the exception of gamma-carboxyglutamic acid, had only a weak affinity. The binding affinity of these acids is thought to be due to their zwitterions being able to adopt conformations in which two carboxyl groups, and possibly the amino group, can interact with the mineral surface without further rotation. The strong binding affinity of di-and tri-carboxylic acids for calcium stone minerals indicates that proteins rich in these amino acids are more likely to play a functional role in stone pathogenesis than those possessing only a few such residues. These findings, as well as the preferential adsorption of the amino acids for calcium oxalate monohydrate rather than calcium phosphate minerals, have ramifications for research aimed at discovering the true role of proteins in stone formation and for potential application in the design of synthetic peptides for use in stone therapy.

Molecular clocks.

A convenient and precise mass spectrometric method for measurement of the deamidation rates of glutaminyl and asparaginyl residues in peptides and proteins has been developed; the rates of deamidation of 306 asparaginyl sequences in model peptides at pH 7.4, 37.0 degrees C, 0.15 M Tris.HCl buffer have been determined; a library of 913 amide-containing peptides for use by other investigators in similar studies has been established; and, by means of simultaneous deamidation rate measurements of rabbit muscle aldolase and appropriate model peptides in the same solutions, the use of this method for quantitative measurement of the relative effects of primary, secondary, tertiary, and quaternary protein structure on deamidation rates has been demonstrated. The measured rates are discussed with respect to the hypothesis that glutaminyl and asparaginyl residues serve, through deamidation, as molecular timers of biological events.

Discovery and design of novel and selective vasopressin and oxytocin agonists and antagonists: the role of bioassays.

Synthetic oxytocin and vasopressin agonists and antagonists have become important tools for research and were instrumental in the identification of the four known receptor subtypes, V1a, V2, V1b (V3) and oxytocin, of these peptide hormones. However, the relative lack of receptor selectivity, particularly of the antagonists, has limited their usefulness as experimental probes and their potential as therapeutic agents. We now present some findings from our continuing studies aimed at the design of more selective oxytocin and vasopressin agonists and antagonists and a structure-activity relationship update on our recently discovered novel hypotensive vasopressin peptides. Bioassays have been, and continue to be, of critical importance in leading to the discovery of the novel agonists, antagonists and hypotensive peptides reported here. This paper highlights three main aspects of these studies. (1) Replacement of the tyrosine2 and/or phenylalanine3 residues in the V2 agonist deamino,[Val4,D-Arg8]arginine-vasopressin (dVDAVP) by thienylalanine resulted in selective V2 agonists with strikingly high potencies. However, the peptide solutions were unstable and lost activity over time. These highly potent V2 agonists, which are devoid of vasopressor activity, are promising leads for improving drugs for treating diabetes insipidus, enuresis and coagulation disorders. (2) Diaminopropionic acid and diaminobutyric acid substitution at position-5 in oxytocin and in V1a antagonists yielded, respectively, the first specific antagonist for the oxytocin receptor, desGly-NH2,d(CH2)5[D-Trp2,Thr4,Dap5]OVT and the first specific antagonist for the vasopressin V1a receptor, d(CH2)5[Tyr(Me)2,Dab5]AVP. The availability of single receptor subtype-specific or selective antagonists will enhance our ability to delineate receptor functions. Utilising these new receptor specific probes, we were able to show that the uterotonic action of vasopressin is mediated principally by oxytocin and not by V1a receptors. (3) Replacement of the phenylalanine3 residue in the V1a/V2/oxytocin antagonist, d(CH2)5[D-Tyr(Et)2,Val4]AVP, with arginine3 yielded the novel, selective, hypotensive vasopressin peptide, d(CH2)5[D-Tyr(Et)2,Arg3,Val4]AVP (Peptide I). Bioassay characterisations of Peptide I show that its vasodepressor action is independent of the peripheral autonomic, bradykinin, nitric oxide and prostaglandin systems and is not mediated by the known classical oxytocin and vasopressin receptors. These findings suggest the existence of a new vasopressin receptor subtype that may be relevant to the vasodilating action of vasopressin in regional vascular beds. Iodinatable hypotensive peptides have been synthesised and could be developed as markers for the putative new receptor. Ongoing structure-activity relationship studies on Peptide I have led to more potent and selective hypotensive peptides for use as new research tools and as leads for the development of a new class of antihypertensive agents.

Exploring immunological specificity using synthetic peptide combinatorial libraries

The definition of epitopes for human B and T cells is fundamental for the understanding of the immune response mechanism and its role in the prevention and cause of human disease. This understanding can be applied to the design of diagnostics and synthetic vaccines. In recent years, the understanding of the specificity of B and T cells has been advanced significantly by the development and use of combinatorial libraries made up of thousands to millions of synthetic peptides. The use of this approach has had four major effects: first, the definition of high affinity ligands both for T cells and antibodies; second, the application of alternative means for identifying immunologically relevant peptides for use as potential preventive and therapeutic vaccines; third, a new appreciation of the requirements for TCR interactions with peptide-MHC complexes in immunogenicity; fourth, the establishment of new principles regarding the level of cross-reactivity in immunological recognition.

ChemInform Abstract: Synthesis of Peptides for Use as Immunogens

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Improved bioavailability of orally delivered peptides and polypeptides in teleost fish

Summary A gonadoliberin analogue (GnRHa) whose biological role is the stimulation of gonadotropin (GTH II) secretion, has been encapsulated together with intestinal absorption enhancers and proteolytic enzyme inhibitors. The capsules have been coated to prevent their solubilization at acid pH in the stomach. After oral delivery of the capsules by forced-feeding, high GnRHa intestinal absorption and high bioavailability in the blood results. GTH II secretion was stimulated in a dose-dependent manner by the orally delivered GnRHa in three species having different digestive tract structures and different feeding habits: the rainbow trout, the common carp and the African catfish. In addition, the oral delivery of GnRHa also results in the induction of ovulation in all three species at doses similar to those usually used to induce spawning in these species. There was no competition of nutrient given together with the microcapsules on GnRHa absorption. From these results, it becomes possible to develop special diets containing microencapsulated bioactive peptides for use in therapy or to modify nonvital functions in fish. In the present case, an orally-induced ovulation treatment will be developed.