Production and characterization of peptide antibodies

Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies are powerful tools in experimental biology and are easily produced to any peptide of choice. A widely used approach for production of peptide antibodies is to immunize animals with a synthetic peptide coupled to a carrier protein. Very important is the selection of the synthetic peptide, where factors such as structure, accessibility and amino acid composition are crucial. Since small peptides tend not to be immunogenic, it may be necessary to conjugate them to carrier proteins in order to enhance immune presentation. Several strategies for conjugation of peptide-carriers applied for immunization exist, including solid-phase peptide-carrier conjugation and peptide-carrier conjugation in solution. Upon immunization, adjuvants such as Al(OH)(3) are added together with the immunogenic peptide-carrier conjugate, which usually leads to high-titred antisera. Following immunization and peptide antibody purification, the antibodies are characterized based on their affinity or specificity. An efficient approach for characterization of peptide antibodies is epitope mapping using peptide based assays. This review describes standard solid-phase approaches for generation of peptide antibodies with special emphasis on peptide selection, generation of peptide conjugates for immunization and characterization of the resulting peptide antibodies.     

Improving mass and liquid chromatography based identification of proteins using bayesian scoring

We present a method for peptide and protein identification based on LC-MS profiling. The method identified peptides at high-throughput without expending the sequencing time necessary for CID spectra based identification. The measurable peptide properties of mass and liquid chromatographic elution conditions are used to characterize and differentiate peptide features, and these peptide features are matched to a reference database from previously acquired and archived LC-MS/MS experiments to generate sequence assignments. The matches are scored according to the probability of an overlap between the peptide feature and the database peptides resulting in a ranked list of possible peptide sequences for each peptide submitted. This method resulted in 6 times more peptide sequence identifications from a single LC-MS analysis of yeast than from shotgun peptide sequencing using LC-MS/MS.     

Limited validity of group additivity for the folding energetics of the peptide group

The principle of group additivity is a standard feature of analyses of the energetics of protein folding, but it is known that it may not always be valid for the polar peptide group. The neighboring residue effect shows that group additivity is not strictly valid for a heteropeptide. We show here that group additivity fails seriously for peptide groups close to either peptide end, even for a homopeptide that has blocked end groups with no formal charges involved. The failure of group additivity is caused by the electrostatic character of the solvation of peptide polar groups and is illustrated with values of the electrostatic solvation free energy (ESF) calculated by DelPhi. Solvation free energies and enthalpies are known experimentally for monoamides and are often used to model the solvation of peptide groups, but ESF results show that monoamide values are very different from those of peptide groups. A main cause of the difference is that peptide solvation depends on the dipole-dipole interactions made between adjacent peptide groups, which vary with peptide conformation. Ligands that interact with the peptide backbone by an electrostatic mechanism could show a similar peptide end effect, and hydrogen exchange results from the literature confirm that exchange rates are position-dependent close to peptide ends.     

Combinatorial solid phase peptide synthesis and bioassays

Solid phase peptide synthesis method, which was introduced by Merrifield in 1963, has spawned the concept of combinatorial chemistry. In this review, we summarize the present technologies of solid phase peptide synthesis (SPPS) that are related to combinatorial chemistry. The conventional methods of peptide library synthesis on polymer support are parallel synthesis, split and mix synthesis and reagent mixture synthesis. Combining surface chemistry with the recent technology of microelectronic semiconductor fabrication system, the peptide microarray synthesis methods on a planar solid support are developed, which leads to spatially addressable peptide library. There are two kinds of peptide microarray synthesis methodologies: pre-synthesized peptide immobilization onto a glass or membrane substrate and in situ peptide synthesis by a photolithography or the SPOT method. This review also discusses the application of peptide libraries for high-throughput bioassays, for example, peptide ligand screening for antibody or cell signaling, enzyme substrate and inhibitor screening as well as other applications.     

The construction of a bioactive peptide database in Metazoa

Bioactive peptides play critical roles in regulating most biological processes in animals, and have considerable biological, medical and industrial importance. A number of peptides have been discovered usually based on their biological activities in vitro or based on their sequence similarities in silico. Through searches in Swiss-Prot and Trembl protein databases using BLAST alignment tools and other in silico methods, all currently known bioactive peptides and their precursor proteins are extracted. In addition, 132 recently discovered putative peptide genes in Drosophila as well as their orthologs in other species are collected. In total, 20 027 bioactive peptides from 19 438 precursor proteins covering 2820 metazoan species are retained, and they, respectively, make up a peptide and a peptide precursor database. The peptides and peptide precursor proteins are further classified into 373 families, 178 of which are represented by Prosite Pfam or Smart motifs, or by typical peptide motifs that have been constructed recently. The remaining 195 families are novel peptide families. The motifs characterizing the 178 peptide families are saved into a peptide motif database. The peptide, peptide precursor and peptide motif databases (version 1.0) are the most complete peptide, precursor and peptide motif collection in Metazoa so far. They are available on the WWW at http://www.peptides.be/.     

Affinity purification of proteins using ligands derived from peptide libraries

Peptide libraries can be used to identify ligands that bind specifically to a desired protein. These peptides may have significant advantages as specific ligands for affinity chromatography separations. This article describes the use of one of such peptide, Try-Asn-Phe-Glu-Val-Leu, as a ligand for the purification of S-protein using affinity chromatography. General strategies for peptide immobilization are discussed and the conditions for peptide immobilization to Emphazetrade mark gel are optimized. The effects of peptide orientation and peptide densities on protein binding are studied. Results indicate that the peptide affinity is not affected by the orientation of the peptide during immobilization, but association constants can be reduced by one order of magnitude when compared with the values in solution.With increased peptide density, the protein binding capacity of the gel increases, but both the percentage of peptide utilization and apparent binding constant between immobilized peptide and S-protein decrease. S-protein is separated from a mixture with BSA via affinity chromatography using specific elution with the peptide in solution.Finally, direct purification of S-protein from an enzymatic digestion mixture of ribonuclease A is demonstrated.(c) 1995 John Wiley & Sons, Inc.     

Peptide heterodimers for molecular imaging

One main issue with peptide-based molecular imaging probes is their relatively low tumor affinity and short retention time. To improve peptide binding affinity, multivalency approach has been introduced. Traditionally, this approach involves the use of peptide homodimers or homomultimers in which peptide ligands of the same type are constructed with suitable linkers. Recently, a new approach using peptide heterodimers has emerged as a promising method for targeting multi-receptor over-expressed tumor cells. Significant affinity enhancements have been observed with peptide heterodimers compared with their parent peptide monomers. In a peptide heterodimer, two different peptide ligands capable of targeting two different receptors are covalently linked. The binding modes of peptide heterodimers can be monovalent or bivalent depending on whether simultaneous binding of two ligands can be achieved. Increased local ligand concentration and improved binding kinetics contribute to enhanced binding in both monovalent- and bivalent binding modes, while multivalency effect also plays an important role in bivalent binding mode. As many tumors overexpress multiple receptors, more peptide heterodimer-based molecular imaging probes are expected to be developed in future. This review article will discuss the peptide homodimers and heterodimers for molecular imaging with special emphasis on peptide heterodimers.     

Peptide crosslinked micelles: a new strategy for the design and synthesis of peptide vaccines

This report presents a new and simple methodology for the synthesis of multicomponent peptide vaccines, named the peptide crosslinked micelles (PCMs). The PCMs are core shell micelles designed to deliver peptide antigens and immunostimulatory DNA to antigen-presenting cells (APCs). They are composed of immunostimulatory DNA, peptide antigen, and a thiopyridal derived poly(ethylene glycol)-polylysine block copolymer. The peptide antigen acts as a crosslinker in the PCM strategy, which allows the peptide antigen to be efficiently encapsulated into the PCMs and also stabilizes them against degradation by serum components. Cell culture studies demonstrated that the PCMs greatly enhance the uptake of peptide antigens into human dendritic cells.     

The Choice of HLA‐Associated Peptide Enrichment and Purification Strategy Affects Peptide Yields and Creates a Bias in Detected Sequence Repertoire

Understanding the most appropriate workflow for biochemical human leukocyte antigen (HLA)‐associated peptide enrichment prior to ligand sequencing is essential to achieve optimal sensitivity in immunopeptidomics experiments. The use of different detergents for HLA solubilization as well as complementary workflows to separate HLA‐bound peptides from HLA protein complex components after their immunoprecipitation including HPLC, C18 cartridge, and 5 kDa filter are described. It is observed that all solubilization approaches tested led to similar peptide ligand identification rates; however, a higher number of peptides are identified in samples lysed with CHAPS compared with other methods. The HPLC method is superior in terms of HLA‐I peptide recovery compared with 5 kDa filter and C18 cartridge peptide purification methods. Most importantly, it is observed that both the choice of detergent and peptide purification strategy creates a significant bias for the identified peptide sequences, and that allele‐specific peptide repertoires are affected depending on the workflow of choice. The results highlight the importance of employing a suitable strategy for HLA peptide enrichment and that the obtained peptide repertoires do not necessarily reflect the true distributions of peptide sequences in the sample.     

Cooperativity during the formation of peptide/MHC class II complexes

To generate an effective immune response, class II major histocompatibility complex molecules (MHCII) must present a diverse array of peptide ligands for recognition by T lymphocytes. Peptide/MHCII complexes are stabilized by hydrophobic anchoring of peptide side chains to pockets in the MHCII protein and the formation of hydrogen bonds to the peptide backbone. Many current models of peptide/MHCII association assume an additive and independent contribution of the interactions between major MHCII pockets and corresponding side chains in the peptide. However, significant conformational rearrangements occur in both the peptide and MHCII during binding. Therefore, we hypothesize that peptide binding to MHCII could be viewed as a folding process in which both molecules cooperate to produce the final conformation. To directly test this hypothesis, we adapt a serial mutagenesis strategy to study cooperativity in the interaction of the human MHCII HLA-DR1 and a peptide derived from influenza hemagglutinin. Substitutions in either the peptide or HLA-DR1 that are predicted to interfere with hydrogen bond formation show cooperative effects on complex stability and affinity. Substitution of a peptide side chain that provides a hydrophobic contact also contributes to the cooperative effect, suggesting a role for all energetic sources in the folding process. We propose that cooperativity throughout the peptide-binding groove reflects the folding of segments of the MHCII molecule into helices around the peptide with a concomitant folding of the peptide into a polyproline helix. The implications of cooperativity for peptide/MHCII structure and epitope selection are discussed.     

Chromatographic Analysis of Gastrin-Releasing Peptide In Heat-Inactivated Fetal Calf Serum

Abstract. Gastrin-releasing peptide and the carboxyterminal fragments gastrin-releasing peptide [14-27] and [18-27] are found by radioimmunology and high performance liquid chromatography to be present in heat-inactivated fetal calf sera. Peptides containing the carboxyterminal sequence of gastrin-releasing peptide are known to display mitogenic activity. Thus, gastrin-releasing peptide represents a new class of mitogenic factors that are present in heat-inactivated fetal calf serum.     

Chromatographic analysis of gastrin-releasing peptide in heat-inactivated fetal calf serum

Gastrin-releasing peptide and the carboxyterminal fragments gastrin-releasing peptide [14-27] and [18-27] are found by radioimmunology and high performance liquid chromatography to be present in heat-inactivated fetal calf sera. Peptides containing the carboxyterminal sequence of gastrin-releasing peptide are known to display mitogenic activity. Thus, gastrin-releasing peptide represents a new class of mitogenic factors that are present in heat-inactivated fetal calf serum.     

Protein kinase C (PKC) isozyme-specific substrates and their design

Protein kinase C (PKC), a phospholipid-dependent serine/threonine kinase, appears to be involved in the signal transduction response to many hormones and growth factors; there are 11 different PKC isozymes. Because PKC isozymes directly and/or indirectly participate in signal transduction pathways of normal and transformed cells through phosphorylation of target proteins, it is critical to understand the diversity of the intracellular signaling pathways regulated by each PKC isozyme. Thus, PKC isozyme-specific substrates are useful to understand the characterization of the intracellular signaling pathways for each PKC isozyme. Consensus sequences and sequence information obtained from PKC target proteins are very important to design PKC isozyme-specific peptide substrates. Moreover, computational prediction programs of phosphorylation sites using a library of peptide substrates aid in the fast design of PKC isozyme-specific peptide substrates. Although a large number of target proteins and synthetic peptides for PKCs are known, only two peptide substrates (peptide 422–426 of murine elongation factor-1α and Alphatomega peptide) have been reported as PKC isozyme-specific peptide substrates. This discussion will review the literature concerning these native and synthetic PKC isozyme-specific peptide substrates and their design.     

Evaluation of absolute peptide quantitation strategies using selected reaction monitoring

The use of internal peptide standards in selected reaction monitoring experiments enables absolute quantitation. Here, we describe three approaches addressing calibration of peptide concentrations in complex matrices and assess their performance in terms of trueness and precision. The simplest approach described is single reference point quantitation where a heavy peptide is spiked into test samples and the endogenous analyte quantified relative to the heavy peptide internal standard. We refer to the second approach as normal curve quantitation. Here, a constant amount of heavy peptide and a varying amount of light peptide are spiked into matrix to construct a calibration curve. This accounts for matrix effects but due to the presence of endogenous analyte, it is usually not possible to determine the lower LOQ. We refer to the third method as reverse curve quantitation. Here, a constant amount of light peptide and a varying amount of heavy peptide are spiked into matrix to construct a calibration curve. Because there is no contribution to the heavy peptide signal from endogenous analyte, it is possible to measure the equivalent of a blank sample and determine LOQ. These approaches are applied to human plasma samples and used to assay peptides of a set of apolipoproteins.     

Lipid-Induced Organization of a Primary Amphipathic Peptide: A Coupled AFM-Monolayer Study

To better understand the nature of the mechanism involved in the membrane uptake of a vector peptide, the interactions between dioleoylphosphatidylcholine and a primary amphipathic peptide containing a signal peptide associated with a nuclear localization sequence have been studied by isotherms analysis of mixed monolayers spread at the air-water interface. The peptide and the lipid interact through strong hydrophobic interactions with expansion of the mean molecular area that resulted from a lipid-induced modification of the organization of the peptide at the interface. In addition, a phase separation occurs for peptide molar fraction ranging from about 0.08 to 0.4 Atomic force microscopy observations made on transferred monolayers confirm the existence of phase separation and further reveal that mixed lipid-peptide particles are formed, the size and shape of which depend on the peptide molar fraction. At low peptide contents, round-shaped particles are observed and an increase of the peptide amount, simultaneously to the lipidic phase separation, induces morphological changes from bowls to filamentous particles. Fourier transform infrared spectra (FTIR) obtained on transferred monolayers indicate that the peptide adopts a β-like structure for high peptide molar fractions. Such an approach involving complementary methods allows us to conclude that the lipid and the peptide have a nonideal miscibility and form mixed particles which phase separate. Received: 31 July 1998/Revised: 4 November 1998     

The human class II MHC protein HLA-DR1 assembles as empty alpha beta heterodimers in the absence of antigenic peptide.

We have produced the human class II histocompatibility protein, HLA-DR1, as a soluble, secreted glycoprotein in insect cells infected with baculoviruses carrying truncated alpha and beta subunit genes. The peptide-binding site is empty, and the empty molecules are fully competent to bind antigenic peptide. We used the empty molecules to measure an intrinsic rate for peptide association, and to investigate the role of peptide in stabilizing the class II structure. Peptide binding kinetics for the empty molecule are only 10-fold faster than for peptide exchange into an occupied site, suggesting that a conformational change may accompany peptide binding. The native alpha beta heterodimer assembles in the absence of antigenic peptide, but peptide binding stabilizes the empty heterodimer against aggregation and against SDS-induced denaturation.     

Processing, secretion, and anti-HIV-1 activity of IL-16 with or without a signal peptide in CD4+ T cells

CD4+ T cells transfected with the C-terminal 130 aa of human IL-16 are rendered resistant to HIV infection. Whether the constitutively expressed IL-16 acts intracellularly, extracellularly, or both is not clear. To address this question and to further study the processing of IL-16, new constructs containing either the C-terminal 130 aa or the C-terminal 100 aa (PDZ-like motif) were constructed with and without a signal peptide. Pulse-chase experiments and treatment of cells with brefeldin A and/or tunicamycin showed that IL-16 is secreted despite the absence of a signal peptide, but with a signal peptide IL-16 is processed through the endoplasmic reticulum-golgi pathway and is glycosylated. Cells expressing IL-16 linked to a signal peptide secrete considerably more IL-16 into the supernatant than cells expressing IL-16 without a signal peptide and are considerably more resistant to HIV replication. Resistance extends to almost 25 days for cells expressing IL-16 with signal peptide as compared with only 15 days for cells without signal peptide. Cells expressing the C-terminal 100 aa not linked to a signal peptide are poor secretors of IL-16 and show little if any resistance to HIV. In contrast, cells expressing the C-terminal 100 aa linked to a signal peptide secrete IL-16 and are resistant to HIV replication. It is concluded that the secretion of IL-16 is required for HIV inhibition.     

Acquisition of an insertion peptide for efficient aminoacylation by a halophile tRNA synthetase

Enzymes of halophilic organisms contain unusual peptide motifs that are absent from their mesophilic counterparts. The functions of these halophile-specific peptides are largely unknown. Here we have identified an unusual peptide that is unique to several halophile archaeal cysteinyl-tRNA synthetases (CysRS), which catalyze attachment of cysteine to tRNA(Cys) to generate the essential cysteinyl-tRNA(Cys) required for protein synthesis. This peptide is located near the active site in the catalytic domain and is highly enriched with acidic residues. In the CysRS of the extreme halophile Halobacterium species NRC-1, deletion of the peptide reduces the catalytic efficiency of aminoacylation by a factor of 100 that largely results from a defect in kcat, rather than the Km for tRNA(Cys). In contrast, maintaining the peptide length but substituting acidic residues in the peptide with neutral or basic residues has no major deleterious effect, suggesting that the acidity of the peptide is not important for the kcat of tRNA aminoacylation. Analysis of general protein structure under physiological high salt concentrations, by circular dichroism and by fluorescence titration of tRNA binding, indicates little change due to deletion of the peptide. However, the presence of the peptide confers tolerance to lower salt levels, and fluorescence analysis in 30% sucrose reveals instability of the enzyme without the peptide. We suggest that the stability associated with the peptide can be used to promote proper enzyme conformation transitions in various stages of tRNA aminoacylation that are associated with catalysis. The acquisition of the peptide by the halophilic CysRS suggests an enzyme adaptation to high salinity.     

HLA-DM Mediates Epitope Selection by a “Compare-Exchange” Mechanism when a Potential Peptide Pool Is Available

Background

HLA-DM (DM) mediates exchange of peptides bound to MHC class II (MHCII) during the epitope selection process. Although DM has been shown to have two activities, peptide release and MHC class II refolding, a clear characterization of the mechanism by which DM facilitates peptide exchange has remained elusive.

Methodology/Principal Findings

We have previously demonstrated that peptide binding to and dissociation from MHCII in the absence of DM are cooperative processes, likely related to conformational changes in the peptide-MHCII complex. Here we show that DM promotes peptide release by a non-cooperative process, whereas it enhances cooperative folding of the exchange peptide. Through electron paramagnetic resonance (EPR) and fluorescence polarization (FP) we show that DM releases prebound peptide very poorly in the absence of a candidate peptide for the exchange process. The affinity and concentration of the candidate peptide are also important for the release of the prebound peptide. Increased fluorescence energy transfer between the prebound and exchange peptides in the presence of DM is evidence for a tetramolecular complex which resolves in favor of the peptide that has superior folding properties.

Conclusion/Significance

This study shows that both the peptide releasing activity on loaded MHCII and the facilitating of MHCII binding by a candidate exchange peptide are integral to DM mediated epitope selection. The exchange process is initiated only in the presence of candidate peptides, avoiding possible release of a prebound peptide and loss of a potential epitope. In a tetramolecular transitional complex, the candidate peptides are checked for their ability to replace the pre-bound peptide with a geometry that allows the rebinding of the original peptide. Thus, DM promotes a “compare-exchange” sorting algorithm on an available peptide pool. Such a “third party”-mediated mechanism may be generally applicable for diverse ligand recognition in other biological systems.