Structural features of the nicotinic acetylcholine receptor revealed by antibodies to synthetic peptides

Antibodies were raised to the amino- and car?y-terminal decapeptides of Torpedo californica acetylcholine receptor. Structural studies of the native receptor using the antipeptide antibodies as probes proved the existence of the car?y terminal sequence in the α subunit predicted from its cDNA sequence and supported structural models of the native receptor that place the car?y termini on the intracellular side. The amino termini of the subunits were not accessible on the surface of native receptor.     

Immunolocalization of intermediate filament proteins using antibodies to synthetic peptides.

Synthetic peptides corresponding to amino acid sequences of amino terminal non-alpha helical domains of human cytokeratin 18 and to low molecular weight human neurofilament subunit were used to obtain monospecific antisera. The results of our immunohistochemical investigations confirmed in general the data previously published on the distribution of cytokeratin 18 in human, rat, and calf tissues. The reactivity of the antiserum was abolished after formalin fixation of specimens. Immunolocalization of the neurofilament subunit using our monospecific antiserum was quite variable from species to species in cells of the central and peripheral nervous systems, and also varied as the result of the tissue fixation procedures. In particular, formalin fixation destroyed the immunoreactivity of the recognized epitope. We discuss the advantages and limits of the use of synthetic peptides as immunogens to produce polyclonal antibodies against intermediate filament proteins, with particular attention to the epitope masking phenomena in cytokeratin polypeptides and the phosphorylation of epitopes in neurofilament subunits.     

Determination of crucial epitopes in the sperm protein calsperin employing synthetic peptides and monoclonal antibodies

The chaperone protein calsperin is exclusively expressed in the testes and is essential for sperm migration from the uterus into the oviduct. During spermatogenesis, calsperin interacts with ADAM3, a spermatozoon membrane protein required for fertilization. In this study, we characterized a calsperin epitope by using two monoclonal antibodies and resin-bound calsperin peptides, which were tested for reactivity using a modified enzyme-linked immunosorbent assay. An epitope located at the C-terminal end of calsperin corresponding to amino acids ²²⁸WEKHFLDAS²³⁷ was identified. Three hot spot amino acids were essential for antibody binding whereas the remaining amino acids in the identified epitope appeared to be essential for bringing the critical contact residues into an α-helix structure. No notable sequence similarity was determined between the identified calsperin epitope and calreticulin, a chaperone homologue with sequence similarity, indicating that the identified epitope was specific for calsperin. Characterization of the calsperin epitope and of the two antibodies tested may be used in assays for further characterization of calsperin, where knowledge about the binding sites is necessary, for example, in sandwich assays. Moreover, studies like these may be used to study the function of calsperin during spermatogenesis and fertilization in detail and to develop new male contraception methods by targeting calsperin and mediating neutralization of its function.     

Conserved epitopes on plant H1 histones recognized by monoclonal antibodies

A series of monoclonal antibodies specific for distinct regions of H1 histone from the plant Nicotiana tabacum were obtained from fusion experiments with spleen cells of mice immunized with tobacco nuclear extracts. These monoclonal antibodies were characterized and the evolutionary conservation of the epitopes in higher plants and animals studied by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Whereas some epitopes appear restricted to the Solanaceae plant family, others are common to all higher eukaryotes tested and even detectable on nuclear proteins of yeast. ELISA experiments performed with isolated tobacco chromatin give some indications of the differential accessibility of the epitopes after interaction of H1 histone with the nucleosome.     

Chimeric epitopes delivered by polymeric synthetic linear peptides induce protective immunity to malaria

Polymeric linear peptide chimeras (LPCs) that incorporate Plasmodium vivax promiscuous T cell epitopes and the P. falciparum circumsporozoite protein B cell epitope have been shown to induce a high level of immunogenicity and overcome genetic restriction when tested as vaccine immunogens in BALB/c mice. The present study evaluates the biological relevance of several LPCs using a well characterized rodent malaria model. Polymeric peptide constructs based on P. berghei and P. yoelii sequences, and orthologous to the human malaria sequences included in the original LPCs, were designed and tested for immunogenicity in mice of different H-2 haplotypes. We demonstrate that robust immune responses are induced and that peptides containing the orthologous rodent Plasmodium sequences exhibited similar immunogenic capabilities. Unique to this report, we show that LPCs can also prime MHC class I-restricted cytotoxic T lymphocytes (CTLs) and, most relevantly, that a peptide construct prototype incorporating single B, T and CTL epitopes induced protection against an experimental challenge with P. berghei or P. yoelii sporozoites. Collectively, these results suggest that polymeric polypeptide chimeras can be used as a platform to deliver subunit vaccines.     

Neoantigens in complement component C3 as detected by monoclonal antibodies. Mapping of the recognized epitopes by synthetic peptides.

The different fragments of the third complement component, C3, generated upon complement activation/inactivation have the ability to bind to several other complement components and receptors as well as to proteins of foreign origin. These multiple reactivities of C3 fragments are associated with a series of conformational changes occurring in the C3 molecule during its degradation. The conformations acquired by the different C3 fragments are also associated with the exposure of neoantigenic epitopes that are specific for (a) particular fragment(s). In order to study these epitopes and thus the conformational changes occurring in C3, monoclonal antibodies (mAbs) recognizing such epitopes were produced in Balb/c mice after immunization with denatured human C3. Two of the three antibodies (7D84.1 and 7D264.6) presented in this study recognized predominantly surface-bound iC3b, and one mAb (7D323.1) recognized both surface-bound and fluid-phase iC3b. Although none of the mAbs recognized any other fluid-phase C3 fragment, all three antibodies detected micro-titre-plate-fixed C3b and iC3b, but not C3c or C3d. In addition to the reaction with human C3, mAb 7D323.1 also bound to micro-titre-plate-fixed rabbit C3. The epitopes recognized by the three mAbs were further localized by using synthetic peptides that were designed on the basis of the differential binding of the mAbs to the C3 fragments. All three antibodies reacted with C3-(924-965)-peptide, which represents the region of C3 between the kallikrein-cleavage site (923-924) and the elastase-cleavage site (965-966). On the basis of the binding of the mAbs to five different overlapping peptides spanning the region between residues 924 and 965 of the human C3 sequence, and the sequence similarity between human C3 and rabbit C3 within this area, the epitopes recognized by these antibodies are mapped. The contribution of the individual amino acid residues in the formation of the epitopes is discussed.     

Plasmodium falciparum: elicitation by peptides and recombinant circumsporozoite proteins of circulating mouse antibodies inhibiting sporozoite invasion of hepatoma cells

The immunodominant epitope region of the circumsporozoite protein of Plasmodium falciparum sporozoites contains 37 tandem repeats of the tetrapeptide Asn-Ala-Asn-Pro and 4 repeats of Asn-Val-Asp-Pro. Synthetic peptides and recombinant proteins of the repeat region were used to immunize mice using different doses and adjuvants. Antisera were tested for inhibition of sporozoite invasion of cultured human hepatoma cells. Synthetic peptides and recombinant proteins elicited high levels of antibodies that inhibited sporozoite invasion when emulsified with complete Freund's adjuvant. Since recombinant proteins with alum elicited a better antibody response to sporozoite invasion than they did without adjuvant, it may be that a recombinant protein containing 32 tandem copies of the tetrapeptide repeat combined with alum could be a candidate malarial vaccine suitable for human trials.     

IgG antibodies from patients with bullous pemphigoid bind to localized epitopes on synthetic peptides encoded by bullous pemphigoid antigen cDNA.

Bullous pemphigoid (BP) is an autoimmune blistering disease characterized in part by the presence of tissue-bound and circulating antibodies specific for basement membrane zone proteins, the BP Ag. The purpose of the present study was to determine seroreactivity of patients with BP to six nonoverlapping synthetic peptides representing sequences in the carboxyl domain of the recently cloned 230-kDa BP Ag. Sera from 40 patients with BP, 57 normal subjects, and 18 patients with other autoimmune blistering skin diseases were examined in an ELISA for binding to six synthetic peptides varying between 17 and 19 amino acids in length. The binding of IgG from patients with BP to three synthetic peptides, P1-2, P1-1, and P3-1, was significantly different from that seen in the normal controls (p less than 0.001, Fisher's exact test). Affinity-purified anti-P1-2 antibody from a patient with BP bound in a characteristic linear band to the epidermal side of 1 M NaCl split skin and immunoprecipitated the native 230-kDa BP Ag. Serum IgG antibodies from a rabbit immunized with a BP fusion protein that contains the sequences for P1-1 and P1-2, bound on ELISA to P1-2 but not to P1-1. These data suggest that multiple epitopes on the 230-kDa BP Ag are recognized by circulating autoantibodies in patients with this disease, and that an epitope encoded within the synthetic peptide P1-2 is expressed on the native molecule and may be relevant in the generation of an immune response both in man and in an animal model.     

Induction of anti-HIV neutralizing antibodies by synthetic peptides.

Two synthetic peptides containing amino acid sequences analogous to the envelope glycoprotein of human T-lymphotropic virus (HTLV) type III (HTLV-III) and lymphadenopathy associated virus (LAV) were produced and used to immunize rabbits. The subsequent rabbit antisera neutralized HTLV-III infectivity in vitro. The two synthetic peptides corresponded to regions associated with the gp120 or gp41 subunits respectively, of human immunodeficiency virus (HIV). This data indicates that at least two neutralizing epitopes are present on the envelope glycoprotein of HIV and these epitopes are associated with two distinct virus envelope glycoproteins. Antisera generated against these peptides neutralized infectivity of two different isolates of HTLV-III. The data is discussed in terms of possible strategy for developing an effective vaccine against the etiologic agents of acquired immune deficiency syndrome (AIDS).     

Cross-reactivity of antibodies against synthetic peptides

Antiserum against the synthetic peptide Lys-Arg-Ser-Arg-His-Phe, corresponding to the carboxy terminus of polyoma virus medium tumor antigen (medium T antigen), immunoprecipitates a protein of 36,000 daltons from polyoma virus-infected and uninfected cell extracts treated with the sulfhydryl group reagent N-ethyl-maleimide. This protein appears to share an antigenic determinant with medium T antigen that is normally buried inside the protein or covered up by another protein or cellular structure. The two-dimensional tryptic fingerprints of the 36K protein and of medium T antigen are apparently unrelated to each other. Antiserum against the octapeptide Ac-Met-Asp-Lys-Val-Leu-Asn-Arg-Tyr, including the amino-terminal heptapeptide sequence of the simian virus 40 (SV40) large tumor (T) and small T antigens, cross-reacts with polyoma virus large T antigen, which has an identical amino-terminal heptapeptide sequence except that Lys is replaced by Arg and Asn by Ser. The problem of cross-reactivities of antipeptide sera is discussed.     

Use of long synthetic peptides to study the antigenicity and immunogenicity of the Plasmodium vivax circumsporozoite protein

Three long synthetic peptides corresponding to amino (N), repeat (R) and carboxyl (C) regions of the Plasmodium vivax circumsporozoite (CS) protein were synthesised and used to assess their potential as vaccine candidates. Antigenicity studies were carried out using human blood samples from residents of a malaria-endemic area of Colombia, and immunogenicity was tested in Aotus monkeys. The N and C peptides spanned the total native amino and carboxyl flanking regions, whereas the R peptide corresponded to a construct based on the first central nona-peptide repeated in tandem three times and colinearly linked to a universal T-cell epitope (ptt-30) derived from tetanus toxin. All three peptides had been shown previously to contain several B-, T-helper (Th) and Cytotoxic T Lymphocytes (CTL) epitopes. Sixty-one percent of the human sera reacted with the R region, whereas 35 and 39% of the samples had antibodies against the N and C peptides, respectively. Human Peripheral Blood Mononuclear Cells (PBMC) showed higher levels of IFN-gamma than IL-4 when stimulated with peptides containing Th epitopes. Aotus monkeys immunised with the peptides formulated in either Montanide ISA720 or Freund's adjuvants produced strong antibody responses that recognised the peptide immunogens and the native circumsporozoite protein on sporozoites. Additionally, high IFN-gamma production was induced when Aotus lymphocytes were stimulated in vitro with each of the three peptides. We observed boosting of antibody responses and IFN-gamma production by exposure to live sporozoites. These results confirm the high antigenicity and immunogenicity of such synthetic polypeptides and underline their vaccine potential.     

Silencing of immunodominant epitopes by contiguous sequences in complex synthetic peptides.

We have previously shown that the T cell response to the synthetic peptide cI12-26:NP365-380 (covalently linked epitopes of lambda repressor (cI) and influenza A nucleoprotein (NP) polypeptides) requires amino acid sequences located in the junctional region between the cI12-26 and NP365-380 epitopes in the H-2d and H-2k haplotypes. In this study, we show that the dominant epitope of cI12-26:NP365-380 in H-2b mice is also located within the junctional region of the peptide, indicating that the same amino acid sequence is immunodominant in three different H-2 haplotypes. Based on results using fixed APC, there was no qualitative difference in epitope recognition due to antigen processing. In addition, antigen presentation by APC expressing mutant I-A molecules constructed by hemiexon shuffling of regions of the molecule containing primarily beta sheet or alpha helix showed that many different substitutions were permissive for at least one of the T hybridomas. More importantly, however, when the junctional sequences are covalently linked in composite synthetic peptides containing additional previously defined T cell epitopes, antigenicity of the immunodominant junctional region was silenced and a new epitope assumed immunodominance. Thus, immunodominance does not correlate with the primary amino acid sequence of the potential epitope. Instead, the immunodominant epitope is determined by complex interactions among the epitopes, which most likely depend on the structural conformation of the composite peptide.     

Tubulin assembly probed with antibodies to synthetic peptides.

Antibodies to synthetic peptides from the alpha and beta-tubulin sequences were employed to study zones of this protein active in microtubule assembly. In purified calf brain tubulin, six short sequences, selected according to their hydrophilicity and conservation, were found to be accessible to their affinity-purified immunoglobulin G (IgG) antibodies, in a competition radioimmunoassay performed under non-assembly native conditions. This indicated that the six sequences are exposed on the surface of the tubulin alpha beta heterodimer. IgG antibodies to the alpha(430-443) and beta(412-431) sequences perturbed substoichiometrically the assembly of purified tubulin, inducing microtubule bundling and the formation of opened up structures. These positions, which are close to the C termini, were accessible to the anti-peptide antibodies in taxol-induced microtubules, Zn2(+)-induced tubulin sheets, Mg2(+)-induced tubulin rings and in PtK2 cell microtubules. This, together with the comparison of the sizes and gross shapes of the antibody probes and microtubules, suggested that these sequences might be located at the protruding parts of the protofilaments. Antibodies to positions alpha(155-168) did not react with microtubules, while the equivalent zone beta(153-165) was accessible. The alpha(214-226) and beta(241-256) sequences were antigenically occluded in the taxol microtubules, Zn2(+)-induced sheets and Mg2(+)-induced ring arrays, as well as in native microtubules from PtK2 cells, though they became reactive by fixation. This result strongly suggested that these two zones are close to tubulin-tubulin contact sites. A working model is proposed in which the positions alpha(214-226) and beta(241-256) are close to the axial contacts between heterodimers, which lead to protofilament formation, while the positions alpha(241-256) and beta(214-226) are suggested to be related to the alpha-beta binding interface within the heterodimer.     

Species specificity of anti-acetylcholine receptor antibodies elicited by synthetic peptides

Two peptides corresponding to amino acid residues 351-368 of the alpha-subunits of Torpedo and human acetylcholine receptor (AChR) were synthesized. These peptides contain a segment (residues 355-364) which displays the greatest variability in amino acid sequence between the two species. Antibodies elicited against the two peptides cross-reacted with the respective native AChRs and were shown to be species specific by radioimmunoassay, immunoblotting, and immunofluorescence microscopy. Thus, antibodies against the Torpedo peptide cross-reacted with Torpedo AChR but did not bind to mammalian or chicken AChR. Antibodies against the human peptide proved to be specific probes for mammalian muscle AChR. They cross-reacted with mammalian AChR (human, calf, mouse, and rat) but not with Torpedo or chicken AChR. These antibodies were also shown to react preferentially with the extrajunctional form of muscle AChR, as compared to their reactivity with junctional muscle AChR. In immunofluorescence experiments, the anti-human peptide antibody stained AChR aggregates in sectioned or ethanol-permeabilized rat and mouse myotubes grown in culture but did not stain living myotubes. This indicates that the sequence 351-368 of the alpha-subunit of mammalian AChR is on the cytoplasmic face of muscle cell membranes, as predicted theoretically.     

Generation of rubella virus-neutralising antibodies by vaccination with synthetic peptides

Abstract Four short peptides from rubella virus proteins E1 and E2, predicted to contain B cell epitopes, were used to vaccinate BALB/c mice. Sera from peptide-vaccinated animals reacted with viral antigens in ELISA and three of the four induced virus-neutralising antibody (nAb) responses. Peptide PY4, in contrast to the others, induced IgG2a responses upon vaccination and stimulated spleen cells in vitro produced IFNγ in the absence of IL-5. It was reasoned that vaccination with PY4 caused Th1 subset activation, the appropriate type of response for anti-viral immunity and hence the efficient neutralising antibody response. Presentation of peptide for vaccination proved to be as important as the sequence. Similar profiles of IgG1 and IgG2a were detected in the sera of mice vaccinated with PY4 in Freund's complete adjuvant or alum; however nAb responses were not found when alum was used.     

Mimicry of dimerization by synthetic peptides designed to target homologous regions of proteins

Rapid progress in sequencing various genomes has highlighted the need for the development of biochemical reagents for the detection of thousands of expressed gene products. The magnitude of this detection problem exceeds current technical capabilities. In an attempt to address this shortcoming, a novel approach has been developed called mimicry of dimerization. Peptide tags have been designed to bind to a specific region of parvalbumin on the basis of amino acid sequence homology with this segment. Multivalent ligands were produced by coupling the synthetic peptides to activated dextran polymers and binding was assessed by chemiluminescence of enhanced avidity reactions using a high density of target protein at the binding surface. Binding of the peptide ligands to parvalbumin was strongest under assay conditions that enriched for native monomeric protein and was affected by pH, temperature and solvent conditions. The results suggest that it should be possible to develop specific reagents for tagging proteins on the basis of sequence and secondary structure information.     

Ro60 peptides induce antibodies to similar epitopes shared among lupus-related autoantigens

The coexistence of autoantibodies to ribonucleoproteins (RNP) in sera of patients with systemic lupus erythematosus has been attributed to intermolecular determinant spreading among physically associated proteins. Recently, we showed that murine Ab responses to rRo60 or Ro60 peptides were diversified unexpectedly to small nuclear RNP. In this investigation, the mechanisms for this autoantibody diversification were examined. Intramolecular determinant spreading was demonstrated in mice immunized with human or mouse Ro60316-335. Immune sera depleted of anti-peptide Ab immunoprecipitated Ro60-associated mY1 and mY3 RNA and remained reactive to a determinant on Ro60128-285. Absorption with the immunogen depleted the immune sera completely of anti-Golgi complex Ab (inducible only with human Ro60316-335) and anti-La Ab, and reduced substantially Ab to SmD and 70-kDa U1RNP. Mouse rRo60 completely inhibited the immune sera reactivity to La, SmD, and 70-kDa U1RNP. However, La, SmD, and 70-kDa U1RNP preferentially inhibited the antiserum reactivities to these Ags, respectively. Affinity-purified anti-La Ab were reactive with Ro60, La, SmD, and 70-kDa U1RNP. These results provide evidence that a population of the induced autoantibodies recognized determinants shared by these autoantigens. Lack of sequence homology between Ro60316-335 and La, SmD, or 70-kDa U1RNP suggests that these determinants are conformational. Interestingly, similar cross-reactive autoantibodies were found in NZB/NZW F1 sera. Thus, a single molecular mimic may generate Ab to multiple RNP Ags. Furthermore, cross-reactive determinants shared between antigenic systems that are not associated physically (Ro/La RNP and small nuclear RNP) may be important in the generation of autoantibody diversity in systemic lupus erythematosus.     

Adsorption equilibrium in immunoaffnity chromatography with antibodies to synthetic peptides

The effects of charged residues in peptide antigens on the binding characteristics of polyclonal antipeptide antibodies were studied using immunoadsorbents prepared by coupling the antibodies to CNBr-activated Sepharose 4B. Among the antipeptide antibodies, an antibody to the peptide without charged residues showed the most stable interaction with the peptide to the changes in pH. Conversely, the binding affinity of antibodies to the pep-tides with histidine residues having a unique pKa value of 6.0 decreased steeply with pH at around 6.0. The binding affinity of an antibody to the peptide with many charged residues decreased steeply with an increase in the ionic strength (adjusted by NaCl). Since circular dichroism (CD) spectrum measurements indicate that these peptides show disordered structures in the pH range of adsorption measurement, the dependence of peptide-antibody interaction on environmental conditions is attributed to the characteristics of side chains of the peptides. These results indicate that the dependence of the binding affinity of antipeptide antibodies on pH and the ionic strength is dominantly affected by the number and the pKa values of charged residues in the peptides.     

Use of long synthetic peptides to study the antigenicity and immunogenicity of the Plasmodium vivax circumsporozoite protein

Three long synthetic peptides corresponding to amino (N), repeat (R) and carboxyl (C) regions of the Plasmodium vivax circumsporozoite (CS) protein were synthesised and used to assess their potential as vaccine candidates. Antigenicity studies were carried out using human blood samples from residents of a malaria-endemic area of Colombia, and immunogenicity was tested in Aotus monkeys. The N and C peptides spanned the total native amino and carboxyl flanking regions, whereas the R peptide corresponded to a construct based on the first central nona-peptide repeated in tandem three times and colinearly linked to a universal T-cell epitope (ptt-30) derived from tetanus toxin. All three peptides had been shown previously to contain several B-, T-helper (Th) and Cytotoxic T Lymphocytes (CTL) epitopes. Sixty-one percent of the human sera reacted with the R region, whereas 35 and 39% of the samples had antibodies against the N and C peptides, respectively. Human Peripheral Blood Mononuclear Cells (PBMC) showed higher levels of IFN-γ than IL-4 when stimulated with peptides containing Th epitopes. Aotus monkeys immunised with the peptides formulated in either Montanide ISA720 or Freund's adjuvants produced strong antibody responses that recognised the peptide immunogens and the native circumsporozoite protein on sporozoites. Additionally, high IFN-γ production was induced when Aotus lymphocytes were stimulated in vitro with each of the three peptides. We observed boosting of antibody responses and IFN-γ production by exposure to live sporozoites. These results confirm the high antigenicity and immunogenicity of such synthetic polypeptides and underline their vaccine potential.     

Characterization of prion proteins with monospecific antisera to synthetic peptides

The prion protein (PrP) 27-30 is the major macromolecular component in highly purified preparations of prions derived from scrapie-infected hamster brain. Immunoblotting studies demonstrated that this protein is generated by partial protease digestion of a larger precursor (PrPSc) with an apparent Mr of 33 to 35 kDa, and that a protease-sensitive cellular PrP isoform, designated PrPC, is present in normal hamster brain. To characterize the relationships among these proteins, ELISA and immunoblotting studies were undertaken with rabbit antisera raised against three synthetic PrP peptides. All three antisera were found to specifically react with the prion proteins, and failed to identify other lower or higher m.w. PrP proteins. Our results provide evidence that the primary structures of PrP 27-30, PrPSc, and PrPC are related; this conclusion supports molecular cloning studies indicating that these proteins are encoded by the same chromosomal gene.