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1.
Diverse procedures for identifying antigenic determinants on proteins have been developed, including experimental as well as computational approaches. However, most of these techniques focus on continuous epitopes, whereas fast and reliable identification and verification of discontinuous epitopes remains barely amenable. In this paper, we describe a computational workflow for the detection of discontinuous epitopes on proteins. The workflow uses a given protein 3D structure as input, and combines a per residue solvent accessibility constraint with epitope to paratope shape complementarity measures and binding energies for assigning antigenic determinants in the conformational context. We have developed the procedure on a given set of 26 antigen-antibody complexes with a known structure, and have further expanded the available paratope shapes by generating a virtual paratope library in order to improve the screening for candidate residues constituting discontinuous epitopes. Applying the workflow on the 26 given antigens with known discontinuous epitopes resulted in the correct identification of the spatial proximity of 12 antigen-antibody interaction sites. Combining solvent accessibility, shape complementarity and binding energies towards the identification of discontinuous epitopes clearly outperforms approaches solely considering accessibility and residue distance constraints.  相似文献   

2.
Neutralizing monoclonal antibodies specific for the fusion (F) glycoprotein of human parainfluenza type 3 virus (PIV3) were used to select neutralization-resistant antigenic variants. Sequence analysis of the F genes of the variants indicated that their resistance to antibody binding, antibody-mediated neutralization or to both was a result of specific amino acid substitutions within the neutralization epitopes of the F1 and F2 subunits. Comparison of the locations of PIV3 neutralization epitopes with those of Newcastle disease and Sendai viruses indicated that the antigenic organization of the fusion proteins of paramyxoviruses is similar. Furthermore, some of the PIV3 epitopes recognized by syncytium-inhibiting monoclonal antibodies are located in an F1 cysteine cluster region which corresponds to an area of the measles virus F protein involved in fusion activity.  相似文献   

3.
The concept and operational definition of protein epitopes   总被引:2,自引:0,他引:2  
The antigenic determinants or epitopes of a protein correspond to those parts of the molecule that are specifically recognized by the binding sites or paratopes of certain immunoglobulin molecules. Epitopes are thus relational entities that require complementary paratopes for their operational recognition. Some authors consider that the concept of epitope necessarily involves the two properties of antigenic reactivity (ability to bind to a paratope) and immunogenicity (ability to induce an immune response). Such a view creates difficulties because it makes the existence of epitopes in a protein depend on immunogenetic and regulatory mechanisms of the immunized host. The delineation of epitopes can be achieved by antigenic cross-reactivity studies or by X-ray crystallography. Both approaches require specific criteria for deciding which residues of the antigen are in contact with the paratope and are functionally part of the epitope. The relative contribution of static accessibility, segmental mobility and induced fit to immune recognition remains controversial. Each of the methods used for analysing antigenic specificity is subject to various operational constraints originating from the type of experimental probe and from the format sensitivity and specificity of the immunoassay used. If a protein is assumed to contain as many epitopes as the number of different monoclonal antibodies that can be raised against it, the delineation of epitopes corresponds to the summation in various hosts of the immune repertoire specific for the antigen. Neutralization epitopes are a special subclass of the epitopes of infectious agents and toxins that are specifically recognized by antibody molecules able to neutralize the biological activity of the antigen. The identification of neutralization epitopes is important for the development of synthetic vaccines because it is this type of epitope that should be mimicked by synthesis and used as a vaccine for eliciting protective immunity. The first demonstration that synthetic peptides could elicit antibodies that neutralized viral infectivity was made by Anderer and his colleagues in the 1960s in their work with tobacco mosaic virus. Nearly 20 years passed before it was shown that antibodies to synthetic peptides were also able to neutralize the infectivity of other viruses such as foot-and-mouth disease, polio and hepatitis B viruses.  相似文献   

4.
Bacterial chemotaxis receptors are posttranslationally modified by carboxyl methylation of specific glutamate residues within their cytoplasmic domains. This highly regulated, reversible modification counterbalances the signaling effects of ligand binding and contributes to adaptation. On the basis of the crystal structure of the gamma-glutamyl methyltransferase CheR, we have postulated that positively charged residues in helix alpha2 in the N-terminal domain of the enzyme may be complementary to the negatively charged methylation region of the methyltransferase substrates, the bacterial chemotaxis receptors. Several altered CheR proteins, in which positively charged arginine or lysine residues were substituted with alanines, were constructed and assayed for their methylation activities toward wild-type receptor and a series of receptor variants containing different glutamates available for methylation. One of the CheR mutant proteins (Arg53Ala) showed significantly lower activity toward all receptor constructs, suggesting that Arg53 may play a general role in catalysis of methyl transfer. The rest of the mutant proteins exhibited different patterns of relative methylation rates toward different receptor substrates, indicating specificity, probably through interaction of CheR with the receptor at sites distal to the specific site of methylation. The findings imply complementarity between positively charged residues of the alpha2 helix of CheR and the negatively charged glutamates of the receptor. It is likely that this complementarity is involved in discriminating different methylation states of the receptors.  相似文献   

5.
Echinococcus granulosus is the parasite responsible for cystic echinococcosis (CE), an important worldwide-distributed zoonosis. New effective vaccines against CE could potentially have great economic and health benefits. Here, we describe an innovative vaccine design scheme starting from an antigenic fraction enriched in tegumental antigens from the protoscolex stage (termed PSEx) already known to induce protection against CE. We first used mass spectrometry to characterize the protein composition of PSEx followed by Gene Ontology analysis to study the potential Biological Processes, Molecular Functions, and Cellular Localizations of the identified proteins. Following, antigenicity predictions and determination of conservancy degree against other organisms were determined. Thus, nine novel proteins were identified as potential vaccine candidates. Furthermore, linear B cell epitopes free of posttranslational modifications were predicted in the whole PSEx proteome through colocalization of in silico predicted epitopes within peptide fragments identified by matrix-assisted laser desorption/ionization-TOF/TOF. Resulting peptides were termed “clean linear B cell epitopes,” and through BLASTp scanning against all nonhelminth proteins, those with 100% identity against any other protein were discarded. Then, the secondary structure was predicted for peptides and their corresponding proteins. Peptides with highly similar secondary structure respect to their parental protein were selected, and those potentially toxic and/or allergenic were discarded. Finally, the selected clean linear B cell epitopes were mapped within their corresponding 3D-modeled protein to analyze their possible antibody accessibilities, resulting in 14 putative peptide vaccine candidates. We propose nine novel proteins and 14 peptides to be further tested as vaccine candidates against CE.  相似文献   

6.
Thermal stability of hepatitis B surface antigen (HBsAg) has been studied by analyzing alterations in the native secondary structure and the antigenic activity. After heating for 19 h, circular dichrosim showed a cooperative transition with a midpoint at 49 degrees C. The conformational changes induced by temperature reduced the helical content of HBsAg S proteins from 49% at 23 degrees C to 26% at 60 degrees C and abolished the antigenic activity, as measured by binding to polyclonal antibodies. Furthermore, the six different antigenic determinants recognized by our panel of monoclonal antibodies were also shown to be dependent on the native structure of HBsAg proteins. Hence, it can be inferred that these epitopes are conformation-dependent. Binding of monoclonal antibodies to HBsAg protected the native structure of the corresponding antigenic determinant from thermal denaturation. In fact, binding of one of the monoclonals tested resulted not only in protection of the corresponding epitope, but also in a consistent increase of antibody binding with increasing temperature. Such an increase in antibody binding occurred simultaneously with an increase in the fluidity of surface lipid regions, as monitored by fluorescence depolarization of 1-(trimethylammoniophenyl)-6-phenyl-1,3,5-hexatriene. This correlation, along with the observation that lipids play an important role in maintaining the structure and antigenic activity of HBsAg (Gavilanes et al. (1990) Biochem. J. 265, 857-864), allow to speculate the certain epitopes of HBsAg which are close to the lipid-protein interface, are dependent on the fluidity of the surface lipid regions. Thus, any change in the physical state of the lipids could confer a different degree of exposure to the antigenic determinants.  相似文献   

7.
Chen H  He X  Wang Z  Wu D  Zhang H  Xu C  He H  Cui L  Ba D  He W 《The Journal of biological chemistry》2008,283(18):12528-12537
Human T lymphocytes, bearing T cell receptor (TCR) gammadelta, play an important role in anti-tumor/microbe immune responses. However, few tumor antigens recognized by TCRgammadelta have been defined so far. To investigate antigenic epitopes/proteins recognized by gammadeltaT cells, we have established a new immunobiochemical strategy that uses complementarity-determining region 3 of TCR delta chain (CDR3delta) peptide-mediated epitope/protein-binding assays. CDR3delta peptides synthesized using the CDR3 region in TCR Vdelta2 chain were validated for their binding specificity to target cells or tissues. These CDR3delta peptides were then employed as probes to pan putative epitopes in a 12-mer random peptide phage-displayed library and to identify putative protein ligands within tumor protein extracts by affinity chromatography and liquid chromatography/electrospray ionization-tandem mass spectrometry analysis. As a result, we have identified nine peptides and two proteins for TCRgammadelta, including human mutS homolog 2 (hMSH2) and heat shock protein (HSP) 60. All nine tested epitope peptides not only bind to gammadeltaT cells but also functionally activate gammadeltaT cells in vitro. Identification of HSP60 confirms the validity of this method as HSP60 is an identified ligand for TCRgammadelta. We show that hMSH2 is expressed on the surface of SKOV3 tumor cells, and cytotoxicity of Vdelta2 gammadeltaT cells to SKOV3 cells was blocked by anti-hMSH2 antibody, suggesting that hMSH2 may be a new ligand for TCRgammadelta. Taken together, our findings provide a novel immunobiochemical strategy to identify epitopes/proteins recognized by gammadeltaT cells.  相似文献   

8.
Abstract The outer membrane proteins of several prominent bacterial pathogens demonstrate substantial variation in their surface antigenic epitopes. To determine if this was also true for Pseudomonas aeruginosa outer membraine protein OprF, gene sequencing of a serotype 5 isolate was performed to permit comparison with the published serotype 12 oprF gene sequence. Only 16 nucleotide substitutions in the 1053 nucleotide coding region were observed; none of these changed the amino acid sequence. A panel of 10 monoclonal antibodies (mAbs) reacted with each of 46 P. aeruginosa strains representing all 17 serotype strains, 12 clinical isolates, 15 environmental isolates and 2 laboratory isolates. Between two and eight of these mAbs also reacted with proteins from representatives of the rRNA homology group I of the Pseudomonadaceae . Nine of the ten mAbs recognized surface antigenic epitopes as determined by indirect immunofluorescence techniques and their ability to opsonize P. aeuroginosa for phagocytosis. These epitopes were partially masked by lipopolysacharide side chains as revealed using a side chain-deficient mutant. It is concluded that OprF is a highly conserved protein with several conserved surface antigenic epitopes.  相似文献   

9.
Plasmodium vivax is the most prevalent species of malaria affecting millions of people annually worldwide and demands effective interventions to develop a successful vaccine. In this milieu, we have dedicated noteworthy efforts to characterize the proteome of P. vivax to give a lead for the epitope-based vaccine development. Membrane proteins of P. vivax were collected from SWISS PROT database and 10 antigenic proteins were identified among them by in silico analysis using multiple servers. T-cell and B-cell epitopes were identified and their immunity was assessed. Their ability to trigger humoral and cell-mediated responses was determined. Three dimensional models were constructed for the antigenic proteins using Modeller, Phyre2, and Modloop tools and their quality was validated using PROCHECK and ProSA-web validation servers. Further, the binding affinity and molecular interactions of these antigenic proteins were characterized by performing protein–protein docking against transmission-blocking anti-malaria antibody Fab2A8 (PDB ID: 3S62) using Z-dock module of Discovery Studio 4.0. The presence of potential B & T-cell epitopes, major histocompatibility complex-binding sites, and their efficient interactions with Fab2A8 antibody suggests the use of predicted antigenic proteins for the construction of multi-epitope peptide vaccine against P. vivax.  相似文献   

10.
Carugo O  Franzot G 《Proteomics》2004,4(6):1727-1736
A method to predict if two proteins interact, based on their three-dimensional structures, is presented. It consists of five steps: (i) the surface of each protein, represented by the solvent accessible atoms, is divided into small patches; (ii) the shape of each patch is described by the atom distributions along its principal axes; (iii) the shape complementarity between two patches is estimated by comparing, through contingency table analysis, their atom distributions along their principal axes; (iv) given protein A, with nA surface patches, and protein B, with nB surface patches, nA x nB shape complementarity values are obtained; and (v) the distribution of the latter allows one to discriminate pairs of interacting and of noninteracting proteins. Only a few seconds are necessary to predict if two proteins interact, with accuracy close to 80%, sensitivity over 70% and specificity close to 50%.  相似文献   

11.
Most of the lipid components of hepatitis B surface antigen (HBsAg) can be removed by treatment with the non-ionic non-denaturing detergent beta-D-octyl glucoside (OG) followed by centrifugation through caesium chloride linear density gradients (density 1.15-1.32 g/ml). The conformational changes induced by the elimination of lipids decreased the helical content of HBsAg proteins from 52 to 28% as indicated by c.d. techniques. Measurements of the extent of quenching of protein fluorescence by iodide showed that half of the tryptophan residues which are buried in the native structure of HBsAg particles are brought close to the surface of the molecule by such conformational changes. The antigenic activity, as measured by binding to polyclonal antibodies, was decreased upon removal of lipids. Moreover, the six different antigenic sites recognized by our panel of monoclonal antibodies decreased their capacity to bind to the corresponding antibody when lipids were removed. However, the extent of this decrease differed for the different antibodies. Thus the apparent dependence of antibody binding on the lipid content seemed to indicate a greater involvement of the lipid-protein interaction for some of the epitopes than for others.  相似文献   

12.
Even in this era of advanced biotechniques, specific antibodies against a protein still prove to be powerful tools to study proteins and their functions. The polyclonal antisera obtained from the immunized rabbits, however, are not always pure, high affinity, antigen-specific polyclonal antibodies. With our new rapid HaloTag-based procedure, specific antibodies are obtained in just two, short steps: (1) simultaneous purification and covalent coupling of the antigen to Sepharose resin via the HaloTag and HaloLink reaction, and (2) affinity column purification of the polyclonal serum (10 μl). The combined antigen purification and coupling step requires only 1 h of room-temperature incubation, plus successive washing steps. Because different regions of an antigen can elicit the production of low affinity antibodies with relatively high cross-reactivity, the best way to produce high affinity antibodies against a protein of interest is to survey all antigenic determinants of that protein and identify the epitopes that result in the production of antibodies with a high affinity and specificity for that protein. Because our HaloTag procedure is quite rapid and simple, potential epitopes can be assessed with relatively little effort for their ability to elicit the production of highly specific antibodies.  相似文献   

13.
Analysis of data from experimentally determined antigenic sites on proteins has revealed that the hydrophobic residues Cys, Leu and Val, if they occur on the surface of a protein, are more likely to be a part of antigenic sites. A semi-empirical method which makes use of physicochemical properties of amino acid residues and their frequencies of occurrence in experimentally known segmental epitopes was developed to predict antigenic determinants on proteins. Application of this method to a large number of proteins has shown that our method can predict antigenic determinants with about 75% accuracy which is better than most of the known methods. This method is based on a single parameter and thus very simple to use.  相似文献   

14.
15.
Background Mycobacterium ulcerans is the fundamental agent of the third most common Mycobacterial disease known as Buruli Ulcer (BU). It is an infection of the skin and soft tissue affecting the human population worldwide. Presently, the vaccine is not available against BU.ObjectiveThis study aimed to investigate the vaccine potential of virulence proteins of M. ulcerans computationally.MethodsChromosome encoded virulence proteins of Mycobacterium ulcerans strain Agy99 were selected, which were available at the VFDB database. These proteins were analyzed for their subcellular localization, antigenicity, and human non-homology analysis. Ten virulence factors were finally chosen and analyzed for further study. Three-dimensional structures for selected proteins were predicted using Phyre2. B cell and T cell epitope analysis was done using methods available at Immune Epitope Database and Analysis Resource. Antigenicity, allergenicity, and toxicity analysis were also done to predict epitopes. Molecular docking analysis was done for T cell epitopes, those showing overlap with B cell epitopes.ResultsSelected virulence proteins were predicted with B cell and T cell epitopes. Some of the selected proteins were found to be already reported as antigenic in other mycobacteria. Some of the predicted epitopes also had similarities with experimentally identified epitopes of M. ulcerans and M. tuberculosis which further supported our predictions.ConclusionIn-silico approach used for the vaccine candidate identification predicted some virulence proteins that could be proved important in future vaccination strategies against this chronic disease. Predicted epitopes require further experimental validation for their potential use as peptide vaccines.  相似文献   

16.
We report here the preparation of BVDV antigen (Ag) to obtain adequate quantities of pure active viral proteins from Madin Darby Bovine kidney (MDBK) cell culture infected with BVDV cytophatic Oregon C24V strain. SDS-PAGE, Immunodot, ECL-Western blot and ELISA showed the best specificity and activity of BVDV Ag obtained from infected cells only by mild experimental conditions. BVDV Ag preparations showed the peculiar BVDV sensitivity to proteases, nonionic surfactants and tend for protein degradation and irreversible loss of conformational antigenic determinants with subsequent inability to detect antibodies against viral Ag in hyperimmune sera. The widest panel of immunologically active and specific polypeptides, that elicit Ab production in hyperimmune sera, was obtained by ultrasonication and subsequent purification on 20%-50% sucrose cushion. We observed that BVDV tend to remain in the infected cells, to associate with components of serum and cellular origin--this is of crucial importance towards the specificity of ELISA. BVDV antigenic properties are determined by the labile conformational antigenic epitopes.  相似文献   

17.
Cognitive features of continuous antigenic determinants   总被引:15,自引:0,他引:15  
We sought to identify the features controlling the specificity of antibody recognition and thus gain insights into molecular recognition between proteins in general. A total of 103 epitopes within 63 well-defined antigenic peptides homologous with the relevant antigen sequence were identified. The contribution of each amino acid residue to the antibody binding activity of each epitope was investigated by ELISA testing of complete sets of peptide analogs containing single amino acid replacements. The data are summarized in a replaceability matrix. Some of the high frequency replaceabilities were expected, such as aspartate for glutamate, serine for threonine, etc., but unexpected relationships were also found, such as a high degree of acceptability of methionine as a replacement. Replaceability with a residue of opposite charge was rare. Glycine and tyrosine were frequently of low acceptability, except for glycine as a replacement for alanine. It was found that on average only about four to five amino acid residues in epitopes were required to determine specificity and provide binding energy. Specificity and binding energy were attributed to amino acid side chains rather than main chain atoms. Propensity factors for occurrence of amino acids in antigenic determinants were calculated. The prominence of certain hydrophobic residues as residues critical to recognition by antibody suggests that the molecular surface of an antigen in its combined form with antibody is altered from that occurring in the absence of antibody. Thus, antigenicity is not a static surface phenomenon but depends on the ability of the antigen to undergo rearrangement, supporting the induced fit concept.  相似文献   

18.
A number of specific Fab and Fv fragments and their complexes with antigens (avian lysozymes), haptens, and anti-idiotopic Fabs have been studied by immunochemical and crystallographic techniques. Antigen and antibody interact through closely complementary contacting surfaces, without major conformational changes. An idiotopic determinant of a monoclonal antibody is shown to include parts of most of its complementarity determining regions. The specificity of antigen recognition resides in the close complementarity of the antigenic determinant with the antibody combining site.  相似文献   

19.
Analysis of the properties for individual hepatitis C virus (HCV) proteins makes it possible to establish their molecular structure and conformation, to localize antigenic and immunogenic determinants, to identify protective epitopes, and to solve applied problems (e.g., design of diagnostic tests, vaccines, and drugs). Linear and conformational epitopes of HCV proteins were localized using the phage display technique, and the peptides exposed on the phages selected with monoclonal antibodies against HCV proteins were tested for immunogenicity. Of the 11 epitopes revealed, three were strongly linear; two depended on the secondary; and one on the tertiary structure of the corresponding protein (conformational epitopes). Amino acid sequences involved in the other epitopes were established. The results can be used to improve the diagnosis of hepatitis C, to study the effect of amino acid substitutions on the antigenic properties of HCV proteins, and to analyze the immune response in patients infected with genotypically different HCV. It was shown with the example of the NS5A epitope that phage particles with epitope-mimicking peptides (mimotopes) induce production of antibodies against the corresponding HCV proteins.  相似文献   

20.
Salmonella flagellar filaments are polymers of a highly antigenic protein, termed flagellin. Eight main subfactors have been identified in the Salmonella phase-1 g. . . series flagellar antigen. To determine the molecular basis for expression of the epitopes by which the g. . . family subfactors are distinguished, 10 members of this series were selected and their fliC (the structural gene for phase-1 flagellin) genes were sequenced. Comparative analyses of the inferred primary structures of these flagellins did not allow the identification of linear epitopes responsible for the antigen subfactors. This suggests that conformational aspects are involved in determining the antigenic specificity in these cases. A phylogenetic analysis of the flagellin sequences showed that members of the g. . . series do not form a single coherent unit.  相似文献   

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