An integrated family of amino acid sequence analysis programs

During the last years abundant sequence data has become availabledue to the rapid progress in protein and DNA sequencing techniques.The exact three-dimensional structures, however, are availableonly for a fraction of proteins with known sequences. For manypurposes the primary amino acid sequence of a protein can bedirectly used to predict important structural parameters. However,mathematical presentation of the calculated values often makesinterpretation difficult, especially if many proteins must beanalysed and compared. Here we introduce a broad-based, user-definedanalysis of amino acid sequence information. The program packageis based on published algorithms and is designed to access standardprotein data bases, calculate hydropathy, surface probabilityand flexibility values and perform secondary structure predictions.The data output is in an ‘easy-to-read’ graphicformat and several parameters can be superimposed within a singleplot in order to simplify data interpretations. Additionally,this package includes a novel algorithm for the prediction ofpotential antigenic sites. Thus the software package presentedhere offers a powerful means of analysing an amino acid sequencefor the purpose of structure/function studies as well as antigenicsite analyses. These algorithms were written to function incontext with the UWGCG (University of Wisconsin Genetics ComputerGroup) program collection, and are now distributed within thatpackage. Received on March 20, 1987; accepted on September 4, 1987     

Analysis of antibody response to synthetic peptides derived from the fusion protein of measles virus

A computer program combining of hydrophilicity, flexibility, surface probability, secondary structure and antigenic index parameters of the amino acid sequence of measles virus (MV) fusion protein was used to select four possible epitopes. Rabbits were immunized with the synthesized peptides conjugated to purified protein derivative using the homobifunctional cross-linker bis-sulfosuccinimidyl suberate. Immune stimulating complexes were prepared with the peptides conjugated to the purified protein derivative carrier using a dialysis method. All antisera raised in rabbits against the peptide conjugates had a high titer to the homologous peptides and reacted well with denatured MV as tested by plate ELISA. None of the sera had neutralizing antibody. Human sera positive for MV antibody reacted strongly with the synthesized peptides indicating that the selected locations function as partial antigenic sites. Antisera against peptide conjugates reacted weakly in immunofluorescence and none of these antisera reacted with purified MV proteins in Western blot. The results obtained in this study indicated that although the computer program could not predict epitopes important for the neutralization of the MV, the predicted epitopes are useful for detecting antibodies against MV.     

A semi-empirical method for prediction of antigenic determinants on protein antigens.

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.     

HCV抗原表位预测

应用网络生物信息资源查找丙型肝炎病毒基因组全序列,用软件Lasergene中的EditSeq将来自中国河北株mRNA序列翻译为氨基酸序列,尔后用程序Protean进行氨基酸序列分析,对HCV各区段的B细胞抗原指数进行预测。同时又在两个网站对中国汉族人中频率较高的HLA基因型进行CD8和CD4T细胞表位预测。B细胞和T细胞抗原表位预测结果对于HCV诊断试剂和疫苗研制有重要的指导意义。     

Identification of mutations at the antigenic and glycosylation sites in hemagglutinin protein of H5N1 strain

Hemagglutinin (HA) is the principal antigen, present on the viral surface. It is the primary target for neutralizing antibodies. In this paper, we have carried out studies on human hemagglutinin protein from H5N1 strain with homologous hemagglutinin from non-human sources of H5N1 strains. In all strains, part of the antigenic site (128-141) predicted by computer program “Antigenic”, corresponds to immunodominant site Sa of H1 subtype. In {"type":"entrez-protein","attrs":{"text":"AAF02304","term_id":"6048753","term_text":"AAF02304"}}AAF02304 strain, A156→S156 mutation lies at the antigenic subsite of site 2 that corresponds to site B in the H3 subtype. In some strains of non-human origins, there are mutations at the antigenic sites. Interestingly, in {"type":"entrez-protein","attrs":{"text":"AAY56367","term_id":"66775625","term_text":"AAY56367"}}AAY56367 strain mutation L138→H138 lies at the receptor binding site, which also overlaps the antigenic site. Therefore, this amino acid substitution may influence both the specificity of receptor recognition and antibody binding. Seven potential glycosylation sites in human HA and in some strains of non-human sources have been predicted by computer program, Scan Prosite. In some strains of HA from non-human sources because of mutation, an additional glycosylation site appeared at the antigenic site. Therefore in these strains the oligosaccharides will mask the surface of HA as well as antigenic site. Hence these strains will not be recognized by host immune system.     

Molecular characterisation of a predominant antigenic region of Giardia lamblia variant surface protein H7

During infection, the intestinal protozoan parasite Giardia lamblia undergoes continuous antigenic variation which is determined by diversification of the parasite's major surface antigen, named VSP (variant surface protein). One member from this protein family, VSP H7, is expressed by G. lamblia clone GS/M-83-H7. In the present study, we characterised a highly antigenic portion of VSP H7 which is positioned inside a 130 amino acid C-terminal region of the protein. This region overlaps with a cysteine-rich motif that is rather conserved within the VSP family. Detailed molecular dissection of the antigenic portion monitored a 12 amino acid peptidyl structure which constitutes a non-conformational epitope of VSP H7. In the murine host, this epitope is recognised relatively early (before day 10 p.i.) during infection and stimulates a strong intestinal immunoglobulin A response. At late infective stages (after day 10 p.i.) this immune reaction is progressively complemented by reactions against 'late' antigenic epitopes which are also located inside the 130 amino acid antigenic portion but in closer proximity to the C-terminal end of VSP H7 than the 12 amino acid epitope. Both the high antigenicity and the conserved character suggest that the 12 amino acid epitope is a key factor within the immunological interplay between G. lamblia and the experimental murine host.     

Prediction of membrane protein types based on the hydrophobic index of amino acids

A new algorithm to predict the types of membrane proteins is proposed. Besides the amino acid composition of the query protein, the information within the amino acid sequence is taken into account. A formulation of the autocorrelation functions based on the hydrophobicity index of the 20 amino acids is adopted. The overall predictive accuracy is remarkably increased for the database of 2054 membrane proteins studied here. An improvement of about 13% in the resubstitution test and 8% in the jackknife test is achieved compared with those of algorithms based merely on the amino acid composition. Consequently, overall predictive accuracy is as high as 94% and 82% for the resubstitution and jackknife tests, respectively, for the prediction of the five types. Since the proposed algorithm is based on more parameters than those in the amino acid composition approach, the predictive accuracy would be further increased for a larger and more class-balanced database. The present algorithm should be useful in the determination of the types and functions of new membrane proteins. The computer program is available on request.     

EB病毒潜伏膜蛋白1的B细胞表位预测

目的 预测EB病毒潜伏膜蛋白1(Latent Membrane Protein 1,LMPl)的B细胞表位.方法 基于EB病毒基因组序列,采用DNAStar Lasergene软件包中的Protean软件,对LMP1的亲水性,表面可能性,抗原指数及其二级结构中的柔性区域进行分析,并结合吴玉章的抗原指数预测法预测其B细胞表位.结果 B细胞表位最有可能位于潜伏膜蛋白N端第356-358,2-19,249-314区段或其附近,而潜伏膜蛋白N端第185-223区段内或附近也可能存在B细胞表位.结论 用多参数预测EB病毒LMP1的B细胞表位,为鼻咽癌的筛查及抗肿瘤转移靶向治疗的分子免疫学研究奠定基础.     

Bioinformatics models for predicting antigenic variants of influenza A/H3N2 virus

MOTIVATION: Continual and accumulated mutations in hemagglutinin (HA) protein of influenza A virus generate novel antigenic strains that cause annual epidemics. RESULTS: We propose a model by incorporating scoring and regression methods to predict antigenic variants. Based on collected sequences of influenza A/H3N2 viruses isolated between 1971 and 2002, our model can be used to accurately predict the antigenic variants in 1999-2004 (agreement rate = 91.67%). Twenty amino acid positions identified in our model contribute significantly to antigenic difference and are potential immunodominant positions.     

Prediction of sequential antigenic regions in proteins

Prediction of antigenic regions in a protein will be helpful for a rational approach to the synthesis of peptides which may elicit antibodies reactive with the intact protein. Earlier methods are based on the assumption that antigenic regions are primarily hydrophilic regions at the surface of the protein molecule. The method presented here is based on the amino acid composition of known antigenic regions in 20 proteins which is compared with that of 314 proteins [(1978) Atlas of Protein Sequence and Structure, vol. 5, suppl. 3, 363-373]. Antigenicity values were derived from the differences between the two data sets. The method was applied to bovine ribonuclease, the B-subunit of cholera toxin and herpes simplex virus type 1 glycoprotein D. There was a good correlation between the predicted regions and previously determined antigenic regions.     

Conservation and variation in the hemagglutinins of Hong Kong subtype influenza viruses during antigenic drift.

The nucleotide sequence was determined for the hemagglutinin gene of the Hong Kong subtype influenza strain A/Bangkok/1/79. The amino acid sequence predicted from these data shows a total of 36 amino acid changes as compared with hemagglutinin for a 1968 Hong Kong strain, 11 more than had occurred in a 1975 strain. The distribution of these changes confirmed that there are conserved and highly variable regions in hemagglutinin as the viral gene evolves during antigenic drift in the Hong Kong subtype. Of the four variable regions found in this study, only two have been seen previously. Correlation of highly variable areas in the hemagglutinins of Hong Kong subtype field strains with sites of amino acid changes in antigenically distinct influenza variants enabled us to predict likely antigenic regions of the protein. The results support and extend similar predictions made recently, based on the three-dimensional arrangement of hemagglutinin from a 1968 influenza strain.     

Characterization of immune response to synthetic peptides derived from the hemagglutinin of measles virus

We describe here four synthetic peptides derived from the hemagglutinin of measles virus. The peptides were predicted by a computer program combining hydrophilicity, flexibility, surface probability, secondary structure and antigenic index parameters of the amino acid sequence of measles virus hemagglutinin. Rabbits were immunized with the synthesized peptides conjugated to purified protein derivative using immunostimulating complex as adjuvant. Anti-peptide antisera raised in rabbits against the peptide conjugates reacted well with the homologous peptides and with measles virus antigen as tested with plate ELISA. None of these sera had either neutralizing or hemagglutination inhibiting antibody or reacted with measles hemagglutinin protein in Western blot and reacted weakly in immunofluorescence. Human sera positive for measles virus antibody reacted with the synthesized peptides indicating that the selected locations function as partial antigenic sites.     

PARA-SITE: a computer algorithm for rapidly analyzing the physical-chemical properties of amino acid sequences at sites of co- and post-translational protein processing

A new algorithm is presented which can be used to examine thephysical-chemical properties of amino acids at sites of co-orpost-translational processing. This algorithm has been incorporatedinto a computer program known as PARA-SITE. Thirty differentparameters can be studied for amino acids which occupy comparablepositions in naturally occurring proteins. PARA-SITE shouldaid in the design and interpretation of protein engineeringexperiments which seek to dissect structure/ activity relationships. Received on August 17, 1987; accepted on November 23, 1987     

Computer-assisted analysis of envelope protein sequences of seven human immunodeficiency virus isolates: prediction of antigenic epitopes in conserved and variable regions.

Independent isolates of human immunodeficiency virus (HIV) exhibit a striking genomic diversity, most of which is located in the viral envelope gene. Since this property of the HIV group of viruses may play an important role in the pathobiology of the virus, we analyzed the predicted amino acid sequences of the envelope proteins of seven different HIV strains, three of which represent sequential isolates from a single patient. By using a computer program that predicts the secondary protein structure and superimposes values for hydrophilicity, surface probability, and flexibility, we identified several potential antigenic epitopes in the envelope proteins of the seven different viruses. Interestingly, the majority of the predicted epitopes in the exterior envelope protein (gp120) were found in regions of high sequence variability which are interspersed with highly conserved regions among the independent viral isolates. A comparison of the sequential viral isolates revealed that changes concerning the secondary structure of the protein occurred only in regions which were predicted to be antigenic, predominantly in highly variable regions. The membrane-associated protein gp41 contains no highly variable regions; about 80% of the amino acids were found to be conserved, and only one hydrophilic area was identified as likely to be accessible to antibody recognition. These findings give insight into the secondary and possible tertiary structure of variant HIV envelope proteins and should facilitate experimental approaches directed toward the identification and fine mapping of HIV envelope proteins.     

The mapping and reconstitution of a conformational discontinuous B-cell epitope of HIV-1

A method for the discovery of the structure of conformational discontinuous epitopes of monoclonal antibodies (mAbs) is described. The mAb is used to select specific phages from combinatorial phage-display peptide libraries that in turn are used as an epitope-defining database that is applied via a novel computer algorithm to analyze the crystalline structure of the original antigen. The algorithm is based on the following: (1) Most contacts between a mAb and an antigen are through side-chain atoms of the residues. (2) In the three-dimensional structure of a protein, amino acid residues remote in linear sequence can juxtapose to one another through folding. (3) Tandem amino acid residues of the selected phage-displayed peptides can represent pairs of juxtaposed amino acid residues of the antigen. (4) Contact residues of the epitope are accessible to the antigen surface. (5) The most frequent tandem pairs of amino acid residues in the selected phage-displayed peptides can reflect pairs of juxtaposed amino acid residues of the epitope. Application of the algorithm enabled prediction of epitopes. On the basis of these predictions, segments of an antigen were used to reconstitute an antigenic epitope mimetic that was recognized by its original mAb.     

ADEPT: a computer program for prediction of protein antigenic determinants

ADEPT, a program that can be used for prediction of proteinantigenic determinants from the amino acid sequence alone forthe cases of humoral and cellular immune response, is described.Most methods presently utilized for this purpose are implementedin the program along with some original parameters for the caseof humoral immune response. There is also a possibility to combineseveral methods for the case of humoral immune response, whichprovides a means to create new predictive methods. ADEPT alsoincludes a literature-derived database of proteins in the SWISS-PROTstandard with experimentally determined antigenic determinants,so the predictive ability of new methods can be assessed withinthe program and corresponding statistical information is calculated.ADEPT may be useful for solving vamious tasks involving thedelineation of antigenic regions on proteins or characterizationof peptide fragments, arising, for examnple, in protein engineering(e.g. selection of protein portions most suitable for insertionof peptide sequences that are antigenically active or possessingother desired characteristics), and in particular for constructionof vaccines containing B- and/or T-cell epitopes, as well asfor other problems of medico-biological research.     

Structural factors modulate the activity of antigenic poliovirus sequences expressed on hybrid hepatitis B surface antigen particles.

We have studied the functional expression of antigenic poliovirus fragments carried by various hybrid hepatitis B surface antigen (HBsAg) particles. Several constructions were made by using two different insertion sites in the HBsAg molecule (amino acid positions 50 and 113) and two different sequences, one derived from poliovirus type 1 (PV-1) and the other from PV-2. The inserted fragments each encompassed residues 93 to 103 of the capsid protein VP1, a segment which includes the linear part of the neutralization antigenic site 1 of the poliovirus. The antigenicity and immunogenicity of the hybrid particles were evaluated and compared in terms of poliovirus neutralization. A high level of antigenic and immunogenic activity of the PV-1 fragment was obtained by insertion at position 113 but not at position 50 of HBsAg. However, a cooperative effect was observed when two PV-1 fragments were inserted at both positions of the same HBsAg molecule. Antibodies elicited by the PV-2 fragment inserted at amino acid position 113 did not bind or neutralize the corresponding poliovirus strain. They did, however, bind a chimeric poliovirus in which the homologous antigenic fragment of PV-1 had been replaced by that of PV-2. The only virions that were neutralized by these antibodies were certain mutants carrying amino acid substitutions within the PV-2 fragment. These results show that position, constraints from the carrier protein, and nature of the inserted sequences are critically important in favoring or limiting the expression of antigenic fragments as viral neutralization immunogens.     

Characterization of the antigenic structure of herpes simplex virus type 1 glycoprotein C through DNA sequence analysis of monoclonal antibody-resistant mutants.

Earlier studies of a group of monoclonal antibody-resistant (mar) mutants of herpes simplex virus type 1 glycoprotein C (gC) operationally defined two distinct antigenic sites on this molecule, each consisting of numerous overlapping epitopes. In this report, we further define epitopes of gC by sequence analysis of the mar mutant gC genes. In 18 mar mutants studied, the mar phenotype was associated with a single nucleotide substitution and a single predicted amino acid change. The mutations were localized to two regions within the coding sequence of the external domain of gC and correlated with the two previously defined antigenic sites. The predicted amino acid substitutions of site I mutants resided between residues Gln-307 and Pro-373, whereas those of site II mutants occurred between amino acids Arg-129 and Glu-247. Of the 12 site II mutations, 9 induced amino acid substitutions within an arginine-rich segment of 8 amino acids extending from residues 143 to 151. The clustering of the majority of substituted residues suggests that they contribute to the structure of the affected sites. Moreover, the patterns of substitutions which affected recognition by antibodies with similar epitope specificities provided evidence that epitope structures are physically linked and overlap within antigenic sites. Of the nine epitopes defined on the basis of mutations, three were located within site I and six were located within site II. Substituted residues affecting the site I epitopes did not overlap substituted residues of site II, supporting our earlier conclusion that sites I and II reside in spatially distinct antigenic domains. A computer analysis of the distribution of charged residues and the predicted secondary structural features of wild-type gC revealed that the two antigenic sites reside within the most hydrophilic regions of the molecule and that the antigenic residues are likely to be organized as beta sheets which loop out from the surface of the molecule. Together, these data and our previous studies support the conclusion that the mar mutations identified by sequence analysis very likely occur within or near the epitope structures themselves. Thus, two highly antigenic regions of gC have now been physically and genetically mapped to well-defined domains of the protein molecule.     

The structure of an antigenic determinant in a protein

The immunogenic and antigenic determinants of a synthetic peptide and the corresponding antigenic determinants in the parent protein have been elucidated. Four determinants have been defined by reactivity of a large panel of antipeptide monoclonal antibodies with short, overlapping peptides (7-28 amino acids), the immunizing peptide (36 amino acids), and the intact parent protein (the influenza virus hemagglutinin, HA). The majority of the antipeptide antibodies that also react strongly with the intact protein recognize one specific nine amino acid sequence. This immunodominant peptide determinant is located in the subunit interface in the HA trimeric structure. The relative inaccessibility of this site implies that antibody binding to the protein is to a more unfolded HA conformation. This antigenic determinant differs from those previously described for the hemagglutinin and clearly demonstrates the ability of synthetic peptides to generate antibodies that interact with regions of the protein not immunogenic or generally accessible when the protein is the immunogen.     

Glycosylation and an amino acid insertion in the head of hemagglutinin independently affect the antigenic properties of H5N1 avian influenza viruses

Antigenic drift forces us to frequently update influenza vaccines; however, the genetic basis for antigenic variation remains largely unknown. In this study, we used clade 7.2 H5 viruses as models to explore the molecular determinants of influenza virus antigenic variation. We generated eight monoclonal antibodies(MAbs) targeted to the hemagglutinin(HA) protein of the index virus A/chicken/Shanxi/2/2006 and found that two representative antigenically drifted clade 7.2 viruses did not react with six of the eight MAbs. The E131 N mutation and insertion of leucine at position 134 in the HA protein of the antigenically drifted strains eliminated the reactivity of the virus with the MAbs. We also found that the amino acid N131 in the H5 HA protein is glycosylated. Our results provide experimental evidence that glycosylation and an amino acid insertion or deletion in HA influence antigenic variation.