AllerTool: a web server for predicting allergenicity and allergic cross-reactivity in proteins

Assessment of potential allergenicity and patterns of cross-reactivity is necessary whenever novel proteins are introduced into human food chain. Current bioinformatic methods in allergology focus mainly on the prediction of allergenic proteins, with no information on cross-reactivity patterns among known allergens. In this study, we present AllerTool, a web server with essential tools for the assessment of predicted as well as published cross-reactivity patterns of allergens. The analysis tools include graphical representation of allergen cross-reactivity information; a local sequence comparison tool that displays information of known cross-reactive allergens; a sequence similarity search tool for assessment of cross-reactivity in accordance to FAO/WHO Codex alimentarius guidelines; and a method based on support vector machine (SVM). A 10-fold cross-validation results showed that the area under the receiver operating curve (A(ROC)) of SVM models is 0.90 with 86.00% sensitivity (SE) at specificity (SP) of 86.00%. Availability: AllerTool is freely available at http://research.i2r.a-star.edu.sg/AllerTool/.     

基于PC/Linux的致敏性评估网站的构建及其应用

利用数据挖掘技术,根据WHO-IUIS变应原列表,从Swiss-prot和NCBI,以及国际上现存的变应原数据库、科学文献等,收集、建立了完整的变应原数据库。基于PC机和Linux操作系统,利用相关软件,搭建了致敏性评估网站。利用FastA算法,通过将未知蛋白与数据库中的已知蛋白进行比对,从而判断其致敏性。结果表明构建的网络平台能够加速数据的分析过程。     

Recombinant allergens for analysing T-cell responses

T-cell responses constitute a central element of allergic disease and a model for studying Th1 and Th2 cytokine pathways. Most studies to date have used extracts of allergens which contain variable quantities of different allergens and non-allergenic antigens. Recombinant allergens provide the tools for studying the responses to allergens in a reproducible and dose-dependent manner and the different T-cell responses of allergic and non-allergic subjects provide a method for verifying the responses and their relationship to allergic sensitisation. Most allergies show dominant responses to one or a few major allergens. These allergens have been described for the common allergies and have been produced as recombinant allergens. A particular problem for allergens is that many are mixtures of proteins from multi-gene families or are highly polymorphic. Information now exists so the sequence variation can be represented. Purified recombinant allergens produced by standard expression systems stimulate the expected T-cell responses from the peripheral blood of allergic and non-allergics to allergen extracts. Although stimulation with recombinant allergens which are not produced with a natural IgE binding activity can provide a measure of allergenicity, the altered tertiary structure can reduce Th2 responses. The sequence information now available provides the means to use PCR to produce cDNA for the production of recombinant allergens from readily available sources. The production of the highly reactive recombinant Der p 2 allergen of house dust mite from natural sources is described.     

A database for allergenic proteins and tools for allergenicity prediction

The AllergenPro database has developed a web-based system that will provide information about allergen in microbes, animals and plants. The database has three major parts and functions:(i) database list; (ii) allergen search; and (iii) allergenicity prediction. The database contains 2,434 allergens related information readily available in the database such as on allergens in rice microbes (712 records), animals (617 records) and plants (1,105 records). Furthermore, this database provides bioinformatics tools for allergenicity prediction. Users can search for specific allergens by various methods and can run tools for allergenicity prediction using three different methods.

Availability

The database is available for free at http://www.niab.go.kr/nabic/     

1H, 13C and 15N resonance assignments and second structure information of Gad m 1: a β-parvalbumin allergen from Atlantic cod (Gadus morhua)

Gad m 1 is the major allergen from Atlantic cod. It belongs to β-parvalbumin protein family and is characterized by the presence of two calcium-binding sites so called EF-hand motifs. β-Parvalbumins such as Gad m 1 are the most important fish allergens and their high cross-reactivity is the cause of the observed polysensitization to various fish species in allergic patients. Despite extensive efforts, the complete elucidation of β-parvalbumin-IgE complexes has not been achieved yet. Allergen structural studies are essential for the development of novel immunotherapy strategies, including vaccination with hypoallergenic derivatives and chimeric molecules. Here, we report for the first time the NMR study of a β-parvalbumin: Gad m 1. This report includes: ¹H, ¹³C and ¹⁵N resonance assignments of Gad m 1 as well as the second structure information based on the ¹³C chemical shifts.     

Nuclear magnetic resonance structure-based epitope mapping and modulation of dust mite group 13 allergen as a hypoallergen

IgE-mediated allergic response involves cross-linking of IgE bound on mast cells by specific surface epitopes of allergens. Structural studies on IgE epitopes of allergens are essential in understanding the characteristics of an allergen and for development of specific allergen immunotherapy. We have determined the structure of a group 13 dust mite allergen from Dermatophagoides farinae, Der f 13, using nuclear magnetic resonance. Sequence comparison of Der f 13 with homologous human fatty acid-binding proteins revealed unique surface charged residues on Der f 13 that may be involved in IgE binding and allergenicity. Site-directed mutagenesis and IgE binding assays have confirmed four surface charged residues on opposite sides of the protein that are involved in IgE binding. A triple mutant of Der f 13 (E41A_K63A_K91A) has been generated and found to have significantly reduced IgE binding and histamine release in skin prick tests on patients allergenic to group 13 dust mite allergens. The triple mutant is also able to induce PBMC proliferation in allergic patients with indices similar to those of wild-type Der f 13 and shift the secretion of cytokines from a Th2 to a Th1 pattern. Mouse IgG serum raised using the triple mutant is capable to block the binding of IgE from allergic patients to wild-type Der f 13, indicating potential for the triple mutant as a hypoallergen for specific immunotherapy. Findings in this study imply the importance of surface charged residues on IgE binding and allergenicity of an allergen, as was also demonstrated in other major allergens studied.     

Structural differences between human proteins and aero- and microbial allergens define allergenicity

The current paradigm suggests that structural homology of allergenic proteins to microbial (particularly helminths) or human proteins underlie their allergenic nature. To examine systematically the structural relationships among allergens and proteins of pathogens (helminths, protozoans, fungi and bacteria) as they relate to allergenicity, we compared the amino acid sequence of 499 molecularly-defined allergens with the predicted proteomes of fifteen known pathogens, including Th2 inducing helminths and Th1-inducing protozoans, and humans using a variety of bioinformatic tools. Allergenicity was assessed based on IgE prevalences using publicly accessible databases and the literature. We found multiple homologues of common allergens among proteins of helminths, protozoans, fungi and humans, but not of bacteria. In contrast, 187 allergens showed no homology with any of the microbial genera studied. Interestingly, allergens without homologues or those with limited levels of sequence conservation were the most allergenic displaying high IgE prevalences in the allergic population. There was an inverse relationship between allergenicity and amino acid conservation levels with either parasite, including helminth, or human proteins. Our results suggest that allergenicity may be associated with the relative "uniqueness" of an antigen, i.e. immunogenicity, while similarity would lead to immunological tolerance.     

Airborne birch and oak pollen grains and birch pollen allergens at a common sampling station in Stockholm

In the present study, the airborne concentrations of birch and oak pollen grains and birch pollen allergens have been recorded in samples from a common sampling station in Stockholm. The sampling period was between April 22^nd and May 31^st 2003. The objectives were to evaluate if analysis of allergen particles in parallel with pollen grains would be relevant to aid subjects suffering from pollinosis. Days with low birch pollen counts had relatively high nominal allergen concentrations in the beginning of the sampling period. The birch pollen grain concentration peaks, during the dry pollination culmination interval in the middle of the period, were associated with correspondingly lower nominal concentrations of allergens than grains. At the end of the sampling period very high nominal amounts of allergen appeared, as reflected by high concentrations of oak pollen grains. The high allergen concentrations were obtained as a result of inherent cross‐reactivity of anti‐ Bet v 1 antibodies with Que a antigens, which are immunologically analogous with Bet v 1. Allergen concentrations increased and decreased after light and heavy rain, respectively. Results obtained indicate that adding a pollen count forecast with allergen concentration data should aid pollen allergic subjects to avoid particulate allergens which might be expected to penetrate more easily than pollen grains into indoor environments.     

Identification of Two Metallothioneins as Novel Inhalative Coffee Allergens Cof a 2 and Cof a 3

BackgroundDust of green coffee beans is known to be a relevant cause for occupational allergic disorders in coffee industry workers. Recently, we described the first coffee allergen (Cof a 1) establishing an allergenic potential of green coffee dust.ObjectiveOur aim was to identify allergenic components of green coffee in order to enhance inhalative coffee allergy diagnosis.MethodsA Coffea arabica pJuFo cDNA phage display library was created and screened for IgE binding with sera from allergic coffee workers. Two further coffee allergens were identified by sequence analysis, expressed in E. coli, and evaluated by Western blots. The prevalence of sensitization to recombinant Cof a 1, Cof a 2, and Cof a 3 and to commercially available extract was investigated by ELISA (enzyme-linked immunosorbent assay) respectively CAP (capacity test) screening in 18 sera of symptomatic coffee workers.ResultsIn addition to the previously described chitinase Cof a 1, two Coffea arabica cysteine-rich metallothioneins of 9 and 7 kDa were identified and included in the IUIS Allergen Nomenclature as Cof a 2 and Cof a 3. Serum IgE antibodies to at least one of the recombinant allergens were found in 8 out of 18 symptomatic coffee workers (44%). Only 2 of the analysed sera (11%) had reacted previously to the commercial allergy test.ConclusionsIn addition to the previously described Cof a 1 we have identified two further coffee proteins to be type I coffee allergens (Cof a 2 and Cof a 3) which may have a relevant potential for the specific diagnosis and/or therapy of coffee allergy.     

花生过敏原iso-Ara h 3的克隆、表达和免疫学鉴定

采用Touchdown PCR技术从花生cDNA中克隆到花生的过敏原iso-Ara h 3基因,并进行重组蛋白表达,再用Western blot技术鉴定重组蛋白过敏原性。结果显示,构建的pET44a-iso—Ara h 3重组菌能表达iso—Ara h 3蛋白。用8例花生过敏的阳性血清鉴定表明,重组的iso—Ara h 3蛋白血清IgE识别率为12．5％,是一种低过敏原性的过敏原蛋白。     

AllerHunter: A SVM-Pairwise System for Assessment of Allergenicity and Allergic Cross-Reactivity in Proteins

Allergy is a major health problem in industrialized countries. The number of transgenic food crops is growing rapidly creating the need for allergenicity assessment before they are introduced into human food chain. While existing bioinformatic methods have achieved good accuracies for highly conserved sequences, the discrimination of allergens and non-allergens from allergen-like non-allergen sequences remains difficult. We describe AllerHunter, a web-based computational system for the assessment of potential allergenicity and allergic cross-reactivity in proteins. It combines an iterative pairwise sequence similarity encoding scheme with SVM as the discriminating engine. The pairwise vectorization framework allows the system to model essential features in allergens that are involved in cross-reactivity, but not limited to distinct sets of physicochemical properties. The system was rigorously trained and tested using 1,356 known allergen and 13,449 putative non-allergen sequences. Extensive testing was performed for validation of the prediction models. The system is effective for distinguishing allergens and non-allergens from allergen-like non-allergen sequences. Testing results showed that AllerHunter, with a sensitivity of 83.4% and specificity of 96.4% (accuracy = 95.3%, area under the receiver operating characteristic curve AROC = 0.928±0.004 and Matthew''s correlation coefficient MCC = 0.738), performs significantly better than a number of existing methods using an independent dataset of 1443 protein sequences. AllerHunter is available at http://tiger.dbs.nus.edu.sg/AllerHunter     

Cross-reactivity of pollen and food allergens: soybean Gly m 4 is a member of the Bet v 1 superfamily and closely resembles yellow lupine proteins

In many cases, patients allergic to birch pollen also show allergic reactions after ingestion of certain fruits or vegetables. This observation is explained at the molecular level by cross-reactivity of IgE antibodies induced by sensitization to the major birch pollen allergen Bet v 1 with homologous food allergens. As IgE antibodies recognize conformational epitopes, a precise structural characterization of the allergens involved is necessary to understand cross-reactivity and thus to develop new methods of allergen-specific immunotherapy for allergic patients. Here, we report the three-dimensional solution structure of the soybean allergen Gly m 4, a member of the superfamily of Bet v 1 homologous proteins and a cross-reactant with IgE antibodies originally raised against Bet v 1 as shown by immunoblot inhibition and histamine release assays. Although the overall fold of Gly m 4 is very similar to that of Bet v 1, the three-dimensional structures of these proteins differ in detail. The Gly m 4 local structures that display those differences are also found in proteins from yellow lupine with known physiological function. The three-dimensional structure of Gly m 4 may thus shed some light on the physiological function of this subgroup of PR10 proteins (class 10 of pathogenesis-related proteins) and, in combination with immunological data, allow us to propose surface patches that might represent cross-reactive epitopes.     

植物类过敏性蛋白(变应原)研究进展

据相关资料最新统计表明,全球大约有25%的人口受到I型变态反应疾病的影响。植物中的花粉、汁液和果实可以分别作为吸入性、接触性和食入性过敏原影响过敏体质的人群。目前,惟一有效的办法是应用特异性变应原进行脱敏治疗(脱敏治疗)。而在应用天然变应原提取物进行传统免疫治疗过程中存在一定的危险性。通过基因工程的方法合成的修饰后的高纯度特异变应原在保持过敏性蛋白完整的免疫活性的同时可降低其本身的过敏原性。这种修饰后的过敏性蛋白的应用将使免疫治疗更趋于标准化操作同时避免应用天然过敏性蛋白天然提取物时的危险性,最终可达到提高治疗的效果的目的。目前,重组这种新的过敏性蛋白已经成为过敏性疾病研究的新的热点。     

A DNA aptamer recognizes the Asp f 1 allergen of Aspergillus fumigatus

Allergies are caused by the binding of IgE antibodies onto specific sites on allergens. However, in the assessment of exposure to airborne allergens, current techniques such as whole spore counts fail to account for the presence of these allergenic epitopes that trigger allergic reactions. The objective of the research is to develop a DNA aptamer for the Asp f 1 allergen of the pathogenic fungus Aspergillus fumigatus, using an IgE-binding epitope of the allergen as the target for aptamer selection. Through in vitro SELEX, an aptamer has been produced that binds with nanomolar affinity to the Asp f 1 IgE-epitope. The aptamer is also able to recognize the native Asp f 1 allergen, and does not bind to allergenic proteins from non-target mold species such as Alternaria alternata. Production of this aptamer provides proof-of-principle that allergen measurement methods can be developed to indicate the potent fraction, or allergenicity, of allergens.     

Molecular characterization of American cockroach tropomyosin (Periplaneta americana allergen 7), a cross-reactive allergen

Inhalation of allergens produced by the American cockroach (Periplaneta americana) induces IgE Ab production and the development of asthma in genetically predisposed individuals. The cloning and expression in Escherichia coli of P. americana tropomyosin allergen have been achieved. The protein shares high homology with other arthropod tropomyosins (80% identity) but less homology with vertebrate ones (50% identity). The recombinant allergen was produced in E. coli as a nonfusion protein with a yield of 9 mg/l of bacterial culture. Both natural and recombinant tropomyosins were purified by isoelectric precipitation. P. americana allergen 1 (Per a 1) and Per a 7 (tropomyosin) are to date the only cross-reacting allergens found in cockroaches. ELISA and Western blot inhibition experiments, using natural and recombinant purified tropomyosins from shrimp and cockroach, showed that tropomyosin induced cross-reactivity of IgE from patients allergic to these allergens, suggesting that this molecule could be a common allergen among invertebrates.     

Assessment of allergen cross-reactivity

The prediction of allergen cross-reactivity is currently largely based on linear sequence data, but will soon include 3D information on homology among surface exposed residues. To evaluate procedures for these predictions, we need ways to quantitatively assess actual cross-reactivity between two allergens. Three parameters are mentioned: 1) the fraction of the epitopes that is cross-reactive; 2) the fraction of IgE that is cross-reactive; 3) the relative affinity of the interaction between IgE and the two allergens. This editorial briefly compares direct binding protocols with the often more appropriate reciprocal inhibition protocols. The latter type of protocol provides information on symmetric versus asymmetric cross-reactivity, and thus on the distinction between complete (= sensitising) allergens versus incomplete, cross-reacting allergens. The need to define the affinity threshold of the assay and a caveat on the use of serum pools are also discussed.     

AllergenPro: an integrated database for allergenicity analysis and prediction

The National Agricultural Biotechnology Information Center (NABIC) reconstructed an AllergenPro database for allergenic proteins analysis and allergenicity prediction. The AllergenPro is an integrated web-based system providing information about allergen in foods, microorganisms, animals and plants. The allergen database has the three main features namely, (1) allergen list with epitopes, (2) searching of allergen using keyword, and (3) methods for allergenicity prediction. This updated AllergenPro outputs the search based allergen information through a user-friendly web interface, and users can run tools for allergenicity prediction using three different methods namely, (1) FAO/WHO, (2) motif-based and (3) epitope-based methods.

Availability

The database is available for free at http://nabic.rda.go.kr/allergen/     

Cytokine gene polymorphisms and atopic disease in two European cohorts. (ECRHS-Basel and SAPALDIA)

Background

Avoidance of allergens is still recommended as the first and best way to prevent allergic illnesses and their comorbid diseases. Despite a variety of attempts there has been very limited success in the area of environmental control of allergic disease. Our objective was to identify a non-invasive, non-pharmacological method to reduce indoor allergen loads in atopic persons' homes and public environments. We employed a novel in vivo approach to examine the possibility of using aluminum sulfate to control environmental allergens.

Methods

Fifty skin test reactive patients were simultaneously skin tested with conventional test materials and the actions of the protein/glycoprotein modifier, aluminum sulfate. Common allergens, dog, cat, dust mite, Alternaria, and cockroach were used in the study.

Results

Skin test reactivity was significantly reduced by the modifier aluminum sulfate. Our studies demonstrate that the effects of histamine were not affected by the presence of aluminum sulfate. In fact, skin test reactivity was reduced independent of whether aluminum sulfate was present in the allergen test material or removed prior to testing, indicating that the allergens had in some way been inactivated.

Conclusion

Aluminum sulfate was found to reduce the in vivo allergic reaction cascade induced by skin testing with common allergens. The exact mechanism is not clear but appears to involve the alteration of IgE-binding epitopes on the allergen. Our results indicate that it may be possible to diminish the allergenicity of an environment by application of the active agent aluminum sulfate, thus producing environmental control without complete removal of the allergen.     

Predicting allergenic proteins using wavelet transform

MOTIVATION: With many transgenic proteins introduced today, the ability to predict their potential allergenicity has become an important issue. Previous studies were based on either sequence similarity or the protein motifs identified from known allergen databases. The similarity-based approaches, although being able to produce high recalls, usually have low prediction precisions. Previous motif-based approaches have been shown to be able to improve the precisions on cross-validation experiments. In this study, a system that combines the advantages of similarity-based and motif-based prediction is described. RESULTS: The new prediction system uses a clustering algorithm that groups the known allergenic proteins into clusters. Proteins within each cluster are assumed to carry one or more common motifs. After a multiple sequence alignment, proteins in each cluster go through a wavelet analysis program whereby conserved motifs will be identified. A hidden Markov model (HMM) profile will then be prepared for each identified motif. The allergens that do not appear to carry detectable allergen motifs will be saved in a small database. The allergenicity of an unknown protein may be predicted by comparing it against the HMM profiles, and, if no matching profiles are found, against the small allergen database by BLASTP. Over 70% of recall and over 90% of precision were observed using cross-validation experiments. Using the entire Swiss-Prot as the query, we predicted about 2000 potential allergens. AVAILABILITY: The software is available upon request from the authors.