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.     

Sequence microheterogeneity of parvalbumin,the major fish allergen

The microheterogeneity of amino acid sequence observed in various allergens may affect immune response, but incidence of sequence microheterogeneity in allergens and its relation to their allergenicity are unclear. The occurrence of sequence microheterogeneity in major fish allergen, parvalbumin (PA), has been explored using bioinformatics approaches. 44% of 111 species with known PA sequence have PA isoforms. 41% of these species exhibit from 1 to 4 cases of PA sequence microheterogeneity, i.e. unique pairs of PA isoforms with sequence identity above 90%. 29% of 210 PA sequences studied are characterized by microheterogeneity. The occurrence of allergens among them is 2.5-fold higher than among other PAs. The incidence of sequence microheterogeneity within more allergenic β isoform of PA is 2.0-fold lower than that for its less allergenic α isoform. 39 residues affected by PA microheterogeneity are concentrated in the region of helices A, B, F, while helices D and E are the most conservative region. 44% and 11% of the microheterogeneous substitutions are located in the species-specific and cross-reactive IgE-binding epitopes of PAs, respectively. 45% and 48% of the substitution cases are predicted to cause notable changes in protein disorder propensity and protein stability, respectively. Hence, the increased allergenicity rate among PAs having microheterogeneous isoforms can be related to differences in their IgE-binding caused directly or in an allosteric manner. Overall, sequence microheterogeneity is shown to be inherent to many of PAs and likely contributes to PA allergenicity.     

Genetically modified soybeans and food allergies

Allergenic reactions to proteins expressed in GM crops has been one of the prominent concerns among biotechnology critics and a concern of regulatory agencies. Soybeans like many plants have intrinsic allergens that present problems for sensitive people. Current GM crops, including soybean, have not been shown to add any additional allergenic risk beyond the intrinsic risks already present. Biotechnology can be used to characterize and eliminate allergens naturally present in crops. Biotechnology has been used to remove a major allergen in soybean demonstrating that genetic modification can be used to reduce allergenicity of food and feed. This provides a model for further use of GM approaches to eliminate allergens.     

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.     

Supervised identification of allergen-representative peptides for in silico detection of potentially allergenic proteins

MOTIVATION: Identification of potentially allergenic proteins is needed for the safety assessment of genetically modified foods, certain pharmaceuticals and various other products on the consumer market. Current methods in bioinformatic allergology exploit common features among allergens for the detection of amino acid sequences of potentially allergenic proteins. Features for identification still unexplored include the motifs occurring commonly in allergens, but rarely in ordinary proteins. In this paper, we present an algorithm for the identification of such motifs with the purpose of biocomputational detection of amino acid sequences of potential allergens. RESULTS: Identification of allergen-representative peptides (ARPs) with low or no occurrence in proteins lacking allergenic properties is the essential component of our new method, designated DASARP (Detection based on Automated Selection of Allergen-Representative Peptide). This approach consistently outperforms the criterion based on identical peptide match for predicting allergenicity recommended by ILSI/IFBC and FAO/WHO and shows results comparable to the alignment-based criterion as outlined by FAO/WHO. AVAILABILITY: The detection software and the ARP set needed for the analysis of a query protein reported here are properties of the Swedish National Food Agency and are available upon request. The protein sequence sets used in this work are publicly available on http://www.slv.se/templatesSLV/SLV_Page____9343.asp. Allergenicity assessment for specific protein sequences of interest is also possible via ulfh@slv.se     

WebAllergen: a web server for predicting allergenic proteins

WebAllergen is a web server that predicts the potential allergenicity of proteins. The query protein will be compared against a set of prebuilt allergenic motifs that have been obtained from 664 known allergen proteins. The query will also be compared with known allergens that do not have detectable allergenic motifs. Moreover, users are allowed to upload their own allergens as alternative training sequences on which a new set of allergenic motifs will be built. The query sequences can also be compared with these motifs. AVAILABILITY: http://weballergen.bii.a-star.edu.sg/     

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/.     

Genetic modification and plant food allergens: risks and benefits

Plant genetic engineering has the potential to both introduce new allergenic proteins into foods and remove established allergens. A number of allergenic plant proteins have been characterized, showing that many are related to proteins which have potentially valuable properties for use in nutritional enhancement, food processing and crop protection. It is therefore important to monitor the allergenic potential of proteins used for plant genetic engineering and major biotechnology companies have established systems for this. Current technology allows gene expression to be down-regulated using antisense or co-suppression and future developments may allow targeted gene mutation or gene replacement. However, the application of this technology may be limited at least in the short term by the presence of multiple allergens and their contribution to food processing or other properties. Furthermore, the long-term stability of these systems needs to be established as reversion could have serious consequences.     

Search for the determinants of allergenicity in proteins of the lipocalin family

Three different lines of analysis have been applied to approach the problem of the allergenicity of certain proteins: biological functions, molecular structures and immunological properties. It is immediately obvious that these three are interdependent. The lipocalin family of proteins includes a significant number of allergens. A considerable amount of data is already available of lipocalins and some insights about allergenic determinants can now be presented. However, more information on the molecular structures and immunological parameters of lipocalin allergens is required.     

IgE binding to proteins from sesame and assessment of allergenicity: implications for biotechnology?

Successful prediction of the potential allergenicity of a protein may be a key factor in the development of novel, genetically modified foods. The use of the decision tree approach for the prediction of allergenicity is discussed. The methods currently used for identifying allergenic proteins (including use of IgE from patient sera for recognition of proteins) are reviewed. Finally, a specific review of the literature concerning identification of allergens from sesame leads to the conclusion that in the absence of validated animal models, identification of allergenicity (and, consequently, prediction of allergenicity) may be problematic.     

Food allergen detection by mass spectrometry: From common to novel protein ingredients

Food allergens are molecules, mainly proteins, that trigger immune responses in susceptible individuals upon consumption even when they would otherwise be harmless. Symptoms of a food allergy can range from mild to acute; this last effect is a severe and potentially life-threatening reaction. The European Union (EU) has identified 14 common food allergens, but new allergens are likely to emerge with constantly changing food habits. Mass spectrometry (MS) is a promising alternative to traditional antibody-based assays for quantifying multiple allergenic proteins in complex matrices with high sensitivity and selectivity. Here, the main allergenic proteins and the advantages and drawbacks of some MS acquisition protocols, such as multiple reaction monitoring (MRM) and data-dependent analysis (DDA) for identifying and quantifying common allergenic proteins in processed foodstuffs are summarized. Sections dedicated to novel foods like microalgae and insects as new sources of allergenic proteins are included, emphasizing the significance of establishing stable marker peptides and validated methods using database searches. The discussion involves the in-silico digestion of allergenic proteins, providing insights into their potential impact on immunogenicity. Finally, case studies focussing on microalgae highlight the value of MS as an effective analytical tool for ensuring regulatory compliance throughout the food control chain.     

Computational analysis of the relationship between allergenicity and digestibility of allergenic proteins in simulated gastric fluid

Background  

Safety assessment of genetically modified (GM) food, with regard to allergenic potential of transgene-encoded xenoproteins, typically involves several different methods, evaluation by digestibility being one thereof. However, there are still debates about whether the allergenicity of food allergens is related to their resistance to digestion by the gastric fluid. The disagreements may in part stem from classification of allergens only by their sources, which we believe is inadequate, and the difficulties in achieving identical experimental conditions for studying digestion by simulated gastric fluid (SGF) so that results can be compared. Here, we reclassify allergenic food allergens into alimentary canal-sensitized (ACS) and non-alimentary canal-sensitized (NACS) allergens and use a computational model that simulates gastric fluid digestion to analyze the digestibilities of these two types.     

Variable expression of pathogenesis-related protein allergen in mountain cedar (Juniperus ashei) pollen

Allergic diseases have been increasing in industrialized countries. The environment is thought to have both direct and indirect modulatory effects on disease pathogenesis, including alterating on the allergenicity of pollens. Certain plant proteins known as pathogenesis-related proteins appear to be up-regulated by certain environmental conditions, including pollutants, and some have emerged as important allergens. Thus, the prospect of environmentally regulated expression of plant-derived allergens becomes yet another potential environmental influence on allergic disease. We have identified a novel pathogenesis-related protein allergen, Jun a 3, from mountain cedar (Juniperus ashei) pollen. The serum IgE from patients with hypersensitivity to either mountain cedar or Japanese cedar were shown to bind to native and recombinant Jun a 3 in Western blot analysis and ELISA. Jun a 3 is homologous to members of the thaumatin-like pathogenesis-related (PR-5) plant protein family. The amounts of Jun a 3 extracted from mountain cedar pollen varied up to 5-fold in lots of pollen collected from the same region in different years and between different regions during the same year. Thus, Jun a 3 may contribute not only to the overall allergenicity of mountain cedar pollen, but variable levels of Jun a 3 may alter the allergenic potency of pollens produced under different environmental conditions.     

Abundance and functional roles of intrinsic disorder in allergenic proteins and allergen representative peptides

The pathological process of allergies generally involves an initial activation of certain immune cells, tied to an ensuing inflammatory reaction on renewed contact with the allergen. In IgE-mediated hypersensitivity, this typically occurs in response to otherwise harmless food- or air-borne proteins. As some members of certain protein families carry special properties that make them allergenic, exploring protein allergens at the molecular level is instrumental to an improved understanding of the disease mechanisms, including the identification of relevant antigen features. For this purpose, we inspected a previously identified set of allergen representative peptides (ARPs) to scrutinize protein intrinsic disorder. The resulting study presented here focused on the association between these ARPs and protein intrinsic disorder. In addition, the connection between the disorder-enriched ARPs and UniProt functional keywords was considered. Our analysis revealed that ～ 20% of the allergen peptides are highly disordered, and that ～ 77% of ARPs are either located within disordered regions of corresponding allergenic proteins or show more disorder/flexibility than their neighbor regions. Furthermore, among the subset of allergenic proteins, ～ 70% of the predicted molecular recognition features (MoRFs that consist of short interactive disordered regions undergoing disorder-to-order transitions at interaction with binding partners) were identified as ARPs. These results suggest that intrinsic disorder and MoRFs may play functional roles in IgE-mediated allergy.     

Microbial extracellular electron transfer and its relevance to iron corrosion

Extracellular electron transfer (EET) is a microbial metabolism that enables efficient electron transfer between microbial cells and extracellular solid materials. Microorganisms harbouring EET abilities have received considerable attention for their various biotechnological applications, including bioleaching and bioelectrochemical systems. On the other hand, recent research revealed that microbial EET potentially induces corrosion of iron structures. It has been well known that corrosion of iron occurring under anoxic conditions is mostly caused by microbial activities, which is termed as microbiologically influenced corrosion (MIC). Among diverse MIC mechanisms, microbial EET activity that enhances corrosion via direct uptake of electrons from metallic iron, specifically termed as electrical MIC (EMIC), has been regarded as one of the major causative factors. The EMIC‐inducing microorganisms initially identified were certain sulfate‐reducing bacteria and methanogenic archaea isolated from marine environments. Subsequently, abilities to induce EMIC were also demonstrated in diverse anaerobic microorganisms in freshwater environments and oil fields, including acetogenic bacteria and nitrate‐reducing bacteria. Abilities of EET and EMIC are now regarded as microbial traits more widespread among diverse microbial clades than was thought previously. In this review, basic understandings of microbial EET and recent progresses in the EMIC research are introduced.     

Purification and characterization of allergens from Xanthium strumarium pollen

The allergenic components present in whole pollen extract of Xanthium strumarium were isolated by sequential ammonium sulphate precipitation, DEAE Sephadex A50 chromatography and gel filtration. The techniques of RAST inhibition and skin test were utilized to check the allergenicity of fractionated proteins revealing the presence of Xan Ib and Xan VIa as the important allergenic components. Xan Ib was found to be devoid of carbohydrate and had a molecular weight of 103,000 daltons. Xan VIa was a glycoprotein of molecular weight 17,000 daltons. The carbohydrate moiety of Xan VIa was found to be associated with allergenicity. The characteristic pattern of whole pollen extract on CIE and TLIEF showed 36 and 21 protein bands, respectively. The use of FPLC in isolation of partially purified allergens from Xanthium is discussed.     

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.