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White-fleshed pitaya (Selenicereus undatus) and red-fleshed pitaya (Selenicereus costaricensis) are becoming increasingly popular because of their nutritional and medicinal benefits. However, in addition to their beneficial properties, allergy to pitaya fruits has occurred in daily life. In this study, we investigated the protein profile of pitaya fruit seeds and focused on the most reactive proteins against immunoglobulin E (IgE) in sera from allergic patients by immunoblotting. A protein band of approximately 20 kDa displayed a clear reaction with the serum IgE. The protein bands of interest were excised, in-gel digested, and analyzed using liquid chromatography–tandem mass spectrometry (LC–MS/MS), followed by data searching against a restricted database (Caryophyllales in UniProtKB) for protein identification. Immunoinformatic tools were used to predict protein allergenicity. The potential allergens included cupin_1 and heat shock protein 70 (HSP70) in white-fleshed pitaya seeds, and cupin_1, heat shock protein 70, and heat shock protein sti1-like in red-fleshed pitaya seeds are potential allergens. The expression of potential allergens was further verified at the transcriptional level in the species of S. undatus and S. costaricensis. [ABSTRACT FROM AUTHOR]

White-fleshed pitaya (Selenicereus undatus) and red-fleshed pitaya (Selenicereus costaricensis) are becoming increasingly popular because of their nutritional and medicinal benefits. However, in addition to their beneficial properties, allergy to pitaya fruits has occurred in daily life. In this study, we investigated the protein profile of pitaya fruit seeds and focused on the most reactive proteins against immunoglobulin E (IgE) in sera from allergic patients by immunoblotting. A protein band of approximately 20 kDa displayed a clear reaction with the serum IgE. The protein bands of interest were excised, in-gel digested, and analyzed using liquid chromatography–tandem mass spectrometry (LC–MS/MS), followed by data searching against a restricted database (Caryophyllales in UniProtKB) for protein identification. Immunoinformatic tools were used to predict protein allergenicity. The potential allergens included cupin_1 and heat shock protein 70 (HSP70) in white-fleshed pitaya seeds, and cupin_1, heat shock protein 70, and heat shock protein sti1-like in red-fleshed pitaya seeds are potential allergens. The expression of potential allergens was further verified at the transcriptional level in the species of S. undatus and S. costaricensis.

Simulium dermatitis is an IgE-mediated skin reaction in animals and humans caused by the bites of black flies. Although Simulium nigrogilvum has been incriminated as the main human-biting black fly species in Thailand, information on its salivary allergens is lacking. Salivary gland extract of S. nigrogilvum females was subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the separated components were applied onto nitrocellulose membranes for immunoblotting, which was performed by probing the protein blots with sera from 17 individuals who were allergic to the bites of S. nigrogilvum. IgE-reactive protein bands were characterized further by liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Nine protein bands (79, 42, 32, 25, 24, 22, 15, 13, and 11 kDa) were recognized in the serum of the subjects. Four of the nine protein bands (32, 24, 15, and 11 kDa) showed IgE reactivity in all (100%) of the tested sera, and they were identified as salivary secreted antigen 5-related protein, salivary serine protease, erythema protein, and hypothetical secreted protein, respectively. Three other proteins, salivary serine protease (25 kDa), salivary D7 secreted protein (22 kDa), and hypothetical protein (13 kDa), reacted with > 50% of the sera. The relevance of the identified protein bands as allergens needs to be confirmed by using pure recombinant proteins, either in the in vivo skin prick test or in vitro detection of the specific IgE in the serum samples of allergic subjects. This will be useful for the rational design of component-resolved diagnosis and allergen immunotherapy for the allergy mediated by the bites of black flies. [ABSTRACT FROM AUTHOR]

Alstonia scholaris or Indian devil tree is a common, evergreen, tropical tree of the Apocynaceae family. The objectives of this study were (a) to observe the seasonal variation of A. scholaris pollen in the atmosphere of an industrial and rural area of West Bengal, India by conducting a 2-year aerobiological survey with a Burkard personal volumetric sampler, (b) to study its allergenicity in the local population by in vivo (skin-prick test) and in vitro tests (enzyme-linked immunosorbent assay and dot blotting), (c) to identify the immunoglobulin E (IgE)-binding proteins present in the pollen extract (sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting), (d) to study its chemical composition. A. scholaris pollen were present in the air from September until November. They contained 14.3 % carbohydrate, 9.2 % lipid, and 4.3 % protein. Among 140 respiratory allergic local patients, 28.57 % showed positive skin reaction to A. scholaris pollen extract. Twelve protein bands in the range of 94.4–13.3 kDa were observed in the pollen extract. Seven IgE-binding proteins were found. Among them, one component of 29.9 kDa was the most important in A. scholaris pollen extract. This component could be purified and would be helpful in the diagnosis and therapy of A. scholaris pollen-susceptible patients. [ABSTRACT FROM AUTHOR]

Abstract: Hazelnuts (Corylus avellana) are one of the most common sources of life-long IgE-mediated food allergies. In this study, we investigated the IgE-reactivity pattern of children with hazelnut allergy (N=15) from Regione Campania, located in Southern Italy, and addressed proteomic strategies for characterizing IgE-binding proteins. For all of the patients (15/15), the predominant IgE-reactive component was a minor ~55kDa protein not previously described. Similar to the hazelnut 11S globulin Cor a 9 allergen, the immunoreactive protein consisted of two subunits linked via a disulfide bridge. In contrast to Cor a 9, only the 20.7kDa alkaline subunit exhibited IgE-affinity. The immunogenic subunit was purified by a two-step chromatographic procedure, but peptide mass fingerprinting was unsuccessful in identifying it, due to the incompleteness of the annotated hazelnut genome. Several tryptic peptides were de novo sequenced by tandem mass spectrometry and showed a high degree of homology with the 11S globulin storage proteins from other seeds, some of which have already been reported as food allergens. The structural characterization suggests that the new putative allergen is a divergent isoform of the hazelnut 11S globulin. These results provide a new platform for developing innovative diagnostic and therapeutic intervention plans. Biological significance: Over the years, at least five proteins have been reported as potential food hazelnut allergens. The predominance of specific allergens appears to be strictly related to the geographical origin of the allergic subjects. The complex patterns of the IgE-reactivity of hazelnut storage proteins result in a poor diagnostic and prognostic accuracy. In the perspective of a component-resolved “molecular approach” to the hazelnut allergy we investigated the immune-reactivity patterns to hazelnuts of 15 patients (14 in the pediatric age range) from Region Campania, located in Southern Italy. For all the patients the predominant IgE-reactive component was a minor ~55kDa protein not previously annotated in either protein or genomic databases. The putative allergen was isolated, partially characterized by MS/MS de novo sequencing and appears to be an isoallergen of the hazelnut 11S globulin Cor a 9. Like this latter, the immunoreactive protein consisted of two subunits linked via a disulfide. In contrast to Cor a 9, only the 20.7kDa alkaline subunit exhibited IgE-affinity, in analogy to 11S allergens from other seeds (pistachio, cashew, soybean). We believe that the application of combined immunochemical and proteomic strategies to characterize the new food allergen could be of interest for the readers of Journal of Proteomics. In addition, the results of this study have functional worth in providing a new platform to plan innovative diagnostic and therapeutic intervention approaches to treat hazelnut allergy. [Copyright &y& Elsevier]

A large number of food allergens, usually proteins capable of inducing allergic symptoms, including severe, even life-threatening reactions in predisposed individuals, have been identified and characterized. As most of these proteins are from our daily dietary intake, they are often difficult to avoid. However, the proteins that cause such immunoglobulin E (IgE)-mediated reactions can be assigned to only a limited number of protein families. Detailed knowledge about the characteristics of food allergens, their structures, biological activity, and stability, may be helpful in improving diagnosis of food allergy, avoiding unnecessary exclusion of diets, and assessing the risk of cross-reactive allergies to other food sources. The purpose of this review is to shed light on the sources and molecular properties of the allergenic proteins, their stability, the mechanisms of the allergenic responses, and recent findings related to prevention of this serious issue. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Soy lecithin is widely used as an emulsifier in processed foods, pharmaceuticals and cosmetics. Soy lecithin is composed principally of phospholipids; however, it has also been shown to contain IgE-binding proteins, albeit at a low level. A few clinical cases involving allergic reactions to soy lecithin have been reported. The purpose of this investigation is to better characterize the IgE-binding proteins typically found in lecithin. Methods: Soy lecithin proteins were isolated following solvent extraction of lipid components and then separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated lecithin proteins were immunoblotted with sera from soy-sensitive individuals to determine the pattern of IgE-binding proteins. The identity of IgE-reactive bands was determined from their N-terminal sequence. Results: The level of protein in six lecithin samples obtained from commercial suppliers ranged from 100 to 1,400 ppm. Lecithin samples showed similar protein patterns when examined by SDS-PAGE. Immunoblotting with sera from soy-sensitive individuals showed IgE binding to bands corresponding to 7, 12, 20, 39 and 57 kD. N-terminal analysis of these IgE-binding bands resulted in sequences for 3 components. The 12-kD band was identified as a methionine-rich protein (MRP) and a member of the 2S albumin class of soy proteins. The 20-kD band was found to be soybean Kunitz trypsin inhibitor. The 39-kD band was matched to a soy protein with unknown function. Conclusions: Soy lecithin contains a number of IgE-binding proteins; thus, it might represent a source of hidden allergens. These allergens are a more significant concern for soy-allergic individuals consuming lecithin products as a health supplement. In addition, the MRP and the 39-kD protein identified in this study represent newly identified IgE-binding proteins.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Peanut kernels contain many allergens able to elicit IgE–mediated type 1 allergic reactions in sensitized individuals. Sera from sensitized patients recognize variable patterns of IgE–binding proteins. The identification of the IgE–binding proteins of peanut extract would faciliate improvement of diagnostic and immunotherapeutic approaches as well as development of sensitive test systems for the detection of hidden peanut allergens present as additives in various industrial food products and the investigation of their stability during processing of food products. Methods: We applied the pJuFo cloning system based on the phage surface display of functional cDNA expression products to clone cDNAs encoding peanut allergens. Sera (n = 40) of peanut–allergic individuals were selected according to case history, radioallergosorbent test and immunoblot analysis to demonstrate IgE binding towards the newly identified recombinant allergens. Results: In addition to the known allergens Ara h 1 and Ara h 2 we were able to identify four allergens with estimated molecular weights of 36, 16, 14.5 and 14 kDa. Three of them formally termed Ara h 4, Ara h6 and Ara h 7 show significant sequence similarities to the family of seed storage proteins and the fourth (Ara h 5) corresponds to the well–known plant allergen profilin. Immunoblotting of the six expressed recombinant allergens with 40 patients sera shows 14 individual recognition patterns and the following frequency of specific IgE binding: Ara h 1 was recognized by 65%, Ara h 2 by 85%, Ara h 4 by 53%, Ara h 5 by 13%, Ara h 6 by 38% and Ara h 7 by 43% of the selected sera. Conclusions: All of the selected peanut–positive sera can detect at least one of the six identified recombinant allergens which can be used to establish individual patients’ reactivity profiles. A comparison of these profiles with the clinical data will possibly allow a further insight into the relationship between clinical severity of the symptoms and specific IgE levels towards the six peanut allergens. [ABSTRACT FROM AUTHOR]

The acidic polypeptide chain of soybean glycinin G1 was identified as one of the several IgE-binding proteins by immuno-blotting with pooled sera from soy-allergic individuals. The region of IgE-binding within glycinin G1 was narrowed by in situ digestion with endoproteinase GluC followed by immuno-blotting of peptide products. This procedure resulted in a single IgE-binding fragment of approximately 15 kDa corresponding to residues 192 to 306 of the acidic chain of glycinin G1. [ABSTRACT FROM AUTHOR]

2S albumin storage proteins are becoming of increasing interest in nutritional and clinical studies as they have been reported as major food allergens in seeds of many mono- and di-cotyledonous plants. This review describes the main biochemical, structural and functional properties of these proteins thought to play a role in determining their potential allergenicity. 2S albumins are considered to sensitize directly via the gastrointestinal tract (GIT). The high stability of their intrinsic protein structure, dominated by a well-conserved skeleton of cysteine residues, to the harsh conditions present in the GIT suggests that these proteins are able to cross the gut mucosal barrier to sensitize the mucosal immune system and/or elicit an allergic response. The flexible and solvent-exposed hypervariable region of these proteins is immunodominant and has the ability to bind IgE from allergic patients sera. Several linear IgE-binding epitopes of 2S albumins spanning this region have been described to play a major role in allergenicity; the role of conformational epitopes of these proteins in food allergy is far from being understood and need to be investigated. Finally, the interaction of these proteins with other components of the food matrix might influence the absorption rates of immunologically reactive 2S albumins but also in their immune response.