Proteomic analysis in the identification of allergenic molecules

Conventional and innovative strategies can be exploited to identify and characterize new allergenic proteins. With the aim of obtaining suggestions for future improvements, this article describes our attempt to understand and describe some of the advantages and pitfalls of the methodologies and procedures often used in this field. The analysis includes the protein extract preparation, starting from the allergenic source, the separation of the proteins contained in a mixture and the detection, identification and characterization of IgE-binding molecules. Classic and emerging proteomic technologies, including mass spectrometry-based methodologies, Edman degradation procedure, microarray-based techniques and bioinformatics search strategies, have been explored. A comparative analysis of biochemistry-based proteomics and molecular biology strategies has also been given.     

Proteomic analysis in the identification of allergenic molecules

Conventional and innovative strategies can be exploited to identify and characterize new allergenic proteins. With the aim of obtaining suggestions for future improvements, this article describes our attempt to understand and describe some of the advantages and pitfalls of the methodologies and procedures often used in this field. The analysis includes the protein extract preparation, starting from the allergenic source, the separation of the proteins contained in a mixture and the detection, identification and characterization of IgE-binding molecules. Classic and emerging proteomic technologies, including mass spectrometry-based methodologies, Edman degradation procedure, microarray-based techniques and bioinformatics search strategies, have been explored. A comparative analysis of biochemistry-based proteomics and molecular biology strategies has also been given.     

Allergome: the characterization of allergens based on a 2D gel electrophoresis approach

Type I hypersensitivity reactions are in constant progression in industrialized countries. The physiopathologic mechanism of these diseases implicates the production of specific immunoglobulin (Ig)E to allergenic molecules, their binding to the Fcepsilon receptor on the surface of mast cells and basophils, and the release of inflammatory mediators when allergens are introduced into the body and crosslink with the IgE bound to the cell surface. An allergen is defined as a molecule that induces the production of, and binds to, IgE. The identification of the allergenic molecules is an important goal to improve diagnosis and treatment of allergy. This characterization aims to extract proteins from the allergenic source, to analyze IgE specificity by immunoblotting and to identify the proteins that bind IgE.     

Allergome: the characterization of allergens based on a 2D gel electrophoresis approach

Type I hypersensitivity reactions are in constant progression in industrialized countries. The physiopathologic mechanism of these diseases implicates the production of specific immunoglobulin (Ig)E to allergenic molecules, their binding to the Fcε receptor on the surface of mast cells and basophils, and the release of inflammatory mediators when allergens are introduced into the body and crosslink with the IgE bound to the cell surface. An allergen is defined as a molecule that induces the production of, and binds to, IgE. The identification of the allergenic molecules is an important goal to improve diagnosis and treatment of allergy. This characterization aims to extract proteins from the allergenic source, to analyze IgE specificity by immunoblotting and to identify the proteins that bind IgE.     

Characterization of latex allergenic components by capillary zone electrophoresis and N-terminal sequence analysis

In a previous study, protein components purified from latex gloves that elicited allergenic reactions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and yielded apparent molecular weights of 14, 22, 30, 34, 46, and 58 kD. These allergenic components were isolated for further characterization by capillary zone electrophoresis and N-terminal amino acid sequence analysis. These components all migrated at approximately 25 and 35 min on capillary zone electrophoresis. Diode array spectral analysis detected indistinguishable characteristics between these two protein peaks. In addition, complex formation of these components with patients' immunoglobulin was demonstrated by capillary zone electrophoresis. Analysis of components separated by SDS-PAGE on a polyvinylidene difluoride membrane showed that the first 13 residues were identical to the sequence of hevein. Based on the criteria of charge-to-mass ratio and N-terminall amino acid sequence, our results suggest that these components of latex proteins are similar in the primary structure.     

Presence of potential allergy-related linear epitopes in novel proteins from conventional crops and the implication for the safety assessment of these crops with respect to the current testing of genetically modified crops

Mitochondria of cytoplasmic male sterile crop plants contain novel, chimeric open reading frames. In addition, a number of crops carry endogenous double-stranded ribonucleic acid (dsRNA). In this study, the novel proteins encoded by these genetic components were screened for the presence of potential binding sites (epitopes) of allergy-associated IgE antibodies, as was previously done with transgenic proteins from genetically modified crops. The procedure entails the identification of stretches of at least six contiguous amino acids that are shared by novel proteins and known allergenic proteins. These stretches are further checked for potential linear IgE-binding epitopes. Of the 16 novel protein sequences screened in this study, nine contained stretches of six or seven amino acids that were also present in allergenic proteins. Four cases of similarity are of special interest, given the predicted antigenicity of the identical stretch within the allergenic and novel protein, the IgE-binding by a peptide containing an identical stretch reported in literature, or the multiple incidence of identical stretches of the same allergen within a novel protein. These selected stretches are present in novel proteins derived from oilseed rape and radish (ORF138), rice (dsRNA), and fava bean (dsRNA), and warrant further clinical testing. The frequency of positive outcomes and the sizes of the identical stretches were comparable to those previously found for transgenic proteins in genetically modified crops. It is discussed whether novel proteins from conventional crops should be subject to an assessment of potential allergenicity, a procedure which is currently mandatory for transgenic proteins from genetically modified crops.     

Immunological identification and characterization of individual food allergens

Food allergies of type-I-allergy are immunoglobulin E (IgE) mediated and caused by certain proteins or glycoproteins, which are called food allergens. An analytical marker of allergens is the IgE-reactivity to these substances. Normally food allergens are minor components in allergenic source material, which consist of a huge number of chemical different substances. Thus allergen extraction, separation and immunological detection methods are described which identify and characterize individual food allergens by a minimum of manipulation. Favoured separation methods of allergenic extracts are electrophoretic ones allowing the combination of highly resolved protein separations with immunological detection methods subsumed by the term immunoblotting. These techniques are a useful basis to characterize allergens by chemical methods. Once the primary protein structure of a food allergen is established, the way is cleared for the identification of epitopes. Epitopes are immunological detectable parts of a protein or glycoprotein generating the interface between chemical structure and immune-system. The nature of epitopes may differ, for instance, can be conformational, continuous, or built up by glycoconjugates, which determine the stability of food allergens, especially in the case of food processing. Progress in identification and characterization of food allergens will improve diagnostics and therapy of food allergy.     

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/     

Evidence of a novel allergenic protein Narcin in the bulbs of Narcissus tazetta

Several plant-derived allergens have been identified which result in the formation of immunoglobulin E antibodies. Primarily, these allergens belong to the protein families including seed storage proteins, structural proteins and pathogenesis-related proteins. Several allergens are also reported from flower bulbs which cause contact dermatitis. Such symptoms are highly common with the bulb growers handling different species of Narcissus. Narcissus toxicity is also reported if the bulbs are consumed accidentally. The present study aimed to characterize the protein from the bulbs of Narcissus tazetta responsible for its allergenic response. A 13 kDa novel allergenic protein, Narcin was isolated from the bulbs of Narcissus tazetta. The protein was extracted using ammonium sulfate fractionation. The protein was further purified by anion exchange chromatography followed by gel filtration chromatography. The N-terminal sequence of the first 15 amino-acid residues was determined using Edman degradation. The allergenicity of the protein was measured by cytokine production using flow cytometry in peripheral blood mononuclear cells. Further estimation of total IgE was performed by ELISA method. This novel protein was found to induce pro-inflammatory cytokines and thus induce allergy by elevating total IgE level. The novel protein, Narcin isolated from Narcissus tazetta was found to exhibit allergenic properties.     

Identification of suberization-associated anionic peroxidase as a possible allergenic protein from tomato

A 45 kDa protein, which is recognized by IgE antibodies in sera of food-allergic patients, was purified and characterized as an allergenic protein from the tomato. The IgE-binding protein purified from tomato extract was found to be a glycoprotein with a molecular weight of approximately 45,000, an isoelectric point of 4.2, and no free N-terminal amino group. Furthermore, it was shown that the purified protein had peroxidase activity. From the amino acid sequence of a peptide fragment prepared by lysylendopeptidase digestion, the allergenic protein was identified to be the tomato suberization-associated anionic peroxidase 1 known as one of the pathogenesis-related proteins widely distributed in plants. These properties suggested the protein isolated from tomato to be a new allergenic protein in plant foodstuffs.     

一种苦荞主要过敏原基因cDNA的克隆及序列分析

为了获得苦荞中主要过敏原的cDNA和由此推导的蛋白质序列 ,分析其结构特点 ,以苦荞幼根根尖为材料 ,提取总RNA并反转录mRNA为cDNA第一链 .通过RT PCR、3′RACE、基因克隆及序列测定 ,获得一种苦荞主要过敏蛋白基因的cDNA片段 (GenBank登录号为AY0 4 4918) .该cDNA片段由 76 8bp组成 ,包括 3′端非编码区 180个bp ,开放阅读框 5 88bp .可编码一个由 195个氨基酸残基组成的功能蛋白及一个终止密码 .苦荞主要过敏原基因与甜荞 2 2kD过敏蛋白、豆球类蛋白的核苷酸序列分别有 95 %和 93%的同源性 .其推导的氨基酸序列与甜荞球蛋白、刀豆蛋白、甜橙柠檬素分别有 93%、83%和 5 7%的同源性 .该过敏蛋白 183～ 188位氨基酸残基KEEEKE在多数不同过敏原中均存在 ,推测可能为其中的抗原决定簇序列     

Analysis of total proteins in pollen of Humulus scandens Lour in Wuhan Region of China by two-dimensional electrophoresis

Total proteins in the pollen of Humulus scandens Lour,one of the most popular aeroallergens in China,were analyzed by two-dimensional electrophoresis in the current study.The proteins were extracted by Trichloracetic acid (TCA) method,and then separated by isoelectric focusing as the first dimension and SDS-PAGE as the second dimension.The spots of proteins were visualized by staining with Coomassie Brilliant Blue.After analysis with software (ImageMaster 2D),122 different proteins were detected;isoelectric point (pI),Molecular weight (MW) and relativevolume of each protein in the pollen were also discovered.This is the first high-resolution,two-dimensional protein map of the pollen ofHumulus scandens Lour in China.Our finding has built a solid foundation for identification,characterization,gene cloning and standardization of allergenic proteins in the pollen ofHumulus scandens Lour for further studies.     

Two-dimensional electrophoresis replica blotting: a valuable technique for the immunological and biochemical characterization of single components of complex extracts

The combination of the high resolution electrophoresis (2-DE) with subsequent transfer onto a protein-binding membrane (blotting), immunological detection, and/or N-terminal sequencing is a powerful tool to identify and characterize single components of complex protein mixtures. Direct comparison of protein staining, immunological detection, and biochemical characterization of single protein spots was achieved by the replica blotting technique. The proteins were transferred from one two-dimensional gel onto several blotting membranes one after another. A canon of methods has been employed to identify and characterize allergens from different allergen sources. We have studied single major allergens as well as related major allergens from different grass species ("allergen groups") using patients' sera and allergen-specific monoclonal antibodies. The biochemical structure of the allergenic components has been analyzed by N-terminal and internal protein sequencing, precise mass determinations by matrix-assisted laser desorption/ionization mass spectrometry and investigations on post-translational modifications such as glycosylation. Here, we give a general survey of methods, and we describe an array of techniques suitable for characterization and identification of components of complex extracts, even if there is little or no previous information available.     

Molecular and Immunological Characterization of the First Allergenic Lipocalin in Hamster: THE MAJOR ALLERGEN FROM SIBERIAN HAMSTER (PHODOPUS SUNGORUS)*

The most frequent pet allergy is to cat and dog, but in recent years, it has become increasingly popular to have other pets, and the risk of exposure to new allergens is more prevalent. The list of new pets includes hamsters, and one of the most popular hamsters is the Siberian hamster (Phodopus sungorus). The aim of this study was the characterization and cloning of the major allergen from this hamster. The study of its allergenicity and cross-reactivity could improve the specific diagnosis and treatment for hamster-allergic patients. Thirteen Siberian hamster-allergic patients were recruited at the outpatient clinic. Protein extracts were prepared from the hair, urine, and salivary glands of four hamster species (European, golden, Siberian, and Roborovski). IgE-binding proteins were detected by immunoblotting and identified by mass spectrometry. The recombinant protein was produced in Escherichia coli and then purified by metal chelate affinity chromatography. The allergenic properties of the recombinant protein were tested by ELISA and immunoblotting, and biological activity was tested according to capacity for basophil activation. Three IgE-binding proteins were identified in extracts obtained from Siberian hamster hair, urine, and salivary glands. All proteins corresponded to the same protein, which was identified as a lipocalin. This lipocalin had no cross-reactivity with common and golden hamsters. The recombinant allergen was cloned and purified, showing similar IgE reactivity in vitro to Siberian hamster protein extracts. Also, the recombinant allergen was capable of producing biological activation in vivo. The major Siberian hamster allergen was cloned, and allergenic properties were characterized, providing a new tool for specific diagnosis of allergy to Siberian hamster.     

中国武汉地区葎草花粉变应原蛋白质组分的双向电泳分析

应用蛋白质双向电泳的分析技术对武汉地区生长的主要过敏原———葎草(Humulus scandensLour.)花粉的蛋白质组分进行了分析。应用三氯乙酸法提取葎草花粉总蛋白质,通过等电聚焦和第二向SDS-聚丙烯酰胺凝胶电泳分析获得了完整的葎草花粉全蛋白质图谱。应用专业分析软件(Im ageM aster 2D)对电泳图谱分析表明:通过等电聚焦和第二向SDS-PAGE电泳,有122个不同的蛋白质组分被检测出来,并进一步确定每个蛋白质相应的分子量、等电点和相对含量。研究中获得的高分辨率的双向电泳图谱是我国葎草花粉变应原蛋白质第一张完整的蛋白质图谱,将为今后葎草花粉中致敏蛋白质的检测、分离、基因克隆和变应原的标准化奠定基础。     

Substrate Preference of γ-Glutamyltransferase from Caps of Fruiting Bodies of Lentinus edodes

Three kinds of proteins (BA-1, BA-2 and BA-3) allergenic to the IgE antibody of allergenic individuals were isolated from buckwheat seeds. These three proteins were essentially homogeneous as judged by both polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The amino acid composition of BA-1 and BA-2 was very similar, and the molecular weight of each allergenic protein was between 8000–9000 by SDS-polyacrylamide gel electrophoresis. One of them was a trypsin inhibitor, and their immunoreactivity was quite stable to heating at 100°C for 60 min.     

High-yield expression in Escherichia coli, purification, and characterization of properly folded major peanut allergen Ara h 2

Allergic reactions to peanuts are a serious health problem because of their high prevalence, associated with potential severity, and chronicity. One of the three major allergens in peanut, Ara h 2, is a member of the conglutin family of seed storage proteins. Ara h 2 shows high sequence homology to proteins of the 2S albumin family. Presently, only very few structural data from allergenic proteins of this family exist. For a detailed understanding of the molecular mechanisms of food-induced allergies and for the development of therapeutic strategies knowledge of the high-resolution three-dimensional structure of allergenic proteins is essential. We report a method for the efficient large-scale preparation of properly folded Ara h 2 for structural studies and report CD-spectroscopic data. In contrast to other allergenic 2S albumins, Ara h 2 exists as a single continuous polypeptide chain in peanut seeds, and thus heterologous expression in Escherichia coli was possible. Ara h 2 was expressed as Trx-His-tag fusion protein in E. coli Origami (DE3), a modified E. coli strain with oxidizing cytoplasm which allows the formation of disulfide bridges. It could be shown that recombinant Ara h 2, thus overexpressed and purified, and the allergen isolated from peanuts are identical as judged from immunoblotting, analytical HPLC, and circular dichroism spectra.     

Expression and epitope analysis of the major allergenic protein Fag e 1 from buckwheat

Seeds of common buckwheat (Fagopyrum esculentum) contain valuable nutritive substances but also allergenic proteins that cause hypersensitive reactions. Thus, the development of hypoallergenic buckwheat would make this important pseudo-cereal available to allergic people. A major allergenic protein of buckwheat is Fag e 1. We isolated the respective cDNA, coding for a 22 kDa protein, from a recently developed autogamous strain of common buckwheat and confirmed its immunoglobulin E (IgE)-binding activity using recombinant Fag e 1 and sera of allergic patients. The derived amino acid sequence from Fag e 1 cDNA was used to synthesize an overlapping peptide library on nitrocellulose membranes for the determination of the Fag e 1 epitopes. We identified eight epitopes and the critical amino acids for IgE-binding within the epitopes. This epitope analysis of a major allergenic protein of buckwheat should help therapeutic efforts and aid in the development of hypoallergenic buckwheat.     

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     

Air pollution exacerbates effect of allergenic pollen proteins of Cassia siamea: a preliminary report

Pollinosis caused by several allergenic proteins in pollen grains has been a major problem of healthcare in most parts of the world. Environmental factors such as air pollution are known to alter the release of allergenic pollen proteins from a variety of the plant species. Cassia siamea is commonly planted along roadsides and in industrialised areas in many parts of India. The present study reports the findings of animal experiments demonstrating the effect of air pollution on the allergenicity of Cassia siamea pollen proteins. Total white blood cell (WBC) count and lymphocyte count were significantly higher in animals that received protein extract of pollen collected from a polluted site compared to those that received protein extract of pollen collected from a non-polluted site. This was concomitant with increased production of IgE antibodies; followed by marked degranulation of mast cell leading to heighten type I hypersensitivity in these animals. These results are important for the development of a consensus linking ever-increasing pollution due to industrialisation and an increase in associated pollinosis.