Comprehensive 3D-modeling of allergenic proteins and amino acid composition of potential conformational IgE epitopes

Similarities in sequences and 3D structures of allergenic proteins provide vital clues to identify clinically relevant immunoglobulin E (IgE) cross-reactivities. However, experimental 3D structures are available in the Protein Data Bank for only 5% (45/829) of all allergens catalogued in the Structural Database of Allergenic Proteins (SDAP, http://fermi.utmb.edu/SDAP). Here, an automated procedure was used to prepare 3D-models of all allergens where there was no experimentally determined 3D structure or high identity (95%) to another protein of known 3D structure. After a final selection by quality criteria, 433 reliable 3D models were retained and are available from our SDAP Website. The new 3D models extensively enhance our knowledge of allergen structures. As an example of their use, experimentally derived "continuous IgE epitopes" were mapped on 3 experimentally determined structures and 13 of our 3D-models of allergenic proteins. Large portions of these continuous sequences are not entirely on the surface and therefore cannot interact with IgE or other proteins. Only the surface exposed residues are constituents of "conformational IgE epitopes" which are not in all cases continuous in sequence. The surface exposed parts of the experimental determined continuous IgE epitopes showed a distinct statistical distribution as compared to their presence in typical protein-protein interfaces. The amino acids Ala, Ser, Asn, Gly and particularly Lys have a high propensity to occur in IgE binding sites. The 3D-models will facilitate further analysis of the common properties of IgE binding sites of allergenic proteins.     

Characteristic motifs for families of allergenic proteins

The identification of potential allergenic proteins is usually done by scanning a database of allergenic proteins and locating known allergens with a high sequence similarity. However, there is no universally accepted cut-off value for sequence similarity to indicate potential IgE cross-reactivity. Further, overall sequence similarity may be less important than discrete areas of similarity in proteins with homologous structure. To identify such areas, we first classified all allergens and their subdomains in the Structural Database of Allergenic Proteins (SDAP, http://fermi.utmb.edu/SDAP/) to their closest protein families as defined in Pfam, and identified conserved physicochemical property motifs characteristic of each group of sequences. Allergens populate only a small subset of all known Pfam families, as all allergenic proteins in SDAP could be grouped to only 130 (of 9318 total) Pfams, and 31 families contain more than four allergens. Conserved physicochemical property motifs for the aligned sequences of the most populated Pfam families were identified with the PCPMer program suite and catalogued in the webserver MotifMate (http://born.utmb.edu/motifmate/summary.php). We also determined specific motifs for allergenic members of a family that could distinguish them from non-allergenic ones. These allergen specific motifs should be most useful in database searches for potential allergens. We found that sequence motifs unique to the allergens in three families (seed storage proteins, Bet v 1, and tropomyosin) overlap with known IgE epitopes, thus providing evidence that our motif based approach can be used to assess the potential allergenicity of novel proteins.     

Probing novel allergenic proteins of commonly consumed legumes

Leguminous crops are the main source of protein in Asian subcontinent including India and their proteins may induce allergic reactions in sensitized individuals. Pepsin resistance of proteins is a characteristic feature of most of the allergens. Simulated gastric fluid (SGF) assay as validated by digestion of purified known allergenic and non-allergenic proteins was the basis of this study. Purified allergenic proteins were stable to SGF digestion contrary to rapidly digested non-allergenic proteins. Crude proteins extracts (CPE) of soybean, peanut, chickpea, black gram, kidney bean and Bengal gram were digested in vitro to detect their non-digestible proteins. Six proteins from soybean and seven from peanut remained undigested after SGF digestion. Likewise, seven proteins from chickpea (70, 64, 55, 45, 35, 20 and 18?kDa), ten from black gram (47, 30, 29, 28, 26, 24, 22, 16, 14 and 12?kDa), five from kidney bean (45, 29, 24, 20 and 6.5?kDa) and one from Bengal gram (20?kDa) remained undigested in SGF. Most of the proteins stable in SGF for more than 2?min showed similarity with characterized allergens on the basis of their molecular weights as in case of soybean, peanut, chickpea and black gram. Also, soybean and chickpea stable proteins showed IgE binding property with respective allergic patient’s sera. The non-digestible proteins from the chickpea, black gram, kidney bean and Bengal gram are being reported for the first time by our group. IgE binding of SGF resistant soybean and chickpea proteins is being reported first time as well.     

Molecular analysis of allergenic proteins in bovine dander

An analytic procedure was established to characterize bovine dander proteins with allergenic properties. The proteins from dander extract were separated by size-exclusion gel filtration, and the fractions were studied with SDS-PAGE followed by immunoblotting. An 11-kDa allergen was found in the same gel filtration fractions as 20- and 22-kDa allergens, and this suggests that the 11-kDa allergen is a dirtier in its native form. Our method also detected two separate 22-kDa allergens. The primary structure of the major bovine dander allergen (BDA20) was also studied. A protein sequencer was used to determine the amino acid sequences of enzymatically cleaved peptides. The homology searches revealed that BDA20 is not a previously known bovine protein.     

The effects of roasting on the allergenic properties of peanut proteins

BACKGROUND: Because of the widespread use of peanut products, peanut allergenicity is a major health concern in the United States. The effect or effects of thermal processing (roasting) on the allergenic properties of peanut proteins have rarely been addressed. OBJECTIVE: We sought to assess the biochemical effects of roasting on the allergenic properties of peanut proteins. METHODS: Competitive inhibition ELISA was used to compare the IgE-binding properties of roasted and raw peanut extracts. A well-characterized in vitro model was used to test whether the Maillard reaction contributes to the allergenic properties of peanut proteins. The allergic properties were measured by using ELISA, digestion by gastric secretions, and stability of the proteins to heat and degradation. RESULTS: Here we report that roasted peanuts from two different sources bound IgE from patients with peanut allergy at approximately 90-fold higher levels than the raw peanuts from the same peanut cultivars. The purified major allergens Ara h 1 and Ara h 2 were subjected to the Maillard reaction in vitro and compared with corresponding unreacted samples for allergenic properties. Ara h 1 and Ara h 2 bound higher levels of IgE and were more resistant to heat and digestion by gastrointestinal enzymes once they had undergone the Maillard reaction. CONCLUSIONS: The data presented here indicate that thermal processing may play an important role in enhancing the allergenic properties of peanuts and that the protein modifications made by the Maillard reaction contribute to this effect.     

Electrophoretic and immunochemical characterization of allergenic proteins in buckwheat

BACKGROUND: Buckwheat allergies are not common, however, it is considered to be a very potent allergen. Ingestion of small amounts has been found to produce anaphylactic reactions, particularly in children. Identification and characterization of the major allergen(s) in buckwheat are currently underway, however, there are some discrepancies in the findings. METHODS: Identification of the major allergen(s) was determined through Western blotting using buckwheat-allergic patients' sera. Once the allergenic proteins were identified, they were purified, their IgE-binding activity assessed through an indirect ELISA and the N-terminal amino acid sequence completed. To assess the stability of the IgE-binding epitopes, protein fractions were exposed to various treatments and assayed using an indirect ELISA. Lastly, the presence of anti-buckwheat IgG in the patients' sera was analyzed through Western blotting and ELISA. RESULTS: IgE binding was detected to proteins with molecular masses of approximately 14 and 18 kDa. N-terminal sequencing was completed and found to share some homology with rice proteins associated with rice allergies and cross-allergenicity with buckwheat proteins. When the water-soluble protein fraction was heated, exposed to acidic and alkaline conditions and fully denatured, IgE-binding activity was reduced. When the fraction was partially denatured through urea, IgE-binding activity increased. Furthermore, IgG-binding activity was detected with proteins only above the 20 kDa region. CONCLUSIONS: Proteins with molecular masses around 14 and 18 kDa were identified as the major allergenic proteins in the buckwheat-allergic patients' sera tested in this study. Results also indicate that these two proteins possess IgE-binding capability.     

Subpollen particles: carriers of allergenic proteins and oxidases

BACKGROUND: Pollen is known to induce allergic asthma in atopic individuals, although only a few inhaled pollen grains penetrate into the lower respiratory tract. OBJECTIVE: We sought to provide evidence that subpollen particles (SPPs) of respirable size, possessing both antigenic and redox properties, are released from weed pollen grains and to test their role in allergic airway inflammation. METHODS: The release of SPPs was analyzed by means of microscopic imaging and flow cytometry. The redox properties of SPPs and the SPP-mediated oxidative effect on epithelial cells were determined by using redox-sensitive probes and specific inhibitors. Western blotting and amino acid sequence analysis were used to examine the protein components of the SPP. The allergenic properties of the SPP were determined in a murine model of experimental asthma. RESULTS: Ragweed pollen grains released 0.5 to 4.5 microm of SPPs on hydration. These contained Amb a 1, along with other allergenic proteins of ragweed pollen, and possessed nicotinamide adenine dinucleotide (reduced) or nicotinamide adenine dinucleotide phosphate (reduced) [NAD(P)H] oxidase activity. The SPPs significantly increased the levels of reactive oxygen species (ROS) in cultured cells and induced allergic airway inflammation in the experimental animals. Pretreatment of the SPPs with NAD(P)H oxidase inhibitors attenuated their capacity to increase ROS levels in the airway epithelial cells and subsequent airway inflammation. CONCLUSIONS: The allergenic potency of SPPs released from ragweed pollen grains is mediated in tandem by ROS generated by intrinsic NAD(P)H oxidases and antigenic proteins. CLINICAL IMPLICATIONS: Severe clinical symptoms associated with seasonal asthma might be explained by immune responses to inhaled SPPs carrying allergenic proteins and ROS-producing NAD(P)H oxidases.     

Suggestions for the assessment of the allergenic potential of genetically modified organisms

The prevalence of allergic diseases has been increasing continuously and, accordingly, there is a great desire to evaluate the allergenic potential of components in our daily environment (e.g., food). Although there is almost no scientific evidence that genetically modified organisms (GMOs) exhibit increased allergenicity compared with the corresponding wild type significant concerns have been raised regarding this matter. In principle, it is possible that the allergenic potential of GMOs may be increased due to the introduction of potential foreign allergens, to potentially upregulated expression of allergenic components caused by the modification of the wild type organism or to different means of exposure. According to the current practice, the proteins to be introduced into a GMO are evaluated for their physiochemical properties, sequence homology with known allergens and occasionally regarding their allergenic activity. We discuss why these current rules and procedures cannot predict or exclude the allergenicity of a given GMO with certainty. As an alternative we suggest to improve the current evaluation by an experimental comparison of the wild-type organism with the whole GMO regarding their potential to elicit reactions in allergic individuals and to induce de novo sensitizations. We also recommend that the suggested assessment procedures be equally applied to GMOs as well as to natural cultivars in order to establish effective measures for allergy prevention.     

Identification of allergenic proteins in condoms by immunoenzymatic methods.

BACKGROUND: A large increase of allergy to latex proteins has been observed lately probably as a result of a great use of latex-containing goods. At present these untoward reactions have led to consideration of this problem as a health and occupational hazard. It is therefore, necessary to identify the allergens contained in latex-manufactured products and to develop effective diagnostic tools to detect sensitized individuals. OBJECTIVE: The objective of this study is to identify antigenic and allergenic components in latex condoms by using chemical, immunochemical, and immunoenzymatic methods. METHODS: The protein content of extracts obtained from several brands of condoms was determined and characterized by using a modified Lowry method, a quantitative ELISA assay and SDS-PAGE. The allergenic behavior of these proteins was studied by IgE immunoblotting, EAST and ELISA techniques, using sera from subjects allergic to latex products, particularly to latex condoms. RESULTS: Wide variations in the protein content (38 to 740 microg/g product) and composition were observed. The SDS-PAGE protein profiles showed components ranging from 7 to 94 kD of relative molecular weights; most of them were also detected in natural rubber latex. The most prominent bands were revealed in the 14 and 30 kD zones. A strong band of 69 kD in the SDS-PAGE profiles would correspond to a neoantigen, since it was not observed in natural latex. The immunoblotting analysis employing sera from 5 patients allergic to latex condoms showed the presence of 4 components with IgE binding capacity (14, 30, 69, and 94 kD). The EAST and ELISA methods showed the presence of allergens in all the condom brands studied. CONCLUSIONS: The presence of allergenic proteins in several condom brands was demonstrated by different immunoenzymatic methods.     

Identification of antigenic and allergenic natural rubber latex proteins by immunoblotting

Quantitation of proteins in finished natural rubber latex (NRL) products is essential in predicting their allergenic potential. The ASTM standard Modified Lowry method for measuring total protein content has been used for several years. Most recently, ASTM published a standard for more sensitive and more specific enzyme immunoassay for quantitation of antigenic NRL proteins. It is an ELISA inhibition assay, using rabbit anti NRL sera. Since the measurement of proteins in this method depends on recognition capacity of rabbit antibodies, the selection of an appropriate protein source for rabbit immunization is crucial for the accuracy of such test. In this study, we evaluated the composition of NRL proteins from ammoniated (AL) and nonammoniated (NAL) raw latex and from finished NRL products, and compared the effectiveness of sera from rabbits immunized with NRL proteins, to react with those extracts. Immune rabbit sera were analyzed by immunoblotting against extracts of several samples of AL, NAL, and glove proteins. In the NAL extracts, we identified 26-28 protein bands by SDS-PAGE. AL samples had between 6 and 9 bands with a great variation in the band positions among the samples. The Western blot analysis showed that anti-AL rabbit serum reacted with 4-9 protein bands in various AL extracts. The highest intensity of reaction was observed with the extract used to immunize the rabbits. Similar reaction was observed with anti-NAL serum. However, when the antisera were blotted against NAL extracts, anti-NAL serum reacted more strongly and with a larger number of proteins than anti-AL serum. In summary, anti-NAL serum recognized an equal number of proteins in AL extract as anti-AL serum. However, anti-AL serum recognized fewer protein molecules in NAL extract than anti-NAL serum. Our findings suggest that NAL extract contains more individual proteins than other extracts, and sera from rabbits immunized with this antigen have a greater capacity to react with a wide spectrum of NRL proteins. This finding may be helpful in selecting the representative reference antigen and antiserum for further efforts in NRL protein quantitation.     

Phage display as a tool for rapid cloning of allergenic proteins

Allergic diseases represent an immune disorder associated with the production of immunoglobulin E (IgE) against normally innocuous antigens (allergens). Almost 20% of the population in industrialized countries suffer from type I allergic symptoms such as allergic rhinitis, conjunctivitis, urticaria or asthma. Although the mechanisms responsible for these allergic reactions are quite well understood, knowledge about the repertoire of molecules able to elicit type I symptoms is still limited. To clone and characterize entire allergen repertoires from complex allergenic sources in a fast and efficient way, new technologies are required. The phage surface display of cDNA libraries described here has proven to be a versatile cloning system to selectively isolate allergens physically linked to their genetic information. The screening of cDNA libraries displayed on phage surfaces with immobilised serum IgE from allergic patients reduces the time required for the selection of candidate clones to a few weeks. Robot-assisted high-throughput screening of the enriched library provides a fast and cost-effective way to isolate complete allergen repertoires. The biotechnological production of recombinant allergens derived from these sequences bears a high potential for the improvement of the diagnosis of allergic diseases.     

Allergenic proteins in Urtica dioica, a member of the Urticaceae allergenic family.

BACKGROUND: Allergy to the pollen of flowering plant species significantly affects the health of people in many parts of the world. Pollens of related genera usually share common antigens and are often, but not always, cross-reactive. Several studies have shown that Parietaria pollen is one of the most common causes of pollinosis in the Mediterranean area, whereas Urtica has no allergenic significance. OBJECTIVES: To report on the localization of Parietaria judaica major allergen in Urtica dioica pollen grains and on the detection of allergenic proteins in U. dioica pollen grains during the hydration-activation process. METHODS: A combination of transmission electron microscopy and immunocytochemical methods was used to locate allergenic proteins in U. dioica pollen grains after different periods of hydration-activation using the anti-Par j 1 (4.1.3.) monoclonal antibody and serum samples from allergic patients. RESULTS: No significant labeling was noted for Parj 1 allergen after 10, 15, and 20 minutes in the walls and cytoplasm. Slight labeling was observed for allergic proteins in the walls of U. dioica after 10 minutes of hydration, and no significant labeling was found after 15 and 20 minutes of hydration. CONCLUSIONS: Immunocytochemical methods confirmed the absence of cross-reactivity between 2 related genera, Parietaria and Urtica, and the lowest allergenic potential of U. dioica.     

Exploiting the avian immunoglobulin system to simplify the generation of recombinant antibodies to allergenic proteins

BaCKGROUND: Monoclonal antibodies are a valuable tool in the study of allergens, but the technology used in their generation can be slow and labour-intensive. Therefore, we have examined recombinant antibody development by phage-display against single allergens and protein mixtures. OBJECTIVE: We used the avian immunoglobulin system (generated from single V(H) and V(L) genes) to provide a rapid method for generating highly specific recombinant antibody fragments from a minimal number of animals. METHODS: A single-chain antibody fragment (scFv) library was generated from a single chicken immunized with model allergens. ScFvs were isolated by phage-display and their properties investigated by ELISA and Western blot. RESULTS: Mono-specific scFvs were generated against recombinant Fel d 1 and native Amb a 1. Pannings against yellow jacket venom extracts only yielded clones that reacted with multiple proteins in the venom extract. The scFvs from each panning type were effectively expressed in Escherichia coli and readily purified. Highly specific and sensitive recognition of Fel d 1 and Amb a 1 was demonstrated in ELISA, with scFvs displaying antibody-concentration-dependent absorbance curves down to picogram levels of antibody. The specificity of selected antibodies for their cognate antigen was further confirmed in Western blot analysis, with scFvs directed to either Fel d 1 or Amb a 1 showing no reactivity for the other antigens used in immunization. Anti-Amb a 1 scFvs also mapped Amb a 1-isoform location in Western blot of ragweed extracts separated by 2D SDS-PAGE. DNA sequence analysis of scFvs showed that multiple different clones had been generated against Fel d 1 and Amb a 1. Using two anti-Fel d 1 scFv for ELISA analysis of Fel d 1 content in crude cat pelt extracts, we could produce data which were highly similar (P=0.33 and 0.89 by paired t-test analysis) to those obtained using conventional assays (radial immunodiffusion). CONCLUSION: Phage-display technology may generate multiple allergen-specific recombinant antibody fragments from a single chicken, to allergens from mammalian, plant and insect sources. The resulting antibody fragments are of demonstrable use in allergen identification and quantification, in comparison with standard immunoassays.     

Identification and characterization of the allergenic proteins of Bahia grass (Paspalum notatum) pollen

BACKGROUND: Pollen of Bahia grass (Paspalum notatum) represents a major cause of type I allergy in diverse geographical areas, particularly in the southeastern coastal plain area of the United States. The aqueous protein extract of Bahia grass pollen contains the allergenically active components that produce skin-test-positive reactions in sensitive patients. OBJECTIVE: The emphasis of this study included the identification and characterization of the allergenic proteins present in the crude protein aqueous extract of Bahia grass pollen. METHODS: The crude extract of Bahia grass pollen, partially purified by isoelectric focusing and fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was electroblotted onto nitrocellulose membranes, probed with sera from patients skin test positive to Bahia grass and detected using anti-human IgE conjugated peroxidase. RESULTS: Four allergenic proteins of Bahia grass pollen with estimated molecular weights of 45, 33, 31 and 28 kD were identified and characterized. Following treatments with deglycosylation enzymes, the 4 allergens retained their antigenic reactivity with Bahia-grass-allergic patient sera containing polyclonal IgE antibodies. CONCLUSION: The crude extract of Bahia grass pollen contains many proteins but only 4 have allergenic reactivity. Following deglycosylation treatment, Bahia grass allergenic proteins have retained their antigenic reactivity with Bahia-grass-allergic patient sera containing polyclonal IgE antibodies. Four proteins reactive with IgE were detected, but the 33-kD protein (pI of 6.59) was the most reactive.     

Evaluation of protein allergenic potential in mice: dose–response analyses

BACKGROUND: With the increasing interest in novel foods derived from transgenic crop plants, there is a growing need for the development of approaches for the characterization of the allergenic potential of proteins. Whereas immunogenicity is a common property of foreign proteins, including food proteins, relatively few are significant dietary allergens with the inherent capacity to provoke IgE antibody production and immediate-type hypersensitivity responses. OBJECTIVE: In order to evaluate an approach for the measurement of the allergenic potential of proteins, detailed dose-response analyses of humoral immune responses induced following systemic exposure of BALB/c strain mice to proteins known to differ in terms of sensitizing activity have been conducted. RESULTS: Mice were exposed to a range of concentrations of ovalbumin, a major allergenic constituent of hen's egg, a purified peanut allergen, Arachis hypogea agglutinin, or to the milk allergen bovine serum albumin, and to materials considered to lack significant allergenicity: a crude potato protein extract and a purified potato protein, potato agglutinin. The specific IgE antibody was measured by homologous passive cutaneous anaphylaxis assay, and the specific IgG antibody was measured by enzyme-linked immunosorbent assay. Each of the five proteins was immunogenic in mice, inducing IgG antibody responses at all doses tested, although there was some variation with respect to the vigour of IgG responses. Marked differences in the capacity of these proteins to induce IgE responses were observed, however, with relatively high-titre IgE antibody provoked by all three allergens over the dose ranges examined, whereas the potato proteins stimulated low-titre IgE antibody at the highest dose (10%) only. Importantly, differences in IgE antibody production have been observed against a background of equivalent immunogenicity (IgG antibody responses). CONCLUSION: The data presented here suggest that the measurement of antibody (IgE) responses in BALB/c mice appears to identify allergens accurately and to distinguish them from those materials that apparently lack allergenicity.     

Identification of important allergenic proteins in extracts of the granary weevil (Sitophilus granarius)

This paper describes the identification of important allergens from the granary weevil ( Sitophilus granaries ) (Sg). Sera from Danish bakers whose skin prick tests were positive to extracts of Sg were screened for IgE against Sg extracts. We found that 54% ( n = 66) had elevated levels of IgE (RAST classes 1–3, by luminescent immunoassay) against whole-body extracts of Sg. The specificity of patient IgE was investigated in an inhibition-dot immunoblotting assay. IgE binding was inhibited in all sera but two, thus indicating that the patients' IgE was indeed specific for the Sg extract. In crossed immunoelectrophoresis, 23 different proteins were identified. All RAST-positive sera were investigated in crossed radioimmuno-electrophoresis. At least 11 proteins in the Sg extract were capable of binding IgE. All individual sera reacted with at least four different proteins. The two most prominent allergens bound IgE from 88% and 100%, respectively, of the patients. These two are considered to be the most important allergens from Sg, and will be useful as markers in environmental immunochemical assays to detect allergens in samples from bakeries, grain stores, etc.     

Gastro-duodenal digestion products of the major peanut allergen Ara h 1 retain an allergenic potential

BACKGROUND: The process of gastro-duodenal digestion may play a role in determining the allergenic properties of food proteins. The sensitizing and allergenic potential of digestion products of highly degraded allergens, such as the major peanut allergen Ara h 1, is currently under debate. We evaluated the effect of in vitro gastro-duodenal digestion of Ara h 1 on T cell reactivity and basophil histamine release. METHODS: An in vitro model of gastro-duodenal digestion was used to investigate changes in the allergenic properties of Ara h 1 using in vitro assays monitoring T cell reactivity (proliferation, cytokine production) and histamine release of basophils from peanut allergic individuals. The digestion process was monitored using an SDS-PAGE gel. RESULTS: In vitro gastric digestion led to rapid degradation of Ara h 1 into small fragments M(r) L5600. Gastric digestion did not affect the ability of Ara h 1 to stimulate cellular proliferation. Gastro-duodenal digestion significantly reduced its ability to stimulate clonal expansion (P<0,05; Wilxocon's signed rank test). The Th-2 type cytokine polarization of T cells from peanut allergic donors (IFN-gamma/IL-13 ratio and IFN-gamma/IL-4 ratio of CFSE(low) CD4(+) T cells) remained unchanged regardless of the level of digestion. Histamine release of basophils from peanut allergic individuals was induced to the same extent by native Ara h 1 and its digestion products. CONCLUSION: Gastro-duodenal digestion fragments of Ara h 1 retain T cell stimulatory and IgE-binding and cross-linking properties of the intact protein.