Effect of Heat Processing on IgE Reactivity and Cross-Reactivity of Tropomyosin and Other Allergens of Asia-Pacific Mollusc Species: Identification of Novel Sydney Rock Oyster Tropomyosin Sac g 1.
ABSTRACT: Shellfish allergy is an increasing global health priority, frequently affecting adults. Molluscs are an important shellfish group causing food allergy but knowledge of their allergens and cross-reactivity is limited. Optimal diagnosis of mollusc allergy enabling accurate advice on food avoidance is difficult. Allergens of four frequently ingested Asia-Pacific molluscs are characterized: Sydney rock oyster (Saccostrea glomerata), blue mussel (Mytilus edulis), saucer scallop (Amusium balloti), and southern calamari (Sepioteuthis australis), examining cross-reactivity between species and with blue swimmer crab tropomyosin, Por p 1.IgE ELISA showed that cooking increased IgE reactivity of mollusc extracts and basophil activation confirmed biologically relevant IgE reactivity. Immunoblotting demonstrated strong IgE reactivity of several proteins including one corresponding to heat-stable tropomyosin in all species (37-40 kDa). IgE-reactive Sydney rock oyster proteins were identified by mass spectrometry, and the novel major oyster tropomyosin allergen was cloned, sequenced, and designated Sac g 1 by the IUIS. Oyster extracts showed highest IgE cross-reactivity with other molluscs, while mussel cross-reactivity was weakest. Inhibition immunoblotting demonstrated high cross-reactivity between tropomyosins of mollusc and crustacean species.These findings inform novel approaches for reliable diagnosis and improved management of mollusc allergy.
Project description:Shellfish allergy is a major cause of food-induced anaphylaxis, but the allergens are not well characterized. This study examined the effects of heating on blue swimmer crab (Portunus pelagicus) allergens in comparison with those of black tiger prawn (Penaeus monodon) by testing reactivity with shellfish-allergic subjects' serum IgE. Cooked extracts of both species showed markedly increased IgE reactivity by ELISA and immunoblotting, and clinical relevance of IgE reactivity was confirmed by basophil activation tests. Inhibition IgE ELISA and immunoblotting demonstrated cross-reactivity between the crab and prawn extracts, predominantly due to tropomyosin, but crab-specific IgE-reactivity was also observed. The major blue swimmer crab allergen tropomyosin, Por p 1, was cloned and sequenced, showing strong homology with tropomyosin of other crustacean species but also sequence variation within known and predicted linear IgE epitopes. These findings will advance more reliable diagnosis and management of potentially severe food allergy due to crustaceans.
Project description:Designer proteins deprived of its IgE-binding reactivity are being sought as a regimen for allergen-specific immunotherapy. Although shrimp tropomyosin (Met e 1) has long been identified as the major shellfish allergen, no immunotherapy is currently available. In this study, we aim at identifying the Met e 1 IgE epitopes for construction of hypoallergens and to determine the IgE inhibitory capacity of the hypoallergens. IgE-binding epitopes were defined by three online computational models, ELISA and dot-blot using sera from shrimp allergy patients. Based on the epitope data, two hypoallergenic derivatives were constructed by site-directed mutagenesis (MEM49) and epitope deletion (MED171). Nine regions on Met e 1 were defined as the major IgE-binding epitopes. Both hypoallergens MEM49 and MED171 showed marked reduction in their in vitro reactivity towards IgE from shrimp allergy patients and Met e 1-sensitized mice, as well as considerable decrease in induction of mast cell degranulation as demonstrated in passive cutaneous anaphylaxis assay. Both hypoallergens were able to induce Met e 1-recognizing IgG antibodies in mice, specifically IgG2a antibodies, that strongly inhibited IgE from shrimp allergy subjects and Met e 1-sensitized mice from binding to Met e 1. These results indicate that the two designer hypoallergenic molecules MEM49 and MED171 exhibit desirable preclinical characteristics, including marked reduction in IgE reactivity and allergenicity, as well as ability to induce blocking IgG antibodies. This approach therefore offers promises for development of immunotherapeutic regimen for shrimp tropomyosin allergy.
Project description:The one-bead-one-compound (OBOC) combinatorial peptide library is a powerful tool to identify ligand and receptor interactions. Here, we applied the OBOC library technology to identify mimotopes specific to the immunoglobulin E (IgE) epitopes of the major shellfish allergen tropomyosin. OBOC peptide libraries with 8-12 amino acid residues were screened with serum samples from patients with shellfish allergy for IgE mimotopes of tropomyosin. Twenty-five mimotopes were identified from the screening and their binding reactivity to tropomyosin-specific IgE was confirmed by peptide ELISA. These mimotopes could be divided into seven clusters based on sequence homology, and epitope mapping by EpiSearch of the clustered mimotopes was performed to characterize and confirm the validity of mimotopes. Five out of six of the predicted epitopes were found to overlap with previously identified epitopes of tropomyosin. To further confirm the mimicry potential of mimotopes, BALB/c mice were immunized with mimotopes conjugated to keyhole limpet hemocyanin and assayed for their capacity to induce tropomyosin-specific antibodies. BALB/c mice that received mimotope immunization were found to have an elevated level of tropomyosin-specific immunoglobulin G, but not mice that received an irrelevant mimotope. This study pioneers the successful application of the OBOC libraries using whole sera to screen and identify multiple shrimp allergen mimotopes and validates their mimicry potential using in vitro, in vivo, and in silico methods.Cellular & Molecular Immunology advance online publication, 14 september 2015; doi:10.1038/cmi.2015.83.
Project description:Shellfish allergy is one of the most common food allergies, with tropomyosin as the major cross-reactive allergen. However, no allergen-specific immunotherapy is clinically available. Recently, we designed two shrimp hypoallergens MEM49 and MED171. This study aimed to examine and compare the efficacy of the MEM49- and MED171-based DNA vaccines (pMEM49 and pMED171) in modulating shrimp allergy in a murine model of shrimp tropomyosin sensitivity. Intradermal immunization of BALB/c mice with pMEM49 or pMED171 effectively down-modulated allergic symptoms, tropomyosin-specific IgE levels, intestinal Th2 cytokines expression, and inflammatory cell infiltration. Both pMEM49 and pMED171 increased the frequency of regulatory T cells, but to a greater extent by pMED171 with upregulation of gut-homing molecules integrin-?4?7. The functionality of the pMED171-induced Treg cells was further illustrated by anti-CD25-mediated depletion of Treg cells and the adoptive transfer of CD4+CD25+Foxp3+Treg cells. Collectively, the data demonstrate that intradermal administration of pMED171 leads to the priming, activation, and migration of dermal dendritic cells which subsequently induce Treg cells, both locally and systemically, to downregulate the allergic responses to tropomyosin. This study is the first to demonstrate the potency of hypoallergen-encoding DNA vaccines as a therapeutic strategy for human shellfish allergy via the vigorous induction of functional Treg cells.
Project description:BACKGROUND:Many types of shellfish including oysters are sometime cooked before ingestion and it has been demonstrated that cooking may affect the allergenicity of food. Therefore, the aim of our present study is to identify major and minor allergens of tropical oyster (Crassostrea belcheri) and to investigate the effect of different cooking processing on the allergenicity of this oyster. METHODS:Raw, boiled, fried and roasted extracts of oyster were prepared. Protein profiles were analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Major and minor allergens and allergenicity patterns of all extracts were then determined by immunoblotting with sera from patients with positive skin prick tests (SPT) to the raw oyster extract. Mass-spectrometry was used to identify the major allergenic proteins of this oyster. RESULTS:SDS-PAGE of the raw extract showed 15 protein bands (20-180 kDa). In contrast, smaller numbers of protein bands were demonstrated in the boiled extract, those ranging between 40-42 and 55-150 kDa were denatured, whereas the protein profiles were altered to a similar degree by frying or roasting. The 37 kDa proteins had the highest frequency of IgE-binding (95 %), thus identified as the major allergen of this tropical oyster. Other minor IgE-binding proteins were observed at various molecular weights. Immunoblot of raw extract yielded 11 IgE-binding proteins. The cooked extracts showed only a single IgE-binding protein at 37 kDa. Mass spectrometry analysis of the 37 kDa major allergen identified this spot as tropomyosin. CONCLUSIONS:Cooked extracts produce lower IgE-binding than raw extract, which suggest that thermal treatment can be used as a tool in attempting to reduce oyster allergenicity by reducing the number of IgE-reactive bands. The degree of allergenicity of this oyster was demonstrated in the order raw > boiled > fried ? roasted. A heat-resistent 37 kDa protein, corresponding to tropomyosin, was identified as the major allergen of this tropical oyster.
Project description:Understanding and predicting an individual's clinical cross-reactivity to related allergens is a key to better management, treatment and progression of novel therapeutics for food allergy. In food allergy, clinical cross-reactivity is observed in patients reacting to unexpected allergen sources containing the same allergenic protein or antibody binding patches (epitopes), often resulting in severe allergic reactions. Shellfish allergy affects up to 2% of the world population and persists for life in most patients. The diagnosis of shellfish allergy is however often challenging due to reported clinical cross-reactivity to other invertebrates including mites and cockroaches. Prediction of cross-reactivity can be achieved utilizing an in-depth analysis of a few selected IgE-antibody binding epitopes. We combined available experimentally proven IgE-binding epitopes with informatics-based cross-reactivity prediction modeling to assist in the identification of clinical cross-reactive biomarkers on shellfish allergens. This knowledge can be translated into prevention and treatment of allergic diseases. To overcome the problem of predicting IgE cross-reactivity of shellfish allergens we developed an epitope conservation model using IgE binding epitopes available in the Immune Epitope Database and Analysis Resource (http://www.iedb.org/). We applied this method to a set of four different shrimp allergens, and successfully identified several non-cross-reactive as well as cross-reactive epitopes, which have been experimentally established to cross-react. Based on these findings we suggest that this method can be used for advanced component-resolved-diagnosis to identify patients sensitized to a specific shellfish group and distinguish from patients with extensive cross-reactivity to ingested and inhaled allergens from invertebrate sources.
Project description:Household arthropods are one of the most common causes of allergic diseases. Four species of cockroaches are found to reside in Korean homes, but published work deals almost exclusively with the German and American cockroaches. This study was undertaken to investigate the cross-reactive allergenic components of the dusky brown cockroach, Periplaneta fuliginosa. Enzyme-linked immunosorbent assay (ELISA) inhibition and immunoblot analyses for the dusky brown cockroach were performed with Blattella germanica and Dermatophagoides farinae allergic sera. cDNA encoding tropomyosin, which is a well known cross-reactive pan-allergen, was cloned by reverse transcriptase PCR, and recombinant protein was produced by using a pET-28b expression system. Native tropomyosin was purified by ammonium sulfate fractionation and electroelution. The immunoglobulin E (IgE) reactivities of native and recombinant tropomyosins were compared by an ELISA inhibition study. All 30 sera tested showed P. fuliginosa-specific IgE, and the IgE-binding reactivity of the P. fuliginosa extract was inhibited as much as 79.4% by a B. germanica extract and as much as 63.3% by a D. farinae extract. The deduced amino acid sequence of cloned cDNA was identical with that of Periplaneta americana tropomyosin (98.5% nucleotide sequence identity). Seven of 26 (26.9%) allergic sera had IgE specific for recombinant protein, and the maximum inhibition of P. fuliginosa-specific IgE achieved with recombinant tropomyosin was 37.7% at an inhibitor concentration of 10 microg/ml. Native tropomyosin inhibited the binding of IgE to the P. fuliginosa, B. germanica, and D. farinae extracts by 65.0, 51.8, and 39% at an inhibitor concentration of 1 microg/ml. P. fuliginosa appears to possess allergens that are highly cross-reactive with allergens of B. germanica and D. farinae. Tropomyosin was found to be a major allergenic component accounting for the cross-reactivity between cockroaches and dust mites.
Project description:BACKGROUND:IgE reactivity to fish allergens in atopic dogs, which are used as models for food allergy, has not been elucidated to date. We investigated IgE reactivity to crude extracts and purified allergens derived from the Pacific cod (Gadus macrocephalus) in atopic dogs to identify the allergenic proteins of cod. RESULTS:The levels of specific IgE to crude cod extracts were measured in the sera of 179 atopic dogs, including 27 dogs with cod allergy, using enzyme-linked immunosorbent assay (ELISA). Specific IgE to crude cod extracts were present in 36 (20%) of the 179 atopic dogs and in 12 (44%) of the 27 dogs with cod allergy. The allergens in crude cod extracts were analyzed by ELISA, immunoblotting, and liquid chromatography-tandem mass spectrometry. In allergen component analysis, IgE reactivity to tropomyosin and enolase was observed in the sera of dogs with cod allergy. IgE reactivity to parvalbumin, collagen, and tropomyosin was evaluated using the sera of atopic dogs that tested positive for specific IgE to crude cod extracts. Among the 36 dogs with IgE reactivity to crude cod extracts, 9 (25%), 14 (39%), and 18 (50%) dogs tested positive for specific IgE to parvalbumin, collagen, and tropomyosin, respectively. CONCLUSIONS:The IgE reactivity to cod allergens observed in dogs was similar to that in humans, and this finding further supports the use of atopic dogs with fish allergy as a model for fish allergy in humans.
Project description:A cDNA from adult female Onchocerca volvulus encoding the C-terminal portion of a tropomyosin isoform (termed MOv-14) has been shown previously to confer protective immunity in rodent models of onchocerciasis. The full-length sequence (designated Ov-tmy-1) obtained by PCR amplification, codes for a protein of 33 kDa and shares 91% identity with tropomyosins from other nematodes, falling to 57% identity with human alpha-tropomyosin. Ov-TMY-1 migrates with an apparent molecular mass of 42 kDa on SDS/PAGE and is present in all life-cycle stages, as determined by immunoblotting. Immunogold electron microscopy identified antigenic sites within muscle blocks and the cuticle of microfilariae and infective larvae. Anti-MOv14 antibodies were abundant in mice exhibiting serum-transferable protection against microfilariae conferred by vaccination with a PBS-soluble parasite extract. In contrast, little or no MOv14-specific antibody was present in mice inoculated with live microfilariae, in which resistance is mediated by antibody-independent mechanisms. In human infections, there was an inverse correlation between anti-tropomyosin IgG levels and densities of microfilariae in the skin. Seropositivity varied with the relative endemicity of infection. An immunodominant B cell epitope within Ov-TMY-1 (AQLLAEEADRKYD) was mapped to the N terminus of the MOv14 protein by using sera from protectively vaccinated mice. Intriguingly, the sequence coincides with an IgE-binding epitope within shrimp tropomyosin, believed to be responsible for hypersensitivity in individuals exhibiting allergy to shellfish. IgG and IgE antibodies reacting with the O. volvulus epitope were detected in human infections. It is concluded that antibody responses to tropomyosin may be important in limiting microfilarial densities in a proportion of individuals with onchocerciasis and have the potential to mediate hypersensitivity reactions to dead microfilariae, raising the possibility of a link with the immunopathology of infection.
Project description:Sensitization to allergens and their peptides varies among patients due to geographical or ethnic differences. The present study aimed to investigate immunoglobulin (Ig)E and IgG4 responses to tropomyosin and its peptides in shrimp allergic patients from northern China. A total of 92 subjects were studied, including 35 shrimp allergic patients, 29 patients with house dust mite (HDM) and/or cockroach allergic patients and 28 healthy volunteers. Serum IgE and IgG4 antibodies to recombinant shrimp tropomyosin (rPen a 1) and its peptides were measured by means of a light?initiated chemiluminescent assay. A total of 9 major sequential epitopes of Pen a 1 reported in the literature were synthesized. Of 35 shrimp allergic patients, 25 (71.4%) had positive Pen 1?specific IgE (sIgE) antibodies and 22 (62.9%) contained measurable rPen a 1?specific IgG4 (sIgG4) antibodies. A strong IgG4 response accompanied the presence of IgE to Pen a 1. None of the patients with HDM and/or cockroach allergy demonstrated IgE reactivity to rPen a 1. The reaction frequency of IgE binding epitope was 20?48%, while that of IgG4 binding epitope was 63.6?90.9%. The IgE and IgG4 recognition patterns of the tropomyosin peptides demonstrated high interpatient heterogeneity. Diversity of IgE binding epitopes was positively correlated with Pen a 1 sIgE levels. In the study population, tropomyosin was a major allergen recognized by the majority of shrimp allergic patients, which is consistent with previous reports. However, none of the 9 epitopes are major (reaction frequency >50%) IgE?binding regions, indicating the epitopes profile may be different in other regions.