Rosalia Ayuso

Tropomyosin: An Invertebrate Pan–Allergen

Among food allergens, crustaceans, such as shrimp, crab, crawfish and lobster, are a frequent cause of adverse food reactions in allergic individuals. The major allergen has been identified as the muscle protein tropomyosin. This molecule belongs to a family of highly conserved proteins with multiple isoforms found in both muscle and nonmuscle cells of all species of vertebrates and invertebrates. Its native structure consists of two parallel alpha–helical tropomyosin molecules that are wound around each other forming a coiled–coil dimer. Allergenic tropomyosins are found in invertebrates such as crustaceans (shrimp, lobster, crab, crawfish), arachnids (house dust mites), insects (cockroaches), and mollusks (e.g. squid), whereas vertebrate tropomyosins are nonallergenic. Studies of cross–reactivities among crustaceans and the high degree of sequence identity among them suggest that tropomyosin is probably the common major allergen in crustaceans. Furthermore, immunological relationships between crustaceans, cockroaches and housedust mites have been established and may suggest tropomyosin as an important cross–sensitizing pan allergen.

Molecular Basis of Arthropod Cross-Reactivity: IgE-Binding Cross-Reactive Epitopes of Shrimp, House Dust Mite and Cockroach Tropomyosins

Background: Shrimp may cross-react with other crustaceans and mollusks and nonedible arthropods such as insects (cockroach and chironomids), arachnids (house dust mites) and even nematodes. Since the muscle protein tropomyosin has been implicated as a possible cross-reacting allergen, this study characterized the IgE-binding epitopes in shrimp tropomyosin, Pen a 1, that cross-react with other allergenic invertebrate tropomyosins in house dust mites (Der p 10, Der f 10) and cockroaches (Per a 7). Pen a 1-reactive sera from shrimp-allergic subjects were used to evaluate the effect on IgE binding of different amino acid substitutions in Pen a 1 epitopes based on homologous sequences in Per a 7 and Der p 10/Der f 10. Methods: Peptides were synthesized spanning the length of Pen a 1 IgE-binding epitopes and amino acid substitutions were performed based on homologous amino acid sequences from Per a 7 and Der p 10/Der f 10. Results: 7/8 individually recognized Pen a 1 epitopes (2, 3a, 3b, 4, 5a, 5b and 5c) had an identical amino acid sequence with lobster allergen, Hom a 1, 4/8 (3a, 3b, 4 and 5a) with Der p 10 and Der f 10, and 5/8 (2, 3a, 3b, 4 and 5a) with Per a 7. In addition, even homologous regions of other arthropod tropomyosins that differ in one or more amino acids from the sequences of Pen a 1 epitopes are still recognized by shrimp-allergic IgE antibodies. In total, shrimp-allergic sera recognize 6/8 peptides homologous to Pen a 1 epitopes in Per a 7, 7/8 in Der p 10/Der f 10, and 7/8 epitopes in Hom a 1. Conclusions: The IgE recognition by shrimp-allergic individuals of identified and/or similar amino acid sequences homologous to Pen a 1 epitopes in mite, cockroach and lobster tropomyosins are the basis of the in vitro cross-reactivity among invertebrate species. Based on amino acid sequence similarity and epitope reactivity, lobster tropomyosin has the strongest and cockroach the least cross-reactivity with shrimp. The clinical relevance of these cross-reactivities in developing allergic reactions to different arthropods needs to be determined.

Identification of Continuous, Allergenic Regions of the Major Shrimp Allergen Pen a 1 (Tropomyosin)

Background: Crustaceans and mollusks are a frequent cause of allergic reactions. The only major allergen identified in shrimp is the muscle protein tropomyosin; at least 80% of shrimp-allergic subjects react to tropomyosin. Furthermore, tropomyosin is an important allergen in other crustaceans such as lobsters, crabs and mollusks, as well as other arthropods such as house dust mites and cockroaches, and has been implied as the cause of clinical cross-sensitivity among invertebrates. In contrast, vertebrate tropomyosins are considered nonallergenic. Objective: The basis of the allergenicity of proteins has not yet been resolved. Thus, tropomyosin molecules provide an excellent opportunity to study the relationship between protein structure and allergenicity. The aim of the current study was to identify the IgE-binding regions of Pen a 1 and compare these regions with homologous sequences in other allergenic and nonallergenic tropomyosins. Methods: Forty-six overlapping peptides (length: 15 amino acids; offset: 6 amino acids) spanning the entire Pen a 1 molecule were synthesized and tested for IgE antibody reactivity with sera from 18 shrimp-allergic subjects to identify the IgE-binding regions of shrimp tropomyosin. Results: Based on the frequency and intensity of the IgE reactivities, five major IgE-binding regions were identified. All five major IgE-binding regions were 15–38 amino acids long. The major IgE-binding regions identified were: region 1: Pen a 1 (43–57); region 2: Pen a 1 (85–105); region 3: Pen a 1 (133–148); region 4: Pen a 1 (187–202), and region 5: Pen a 1 (247–284). In addition, 22 peptides were categorized as minor IgE-binding regions, and 12 peptides did not bind any IgE antibodies. No substantial differences in amino acid group composition in the five IgE-binding regions compared to the whole molecule were detected. Sequence identities and similarities of the Pen a 1 IgE-binding regions with homologous regions of allergenic arthropod tropomyosins were as high as 100%, whereas identities and similarities with homologous vertebrate sequences ranged from 36 to 76% and 53 to 85%, respectively. Conclusion: Five major IgE-binding regions of the allergenic shrimp tropomyosin, Pen a 1, were identified which are positioned at regular intervals of approximately 42 amino acids (7 heptads), suggesting a relationship with the repetitive coiled-coil structure of the tropomyosin molecule. The high degree of similarity between Pen a 1 IgE-binding regions and homologous sequences in invertebrate tropomyosins and the lower percentage of similarity with homologous regions of vertebrate tropomyosins supports a structural basis for cross-reactivity of allergenic tropomyosins.

Immunoglobulin E antibody reactivity to the major shrimp allergen, tropomyosin, in unexposed Orthodox Jews

Background Assessment of allergic (IgE antibody‐mediated) reactions to foods may become complicated by cross‐reactivity that can occur among certain food families and between foods and seemingly unrelated allergens.

Seafood Allergy and Allergens: A Review

Seafoods are composed of diverse sea organisms and humans are allergic to many of them. Tropomyosin is a major allergen in many shellfish, especially crustacea and mollusks. Interestingly, tropomyosin has also been identified as an important allergen in other invertebrates including dust mites and cockroaches, and it has been proposed by some to be an invertebrate pan allergen. Different regions of shrimp tropomyosin bind IgE; 5 major IgE-binding regions have been identified in shrimp tropomyosin containing 8 epitopes. Mutations of these shrimp allergenic epitopes can reduce seafood allergenicity; methods utilizing such mutations will provide safer vaccines for more effective treatment of seafood-allergic patients, and in the future less-allergenic seafood products for consumption.

Current Understanding of Food Allergens

Abstract: Food allergies are IgE‐mediated immunological reactions; this distinguishes them from other adverse reactions to foods. Most (>90%) of the recognized food allergies are generally thought to be caused by eight foods or food groups. A number of factors can affect food allergy development, including diet and culture, route of exposure, processing, cooking, and digestion. In addition, it is thought that the properties of certain food proteins render them more likely to be allergenic than other proteins. Most food allergens are major proteins, polyvalent molecules with at least two or more IgE‐binding sites, and are recognized as foreign molecules (hence immunogenic). A number of major food allergens have been recently characterized, and amino acid sequences determined. Tropomyosin is the only major allergen of shrimp. A number of IgE‐binding epitopes have been identified in this molecule, though they may vary from one shrimp‐allergic individual to another. Single amino acid substitutions within epitopes based on that of homologous, nonreactive tropomyosins can substantially enhance or abolish IgE antibody binding. Using the accumulated knowledge of food allergen protein structure, the allergenicity of novel proteins to which there has been no prior human exposure has been assessed. This has been based primarily on the lability or resistance of a protein to enzymatic degradation. Clearly, further criteria must be developed to refine this process. In this regard, the development of animal models that have been sufficiently validated as surrogates of human IgE antibody responses is needed for more precise assessment of the allergenic potential of proteins.

Reduced Allergenic Potency of VR9-1, a Mutant of the Major Shrimp Allergen Pen a 1 (Tropomyosin)

The major shrimp allergen, tropomyosin, is an excellent model allergen for studying the influence of mutations within the primary structure on the allergenic potency of an allergen; Pen a 1 allows systematic evaluation and comparison of Ab-binding epitopes, because amino acid sequences of both allergenic and nonallergenic tropomyosins are known. Individually recognized IgE Ab-binding epitopes, amino acid positions, and substitutions critical for IgE Ab binding were identified by combinatorial substitution analysis, and 12 positions deemed critical were mutated in the eight major epitopes. The mutant VR9-1 was characterized with regard to allergenic potency by mediator release assays using sera from shrimp-allergic subjects and sera from BALB/c, C57BL/6J, C3H/HeJ, and CBA/J mice sensitized with shrimp extract using alum, cholera toxin, and Bordetella pertussis, as adjuvants. The secondary structure of VR9-1 was not altered; however, the allergenic potency was reduced by 90–98% measuring allergen-specific mediator release from humanized rat basophilic leukemia (RBL) cells, RBL 30/25. Reduced mediator release of RBL-2H3 cells sensitized with sera from mice that were immunized with shrimp extract indicated that mice produced IgE Abs to Pen a 1 and to the same epitopes as humans did. In conclusion, data obtained by mapping sequential epitopes were used to generate a Pen a 1 mutant with significantly reduced allergenic potency. Epitopes that are relevant for human IgE Ab binding are also major binding sites for murine IgE Abs. These results indicate that the murine model might be used to optimize the Pen a 1 mutant for future therapeutic use.

IgE antibody response to vertebrate meat proteins including tropomyosin

BACKGROUND Although meat is a main source of proteins in western diets, little information is available regarding allergy to vertebrate meats or the allergens implicated in these reactions. OBJECTIVE To evaluate the in vitro IgE antibody response to different vertebrate meats in suspected meat-allergic subjects, as well as the possible role of tropomyosin in meat allergy and to analyze the cross-reactivity between vertebrate meats and the effect of heating on the IgE-binding to meat proteins. METHODS Fifty-seven sera from suspected meat-allergic subjects were tested by grid blot to extracts of beef, lamb, pork, venison, chicken, and turkey and to four mammalian tropomyosins of different origins. RESULTS Meat-allergic subjects have IgE antibodies to proteins in different mammalian meats (43/57 subjects); cross-reactivity with avian meat was limited: less than 50% (19/43) of meat positive sera reacted to chicken. In contrast, most of the poultry-positive sera also reacted to different mammalian meats. In general, there was stronger IgE reactivity to raw meats in comparison to cooked meats; an exception was six cases in which IgE reactivity to cooked poultry was stronger. Weak IgE reactivity to tropomyosin was detected in only 2/57 sera tested. CONCLUSIONS Suspected meat-allergic subjects have serum IgE directed to meat proteins. In vitro cross-reactivity among mammalian meats appears to be important, while cross-reactivity to poultry is limited indicating mammalian-specific proteins. Although cooking in general denatures meat proteins rendering them less allergenic, in some cases the process of cooking may result in the formation of new allergenic moieties. The muscle protein tropomyosin is not an important vertebrate meat allergen.

Identification of bovine IgG as a major cross-reactive vertebrate meat allergen.

Background: Although beef is a main source of protein in Western diets, very little has been published on allergic reactions to beef or the main allergens implicated in these reactions. The aim was to evaluate the IgE antibody response to beef in suspected meat‐allergic subjects and assess cross‐reactivity of beef with other vertebrate meats.

Characterization and identification of allergen epitopes: recombinant peptide libraries and synthetic, overlapping peptides.

For the understanding of the relationship between protein structure and allergenicity, it is important to identify allergenic epitopes. Two methods to characterize primarily linear epitopes are compared using the major allergen from brown shrimp (Penaeus aztecus), Pen a 1, as an example. A recombinant peptide library was constructed and synthetic, overlapping peptides, spanning the entire Pen a 1 molecule, were synthesized and tested for specific IgE reactivity. Both methods identified IgE-binding of Pen a 1, however, the SPOTs procedure resulted in the identification of more epitopes of the major shrimp allergen Pen a 1 than the usage of the recombinant peptide library. For detection of specific IgE antibodies, the usage of 125I-labeled detection antibody seems to be superior over enzyme-labeled anti IgE antibodies. The regeneration of SPOTs membranes is possible, but it is prudent to test regenerated membranes for residual activity. If a given food allergen contains significant linear epitopes, which seems to be true for stable major allergens such as those of peanut and shrimp the SPOTs system may be more advantageous than the use of recombinant peptides libraries. However, if allergens are studied that contain more conformational epitopes, recombinant peptide libraries may help to identify the relevant epitopes. It has to be emphasized that no system for epitope identification will detect all epitopes and that the relevance of identified epitopes has to be confirmed with other methods such as inhibition studies, crystallographic analysis or the immunological evaluation of modified whole allergens.