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Les tropomyosines
dimanche 22 mars 2009, par
La base Allerdata recense près d’une centaine de produits allergisants contenant une tropomyosine IgE-réactive. C’est la seconde famille de protéines, après les profilines, en nombre d’allergènes identifiés à ce jour.
Hoffman avait dès 1981 repéré ce type de protéine comme étant un allergène dans la crevette. Et Witteman montrait en 1994 le rôle des tropomyosines dans la réactivité croisée entre crevette et acariens 
. Depuis, il a été montré que les tropomyosines méritaient leur qualification de « panallergènes » car elles présentent une IgE-réactivité dans l’ensemble des catégories d’animaux invertébrés : crustacés, mollusques, insectes, acariens, nématodes, ...
Connues principalement pour rendre compte de la majorité des allergies aux crustacés, les tropomyosines participent à des degrés divers à des associations cliniques ou immunologiques entre ces diverses catégories d’animaux.
Un concept plus large de « panallergie » aux invertébrés a été avancé par Panzani , au sein duquel les tropomyosines ont probablement un rôle non négligeable. Il a le mérite de rappeler la place des allergènes issus d’insectes. En dehors des phénomènes d’émergence aigüe d’insectes (ex. trichoptères, mouches du Soudan, ..) et des contacts professionnels (ex. papillon de la farine, charançons, ..), une source sous-estimée d’allergènes est en effet constituée de débris d’insectes vivant dans les habitations et dont la présence est négligée ou méconnue . C’est le cas, par exemple, pour les poissons d’argent (Lepisma saccharina) qui possèdent une tropomyosine douée d’IgE-réactivité .
Que sont les tropomyosines ?
Les tropomyosines sont connues avant tout comme des allergènes présents dans les muscles des invertébrés (ex. la chair des crustacés). De fait, elles jouent rôle indispensable dans la contractilité de la cellule musculaire . Cependant, on trouve aussi des tropomyosines dans d’autres types cellulaires, comme le cerveau ou les fibroblastes. Et les tropomyosines ne sont pas l’apanage des invertébrés : les animaux vertébrés, y compris l’homme, possèdent aussi des tropomyosines.
L’importance physiologique de ces protéines justifie en partie leur bonne « conservation » au cours de l’évolution des espèces. Pour d’autres mécanismes cellulaires soit la famille de protéines a changé (ex. arginine kinases chez les invertébrés et créatine kinases chez les vertébrés), soit les disparités au sein de la famille se sont amplement accumulées. Les tropomyosines ont dans leur ensemble des % d’identité séquentielle plutôt élevés.
Structure des tropomyosines
Les tropomyosines ont une structure assez particulière : elles forment une sorte de filament torsadé double-brin (cf. figure ci-dessous). Ce sont donc des dimères à l’état naturel, les 2 molécules étant assemblées tête-bêche. A noter que cet assemblage risque de ne pas être reproduit avec une protéine recombinante exprimée par E. coli (avec perte de pertinence clinique ?).
Structure tridimensionnelle d’une tropomyosine (réf. PDB 2b9c )
Au sein de la cellule musculaire les tropomyosines sont associées avec d’autres protéines comme l’actine, la myosine et des troponines pour produire la contraction. Il est intéressant de souligner que nombre de ces protéines sont également douées d’IgE-réactivité.
La structure globale des tropomyosines n’est donc pas globulaire. Leurs épitopes B seront ainsi plutôt « linéaires » que conformationnels.
Un certain degré de répétition a été détecté sur l’enchaînement des acides aminés (AA) des tropomyosines, notamment au niveau de leurs épitopes . Ces séquences répétitives d’AA pourraient contribuer à l’allergénicité des tropomyosines en facilitant un pontage d’IgE contigües sur les cellules effectrices par réactivité croisée intra-moléculaire. Cependant, ces motifs répétitifs sont trop courts (5 AA) pour constituer en eux-mêmes des épitopes et quand on intègre les autres AA des épitopes, on n’obtient pas un degré d’homologie suffisant entre épitopes pour assurer une telle réactivité croisée intra-moléculaire .
L’homologie séquentielle des tropomyosines
Les tropomyosines constituent un exemple très rare de famille protéique où une identité totale (100%) est possible entre 2 protéines issues d’espèces vivantes différentes. Bien sûr, ces espèces sont très proches taxonomiquement et, dans le cas d’espèce il s’agit de crevettes : Farfantepeaeus aztecus (tropomyosine Pen a 1), Penaeus monodon (Pen m 1), Litopenaeus vannamei (Lit v 1), et Penaeus japonicus.
Des % d’identité très proches de 100% ne sont pas rares également, comme 99,6% entre des tropomyosines de blattes Periplaneta (Per a 7 et Per f 7) ou 98,9% entre des tropomyosines de seiche et de calamar .
Comme beaucoup d’autres sortes de protéines, les tropomyosines existent à l’état naturel, sous différentes variantes et isoformes. On distingue ainsi des tropomyosines « fast », des « slow », etc, ... On pourrait s’attendre à ce que les % d’identité entre ces variantes soient très élevés : ce n’est pas toujours le cas et certaines « fast » n’ont que 88-95% d’identité avec les « slow » du même animal .
Il est possible que ces petites différences participent à l’individualité des réponses IgE des patients.
Au sein d’un même groupe taxonomique (ex. les Crustacés) les % d’identité entre tropomyosines sont très supérieurs à ceux existant d’un groupe à un autre. On constate donc un certain parallélisme entre proximité taxonomique et homologie. Cependant, des divergences sont possibles :
- bien qu’étant classés comme les Décapodes parmi les Crustacés, les pouce-pieds ont des tropomyosines plus homologues de celles des Gastéropodes (calamar, ormeau, etc..) que de celles des Décapodes (crevettes, crabes, etc..)
- le krill est composé de mini-crevettes et appartient à une branche proche des Décapodes, tandis que les squilles en sont plus éloignées. Mais c’est le contraire pour leurs tropomyosines
- les tropomyosines de poulpe et d’escargot ont un % d’identité équivalent vis-à-vis de Pen m 1 (crevette P. monodon) : pourtant la tropomyosine de poulpe est immunologiquement plus proche de Pen m 1 que n’est celle d’escargot .
Homologies et épitopes
Les tropomyosines n’adoptant pas une structure tertiaire globulaire se prêtent bien à l’étude des épitopes par la technique de « peptides chevauchants ».
Plusieurs travaux se sont ainsi attachés à positionner les zones épitopiques sur la chaîne polypeptidique et à comparer ces positions d’une tropomyosine à une autre, ou selon les patients :
- on constate une variabilité partielle du positionnement des épitopes sur une même tropomyosine, selon les patients
- de même, le pourcentage de patients reconnaissant tel ou tel épitope est variable : par exemple entre 27 et 83% selon les épitopes sur Pen a 1
- un % d’identité très élevé entre deux tropomyosines n’est pas une garantie pour que les épitopes de ces tropomyosines soient eux-mêmes à des emplacements équivalents , ni que les épitopes soient 100% identiques : par exemple l’épitope en position 249-259 sur Pen m 1 est très bien conservé sur les différentes catégories de mollusques, mais l’épitope 88-101 sur Pen m 1 n’est fidèlement retrouvé que chez les Céphalopodes seulement .
La relation taxonomie -> homologie est globalement valide au niveau des épitopes : l’homologie chute à mesure que la distance taxonomique croît :
- pour l’épitope 47-63 de Pen i 1 (crevette Penaeus indicus) on a 94% d’identité au même endroit sur Met e 1 (crevette Metapenaeus ensis), mais 53% sur la tropomyosine de mouche drosophile, et 20-25% sur des tropomyosines de mammifères
- Trois des 6 zones épitopiques partagées par les Crustacés présentent des modifications significatives sur les tropomyosines de blattes ou d’acariens
- il en est de même pour les acariens : des épitopes sont plus ou moins spécifiques de tel ou tel acarien, malgré des % d’identité globaux très élevés entre leurs tropomyosines
Le tableau ci-dessous montre l’homologie entre les 5 épitopes de Pen a 1 et les zones correspondantes sur différentes tropomyosines : on peut constater que la chute d’identité globale, générée par la distance taxonomique entre les animaux, ne touche pas de manière parallèle les zones équivalentes aux épitopes de Pen a 1 sur les tropomyosines de ces animaux. Ainsi on a jusqu’à 71-76% d’identité pour le lapin ou le poulet, alors que ces tropomyosines sont réputées ne pas croiser avec celles des Crustacés, des taux atteints plus difficilement sur la tropomyosine de moule qui, elle, est connue pour croiser .
Identité (en %) entre Pen a 1 et d’autres tropomyosines, au niveau global et au niveau des épitopes de Pen a 1
| % id. | % id. au niveau des épitopes | |||||
|---|---|---|---|---|---|---|
| global | 1 | 2 | 3 | 4 | 5 | |
| Homard | 98 | 100 | 100 | 100 | 100 | 100 |
| Blatte | 82 | 60 | 100 | 80 | 100 | 81 |
| Acarien | 81 | 60 | 95 | 73 | 100 | 89 |
| Moule | 57 | 53 | 52 | 40 | 57 | 76 |
| Poulet | 58 | 33 | 71 | 47 | 76 | 44 |
| Lapin | 56 | 33 | 71 | 47 | 71 | 44 |
Malgré tout, l’étude des épitopes IgE-réactifs des tropomyosines a fourni globalement des résultats assez cohérents avec la relation taxonomie -> homologie. En est-il de même pour l’interprétation à accorder à ces résultats ? Il n’est pas inutile de rappeler à ce sujet 2 points concernant les épitopes :
- les techniques de placement des épitopes par « peptides chevauchants », même si elles sont bien adaptées à des protéines non globulaires comme les tropomyosines, traduisent mal la physiologie épitopique : l’affinité des ces petits peptides artificiels est considérablement plus faible que celle de l’allergène natif . Dès lors, les liaisons observées in vitro entre ces peptides et des IgE de patients sont-elles cliniquement relevantes ?
- il suffit parfois de modifier un seul AA au sein d’un épitope pour que l’IgE-réactivité soit radicalement changée . Aussi, le risque est grand de voir contredite la quasi-certitude qu’il y aura IgE-réactivité du fait d’une quasi-identité avec un épitope connu.
Et cela pose la question de notre aptitude à prédire si une protéine est ou pourrait être un allergène (cf. Prédiction de l’allergénicité).
Homologie, épitopes et réactions croisées in vitro
Comment ces ressemblances ou disparités au niveau des épitopes se traduisent-elles en termes de réactivité croisée entre tropomyosines ?
Les travaux visant à démontrer l’existence de réactions croisées (RC) entre tropomyosines sont assez cohérents avec la relation taxonomie -> homologie :
- chez des patients allergiques aux crustacés, des RC sont vues entre crustacés , entre crustacés et mollusques et/ou blattes
- chez des patients avec allergie aux acariens on observe des RC entre tropomyosines d’acariens et entre celles d’acariens ou blattes et crustacés
- chez des patients avec rhinite/asthme une réactivité croisée a été montrée entre crustacés et/ou acariens et insectes (ex. blattes)
- chez des allergiques aux escargots, des RC sont possibles avec des mollusques marins, mais sont plus limitées entre escargots et crustacés, acariens et/ou blattes
- chez des allergiques aux blattes, les RC avec les acariens ou les crustacés semblent très restreintes .
Existe-t-il un « syndrome tropomyosines » ?
On connaît un grand nombre de tropomyosines ainsi que de multiples réactions croisées entre elles : est-ce que cela se traduit également en clinique, à l’image par exemple du « syndrome bouleau-pomme » ? Est-ce qu’une sensibilisation à des tropomyosines d’acariens peut se montrer capable de générer une allergie aux crevettes ?
L’étude de Fernandes est emblématique à ce sujet : il s’agit de patients présentant un TC positif pour la crevette mais n’ayant jamais mangé de fruits de mer. Les 9 patients en question avaient tous un TC positif pour les acariens. De plus, la tropomyosine de crevette Pen a 1 était inhibable par un extrait d’acariens. On serait donc en droit de suspecter une sensibilisation initiale vis-à-vis des acariens (et de leurs tropomyosines) ayant induit une réactivité secondaire aux crevettes.
Il aurait été intéressant de savoir si cette réactivité croisée, sérologique et cutanée, s’exprimait cliniquement, c’est-à-dire allait jusqu’à une véritable llergie croisé. Mais les pratiques religieuses de ces sujets leur interdisait d’ingérer des fruits de mer et donc de pratiquer un TPO.
Même sans cette vérification, il semble peu vraisemblable que les allergies aux crustacés soient communément générées par une sensibilisation préalable aux acariens. En effet, la prévalence de l’allergie aux crustacés (ou aux mollusques et/ou escargots) chez les allergiques aux acariens n’est pas de 40-60% comme c’est le cas pour la pomme chez les polliniques au bouleau.
Et c’est plutôt l’inverse qui est observé : beaucoup de patients avec allergie alimentaire aux crustacés ou mollusques sont également réactifs aux acariens.
On serait donc plutôt en présence d’un effet de sélection, les allergiques aux crustacés/mollusques étant plus souvent des atopiques.
Si certains travaux concluent en estimant possible une induction d’allergie aux crevettes du fait d’une sensibilisation initiale aux tropomyosines d’acariens , pour d’autres auteurs, ce « syndrome tropomyosines » associant aliments et acariens et/ou blattes n’est pas cliniquement démontré . Et dans le cas des escargots, si l’association clinique est relativement admise, le rôle des tropomyosines est, lui, contesté.
Pour les tropomyosines parasitaires, le lien entre allergie respiratoire, réactivité aux acariens et/ou blattes et parasitose est également mal élucidé, malgré les fortes prévalences de positivité pour les tropomyosines d’acariens et de blattes (40 à 75%) dans les régions d’endémie parasitaire . La sensibilisation précoce à une tropomyosine parasitaire peut-elle jouer un rôle d’adjuvant ?
Une analyse plus détaillée de ces relations entre invertébrés est donnée dans divers chapitres d’Allerdata, lesquels sont accessibles avec les liens du tableau ci-dessous :
| Crustacés | Acariens | Mollusques marins | |
|---|---|---|---|
| Crustacés | voir | ||
| Acariens | voir | voir | |
| Moll. Marins | voir | voir | voir |
| Escargots | voir | voir | voir |
| Blattes | voir | voir |
Les tropomyosines sont un support intéressant pour aborder les questions de l’origine des réactions croisées et de la prédictibilité de l’allergénicité d’une protéine. Aussi, on trouvera des notions complémentaires sur les tropomyosines en se référant à ces 2 thèmes.
[1] - Witteman AM, Akkerdaas JH, Van Leeuwen J, Van der Zee JS, Aalberse RC. Identification of a crossreactive allergen (presumably tropomyosin) in shrimp, mite and insects. Int Arch Allergy Immunol 1994;105:56-61
A monoclonal antibody to Dermatophagoides pteronyssinus is described that cross-reacts with an IgE-binding antigen present in insects, Crustacea (e.g.
shrimp) and other invertebrates. By means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration and immunofluorescence it was shown that this monoclonal antibody presumably recognizes tropomyosin. Tropomyosin was shown to be involved in cross-reactivity between mite, shrimp and insects in shrimp-allergic patients.
↩[2] - Panzani RC, Ariano R. Arthropods and invertebrates allergy (with the exclusion of mites): the concept of panallergy. Allergy 2001;56(suppl. 69):1-22 ↩
[3] - Martinez A, Martinez J, Palacios R, Panzani R. Importance of tropomyosin in the allergy to household arthropods: cross-reactivity with other invertebrate extracts. Allergol Immunopathol (Madr) 1997;25:118-126
The aim of the study was to investigate the involvement of the actin binding protein tropomyosin in the allergic sensitization of patients to household arthropods, as well as to study its panallergenic character in relation to other invertebrate extracts. Three arthropod extracts were prepared, namely fly (Musca domestica), moth (Ephestia spp.) and spider (Tegenaria spp.), and used to evaluate by cutaneous and RAST tests a population of 100 household arthropod allergic patients. Twenty-nine sera were selected for the subsequent SDS-PAGE Immunoblotting assays. IgE binding bands at 36, 34, 31, 27 and 17 kDa were detected in the fly extract by more than 50% of tested sera. In moth and spider extracts, the more relevant allergens were found at 34, 31, 24 and 110, 38, 35, 26, 19 kDa, respectively. A commercial polyclonal antiserum anti-chicken muscle tropomyosin was used for tropomyosin identification. Cross-reactivity studies performed by SDS-PAGE Immunoblotting, using a pool of household arthropod allergic patients and tropomyosin antiserum, preliminarily demonstrated the presence of such protein as a cross-reacting allergen in a large variety of extracts obtained from insects, mites, crustaceans, mollusks and parasites.
↩[4] - Barletta B, Butteroni C, Puggioni EM, Iacovacci P, Afferni C, Tinghino R, et al. Immunological characterization of a recombinant tropomyosin from a new indoor source, Lepisma saccharina. Clin Exp Allergy 2005;35:483-489
Summary Background The presence of specific IgE antibodies to invertebrates is common among patients with rhinitis and asthma. Tropomyosin has been described as an invertebrate cross-reactive allergen. We have recently characterized an allergenic extract from silverfish (Lepisma saccharina). Since this insect could be a new source of tropomyosin in the indoor environment, we have thought important to clone and characterize the tropomyosin from it. Methods Recombinant tropomyosin was cloned and characterized by means of immunoblotting with tropomyosin-specific monoclonal antibodies, rabbit polyclonal antibodies and IgE from allergic patients. Its allergenic activity was investigated in histamine release assays. Immunoblotting and ELISA inhibition were carried out to identify the natural tropomyosin in the silverfish extract and to study the cross-reactivity among other arthropod tropomyosins. Results Tropomyosin-specific antibodies recognized in immunoblotting the natural tropomyosin in the insoluble fraction of silverfish extract. The silverfish tropomyosin (Lep s 1) was cloned and fully expressed. It shared high homology with other arthropod tropomyosins. rLep s 1 was recognized by tropomyosin-specific monoclonal and polyclonal antibodies and by IgE of allergic patients. It was able to inhibit the IgE binding to the insoluble fraction of silverfish extract, and to induce histamine release by an arthropod-allergic serum. Inhibition experiments revealed IgE cross-reactivity between rLep s 1 and other arthropod tropomyosins. Conclusion rLep s 1 is the first allergen cloned and characterized from silverfish extract. It enabled us to identify the natural counterpart in the insoluble fraction of silverfish extract, suggesting that the tropomyosin is not readily extractable with a classic aqueous extraction procedure. rLep s 1 displayed biological activity, suggesting that it could be regarded as a useful tool to study the role of silverfish tropomyosin in the sensitization to invertebrate allergic sources.
↩[5] - Jeong KY, Hong CS, Yong TS. Allergenic tropomyosins and their cross-reactivities. Protein Pept Lett 2006;13:835-845
The ingestion or inhalation of some proteins may lead to adverse immune reactions. Allergens may trigger allergic reactions in genetically predisposed individuals when they are absorbed through the skin or make contact with mucous membranes. An allergic disease often deteriorates the quality of life and may sometimes be life-threatening due to anaphylactic shock. A number of allergens have been characterized from various multicellular organisms to date. It is thought to be reasonable to pay a special attention to the substance which is highly cross-reactive and which causes adverse responses in the molecules that are not sensitized but similar to the sensitized allergen. Tropomyosin has been described as an important food allergen in shrimp, lobster, crab, oysters, squid, and other invertebrates. Allergic reactions to shellfish and mollusks are often cross-reactive, which may be explained by the highly conserved amino acid sequences of tropomyosins among invertebrates, but vertebrate tropomyosins are not known to be allergenic. Several tropomyosins from domestic arthropods have been reported to be allergenic. Recently, it was suggested that an infection of helminthic parasites might lead to sensitization to tropomyosin and elicit allergic reactions to other invertebrates. Much effort has been made to characterize these allergenic tropomyosins from various sources. We will discuss the physicochemical characteristics and the potential application of tropomyosin for the diagnosis and therapeutics of allergic disorders.
↩[6] - Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, et al. The Protein Data Bank. Nucleic Acids Res 2002;28:235-242 ↩
[7] - Ayuso R, Lehrer SB, Reese G. Identification of Continuous, Allergenic Regions of the Major Shrimp Allergen Pen a 1 (Tropomyosin). Int Arch Allergy Immunol 2002;127:27-37
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.
↩[8] - Reese G, Ayuso R, Leong-Kee SM, Plante M, Lehrer SB. The IgE-binding regions of the major allergen Pen a 1: multiple epitopes or intramolecular cross-reactivity ? Int Arch Allergy Immunol 2001;124:103-106 ↩
[9] - Jeong KY, Hong CS, Yong TS. Allergenic tropomyosins and their cross-reactivities. Protein Pept Lett 2006;13:835-845
The ingestion or inhalation of some proteins may lead to adverse immune reactions. Allergens may trigger allergic reactions in genetically predisposed individuals when they are absorbed through the skin or make contact with mucous membranes. An allergic disease often deteriorates the quality of life and may sometimes be life-threatening due to anaphylactic shock. A number of allergens have been characterized from various multicellular organisms to date. It is thought to be reasonable to pay a special attention to the substance which is highly cross-reactive and which causes adverse responses in the molecules that are not sensitized but similar to the sensitized allergen. Tropomyosin has been described as an important food allergen in shrimp, lobster, crab, oysters, squid, and other invertebrates. Allergic reactions to shellfish and mollusks are often cross-reactive, which may be explained by the highly conserved amino acid sequences of tropomyosins among invertebrates, but vertebrate tropomyosins are not known to be allergenic. Several tropomyosins from domestic arthropods have been reported to be allergenic. Recently, it was suggested that an infection of helminthic parasites might lead to sensitization to tropomyosin and elicit allergic reactions to other invertebrates. Much effort has been made to characterize these allergenic tropomyosins from various sources. We will discuss the physicochemical characteristics and the potential application of tropomyosin for the diagnosis and therapeutics of allergic disorders.
↩[10] - Motoyama K, Ishizaki S, Nagashima Y, Shiomi K. Cephalopod tropomyosins: Identification as major allergens and molecular cloning. Food Chem Toxicol 2006;44:1997-2002
Heated extracts prepared from the mantle muscles (for decapods) or leg muscles (for octapods) of nine species of cephalopods were shown to be all reactive with serum IgE in crustacean-allergic patients. No marked difference in the reactivity with IgE was recognized among the cephalopods, suggesting that they are almost equally allergenic. Immunoblotting and inhibition immunoblotting data revealed that the major allergen is tropomyosin in common with the nine species of cephalopods and that the cephalopod tropomyosins are cross-reactive with one another and also with crustacean tropomyosins. Molecular cloning experiments first elucidated the primary structures of tropomyosins from five species of cephalopods. The cephalopod tropomyosins show high sequence identity (more than 92% identity) with one another, being the molecular basis for their cross-reactivity. Although the sequence identity between cephalopod and crustacean topomyosins is only about 63-64%, some of the IgE-binding epitopes proposed for brown shrimp Penaeus aztecus tropomyosin (Pen a 1) are well conserved in the cephalopod tropomyosins, supporting the cross-reactivity between cephalopod and crustacean tropomyosins.
↩[11] - Motoyama K, Suma Y, Ishizaki S, Nagashima Y, Shiomi K. Molecular cloning of tropomyosins identified as allergens in six species of crustaceans. J Agric Food Chem 2007;55:985-991
Although tropomyosin is known to be a major allergen of crustaceans, its structural information is limited to only five species. In this study, tropomyosin was confirmed to be a major allergen in six species of crustaceans (black tiger prawn, kuruma prawn, pink shrimp, king crab, snow crab, and horsehair crab) by immunoblotting. Then, the amino acid sequences of tropomyosins from these crustaceans were elucidated by a cDNA cloning technique. Sequence data for crustacean tropomyosins including the obtained results reveal that fast tropomyosins are contained in shrimps (or prawns) and lobsters, slow tropomyosins in crabs, and both tropomyosins in crayfishes and hermit crabs. Although fast and slow tropomyosins share a high sequence identity (about 90%) with each other, significant differences are observed in specific regions between both tropomyosins.
↩[12] - Suma Y, Ishizaki S, Nagashima Y, Lu Y, Ushio H, Shiomi K. Comparative analysis of barnacle tropomyosin: Divergence from decapod tropomyosins and role as a potential allergen. Comp Biochem Physiol B Biochem Mol Biol 2007;147:230-236
Tropomyosin, a myofibrillar protein of 35-38 kDa, represents a major and cross-reactive allergen in decapod crustaceans. This study was initiated to clarify whether decapod-allergic patients also recognize tropomyosins of barnacles, crustaceans phylogenetically remote from decapods, which are locally consumed as a delicacy. On SDS-PAGE, a 37 kDa protein was observed in all the heated extracts prepared from two species of decapods (American lobster Homarus americanus and black tiger prawn Penaeus monodon) and two species of barnacles (acorn barnacle Balanus rostratus and goose barnacle Capitulum mitella). In immunoblotting, the 37 kDa protein was found to react with monoclonal antibodies against American lobster tropomyosin and hence identified as tropomyosin. The patient sera reacted to tropomyosins from both decapods and barnacles and the reactivity was abolished by preincubation with American lobster tropomyosin, demonstrating that barnacle tropomyosins are allergens cross-reactive with decapod tropomyosins. However, the amino acid sequence of acorn barnacle tropomyosin, deduced by cDNA cloning experiments, shares higher sequence identity with abalone tropomyosins than with decapod tropomyosins. In accordance with this, the phylogenetic tree made for tropomyosins from various animals showed that the acorn barnacle tropomyosin is evolutionally classified not into the decapod tropomyosin family but into the molluscan tropomyosin family.
↩[13] - Motoyama K, Suma Y, Ishizaki S, Nagashima Y, Lu Y, Ushio H, et al. Identification of Tropomyosins as Major Allergens in Antarctic Krill and Mantis Shrimp and Their Amino Acid Sequence Characteristics. Mar Biotechnol (NY) 2008;10:709-718
Tropomyosin represents a major allergen of decapod crustaceans such as shrimps and crabs, and its highly conserved amino acid sequence (>90% identity) is a molecular basis of the immunoglobulin E (IgE) cross-reactivity among decapods. At present, however, little information is available about allergens in edible crustaceans other than decapods. In this study, the major allergen in two species of edible crustaceans, Antarctic krill Euphausia superba and mantis shrimp Oratosquilla oratoria that are taxonomically distinct from decapods, was demonstrated to be tropomyosin by IgE-immunoblotting using patient sera. The cross-reactivity of the tropomyosins from both species with decapod tropomyosins was also confirmed by inhibition IgE immunoblotting. Sequences of the tropomyosins from both species were determined by complementary deoxyribonucleic acid cloning. The mantis shrimp tropomyosin has high sequence identity (>90% identity) with decapod tropomyosins, especially with fast-type tropomyosins. On the other hand, the Antarctic krill tropomyosin is characterized by diverse alterations in region 13-42, the amino acid sequence of which is highly conserved for decapod tropomyosins, and hence, it shares somewhat lower sequence identity (82.4-89.8% identity) with decapod tropomyosins than the mantis shrimp tropomyosin. Quantification by enzyme-linked immunosorbent assay revealed that Antarctic krill contains tropomyosin at almost the same level as decapods, suggesting that its allergenicity is equivalent to decapods. However, mantis shrimp was assumed to be substantially not allergenic because of the extremely low content of tropomyosin.
↩[14] - Maranon M, Wang A, Whitters E, Salazar L, Campbell B, Hall G, et al. IgE reactivity to native and recombinant tropomyosin (Pen m 1) from Peneus monodon: implications in the development of allergy diagnostic tests. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°510
Background: Current research in allergy diagnosis suggests the potential utility of panallergens representing families of proteins with structural similarities. Many allergies could thus be diagnosed using a discrete number of proteins occurring in distantly related species. Recombinant proteins are chosen because they are easy to produce in large quantities. However, posttranslational modifications and differences in secondary and tertiary structures (folding) make them different from their native homologues. In this study we assess differences in reactivity between native and recombinant tropomyosins from black tiger shrimp (Peneus monodon) using the chemiluminescent IMMULITE 2000® 3gAllergyTM assay for allergen-specific IgE.
Methods: Recombinant Pen m 1 (rPen m 1) was cloned and expressed in E. coli, affinity purified and refolded by dialysis against 0.2 M bicarbonate buffer at pH 9.5. The cDNA sequence was identical to the tropomyosin from Peneus aztecus (Pen a 1). Native Pen m 1 (nPen m 1) was extracted from black tiger shrimp and purified to homogeneity. Both nPen m 1 and rPen m 1 were indistinguishable by SDS-PAGE and reacted with a commercial monoclonal antibody against arthropod tropomyosin (MAb 1A6, Indoor Biotechnologies).
Results: The reactivity of IgE from the sera of patients known to be allergic to shrimp was tested with both nPen m 1 and rPen m 1 by IMMULITE 2000 and compared to Pharmacia's UniCAP® which uses rPen a 1. rPen m 1 and rPen a 1 showed similar results in detecting serum specific IgE. However, the recombinant proteins were less sensitive than the native one. nPen m 1 reacted with serum from a shrimp-allergic patient (class 3) missed by both rPen m 1 and rPen a 1. nPen m 1 previously incubated with an IgE-reactive pool inhibited the reactivity to rPen a 1 (UniCAP) by 86%. Mouse antisera against nPen m 1 and rPen m 1 reacted to tropomyosins from shrimp, lobster, squid, cockroach and house dust mites. Reactivity differences may be related to the formation of 70 kDa dimers in nPen m 1 absent from rPen m 1. By Western blot, 3 shrimp-allergic patients out of 30 reacted with the dimer, but not the monomer, of nPen m 1 and were negative against rPen m 1.
Conclusions: This study demonstrates the validity of using panallergens in the diagnosis of allergies and the determination of cross-reactivity. It also suggests that recombinant proteins may not always be as effective as native proteins in allergy diagnosis.
↩[15] - Lehrer SB, Ayuso R, Reese G. Current Understanding of Food Allergens. Ann N Y Acad Sci 2002;964:69-85
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
↩[16] - Ayuso R, Lehrer SB, Reese G. Identification of Continuous, Allergenic Regions of the Major Shrimp Allergen Pen a 1 (Tropomyosin). Int Arch Allergy Immunol 2002;127:27-37
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.
↩[17] - Jeong KY, Hong CS, Yong TS. Allergenic tropomyosins and their cross-reactivities. Protein Pept Lett 2006;13:835-845
The ingestion or inhalation of some proteins may lead to adverse immune reactions. Allergens may trigger allergic reactions in genetically predisposed individuals when they are absorbed through the skin or make contact with mucous membranes. An allergic disease often deteriorates the quality of life and may sometimes be life-threatening due to anaphylactic shock. A number of allergens have been characterized from various multicellular organisms to date. It is thought to be reasonable to pay a special attention to the substance which is highly cross-reactive and which causes adverse responses in the molecules that are not sensitized but similar to the sensitized allergen. Tropomyosin has been described as an important food allergen in shrimp, lobster, crab, oysters, squid, and other invertebrates. Allergic reactions to shellfish and mollusks are often cross-reactive, which may be explained by the highly conserved amino acid sequences of tropomyosins among invertebrates, but vertebrate tropomyosins are not known to be allergenic. Several tropomyosins from domestic arthropods have been reported to be allergenic. Recently, it was suggested that an infection of helminthic parasites might lead to sensitization to tropomyosin and elicit allergic reactions to other invertebrates. Much effort has been made to characterize these allergenic tropomyosins from various sources. We will discuss the physicochemical characteristics and the potential application of tropomyosin for the diagnosis and therapeutics of allergic disorders.
↩[18] - Motoyama K, Ishizaki S, Nagashima Y, Shiomi K. Cephalopod tropomyosins: Identification as major allergens and molecular cloning. Food Chem Toxicol 2006;44:1997-2002
Heated extracts prepared from the mantle muscles (for decapods) or leg muscles (for octapods) of nine species of cephalopods were shown to be all reactive with serum IgE in crustacean-allergic patients. No marked difference in the reactivity with IgE was recognized among the cephalopods, suggesting that they are almost equally allergenic. Immunoblotting and inhibition immunoblotting data revealed that the major allergen is tropomyosin in common with the nine species of cephalopods and that the cephalopod tropomyosins are cross-reactive with one another and also with crustacean tropomyosins. Molecular cloning experiments first elucidated the primary structures of tropomyosins from five species of cephalopods. The cephalopod tropomyosins show high sequence identity (more than 92% identity) with one another, being the molecular basis for their cross-reactivity. Although the sequence identity between cephalopod and crustacean topomyosins is only about 63-64%, some of the IgE-binding epitopes proposed for brown shrimp Penaeus aztecus tropomyosin (Pen a 1) are well conserved in the cephalopod tropomyosins, supporting the cross-reactivity between cephalopod and crustacean tropomyosins.
↩[19] - Subba Rao PV, Rajagopal D, Ganesh KA. B- and T-cell epitopes of tropomyosin, the major shrimp allergen. Allergy 1998;53(suppl. 46):44-47
The major crustacean allergen characterized from different species of shrimp is the muscle protein tropomyosin. Two shared epitopes corresponding to 47-63 and 150-158 of the deduced amino-acid sequence of the brown shrimp, M. ensis, were identified as IgE-binding B-cell epitopes. A 21-mer peptide spanning the amino-acid residues 261-281 was identified as a putative T-cell epitope capable of reducing ongoing tropomyosin-specific IgG and IgE responses in a mouse model. These observations suggest that peptide immunotherapy may also be effective in the treatment of food hypersensitivity.
↩[20] - Ayuso R, Reese G, Leong-Kee S, Plante M, Lehrer SB. Molecular Basis of Arthropod Cross-Reactivity: IgE-Binding Cross-Reactive Epitopes of Shrimp, House Dust Mite and Cockroach Tropomyosins. Int Arch Allergy Immunol 2002;129:38-48
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.
↩[21] - Yi FC, Cheong N, Shek PCL, Wang DY, Chua KY, Lee BW. Identification of shared and unique immunoglobulin E epitopes of the highly conserved tropomyosins in Blomia tropicalis and Dermatophagoides pteronyssinus. Clin Exp Allergy 2002;32:1203-1210
Background Tropomyosin belongs to a class of highly conserved proteins in invertebrates and vertebrates. The invertebrate tropomyosins are allergenic in man with high IgE cross-reactivity and have been therefore referred to as pan-allergens._Objectives This study aimed to clone and identify the IgE epitopes of tropomyosin from Blomia tropicalis (Blo t 10) mite. Cross-reactivity between the IgE epitopes of Blo t 10 and Der p 10 was also evaluated._Methods Blo t 10 was isolated using mouse anti-Der p 10 antibodies. Allergenicity of the cloned Blo t 10 was confirmed by skin prick test (SPT) and enzyme-linked immunosorbent assay (ELISA). Dose-dependent inhibition assay was performed to determine the degree of IgE cross-reactivity between Blo t 10 and Der p 10. Overlapping polymerase chain reaction-derived cDNA were generated and expressed as glutathione-S-transferase (GST) recombinant proteins in Escherichia coli and used to identify shared and unique IgE epitopes of Blo t 10 and Der p 10._Results The cloned Blo t 10 shared up to 96% amino acid identity to tropomyosin of other mites. SPT and ELISA IgE-immunoassay showed recombinant Blo t 10 sensitization rates of between 20% and 29% in atopic subjects. Results of SPT and dose-dependent inhibition assays showed that some allergic individuals had unique IgE epitopes for Blo t 10. IgE epitope mapping of Blo t 10 revealed that the epitopes were mainly located at N- and C-termini of the molecule. The results of ELISA inhibition assays of overlapping recombinant fragments indicated that the unique IgE epitopes of Blo t 10 were located at the C-terminal._Conclusion Although Blo t 10 and Der p 10 are highly conserved (shared 95% amino acids identity) and significantly cross-reactive, unique IgE epitopes do exist. The results suggest the potential deficiency of using only one of these highly conserved allergens as diagnostic or therapeutic reagents.
↩[22] - Nah HL, Tay ASL, Loo AHB, Chew FT. Differential IgE Binding to Recombinant Tropomyosin Orthologs from Different Dust Mite Species Localizes Critical Epitope Regions. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°454
RATIONALE: Tropomyosin is a pan-allergen which exists across diverse invertebrate lineages and plays an important role in seafood, dust mite and insect allergy
METHODS: Tropomyosin orthologs from nine species of dust mites were isolated and expressed as his-tagged recombinant proteins. The proteins were then used to screen sera of 181 Singaporean atopic individuals for tropomyosin-specific IgE. Phylogenetic methods incorporating sequence alignments, parsimony analysis and the identification of evolutionary constrained regions (ECR) were used to understand their sequence similarities. IgE inhibition assays were performed using competitive ELISA to evaluate the degree of cross reactivity between these allergens
RESULTS: Among the 181 atopic individuals, 73/181 had specific IgE to Dermatophagoides spp. tropomyosin (Der p 10 and Der f 10) and 50/181 responded to Blo t 10. Tropomyosin from the other storage mites (Lep d 10, Gly d 10, Sui m 10, Tyr p 10, Aca s 10 and Alu o 10) had between 41 and 52 responders. Cross IgE inhibition studies show that, on selected sera, the Dermatophagoides spp. tropomyosins and Blo t 10, did not totally inhibit IgE binding responses of each other. Cross comparison between allergens with strong- and those with low- responses revealed specific sites which modified IgE binding responses. Two critical changes between Der p 10 and other tropomyosins were found to be crucial for IgE binding: a change from A to G at position 45 and N to S at position 146
CONCLUSIONS: Our data suggests the presence of distinct IgE binding epitopes between even highly conserved tropomyosins Funding: Biomedical Research Council (BMRC) Singapore
↩[23] - Leung PS, Chow WK, Duffey S, Kwan HS, Gershwin ME, Chu KH. IgE reactivity against a cross-reactive allergen in crustacea and mollusca: evidence for tropomyosin as the common allergen. J Allergy Clin Immunol 1996;98:954-961
Although cross-reactivity between mollusks and other crustaceans in shrimp-sensitive subjects has been reported, the mechanism of this allergenic cross-reactivity has not been studied in detail. OBJECTIVE: To investigate this cross-reactivity in vitro, we have taken advantage of a complementary DNA that expresses tropomyosin, the immunodominant shrimp allergen.
METHODS: Serum IgE from nine patients with known anaphylaxis to shrimp and five normal volunteers were analyzed by immunoblotting against 13 distinct crustaceans and mollusks. As additional antigens, muscle preparations were isolated from grasshopper, cockroach, fruit fly, chicken, and mouse.
RESULTS: Sera from all patients, but not control subjects, reacted specifically with a 38 kd protein in all crustaceans and mollusks studied. In addition, 8 of 9 sera from patients, but from none of the normal control subjects, recognized proteins of various other molecular weights among the mollusk extracts studied. The 38 kd protein was identified as tropomyosin and was shown to share immunodominant epitopes among all species of crustaceans and mollusks tested by specific absorption studies. Moreover, sera from all nine subjects with shrimp allergy demonstrated IgE reactivities against grasshopper, cockroach, and fruit fly but not chicken or murine muscle. CONCLUSION: The allergic epitopes on tropomyosin are conserved among invertebrates including not only shellfish but also insects. This latter observation suggests that persons sensitive to shrimp should undergo further study for potential cross-reactive inhalant or ingested insect sensitivity.
↩[24] - Aalberse RC. Structural biology of allergens. J Allergy Clin Immunol 2000;106:228-238
One of the major challenges of molecular allergy is to predict the allergenic potential of a protein, particularly in novel foods. Two aspects have to be distinguished: immunogenicity and cross-reactivity.
Immunogenicity reflects the potential of a protein to induce IgE antibodies, whereas cross-reactivity is the reactivity of (usually preexisting) IgE antibodies with the target protein. In addition to these two issues, the relation between IgE-binding potential and clinical symptoms is of interest. This is influenced by physical properties (eg, stability and size) and immunologic properties (affinity and epitope valence). Discussions on immunogenicity and cross-reactivity of allergens rely on the establishment of structural similarities and differences among allergens and between allergens and nonallergens. For comparisons between the 3-dimensional protein folds, the representation as 2-dimensional proximity plots provides a convenient visual aid. Analysis of approximately 40 allergenic proteins (or parts of these proteins), of which the protein folds are either known or can be predicted on the basis of homology, indicates that most of these can be classified into 4 structural families: (1) antiparallel beta-strands: the immunoglobulin-fold family (grass group 2, mite group 2), serine proteases (mite group 3, 6, and 9), and soybean-type trypsin inhibitor (Ole e 1, grass group 11); (2) antiparallel beta-sheets intimately associated with one or more alpha-helices: tree group 1, lipocalin, profilin, aspartate protease (cockroach group 2); (3) (alpha+beta) structures, in which the alpha- and beta-structural elements are not intimately associated: mite group 1, lysozyme/lactalbumin, vespid group 5; and (4) alpha-helical: nonspecific lipid transfer protein, seed 2S protein, insect hemoglobin, fish parvalbumin, pollen calmodulin, mellitin from bee venom, Fel d 1 chain 1, serum albumin. Allergens with parallel beta-strands (in combination with an alpha-helix linking the two strands, a motif commonly found in, for example, nucleotide-binding proteins) seem to be underrepresented. The conclusion is that allergens have no characteristic structural features other than that they need to be able to reach (and stimulate) immune cells and mast cells. Within this constraint, any antigen may be allergenic, particularly if it avoids activation of T(H)2-suppressive mechanisms (CD8 cells and T(H)1 cells). [References: 40]
↩[25] - Lehrer SB, Ayuso R, Reese G. Current Understanding of Food Allergens. Ann N Y Acad Sci 2002;964:69-85
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
↩[26] - Reese G, Viebranz J, Leong-Kee SM, Plante M, Lauer I, Randow S. Reduced allergenic potency of VR9-1, a mutant of the major shrimp allergen Pen a 1 (tropomyosin). J Immunol 2005;175:8354-8364
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.
↩[27] - Motoyama K, Suma Y, Ishizaki S, Nagashima Y, Shiomi K. Molecular cloning of tropomyosins identified as allergens in six species of crustaceans. J Agric Food Chem 2007;55:985-991
Although tropomyosin is known to be a major allergen of crustaceans, its structural information is limited to only five species. In this study, tropomyosin was confirmed to be a major allergen in six species of crustaceans (black tiger prawn, kuruma prawn, pink shrimp, king crab, snow crab, and horsehair crab) by immunoblotting. Then, the amino acid sequences of tropomyosins from these crustaceans were elucidated by a cDNA cloning technique. Sequence data for crustacean tropomyosins including the obtained results reveal that fast tropomyosins are contained in shrimps (or prawns) and lobsters, slow tropomyosins in crabs, and both tropomyosins in crayfishes and hermit crabs. Although fast and slow tropomyosins share a high sequence identity (about 90%) with each other, significant differences are observed in specific regions between both tropomyosins.
↩[28] - Leung PS, Chow WK, Duffey S, Kwan HS, Gershwin ME, Chu KH. IgE reactivity against a cross-reactive allergen in crustacea and mollusca: evidence for tropomyosin as the common allergen. J Allergy Clin Immunol 1996;98:954-961
Although cross-reactivity between mollusks and other crustaceans in shrimp-sensitive subjects has been reported, the mechanism of this allergenic cross-reactivity has not been studied in detail. OBJECTIVE: To investigate this cross-reactivity in vitro, we have taken advantage of a complementary DNA that expresses tropomyosin, the immunodominant shrimp allergen.
METHODS: Serum IgE from nine patients with known anaphylaxis to shrimp and five normal volunteers were analyzed by immunoblotting against 13 distinct crustaceans and mollusks. As additional antigens, muscle preparations were isolated from grasshopper, cockroach, fruit fly, chicken, and mouse.
RESULTS: Sera from all patients, but not control subjects, reacted specifically with a 38 kd protein in all crustaceans and mollusks studied. In addition, 8 of 9 sera from patients, but from none of the normal control subjects, recognized proteins of various other molecular weights among the mollusk extracts studied. The 38 kd protein was identified as tropomyosin and was shown to share immunodominant epitopes among all species of crustaceans and mollusks tested by specific absorption studies. Moreover, sera from all nine subjects with shrimp allergy demonstrated IgE reactivities against grasshopper, cockroach, and fruit fly but not chicken or murine muscle. CONCLUSION: The allergic epitopes on tropomyosin are conserved among invertebrates including not only shellfish but also insects. This latter observation suggests that persons sensitive to shrimp should undergo further study for potential cross-reactive inhalant or ingested insect sensitivity.
↩[29] - Leung PS, Chen YC, Mykles DL, Chow WK, Li CP, Chu KH. Molecular identification of the lobster muscle protein tropomyosin as a seafood allergen. Mol Mar Biol Biotechnol 1998;7:12-20
Crustaceans are a major cause of seafood allergy. Recent studies have identified tropomyosin as the major allergen in shrimp. However, such data are lacking in other crustaceans. In the present study lobster allergens were identified and characterized by molecular cloning, sequencing, and expression. An IgE-reactive complementary DNA clone of 2 kilobase pairs (kb) was identified by screening an expression library of the spiny lobster Panulirus stimpsoni using sera from subjects with crustacean allergy. Expression and sequencing of this clone showed that it has an opening reading frame of 274 amino acids, coding for a 34-kDa protein designated as Pan s I. In addition, we expressed the fast muscle tropomyosin from the American lobster Homarus americanus and found that this protein, coined Hom a I, was also recognized by IgE from patients with crustacean allergies. The deduced amino acid sequences of Pan s I and Hom a I, which are the first identified lobster allergens, show significant homology to shrimp tropomyosin. Sera from subjects with crustacean allergies, when preabsorbed with recombinant proteins Pan s I or Hom a I, lost their IgE reactivity to muscle extract of P. stimpsoni and H. americanus. Preincubation of crustacean allergy sera with the recombinant shrimp tropomyosin Met e I also removed their IgE reactivity to lobster muscle extracts. The results suggest that patients with allergic reactions to crustaceans have common and possibly cross-reactive IgE-reactive epitopes in lobster and shrimp.
↩[30] - Motoyama K, Ishizaki S, Nagashima Y, Shiomi K. Cephalopod tropomyosins: Identification as major allergens and molecular cloning. Food Chem Toxicol 2006;44:1997-2002
Heated extracts prepared from the mantle muscles (for decapods) or leg muscles (for octapods) of nine species of cephalopods were shown to be all reactive with serum IgE in crustacean-allergic patients. No marked difference in the reactivity with IgE was recognized among the cephalopods, suggesting that they are almost equally allergenic. Immunoblotting and inhibition immunoblotting data revealed that the major allergen is tropomyosin in common with the nine species of cephalopods and that the cephalopod tropomyosins are cross-reactive with one another and also with crustacean tropomyosins. Molecular cloning experiments first elucidated the primary structures of tropomyosins from five species of cephalopods. The cephalopod tropomyosins show high sequence identity (more than 92% identity) with one another, being the molecular basis for their cross-reactivity. Although the sequence identity between cephalopod and crustacean topomyosins is only about 63-64%, some of the IgE-binding epitopes proposed for brown shrimp Penaeus aztecus tropomyosin (Pen a 1) are well conserved in the cephalopod tropomyosins, supporting the cross-reactivity between cephalopod and crustacean tropomyosins.
↩[31] - Leung PS, Chow WK, Duffey S, Kwan HS, Gershwin ME, Chu KH. IgE reactivity against a cross-reactive allergen in crustacea and mollusca: evidence for tropomyosin as the common allergen. J Allergy Clin Immunol 1996;98:954-961
Although cross-reactivity between mollusks and other crustaceans in shrimp-sensitive subjects has been reported, the mechanism of this allergenic cross-reactivity has not been studied in detail. OBJECTIVE: To investigate this cross-reactivity in vitro, we have taken advantage of a complementary DNA that expresses tropomyosin, the immunodominant shrimp allergen.
METHODS: Serum IgE from nine patients with known anaphylaxis to shrimp and five normal volunteers were analyzed by immunoblotting against 13 distinct crustaceans and mollusks. As additional antigens, muscle preparations were isolated from grasshopper, cockroach, fruit fly, chicken, and mouse.
RESULTS: Sera from all patients, but not control subjects, reacted specifically with a 38 kd protein in all crustaceans and mollusks studied. In addition, 8 of 9 sera from patients, but from none of the normal control subjects, recognized proteins of various other molecular weights among the mollusk extracts studied. The 38 kd protein was identified as tropomyosin and was shown to share immunodominant epitopes among all species of crustaceans and mollusks tested by specific absorption studies. Moreover, sera from all nine subjects with shrimp allergy demonstrated IgE reactivities against grasshopper, cockroach, and fruit fly but not chicken or murine muscle. CONCLUSION: The allergic epitopes on tropomyosin are conserved among invertebrates including not only shellfish but also insects. This latter observation suggests that persons sensitive to shrimp should undergo further study for potential cross-reactive inhalant or ingested insect sensitivity.
↩[32] - Yi FC, Cheong N, Shek PCL, Wang DY, Chua KY, Lee BW. Identification of shared and unique immunoglobulin E epitopes of the highly conserved tropomyosins in Blomia tropicalis and Dermatophagoides pteronyssinus. Clin Exp Allergy 2002;32:1203-1210
Background Tropomyosin belongs to a class of highly conserved proteins in invertebrates and vertebrates. The invertebrate tropomyosins are allergenic in man with high IgE cross-reactivity and have been therefore referred to as pan-allergens._Objectives This study aimed to clone and identify the IgE epitopes of tropomyosin from Blomia tropicalis (Blo t 10) mite. Cross-reactivity between the IgE epitopes of Blo t 10 and Der p 10 was also evaluated._Methods Blo t 10 was isolated using mouse anti-Der p 10 antibodies. Allergenicity of the cloned Blo t 10 was confirmed by skin prick test (SPT) and enzyme-linked immunosorbent assay (ELISA). Dose-dependent inhibition assay was performed to determine the degree of IgE cross-reactivity between Blo t 10 and Der p 10. Overlapping polymerase chain reaction-derived cDNA were generated and expressed as glutathione-S-transferase (GST) recombinant proteins in Escherichia coli and used to identify shared and unique IgE epitopes of Blo t 10 and Der p 10._Results The cloned Blo t 10 shared up to 96% amino acid identity to tropomyosin of other mites. SPT and ELISA IgE-immunoassay showed recombinant Blo t 10 sensitization rates of between 20% and 29% in atopic subjects. Results of SPT and dose-dependent inhibition assays showed that some allergic individuals had unique IgE epitopes for Blo t 10. IgE epitope mapping of Blo t 10 revealed that the epitopes were mainly located at N- and C-termini of the molecule. The results of ELISA inhibition assays of overlapping recombinant fragments indicated that the unique IgE epitopes of Blo t 10 were located at the C-terminal._Conclusion Although Blo t 10 and Der p 10 are highly conserved (shared 95% amino acids identity) and significantly cross-reactive, unique IgE epitopes do exist. The results suggest the potential deficiency of using only one of these highly conserved allergens as diagnostic or therapeutic reagents.
↩[33] - Fernandes J, Reshef A, Patton L, Ayuso R, Reese G, Lehrer SB. IgE antibody reactivity to the major shrimp allergen, tropomyosin, in unexposed Orthodox Jews. Clin Exp Allergy 2003;33:956-961
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 .
OBJECTIVE: The allergenic properties of tropomyosin (muscle-derived protein) have been recently demonstrated in invertebrates such as cockroaches, dust mites, and shrimp. In view of a possible cross-reactivity between food allergens and related allergens from animal sources, we designed a study to assess IgE antibody reactivity to the major shrimp allergen, Pen a 1, in an unexposed population of Orthodox Jews, who observe Kosher dietary laws that prohibit eating shellfish .
METHODS: Nine subjects, who reacted positively by skin tests to shrimp (Penaeus setiferous), were selected for the study. Subjects (two females, seven males) ranged in age from 14 to 32 years (mean 20.4). All subjects were strictly observant of Jewish tradition and had no prior exposure to seafood (regarded as a non-Kosher food). Serum was obtained from all the subjects and tested for IgE antibody reactivity to shrimp and dust mite .
RESULTS: All subjects reported symptoms of perennial allergic rhinitis, five had history of asthma, atopic dermatitis, and/or sinusitis. All had positive skin prick tests to shrimp and house dust mite (HDM) (Dermatophagoides farinae, D. pteronyssinus, or both); 2/7 subjects were positive to cockroach mix (Blattella germanica and Periplaneta americana). Sera of 4/9 subjects demonstrated specific IgE antibodies by RAST to shrimp (7.0-20.0%), 3/9 to Pen a 1 (6.3-24.1%), and 3/9 to shrimp or Pen a 1 by immunoblot. IgE binding to Pen a 1 was inhibited with either mite or cockroach extracts as demonstrated by RAST and/or immunoblot inhibition analysis .
CONCLUSIONS: These studies indicate that IgE antibody reactivity to a major food allergen, shrimp, can occur in an unexposed population of individuals; some subjects allergic to HDM and/or cockroach show substantial IgE antibody reactivity to the major shrimp allergen Pen a 1 (tropomyosin). Based on inhibition with cockroach and/or dust mite extracts, this reactivity appears to be due to cross-reacting tropomyosins.
↩[34] - Barletta B, Butteroni C, Puggioni EM, Iacovacci P, Afferni C, Tinghino R, et al. Immunological characterization of a recombinant tropomyosin from a new indoor source, Lepisma saccharina. Clin Exp Allergy 2005;35:483-489
Summary Background The presence of specific IgE antibodies to invertebrates is common among patients with rhinitis and asthma. Tropomyosin has been described as an invertebrate cross-reactive allergen. We have recently characterized an allergenic extract from silverfish (Lepisma saccharina). Since this insect could be a new source of tropomyosin in the indoor environment, we have thought important to clone and characterize the tropomyosin from it. Methods Recombinant tropomyosin was cloned and characterized by means of immunoblotting with tropomyosin-specific monoclonal antibodies, rabbit polyclonal antibodies and IgE from allergic patients. Its allergenic activity was investigated in histamine release assays. Immunoblotting and ELISA inhibition were carried out to identify the natural tropomyosin in the silverfish extract and to study the cross-reactivity among other arthropod tropomyosins. Results Tropomyosin-specific antibodies recognized in immunoblotting the natural tropomyosin in the insoluble fraction of silverfish extract. The silverfish tropomyosin (Lep s 1) was cloned and fully expressed. It shared high homology with other arthropod tropomyosins. rLep s 1 was recognized by tropomyosin-specific monoclonal and polyclonal antibodies and by IgE of allergic patients. It was able to inhibit the IgE binding to the insoluble fraction of silverfish extract, and to induce histamine release by an arthropod-allergic serum. Inhibition experiments revealed IgE cross-reactivity between rLep s 1 and other arthropod tropomyosins. Conclusion rLep s 1 is the first allergen cloned and characterized from silverfish extract. It enabled us to identify the natural counterpart in the insoluble fraction of silverfish extract, suggesting that the tropomyosin is not readily extractable with a classic aqueous extraction procedure. rLep s 1 displayed biological activity, suggesting that it could be regarded as a useful tool to study the role of silverfish tropomyosin in the sensitization to invertebrate allergic sources.
↩[35] - Binder M, Mahler V, Hayek B, Sperr WR, Schöller M, Prozell S, et al. Molecular and Immunological Characterization of Arginine Kinase from the Indianmeal Moth, Plodia interpunctella, a Novel Cross-Reactive Invertebrate Pan-Allergen. J Immunol 2001;167:5470-5477
IgE recognition of indoor allergens represents a major cause of allergic asthma in atopic individuals. We found that 52 of 102 patients suffering from allergic symptoms indoors contained IgE Abs against allergens from the Indianmeal moth (Plodia interpunctella), a ubiquitous food pest. Using serum IgE from a moth-sensitized patient we screened an expression cDNA library constructed from P. interpunctella larvae. cDNAs coding for arginine kinase (EC 2.7.3.3), a 40-kDa enzyme commonly occurring in invertebrates that is involved in the storage of such high-energy phosphate bonds as phosphoarginine, were isolated. Recombinant moth arginine kinase, designated Plo i 1, was expressed in Escherichia coli as a histidine-tagged protein with enzymatic activity, and purified to homogeneity by nickel chelate affinity chromatography. Purified recombinant arginine kinase induced specific basophil histamine release and immediate as well as late-phase skin reactions. It reacted with serum IgE from 13 of the 52 (25%) moth-allergic patients and inhibited the binding of allergic patients‚ IgE to an immunologically related 40-kDa allergen present in house dust mite, cockroach, king prawn, lobster, and mussel. Our results indicate that arginine kinases represent a new class of cross-reactive invertebrate pan-allergens. Recombinant arginine kinase may be used to identify a group of polysensitized indoor allergic patients and for immunotherapy of these individuals
↩[36] - Asturias JA, Eraso E, Arilla C, Gómez-Bayón N, Inácio F, Martínez A. Cloning, Isolation, and IgE-Binding Properties of Helix aspersa (Brown Garden Snail) Tropomyosin. Int Arch Allergy Immunol 2002;128:90-96
Background: Gastropod consumption is quite frequent in the Mediterranean countries and cross-reactivities with crustaceans have been described, but the mechanism of this allergenic cross-reactivity has not been studied in detail. This study aimed to produce recombinant Helix aspersa (brown garden snail) tropomyosin and investigate its implication for cross-reactivity among invertebrates.
Methods: A tropomyosin-specific cDNA encoding H. aspersa tropomyosin was synthetized, and recombinant allergen was overexpressed in Escherichia coli as nonfusion protein. IgE-binding reactivity was studied by immunoblotting and immunoblot inhibition experiments with sera from snail-allergic patients.
Results: Cloned brown garden snail tropomyosin shares high homology with other edible mollusk tropomyosins (84-69% identity) as well as with those from arthropods (65-62%), and less homology with vertebrate ones (56% identity). Tropomyosin reacted with 18% of the sera from patients with snail allergy. Inhibition experiments, using natural and recombinant tropomyosins, showed different degrees of cross-reactivity between invertebrate tropomyosins. Sera from snail-allergic subjects recognized tropomyosins in both mollusks and crustacean extracts.
Conclusions: Tropomyosin represents a minor allergen in snail extracts, but it is clearly involved in invertebrate cross-reactivity
↩[37] - Asturias JA, Gomez-Bayon N, Arilla MC, Martinez A, Palacios R, Sanchez-Gascon F, et al. Molecular characterization of American cockroach tropomyosin (Periplaneta americana allergen 7), a cross-reactive allergen. J Immunol 1999;162:4342-4348
Inhalation of allergens produced by the American cockroach (Periplaneta americana) induces IgE Ab production and the development of asthma in genetically predisposed individuals. The cloning and expression in Escherichia coli of P. americana tropomyosin allergen have been achieved.
The protein shares high homology with other arthropod tropomyosins (80% identity) but less homology with vertebrate ones (50% identity). The recombinant allergen was produced in E. coli as a nonfusion protein with a yield of 9 mg/l of bacterial culture. Both natural and recombinant tropomyosins were purified by isoelectric precipitation. P. americana allergen 1 (Per a 1) and Per a 7 (tropomyosin) are to date the only cross-reacting allergens found in cockroaches. ELISA and Western blot inhibition experiments, using natural and recombinant purified tropomyosins from shrimp and cockroach, showed that tropomyosin induced cross-reactivity of IgE from patients allergic to these allergens, suggesting that this molecule could be a common allergen among invertebrates.
↩[38] - Jeong KY, Hwang H, Lee J, Lee IY, Kim DS, Hong CS, et al. Allergenic characterization of tropomyosin from the dusky brown cockroach, Periplaneta fuliginosa. Clin Diagn Lab Immunol 2004;11:680-685
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.
↩[39] - Purohit A, Shao J, Degreef JM, van Leeuwen A, van Ree R, Pauli G, et al. Role of tropomyosin as a cross-reacting allergen in sensitization to cockroach in patients from Martinique (French Caribbean island) with a respiratory allergy to mite and a food allergy to crab and shrimp. Eur Ann Allergy Clin Immunol 2007;39:85-88
BACKGROUND: Tropomyosin has been described as cross-reacting allergen between mite, cockroach and shrimp.
METHODS: In 13 patients with asthma and/or rhinitis sensitized to mite and/or German cockroach and presenting urticaria, oral allergy syndrome or angio-edema upon eating shrimp and/or crab, we measured specific IgE to mite, cockroach, crab and shrimp tropomyosin.
RESULTS: Ten patients had specific IgE to tropomyosin from mite, 8 from shrimp, 6 from crab and 5 from cockroach. AST inhibition tests indicated that mite allergen is a primary sensitizer and is cross-reacting with shrimp, crab and cockroach allergens. CONCLUSION: Tropomyosin could be the cross-reacting allergen relevant for clinical symptoms to mite, cockroach, shrimp and crab.
↩[40] - Arruda LK. Tropomyosin in Parasites - A Crossreactive IgE-binding Protein ? Allergy Clin Immunol Int 2005;17:243-245
Background: The role of infections with intestinal parasites in the development of allergy and asthma is still controversial, with some studies pointing towards protective effects of parasitic infection and others suggesting a heightened risk of allergic sensitization. Methods/Data base: A review of the literature.
Results: Tropomyosins from mites, cockroach, shrimp, and parasites including Ascaris lumbricoides show a high degree of sequence identity. Studies on IgE-binding epitopes have provided molecular evidence for crossreactivity among these invertebrate tropomyosins, which might have clinical relevance.
Conclusion: Several issues including age at the time of initial parasitic infection, parasite load, type of parasite, socioeconomic conditions, life-style, and environmental allergen exposure may play a role in the complex relationship between parasite infections and the development of allergic diseases. Our results prompted us to speculate that crossreactive allergens such as tropomyosin at the time of the initial Ascaris infections could facilitate subsequent development of crossreactive IgE antibody responses upon exposure to mite or cockroach, which could lead to airway inflammation and asthma. Therefore, infection with Ascaris could have an adjuvant effect on the development of asthma, in the subset of Ascaris-infected individuals who develop IgE responses to tropomyosin.
↩[41] - Jeong KY, Hong CS, Yong TS. Allergenic tropomyosins and their cross-reactivities. Protein Pept Lett 2006;13:835-845
The ingestion or inhalation of some proteins may lead to adverse immune reactions. Allergens may trigger allergic reactions in genetically predisposed individuals when they are absorbed through the skin or make contact with mucous membranes. An allergic disease often deteriorates the quality of life and may sometimes be life-threatening due to anaphylactic shock. A number of allergens have been characterized from various multicellular organisms to date. It is thought to be reasonable to pay a special attention to the substance which is highly cross-reactive and which causes adverse responses in the molecules that are not sensitized but similar to the sensitized allergen. Tropomyosin has been described as an important food allergen in shrimp, lobster, crab, oysters, squid, and other invertebrates. Allergic reactions to shellfish and mollusks are often cross-reactive, which may be explained by the highly conserved amino acid sequences of tropomyosins among invertebrates, but vertebrate tropomyosins are not known to be allergenic. Several tropomyosins from domestic arthropods have been reported to be allergenic. Recently, it was suggested that an infection of helminthic parasites might lead to sensitization to tropomyosin and elicit allergic reactions to other invertebrates. Much effort has been made to characterize these allergenic tropomyosins from various sources. We will discuss the physicochemical characteristics and the potential application of tropomyosin for the diagnosis and therapeutics of allergic disorders.
↩[42] - Santos AB, Chapman MD, Aalberse RC, Vailes LD, Ferriani VP, Oliver C, et al. Cockroach allergens and asthma in Brazil: identification of tropomyosin as a major allergen with potential cross-reactivity with mite and shrimp allergens. J Allergy Clin Immunol 1999;104:329-337
BACKGROUND: Cockroaches produce several proteins that induce IgE antibody responses. Although cockroaches are abundant in warm and humid areas, sensitization to cockroach allergens has not been investigated in Brazil.
OBJECTIVE: The aims of this study were to investigate the frequency of cockroach allergy among patients with asthma, rhinitis, or both in Brazil and to identify American cockroach allergens.
METHODS: Skin tests using cockroach extracts were performed on children and young adults with asthma, rhinitis, or both. A Periplaneta americana complementary (c)DNA library was screened by using IgE antibodies from Brazilian patients allergic to cockroaches. Reactivity of an mAb directed to Dermatophagoides pteronyssinus tropomyosin against cockroach tissue was examined by immunofluorescence.
RESULTS: Cockroach allergy was present in 55% and 79% of the patients, as determined by using skin prick tests alone or combined prick and intradermal tests, respectively. Five cDNA clones reacted with IgE antibody and contained the same sequence. A representative clone (1300 bp), pa 12, coded for a protein that reacted with 50% of the sera from patients allergic to cockroaches on plaque immunoassay and showed a high degree of homology to tropomyosins, particularly those from invertebrates.
P americana tropomyosin showed 80%, 81%, and 82% sequence identity to tropomyosins from D pteronyssinus, D farinae, and shrimp, respectively, which have been previously defined as important allergens. An mAb directed against D pteronyssinus tropomyosin, which also recognizes shrimp tropomyosin, showed binding to cockroach striated muscle. CONCLUSION: Our results support the recommendation that cockroach extracts should be routinely used for the evaluation of patients with asthma, rhinitis, or both in Brazil. The identification of P americana tropomyosin as an important allergen will make it possible to investigate cross-reactivity among cockroaches, mites, and food derived from invertebrates.
↩[43] - Satinover SM, Reefer AJ, Pomes A, Chapman MD, Platts-Mills TAE, Woodfolk JA. Specific IgE and IgG antibody-binding patterns to recombinant cockroach allergens. J Allergy Clin Immunol 2005;115:803-809
BACKGROUND: The specificity of serum antibody responses to different cockroach allergens has not been studied .
OBJECTIVE: We sought to quantitate serum IgE and IgG antibodies to a panel of purified cockroach allergens among cockroach-sensitized subjects .
METHODS: IgE antibodies to recombinant cockroach allergens (rBla g 1, rBla g 2, rBla g 4, rBla g 5, and rPer a 7) were measured in sera containing IgE antibodies to Blattella germanica extract (n = 118) by using a streptavidin CAP assay and a multiplex flow cytometric assay. Specific IgG antibodies were determined by using radioimmunoprecipitation techniques .
RESULTS: Specific IgE antibodies measured by means of CAP assay and multiplex assay were strongly correlated ( r = 0.8, P < .001). The sum of IgE antibodies (in international units per milliliter) against all 5 allergens equated to IgE antibodies to cockroach extract. Although the prevalence of IgE antibodies was highest for rBla g 2 (54.4%) and rBla g 5 (37.4%), patterns of IgE antibody binding were unique to each subject. Surprisingly, only 16% of cockroach-sensitized subjects with IgE antibodies to house dust mite exhibited IgE antibody binding to cockroach tropomyosin (rPer a 7). Specific IgE antibodies were associated with increased IgG antibody levels, although detection of IgG in the absence of IgE was not uncommon .
CONCLUSION: The techniques described offer a new approach for defining the hierarchy of purified allergens. IgE antibodies directed against 5 allergens constitute the majority of the IgE antibody repertoire for cockroach. Such distinct patterns of IgE-IgG responsiveness to different cockroach allergens highlight the complexity of B-cell responses to environmental allergens.
↩[44] - Westritschnig K, Sibanda E, Thomas W, Auer H, Aspöch H, Pittner G, et al. Analysis of the sensitization towards allergens in central Africa. Clin Exp Allergy 2003;33:22-27
BACKGROUND: Almost no information is available regarding the prevalence of IgE-mediated allergies and the disease-eliciting allergens in tropical Africa .
OBJECTIVE: To study IgE-mediated allergies and the allergen profile in allergic patients from Zimbabwe .
METHODS: The frequency of sensitization to common environmental allergen sources was determined by skin prick testing in 650 allergic patients from Zimbabwe. Fifty representative sera were analysed for IgE reactivity to 20 respiratory and 20 food allergen extracts by multiallergen extract testing. The IgE reactivity profiles to recombinant pollen and mite allergens were compared between grass pollen- and mite-sensitized patients from Zimbabwe and central Europe. Sera from grass pollen-allergic patients were also analysed for IgE reactivity to nitrocellulose-blotted natural timothy grass and Bermuda grass pollen allergens .
RESULTS: IgE-mediated allergies were found to be common in Zimbabwe. Similar to the situation in central Europe, mites and grass pollens represented the most prevalent allergen sources. However, the IgE reactivity profiles determined with single recombinant pollen and mite allergens revealed interesting differences between the European and African patients, which most likely reflect the local allergen exposure .
CONCLUSIONS: The striking differences regarding sensitization to grass pollen and mite allergens between African and European patients revealed by recombinant allergen-based testing emphasize the need for component-resolved allergy testing to optimize allergy prevention and therapy in different populations.
↩[45] - Togias A, Poyser J, Satinover S, Collins D, Richards T, Green T, et al. IgE to Cockroach Allergens and Asthma. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°834
RATIONALE: Although allergy to cockroach has been strongly associated with asthma severity, only one study has found an association between cockroach allergy and asthma. Our goal was to examine whether such an association also exists within a homogeneous community that has ubiquitous exposure to cockroaches. Furthermore, we wanted to identify which cockroach allergens are the most common sensitizers
METHODS: We measured cockroach and dust mite serum IgE using CAP in 47 public housing residents from two Baltimore City communities We also measured specific IgE levels to various cockroach allergens Our subjects underwent extensive clinical evaluation including methacholine bronchoprovocation or albuterol reversibility testing for asthma phenotyping
RESULTS: Based on the clinical evaluation, we categorized the cohort in 3 groups: asthma (A, N=13), possible asthma (PA, N=11) and no asthma (NA, N=23). Cockroach IgE (IU/ml, mean ±SEM): 17.4±8.1 (A), 2.28±1.4 (PA), 3.0±2.0 (NA), p = 0.039. In contrast to cockroach, we found no difference between the 3 groups in D. pteronyssinus or D. farinae serum IgE. Positive IgE against Bla g 5 was the most common finding with Bla g 4, Blag 1, Bla g 2 and Per a 7 following in rank order
CONCLUSIONS: In Baltimore public housing residents, serum IgE against cockroach is a strong risk factor for asthma whereas IgE against dust mites is not. Bla g 5, a glutathione transferase protein, appears to be the major allergen in this population.
↩[46] - Santos AR, Rodrigues MC, Thiesen M, Zampolo AS, Ferriani VPL, Arruda L. Use of Recombinant Proteins for Diagnosis of Cockroach Allergy in Patients with Asthma and/or Rhinitis Living in Brazil. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°843
RATIONALE: Several cockroach allergens have been produced as recombinant proteins, however skin test reactivity to these allergens has been established in a limited number of patients
METHODS: Frequency of positive skin tests to recombinant allergens of Blattella germanica (rBlag 2, rBlag 4, rBlag 5) and Periplaneta americana (rPera 1 and rPera 7) was evaluated in 50 cockroach allergic patients with asthma and/or rhinitis, aged 7 to 70 years-old. Patients presented positive skin tests to P. americana and B. germanica. Skin prick tests with recombinant allergens were carried out at 10mcg/ml or 5mcg/ml (rPera 1) concentrations. Recombinant tropomyosins from P. americana (rPera 7) and A. lumbricoides, expressed in Pichia pastoris, were used in chimeric ELISA to quantitate IgE antibodies in sera of 48/50 patients
RESULTS: Positive skin tests to rBlag 2, rBlag 4, rBlag 5, rPera 1 and rPera 7 were found in 4(8%), 3(6%), 3(6%), 3(6%), and 18(36%) patients, respectively. Positive test to at least one allergen was found in 46% of patients. IgE antibodies to P. americana and A. lumbricoides tropomyosins were detected in 26(54.2%) and 25(52%) patients, respectively Levels of IgE to rPer a 7 varied from 0.6 to 200IU/mL (geometric mean GM 2.3IU/mL) and to A. lumbricoides tropomyosin, from 0.6 to 500IU/mL (GM 1.8IU/mL). There was a significant correlation of levels of IgE to Ascaris and P. americana tropomyosin (r=0.41; p=0.0034)
CONCLUSIONS: Recombinant allergens could be safely used for diagnosis of cockroach allergic patients living in Brazil. This approach could be improved by inclusion of additional cockroach allergens in the panel Funding:
↩[47] - Santos ABR, Rocha GM, Oliver C, Ferriani VPL, Lima RC, Palma MS, et al. Cross-reactive IgE antibody responses to tropomyosins from Ascaris lumbricoides and cockroach. J Allergy Clin Immunol 2008;121:1040-1046
BACKGROUND: Evidence indicates that infection with Ascaris lumbricoides may promote development of allergy and asthma .
OBJECTIVE: To study the role of tropomyosin, a pan-allergen in invertebrates, in IgE responses to A lumbricoides .
METHODS: Recombinant A lumbricoides and Periplaneta americana tropomyosins were expressed in Pichia pastoris. Levels of IgE to tropomyosins from A lumbricoides and P americana were determined by chimeric ELISA in sera from 119 children living in a parasite-endemic area and 112 patients with cockroach allergy from the allergy clinics. Presence of tropomyosin in A lumbricoides larvae at L3 stage was evaluated by immunofluorescence using mAb 1A6, directed against mite tropomyosin. Molecular modeling of P americana and A lumbricoides tropomyosins was performed by using the MODELLER program .
RESULTS: A lumbricoides tropomyosin showed 69% to 98% sequence identity to tropomyosins from other invertebrates. The predicted structure of A lumbricoides tropomyosin was similar to that of P americana tropomyosin and showed the characteristic coiled-coil structure. Strong correlation was found for IgE antibodies to tropomyosins from A lumbricoides and P americana in sera from children living in a parasite-endemic area and from patients with cockroach allergy. Larvae of A lumbricoides reacted strongly with mAb 1A6 .
CONCLUSION: Tropomyosin induces IgE responses in A lumbricoides-infected children and in patients allergic to cockroach.
↩[48] - Arruda LK. Tropomyosin in Parasites - A Crossreactive IgE-binding Protein ? Allergy Clin Immunol Int 2005;17:243-245
Imprimer la bibliographieBackground: The role of infections with intestinal parasites in the development of allergy and asthma is still controversial, with some studies pointing towards protective effects of parasitic infection and others suggesting a heightened risk of allergic sensitization. Methods/Data base: A review of the literature.
Results: Tropomyosins from mites, cockroach, shrimp, and parasites including Ascaris lumbricoides show a high degree of sequence identity. Studies on IgE-binding epitopes have provided molecular evidence for crossreactivity among these invertebrate tropomyosins, which might have clinical relevance.
Conclusion: Several issues including age at the time of initial parasitic infection, parasite load, type of parasite, socioeconomic conditions, life-style, and environmental allergen exposure may play a role in the complex relationship between parasite infections and the development of allergic diseases. Our results prompted us to speculate that crossreactive allergens such as tropomyosin at the time of the initial Ascaris infections could facilitate subsequent development of crossreactive IgE antibody responses upon exposure to mite or cockroach, which could lead to airway inflammation and asthma. Therefore, infection with Ascaris could have an adjuvant effect on the development of asthma, in the subset of Ascaris-infected individuals who develop IgE responses to tropomyosin.
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