Gerald Reese

Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment

Type I allergy is an immunoglobulin E (IgE)‐mediated hypersensitivity disease affecting more than 25% of the population. Currently, diagnosis of allergy is performed by provocation testing and IgE serology using allergen extracts. This process defines allergen‐containing sources but cannot identify the disease‐eliciting allergenic molecules. We have applied microarray technology to develop a miniaturized allergy test containing 94 purified allergen molecules that represent the most common allergen sources. The allergen microarray allows the determination and monitoring of allergic patients’ IgE reactivity profiles to large numbers of disease‐causing allergens by using single measurements and minute amounts of serum. This method may change established practice in allergy diagnosis, prevention, and therapy. In addition, microarrayed antigens may be applied to the diagnosis of autoimmune and infectious diseases.

Tropomyosin: An Invertebrate Pan–Allergen

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

Identification of the Major Brown Shrimp (Penaeus aztecus) Allergen as the Muscle Protein Tropomyosin

Shrimp, a major seafood allergen, was investigated as a model food allergen. Extracts from both shrimp (Penaeus aztecus) meat and cooking fluid contain a substantial and similar amount of allergenic activity. A 36-kD allergen, demonstrated in both extracts by SDS-PAGE/Western blot analysis, reacted with 28/34 (82%) sera from shrimp-sensitive, skin test and RAST-positive, individuals. This allergen, named Pen a I, was isolated by SDS-PAGE; its amino acid composition was rich in aspartic and glutamic acids. A 21-residue peptide, obtained from endoproteinase Lys-C digested Pen a I by high-performance liquid chromatography, demonstrated significant homology (60-87%) with the muscle protein tropomyosin from various species and origins. The greatest homology (87%) was noted with tropomyosin of the fruit fly (Drosophila melanogaster) reflecting the phylogenic relationship between these two arthropods. These studies demonstrate that tropomyosin is the major shrimp allergen. Although the amino acid sequence of this shrimp muscle protein shares considerable homology with tropomyosins of other species including man, significant differences remain in allergenic activity.

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

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

Lipid transfer protein (Ara h 9) as a new peanut allergen relevant for a Mediterranean allergic population

BACKGROUND Nonspecific lipid transfer proteins (LTPs) represent potent pollen and food allergens. However, the allergenic properties of peanut LTP have not been studied. OBJECTIVE To identify LTP in peanut extract using sera from subjects with peanut allergy and Pru p 3-sensitized subjects from Southern Europe, clone and express this protein, and obtain information on the importance as allergen for these selected patients. METHODS Peanut LTP (Ara h 9) was cloned and sequenced by using a combination of bioinformatic and molecular biology tools (PCR, immunoblotting, Basic Local Alignment Search Tool [BLAST] searches). The immunologic properties of Ara h 9, Ara h 1, Ara h 2, and Ara h 3 were studied by using sera from subjects with peanut and peach allergy from Italy by immunoblotting and allergen microarray technology. RESULTS Two Ara h 9 isoforms-Ara h 9.01 and Ara h 9.02-were cloned and expressed. Ara h 9 represented a minor allergen for subjects with peanut allergy. However, including Ara h 9 as single component for serologic detection of sensitization to peanut by component-resolved diagnosis seems crucial, because the frequency of sensitization to the classic major peanut allergens Ara h 1, Ara h 2, and Ara h 3 was low in these patients from Southern Europe. CONCLUSION Ara h 9 is a new member of the LTP allergen family that seems to play an important role in peanut allergy for patients from the Mediterranean area.

Structure and stability of 2S albumin-type peanut allergens: implications for the severity of peanut allergic reactions.

Resistance to proteolytic enzymes and heat is thought to be a prerequisite property of food allergens. Allergens from peanut (Arachis hypogaea) are the most frequent cause of fatal food allergic reactions. The allergenic 2S albumin Ara h 2 and the homologous minor allergen Ara h 6 were studied at the molecular level with regard to allergenic potency of native and protease-treated allergen. A high-resolution solution structure of the protease-resistant core of Ara h 6 was determined by NMR spectroscopy, and homology modelling was applied to generate an Ara h 2 structure. Ara h 2 appeared to be the more potent allergen, even though the two peanut allergens share substantial cross-reactivity. Both allergens contain cores that are highly resistant to proteolytic digestion and to temperatures of up to 100 degrees C. Even though IgE antibody-binding capacity was reduced by protease treatment, the mediator release from a functional equivalent of a mast cell or basophil, the humanized RBL (rat basophilic leukaemia) cell, demonstrated that this reduction in IgE antibody-binding capacity does not necessarily translate into reduced allergenic potency. Native Ara h 2 and Ara h 6 have virtually identical allergenic potency as compared with the allergens that were treated with digestive enzymes. The folds of the allergenic cores are virtually identical with each other and with the fold of the corresponding regions in the undigested proteins. The extreme immunological stability of the core structures of Ara h 2 and Ara h 6 provides an explanation for the persistence of the allergenic potency even after food processing.

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

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

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

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

Seafood Allergy and Allergens: A Review

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

Identification of the first major allergen of a squid (Todarodes pacificus)

BACKGROUND In Japan, squid is an important seafood, and some patients with food allergies are sensitive to squid. There has been no report, however, describing the major allergens of squid. OBJECTIVE To characterize squid allergens, we isolated a major allergen from the Pacific flying squid (Todarodes pacificus) and compared it with a major allergen from a shrimp (Penaeus orientalis). METHODS The major squid and shrimp allergens were isolated by column chromatography on diethylaminoethyl-Sepharose (Pharmacia, Uppsala, Sweden), hydroxylapatite, and Sephacryl S-300 (Pharmacia). The IgE reactivity of the isolated allergens was assessed by immunoblotting. The cross-reactivity between the squid and shrimp allergens was examined by use of mouse polyclonal and monoclonal antibodies to the major allergens. Amino acid sequence analyses of the isolated allergens were done. RESULTS The isolated squid allergen is a 38 kd, heat-stable protein. IgE antibody binding to the purified squid allergen was demonstrated by immunoblotting. Cross-reactivity between major squid and shrimp allergens was demonstrated with sera from patients allergic to squid or shrimp or with allergen-specific monoclonal antibodies. The amino acid sequence analysis of the major squid allergen showed a marked homology with tropomyosin from blood fluke planorbid (Biomphalaria glabrata), which is a common vector snail of Schistosoma mansoni. CONCLUSION This 38 kd protein is a major allergen of the squid, Todarodes pacificus, and is believed to be squid muscle protein tropomyosin. We named it Tod p 1 according to International Union of Immunological Societies allergen nomenclature regulation.