Paramyosin of the disc abalone Haliotis discus discus: Identification as a new allergen and cross-reactivity with tropomyosin
Introduction
Food allergy mediated by immunoglobulin E (IgE) is a public concern in industrialised countries. In sensitised subjects with high levels of specific IgE to a certain food, adverse reactions such as urticaria, asthma, diarrhoea and anaphylaxis are induced immediately after ingestion of the food; in severe cases, anaphylactic shock leads to even death. Of a number of allergenic foodstuffs, seafoods (fish, crustaceans and molluscs) are recognised as an important cause of food allergy especially in coastal countries, where their consumption is high. Previous studies showed that parvalbumin, a 12 kDa sarcoplasmic protein, is a major cross-reactive allergen in fish (Lehrer et al., 2003, Van et al., 2005, Wild and Lehrer, 2005). Besides parvalbumin, collagen has been additionally identified as a fish allergen although not major (Hamada et al., 2001, Sakaguchi et al., 2000). As for crustaceans and molluscs, tropomyosin, a 35–38 kDa myofibrillar protein, is a common major allergen (Chuo et al., 2000, Emoto et al., 2009, Leung et al., 1996, Motoyama et al., 2006, Motoyama et al., 2007, Reese et al., 1999, Wild and Lehrer, 2005). IgE cross-reactivity of tropomyosin can be seen amongst crustaceans, amongst molluscs, between crustaceans and molluscs, and even between crustaceans and terrestrial arthropods, such as cockroaches and mites (Arlian et al., 2009, Ayuso et al., 2002). Recently, arginine kinase (García-Orozco et al., 2007, Yu et al., 2003), a sarcoplasmic calcium-binding protein (Ayuso et al., 2009, Shiomi et al., 2008) and myosin light chain (Ayuso et al., 2008) have also been confirmed to be new allergens in crustaceans.
To prevent allergic accidents, labelling systems for processed food products containing allergenic food materials have recently been established in some countries. In Japan, 25 kinds of food materials are considered to be especially allergenic, and therefore are obligated or recommended to put labels on packages or bottles of processed food products containing them as raw materials. Abalone, together with other six kinds of seafoods (salmon, mackerel, shrimp, crab, squid and salmon roe), is amongst the 25 kinds of allergenic food materials. Molecular studies with two species of abalones, the Japanese abalone Haliotis diversicolor (Chuo et al., 2000), and the disc abalone Haliotis discus discus (Emoto et al., 2009), proved that their major allergen is tropomyosin, as in the other molluscs. During our study for allergens in disc abalone by immunoblotting, however, not only tropomyosin but also a 100 kDa allergen was found to be allergenic. This finding prompted us to purify and identify the 100 kDa allergen in the disc abalone. As a result, the 100 kDa allergen was demonstrated to be paramyosin by partial amino acid sequencing of the purified preparation. Subsequent ELISA experiments showed that paramyosin, as well as tropomyosin, is a major allergen in disc abalone. Interestingly, the IgE cross-reactivity between paramyosin and tropomyosin was also verified by inhibition experiments. We report here the identification of the 100 kDa allergen in the disc abalone, as paramyosin, and the cross-reactivity of paramyosin with tropomyosin.
Section snippets
Animal samples
Live or fresh specimens of the disc abalone, turban shell Turbo cornutus, Mediterranean mussel Mytilus galloprovincialis, Yesso scallop Patinopecten yessoensis, Japanese flying squid Todarodes pacificus, and common octopus Octopus vulgaris, were purchased at the Tokyo Central Whoelsale Market. Columellar muscle was collected from disc abalone, foot muscle from turban shell and Mediterranean mussel, adductor muscle from Yesso scallop, mantle muscle from Japanese flying squid, and leg muscle from
Purification and identification of a 100 kDa allergen in disc abalone
The 100 kDa allergen was mostly recovered in the myofibrillar protein fraction, suggesting that it is one of myofibrillar proteins. Its purification from the myofibrillar protein fraction was achieved by salting-out with ammonium sulphate (10–20% saturation), followed by hydroxyapatite HPLC on a Bio-Scale CHT2-I column. In hydroxyapatite HPLC, the 100 kDa allergen was eluted in a symmetrical peak at a retention time of 23.8 min (Fig. 1A). The 100 kDa allergen thus obtained was confirmed to be
Discussion
Tropomyosin, a 35–38 kDa myofibrillar protein, is a major allergen in molluscs as well as in crustaceans (Chuo et al., 2000, Emoto et al., 2009, Leung et al., 1996, Motoyama et al., 2006, Motoyama et al., 2007, Reese et al., 1999, Wild and Lehrer, 2005). In this study, however, a 100 kDa allergen differing from tropomyosin was purified from the muscle of the disc abalone Haliotis discus discus by salting-out and hydroxyapatite HPLC, and was clearly identified as paramyosin, one of the
Acknowledgments
The authors would like to thank Drs. A. Urisu and Y. Kondo, Fujita Health University School of Medicine, and Drs. H. Ohsuna and Z. Ikezawa, Department of Dermatology, Yokohama City University, for providing the patient sera. This study was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and a grant from the Ministry of Health, Labour and Welfare of Japan.
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