Arginine Kinase as a Pan-allergenic Component in Shellfish Wong S.M. Jane 1 and Chew F.T. 2 Department of Biological Sciences, The National University of Singapore. ABSTRACT Studies have shown that allergic disorders are in a rising trend in many developed countries. Food allergy, one of the causes of life-threatening allergic responses like anaphylaxis, became a main concern. An earlier study on seafood-induced allergic response in Singapore concluded that the molecular weight profiles of IgE-binding bands were mainly in the high molecular range (>80kDa) for mollusc species, which is also the most allergenic species to the population. This corresponds to the size of arginine kinase (AK) in molluscs (~81kDa). Thus, the objective of this study is to evaluate the allerginicity of AK from different species of shellfish, crustacean, dust mite and cockroach and identify possible cross-reactivity between them. Methods used in this study were cloning of AK cds from raw seafood, expression of recombinant AK protein, immuno-blotting and competitive ELISA inhibition reaction. Results showed 76.4% of the patients’ sera contain IgE binding to purified AK. Four main groups of AKs were derived from the cross-reactivity reactions namely the dust mites, cockroach, bivalves and crustaceans. Within the group, AKs are able to cross-react almost 100% (except for dust mite) but between the groups, AKs are either partially cross-reactive or independently sensitized. INTRODUCTION The prevalence and severity of asthma and other allergic symptoms like wheezing, eczema and rhinitis show a rising trend in many developed countries. (Ninan TK, Russel G., 1992) Local study also suggests that atopic disorders are an increasing problem not only in the West but also in the Asian population. (Goh D.Y.T. et al. 1996) Food allergens could cause allergic reactions through very different means. Therefore, allergen components, ways of food processing, genetic makeup and even gastrointestinal conditions of an individual contribute to the different manifestation of food allergic response. Some allergens are allergenic due to its stability, some become allergenic after heat-induced modifications while others may contain linear IgE-reactive epitopes which may cause an allergic response with or without external modification. Food is one of the most common causes of allergy reaction (especially severe responses). A study referred to a clinical immunology/allergy centre in Singapore shows that food (molluscs followed by crustacean) was the most common cause of anaphylaxis in Singapore, followed by insect stings. (Thong H., et al, 2005) There are two stages in the mechanism of allergic responses namely the sensitization stage and the effector stage. In the first stage, an individual is exposed to a specific allergen for the 1 Student 2 Assistant Professor first time and that results in production of IgE specific to the allergen. In the second stage, the same individual is exposed to the same allergen for the second time. Presence of mast cells coated with IgE specific to the allergen result in mast cells degranulation causing release of inflammatory mediators like histamine, leukotriene, prostaglandin and cytokines. These mediators cause increase in vascular permeability, vasodilation and smooth muscle contraction. In some cases, the stage one, sensitization may be triggered by a similar yet different allergen from the one which triggers the stage two, mast cells degranulation. This is referred to as cross-reactivity and these cross-reacting allergens may enter the body through different routes. For instance, a cross-reactivity between inhalant and food allergen. A study on allergens in seafood-induced allergic responses was done and it was found that a high prevalence of Singapore population being allergic to mollusc species and the molecular weight profiles of the IgE-binding bands were mostly in the high molecular range (>80kDa). (W.J., 2006) This molecular size correspond to the arginine kinase of mollusc which is about 81-82 kDa. Arginine kinase belongs to the family of metabolic enzymes that catalyze the buffereing of ATP in cells with fluctuating energy requirements. (Zhou G. et al. 1998) It transfers phosphorus-containing groups with nitrogenous group as acceptor: ATP + l-arginine ⇌ ADP + Nomega-phospho-L-arginine It is through this buffering reaction that cells can support burst or nerve or muscle activity that would otherwise depletes ATP to levels that would not sustain other essential functions. MATERIALS AND METHODS Seafood Specimens Table 1: Taxonomy of specimens obtained Phylum Class Order Family Scientific Name Common Name Dermatophagoides American house dust Arthropoda Arachnida Astigmata Pyroglyphidae farinae mite Brown legged grain Acaridae Aleuroglyphus ovatus mite Insecta Blattaria Blattidae Periplaneta Americana Cockroach Malacostraca Decapoda Penaeidae Penaeus monodon Tiger prawn Fenneropenaeus merguiensis Banana prawn Portunidae Scylla olivacea Mud crab Mollusca Bivalvia Veneroida Veneridae Meretrix meretrix Flower clam Meretrix lyrata Vietnamese White clam Paphia undulata Mango clam Glauconomidae Glaucomone rugosa Big Head Mussel Myoida Pholadidae Pholas orientalis "Ya Zui" clam Arcoida Arcidae Anadara granosa Blood cockle Human Sera A total of 55 patient sera from KK Women’s and Children’s hospital collected between 23rd December 2008 to18th February 2009 were used to screen the purified arginine kinase. Patients were of age six to nineteen at the point of sera collection and 92.7% of the patients are atopic patients Methods Total RNA was extracted from live seafood using TRIzol® reagent, mRNA was then extracted from the pool of total RNA Oligotex® mRNA Kit. Oligotex® mRNA Kit. Cloning and sequencing of AK sequences were done using various bioinformatics software, namely BLASTX and BioEdit Sequence Alignment Editor software. Small scale protein expression was done to validate solubility of AK after which large scale expression is carried out. Protein purification was done using resin which binds to the His-tagged recombinant AK protein. Immuno-blotting was done to evaluate allergenicity of AK while ELISA inhibition reactions were carried out to identify possible cross- reactivity between the AK of differe sources. RESULTS AND DISCUSSION Total RNA extraction, mRNA extraction and AK cds amplification Figure1. Gel photo of Total RNA extraction kb Figure 3. Gel from the Scylla olivacea photo of amplified kb kb crude. full length 10 arginine kinase 8 6 Figure2. Gel photo of 4 2.5 for Scylla 5 mRNA extraction from 3 2 4 3 2 olivacea. 2.5 Scylla olivacea total 1.5 1.5 2 RNA. E1 - First elution- 1 1 1.5 0.8 0.75 contains the highest 0.6 0.5 1 amount of mRNA; E2 - 0.8 0.4 Second elution; E3 - 0.25 0.6 Third elution (contains 0.2 0.4 the least amount of 1kb E1 E2 E3 1kb 0.2 mRNA) ladder ladder 1kb ladder Protein expression and purification Figure 4: SDS-PAGE gel 250 150 100 75 50 37 25 20 15 Scylla olivacea kDa Immuno-blotting and ELISA inhibition reactions. Graph1: Scatterplot Graph2: ELISA inhibition reactions From the scatterplots, it is observed that the correlation of AK reactivity of certain pairs of species was quite significant especially those within the same class or order. From those relationships, AKs can be categorized into four groups namely: dustmite, cockroach, bivalves and crab/prawn. Within the group, AKs are able to cross react almost 100% (except for dust mite) but between the groups, AKs are either partially cross-reactive or independently sensitized. CONCLUSION The present study provides only a basis of identifying allergenic protein based on specific IgE binding of patient’s sera which can only prove that the patient is sensitized to the protein. There is yet to be evidence that shows manifestation of clinical symptoms in patients. Nevertheless, arginine kinase is portrayed to be an important pan-allergen in this study. As much as 76.4% of patients’ sera (92.7% atopic) react with at least one of the arginine kinase expressed in this study. This study shows that although AK homologs are well conserved across different species, prominent cross-reactivity only occurs among species within the same group. REFERENCES Chua W.J. (2006). Identification and characterization of the allergens involved in seafood- induced allergic responses and anaphylaxis reactions. 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