Sample NUROP Congress Paper Arginine

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					               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.


   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.


    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

    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.


           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
              Insecta        Blattaria   Blattidae       Periplaneta Americana   Cockroach

              Malacostraca   Decapoda    Penaeidae       Penaeus monodon         Tiger prawn
                                                         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


              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.


          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
                          Figure2. Gel photo of      4
                                                                                                         for Scylla
 5                        mRNA extraction from       3                                   2
                                                     2                                                   olivacea.
 2.5                      Scylla olivacea total      1.5                                 1.5
 2                        RNA. E1 - First elution-                                        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
                          Third elution (contains    0.2
                          the least amount of               1kb   E1      E2     E3
 0.2                      mRNA)                            ladder

          Protein expression and purification
                                   Figure 4: SDS-PAGE gel



                                    15                                 Scylla olivacea
       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


   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.


Chua W.J. (2006). Identification and characterization of the allergens involved in seafood-
   induced allergic responses and anaphylaxis reactions. A thesis submitted to the Department
   of Biological Sciences, NUS.
Goh D.Y.T., Chew F.T., Quek S.C., Lee B.W. (1996). Prevalence and severity of asthma,
   rhinitis, and eczema in Singapore schoolchildren. Archives of Disease in Childhood ,
Ninan T.K., Russel G. (1992). Respiratory symptoms and atopy in Aberdeen schoolchildren:
   evidence from two surveys 25 years apart. BMJ , 304: 873-5.

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