Studies on the immunoreactivity of the major allergen tropomyosin in mantis shrimp (<I>Squilla oratoria</I>)

Abstract

Crustacean is one of the eight kinds of allergen sources in coastal areas,which causes the IgE-mediated hypersensitive reactions with clinical manifestations including urticaria,angioedema,asthma,and even fatal anaphylaxis. Tropomyosin (TM) is the major allergen of decapod crustaceans with highly conserved amino acid sequences. The purpose of this study is to confirm whether TM is a major cross-reactive allergen in mantis shrimp (Squilla oratoria) which is taxonomically distinct from decapods and largely consumed as a delicacy in China. Quantification of TM in different species of crustaceans was also conducted. Muscle sample from mantis shrimp was homogenized with phosphate buffer to prepare heated extracts and separated by 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A protein band with molecular mass of about 36 ku was detected by IgE-immunoblotting by all of the sera with crustacean allergy,suggesting it is the major allergen of mantis shrimp. This protein was further purified to homogeneity by acetone powder preparation,isoelectric point precipitation,ammonium sulfate fractionation (40%-60% saturation),and heat treatment. Purified protein was demonstrated to be TM by Western blot using polyclonal antibody against TM from Chinese mitten crab (Eriocheir sinensis). Heated extracts from other crustaceans including mud crab (Scylla serrata),Pacific white shrimp (Penaeus vannamei) and short necked clam (Venerupis variegata) were also prepared,and the cross-reactivity of TM from mantis shrimp with TMs from these crustaceans was confirmed by inhibition immunoblotting using polyclonal antibody against TM and inhibition ELISA using sera with crustacean allergy. Quantification by ELISA using polyclonal antibody against TM revealed that TM content in mantis shrimp muscle is much lower than that in Pacific white shrimp and mud crab muscle,which was about 45 times lower in mantis shrimp muscle than that in Pacific white shrimp muscle. However,its allergenicity seems equivalent to other decapod crustaceans as showed by inhibition ELISA using sera with crustacean allergy. This may be due to the degradation of TM in mantis shrimp by its endogenous serine proteinases and cathepsins which destroyed the IgG-binding epitope but not the IgE-binding epitopes. In conclusion,this study demonstrated that allergenicity of mantis shrimp is almost equivalent to decapods and TM is the major allergen in mantis shrimp with high cross-reactivity to decapod crustaceans.