title: Identification of tropomyosin as the major shrimp allergen and characterization of its IgE-binding epitopes.
abstract: The major heat-stable shrimp allergen (designated as Sa-II), capable of provoking IgE-mediated immediate type hypersensitivity reactions after the ingestion of cooked shrimp, has been shown to be a 34-kDa heat-stable protein containing 300 amino acid residues. Here, we report that a comparison of amino acid sequences of different peptides generated by proteolysis of Sa-II revealed an 86% homology with tropomyosin from Drosophila melanogaster, suggesting that Sa-II could be the shrimp muscle protein tropomyosin. To establish that Sa-II is indeed tropomyosin, the latter was isolated from uncooked shrimp (Penaeus indicus) and its physicochemical and immunochemical properties were compared with those of Sa-II. Both tropomyosin and Sa-II had the same molecular mass and focused in the isoelectric pH range of 4.8 to 5.4. In the presence of 6 M urea, the mobility of both Sa-II and shrimp tropomyosin shifted to give an apparent molecular mass of 50 kDa, which is a characteristic property of tropomyosins. Shrimp tropomyosin bound to specific IgE antibodies in the sera of shrimp-sensitive patients as assessed by competitive ELISA inhibition and Western blot analysis. Tryptic maps of both Sa-II and tropomyosin as obtained by reverse phase HPLC were superimposable. Dot-blot and competitive ELISA inhibition using sera of shrimp-sensitive patients revealed that antigenic as well as allergenic activities were associated with two peptide fractions. These IgE-binding tryptic peptides were purified and sequenced. Mouse anti-anti-idiotypic antibodies raised against Sa-II specific human idiotypic antibodies recognized not only tropomyosin but also the two allergenic peptides, thus suggesting that these peptides represent the major IgE binding epitopes of tropomyosin. A comparison of the amino acid sequence of shrimp tropomyosin in the region of IgE binding epitopes (residues 50-66 and 153-161) with the corresponding regions of tropomyosins from different vertebrates confirmed lack of allergenic cross-reactivity between tropomyosins from phylogenetically distinct species.
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore.
authors.forename: K N
authors.initials: KN
authors.lastname: Shanti
authors.forename: B M
authors.initials: BM
authors.lastname: Martin
authors.forename: S
authors.initials: S
authors.lastname: Nagpal
authors.forename: D D
authors.initials: DD
authors.lastname: Metcalfe
authors.forename: P V
authors.initials: PV
authors.lastname: Rao
mesh_terms: D000485:Allergens / Q000032:analysis / Q000737:chemistry / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D003429:Cross Reactions; D033364:Decapoda / Q000276:immunology*; D000939:Epitopes; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D014335:Tropomyosin / Q000032:analysis / Q000737:chemistry / Q000276:immunology*; D014357:Trypsin / Q000494:pharmacology
chemical_list: D000485:Allergens; D000939:Epitopes; D010446:Peptide Fragments; D014335:Tropomyosin; D007073:Immunoglobulin E; D014357:Trypsin