Allergenicity can survive cooking, possibly because tropomyosin have a very simple helical structure which can rapidly refold after denaturation.
Nature of main cross-reacting proteins:
Miyazawa et al. (1996) [1558] demonstrated that shrimp tropomyosin completely inhibits IgE binding to squid tropomyosin. Carrillo et al. (1992) [1556], Castillo et al. (1994) [1759] and Castillo et al. (1996) [1758] show that almost all squid allergics in their study were allergic to shrimp.
Allergen properties & biological function:
Tropomyosins bind to actin in muscle increasing thin filament stability and rigidity. Depolymerization from the pointed end is inhibited, without affecting elongation (Broschat, 1990 [1589]). As tropomyosin prevents the binding of myosin, it may play an important role with troponin in controlling muscle contraction. The sequence exhibits a prominent seven-residues periodicity and this is reflected in the interactions of the 2 polypeptide chains which form a coiled coil structure of two alpha-helices as originally proposed by Crick in 1952 (see the porcine structure 1C1G). Some tropomyosins are N-acetylated modifying the structure of the N terminal region and increasing the affinity for the thin filaments (Greenfield & Fowler, 2002 [1590]).
Allergen purification:
Miyazawa et al. (1996) [1558] used 100 g fresh squid, cut into small pieces with scissors and boiled in distilled water (300 ml) at 100° C for 10 minutes, then centrifuged at 20,000 g for 20 minutes. The supernatant was used as the crude extract. Ammonium sulfate was added with stirring to 50% saturation. After centrifugation at 20,000 g for 20 minutes, the resulting precipitate was collected and dissolved in 0.05 mol/L Tris-HCl buffer, pH 8.0, and then dialyzed against the same buffer. The sample was applied to a DEAE-Sepharose column (Pharmacia). Fractions were collected with a linear gradient of 0.18 to 0.6 M NaCl in Tris buffer and those with allergenic activity were pooled and dialyzed overnight in 0.01 M phosphate buffer (pH 6.8). This was applied to a hydroxylaptite column (Bio-Rad Laboratories, Hercules, Calif.). Fractions were collected with a 0.01 to 0.5 M NaCl gradient in the same buffer, and those with allergenic activity were pooled and dialyzed against phosphate-buffered saline. This was applied to a Sephacryl S-300 column (Pharmacia) equilibrated with PBS and the purification of the 38 kDa protein Tod p 1 was verified by SDS-PAGE and immunoblotting.
Other biochemical information:
Miyazawa et al. (1996) [1558] isolated 5 peptides by reverse-phase high-performance liquid chromatography and reported the peptide sequences as NTIEEDLSTLQK, YSNLENDFDNA, KENLTVANTNLEASEK, VLENRSQGDEERIDLLEK, EVDRLEDELLAEK. These differ from the sequence http://www.expasy.org/uniprot/Q6E216 which is only distantly related to allergenic tropomyosins.
References (6)
Broschat KO.
Tropomyosin prevents depolymerization of actin filaments from the pointed end.
J Biol Chem. 265(34):21323-21329. 1990
PUBMED ID:
2250026
Jeoung BJ, Reese G, Hauck P, Oliver JB, Daul CB, Lehrer SB.
Quantification of the major brown shrimp allergen Pen a 1 (tropomyosin) by a monoclonal antibody-based sandwich ELISA.
J Allergy Clin Immunol. 100(2):229-234. 1997
PUBMED ID:
9275145
Miyazawa H, Fukamachi H, Inagaki Y, Reese G, Daul CB, Lehrer SB, Inouye S, Sakaguchi M.
Identification of the first major allergen of a squid (Todarodes pacificus).
J Allergy Clin Immunol. 98(5 Pt 1):948-953.
1996
PUBMED ID:
8939158
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