Not known for Cha f 1. However, the data on shrimp allergens Pen a 1 and Pen i 1 might be relevant.
Allergen stability: Process, chemical, enzymatic
The allergenicity of crustacea can survive cooking, possibly because tropomyosin have a very simple helical structure which can rapidly refold after denaturation. Extracts from boiled crustacea are frequently used in allergen purification and for extract preparation.
Nature of main cross-reacting proteins:
Leung et al (1998) [1554] reported the percentage of amino acid identity of crab Cha f 1 with Met e 1, lobster Pan s 1 and Hom a 1 and Homarus americanus slow muscle, fruit fly and chicken tropomyosins as 90%, 91%, 92%, 96%, 69% and 60% respectively. They found that IgE from the sera from 10/10 crab allergic subjects bound rMet e 1, rPan s 1 and rHom a 1.
DeWitt et al. (2004) [1536] report that the level of sequence identity of tropomyosins with Pen a 1 is 99% for lobster (Homarus americanus), 92% for crab (Charybdis feriatus), 78-82% for insects and dust mites, 71% for a nematode (Caenorhabditis elegans) and 57% for both blue mussel (Mytilus edulis) and human, suggesting that IgE cross-reactivity is very likely for the invertebrate tropomysosins. DeWitt et al. (2004) [1536] also showed specific IgE binding to recombinant Pen a 1 and seven invertebrate extracts with 9 sera. 6 sera bound extracts from crustacea most strongly, 2 bound dust mite extract more strongly and one serum showed similar binding with both extracts. rPen a 1 bound 94% of the IgE from the 6 crustacea specific sera and gave 50% inhibition of the binding of extracts at about 0.1 µg/ml. However, the crab used by DeWitt et al. (2004) was Cancer pagurus.
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:
Leung et al (1998) [1554] identified cDNA encoding Cha f 1 by screening against allergic sera and cloned it into the EcoRI site of plasmid expression vectors pGEX (as fusions with glutathione S-transferase). Recombinant Cha f 1 was purified from cells harvested from a 500-mL overnight culture which had been induced with 1 mM IPTG. The cells were suspended in 5 mL of lysis buffer (1% Triton X-100, 1% Tween 20, 10 mM dithiothreitol) and sonicated. The cell lysate was then spun to pellet the debris, and the supernatant was added to glutathione agarose. After incubating for 2 hours on a rocker, the glutathione agarose was washed 3 times with PBST (PBS containing 1% Triton X-100, pH 7.3) and the bound protein eluted with 2 mL of 5 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0. The purified protein was quantified and the purity determined by SDS-PAGE and immunoblotting, revealing a pure protein band of 60-kDa as expected for a fusion protein.
Other biochemical information:
The sequence of Cha f 1 is shorter than the shrimp tropomyosin sequences, lacking the 20 C-terminal residues. Leung et al. (1998) [1554] argue that this is a genuine difference. However, Swissprot has marked the sequence as partial. Over the 254 residues of the common sequence, Cha f 1 and Met e 1 have 91% sequence identity.
Most of the articles on IgE cross-reactivity of crustacean tropomyosins explored the cross-reactivity of shrimp rather than crab tropomyosins. However, the high level of sequence identity suggests that patients sensitized by Cha f 1 will show very similar IgE cross-reactivity and that patients sensitized to other tropomyosins will generally react to crab as well as shrimp (see shrimp Met e 1 data)
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