Fish

1. Major Allergens

     Gad c 1:     The most comprehensive analysis of a food allergen was done by Aas and Elsayed and colleagues, which resulted in the purification and characterization of  the major codfish allergen, Gad c 1 (originally designated Allergen M). Several studies have documented that Gad c1 is the  major codfish allergen[58-60]. Belonging to a group of muscle tissue proteins known as parvalbumins[8]. Parvalbumins control the flow of calcium in and out of cells and are only found in the muscles of amphibians and fish. The existence of structural related parvalbumins in different fish species may explain cross-reactivity in fish-allergic individuals. because Gad c1 shares approximately 34% homology with similar proteins from hake, carp, pile and whiting. The proportion of Gad c1 in frish white cod muscle tissue is 0.05 to 0.1%.

2. Minor Allergens

      Ag-17-cod:     CRIE studies indicate that there are other minor codfish allergens distinct from Gad c 1[58,61], but these have not been characterized. One of these minor allergens was designated Ag-17-cod, and two of eight serum samples from cod-allergic subjects demonstrated IgE binding to this allergen[58]. It has also been reorted that approximaedly 10% of cod-allergic individuals react to a distinct ptotein gound in cod blood serum, but none appear to react exclusively to it[62].

      Protamine sulfate:     A low-molecular-weight protein widely used as a hyparin antagonistm, is a pperm protein of salmon and related fish species belonging to the families Salmonidae and Clupeidae (herring, sardimes, trout)[63]. Oe study revealed the presence of antiprotamine sulfate IgE in the serum of one fish-allergic subject. This subject also exhibited a positive skin test against protamine sulfate[64]. However, another study found no cross-reactivity between IgE to salmon and protamine sulfate in two fish-allergec subjects[65]. Although protamine sulfate has been isentified as an allergen in some reports of individuals with fish hypersensitivity[66], one study showed that protamine administration did not cause any adverse reaction in 16 fish-allergic subjects[67]. Therefore, it can be concluded that lprotamine sulfate is rarely allergenic for individuals with fish hypersensitivity.

      Surimi 63-kDa protein:     Mata et al.[68] found that surimi, a collection of one or many different varieties of small fish taht are minced and washed extensively, gave a single protein band of 63.5 kDa in SDS-PAGE, Using RAST, they showed that six of sera from fish-allergic patients showed slightly positive responses to surumu, although skin-peick tests were only positive in two of six patients. No further conclusions can be reached on the significance of this allergen until more research of completed.

3. Structure-Epitopes

  a. Gad c 1      Gad c 1 is an acidic protein (pI 4.75) with a molecular wright 12,328 Da, and is composed of 113 amino acids and one glucose molecule[69]. As a parvalbumin, the tertiary structure of Gad c 1 comprises three domains: AB, CD, and EF. The CD and EF domains coordinate one Ca²-binding site each, whereas the AB domain does not have this property.

       Gad c 1 contains:   at least five IgE-binding sites[70]. The single arginine at position 75 in  Gad c 1 plays a major role in the tertiary strusture of the allergen, but modification of the arginine residue  resulted in no difference in IgE reactivity[71]. Tryptic cleavage at the arginine residue resulted in two allergenic fragments[72], TM1 and TM2, which were equally active in skin-prick tests, Prausnitz-Kustner (passive sensitization) tests, and RAST inhibition studies. TM1 comprises amino acid residues 1 to 75 and envelopes the AB and CD domain[8]. The acidic residues 59 to 62 are associated with the Ca² binding of the CD domain. The single glucose molecule presint in Gad c 1 is located at the Cys-18 position[73]. This carbohydrate moiety does not appear to be involved in allergenicity, as the allergenic activity of TM1 without the carbohydrate was equal to that of TM2. Fragment TM2 comprises amino acid residues 76 to 113 and envelopes the EF domain; residues 90 to 101 constitute the Ca²- binding region of the EF domain[74], Fragmint TM2 also contains one residue of tryptophan, which seems to have no bearing on allergenicity[75]. Further tryptic hydrolysis studies of fragment TM1 followed by skin tests and Prausnitz-Kusmer tests showed that region 33 to 44 was important for allergenicity[76]. Tryptic hyptic hydrolysis studies of TM2 showed that region 88 to 96 is partially responsible for the allergenicity[77].

      Studies using synthetic peptides established that region 49 to 64 encirled two repetitive sequences (Asp-Glu-Asp-Lys and Asp-Glu-Leu-Lys). These two tereapeptides are mutually important for antibody binding, as region 49 to 64 showed relatively high RAST inhibition (39%) compared with Gad c 1 (68%), and produced positive Prausnitz-Kustner tests. Eegion 57 to 64 did not show any allergenic activity[78]. In a subsequent article, it was shown that region 41 to 64 contained three homologous tetrapeptides, repeated in three sites, interspaced by six amino acids in a segment of 24 residues. A series of synthetic peptides of this region showed taht at least two of the tereapeptides were necessary for interaction with antibody, as all peptides encompassing a minimum of two of the tetrapeptides produced positive RAST inhibition and Prausniz-Kustner tests[79]. IgE-binding capability is independent of both the constitution and sequence of the spacer amino acids.

      Studies of synthetic peptides also showed that residues 88 to 103 in the EF domain have 37.5% amino acid sequence homology with the CD domain peptide, but lack the essential terminally located tetrapeptides responsivle for antivohy vinding in region 41 to 64. However, this residue bound specifically to IgE both in vivo (Prausnitz-Kustner tests) and in vitro (RAST inhibition tests)[80]. Therefore, region 88 to 103 was proposed to have a monovalent binding function that can consequently block, but not elicit allergic reactions.

      The AB domain, which does not bond calcium, nonetheless shares more than 30% amino acid sequence homology with the CD and EF domains, and comptises residues 13 to 32. Synthetic peptides of this region showed that the AB domain is functionally divalent in Prausnitz-Kustner and RAST inhibition in equimolar concentration at a retio of 6:1 in comparison with  Gad c 1[81].

      Regions 13 to 32 and 49 to 64 possess the divalent determinants necessary to elicit an allergic reaction. Repeating amino acid sequenes are abundant in Gad c 1[8]. In regions 35 to 41and 67 to 73, five of the seven residues are identical  (Leu-X-Ala-Phe-X-Ala-Asp), suggesting repeating IgE-binding sites. Region 65 to83 is found between the CD and EF domains. A similar region jions the AB and CD domains and embraces section 33 to 44. These two regions possess a high degree of homology[71]. This, plus the high degree of immunologic cross-reactivity between TM1 and TM2, all point to IgE-binding sites distributed in a repetitive manner along the polypeptide chain.

  b. Protamine Sulfate

      Salmine AI, a protamine from salmon, has the amino acid sequence

                               10                               20                               30

H-P-R-R-R-S-S-S-R-P-V-R-R-R-R-R-P-R-V-S-R-R-R-R-R-R-G-G-R-R-R-R-OH. Iridine Ia, a protamine from rainbow trout, differs from salmine AI only in that there is an insertion of an additional Arg at position 19 (underlined).

      Although there is not much information available on the amount of fish required to produce an allergic response in fish-allergic individuals, it was reported that cod-allergic patients reacted to less than 1 mg of purified Gad c 1 disguised in a 50-g meatball[59]. O'Neil et al.[82] found that 1 g or less of catfish and 4 g or less of other species tested (codfish or snapper) elicited allergic reactions in fish-sensitive individuals in double-blind placebo-controlled food challenge (DBPCFC). Positive reactions were obtained in DBPCFC using 2 oz (uncooked weight) of fish[83]. Another study reported that 8 g dehydrated fish administered over 1 h could elicit reactions[84]. However, these last two studies were not done in subjects with a history of anaphylaxis to fish.