Crustacea 

1. Shrimp: Major Allergens

  a. Antigens I and II

      Shrimp is the most studied of the crustacea allergens. Hoffman et al. were the first to partially characterize allergens from shrimp. Two allergenic proteins were found in the body and shell extracts of raw shrimp, and were calked antigen I and II. IN a study of 11 shrimp-sensitive subjects, 7 of 11 serum samples bound to antigen I. As only a trace of antigen I was found in raw shrimp and shell extracts, it was thought to be a heat-labile protein composed of two noncovalently bound polypeptide chains with a molecular weight of 21 kDa. Purification by gel filtration of antigen I resulted in a molecular weight of 45 kDa, suggesting it was a dimer. Antigen I had an pI of 4.75 to 5 and contained 189 amino acid residues and 0.5% carbohydrate.

      Antigen II, isolated easily from boiled shrimp, was an acidic, heat-stable glycoprotein with a molecular weight of 38 kDa and a pI of 5.4 to 5.8, composed of 341 amino acid residues and 45 carbohydrate. It appeared to be the major allergen for the subjects in this study, as it bound IgE in all of the 11 shrimp-allergic serum samples. Antigen II gave a correlation coefficient of 0.98 with cooked shrimp in RAST inhibition studies. The allergens were not evaluated using skin tests, so divalent binding ablity was not assessed. Antigens I snd II were considered to be unrelated, based on amino acid composition and immunologic studies.

  b. SA-I and SA-II

      Nagpal et al. described twoallergenic polypeptides isolated from boiled shrimp. Allergen SA-I had a molecular weight of 8.2 kDa and was not analyzed further. The second allergen, SA-II, was composed of 301 amino acid residues, had a molecular weight of 34 kDa, and appeared to be similar to antjgen I isolated by Hoffman and colleagues, but was reported not to possess any carbohydrate.

      Nagpal et al. stated that approximately 545 of the allergenic epitopes of SA-I and SA-II were shared, suggesting that SA-I was a fragment of SA-II. Taking this into consideration, SA-I contributed approximately 33% and SA-II approximately 56% of the totle IgE-binding activity of crude boiled shrimp extract. The authors suggested that the remaining IgE-binding activity (11%) was contained in the shrimp tRNA allergen discussed below. These allergens were not evaluated using skin-test methods.

  c. Pen a 1 and Pen i 1

      Daul et al. isolated a major shrimp allergen, Pen a 1, from boiled brown shrimp (P.aztecus) and reported that its sequence was similar to fruit fly tropomyosin. Pen a 1 has a molecular weight of 36 kDa, is readily isolated from the boiling water and meat of cooked shrimp, and is similar to SA-II. It constitutes 29% of the soluble protein in crude cooked shrimp extract and inhibited the RAST reactivity of pooled shrimp-sensitive subjects' serum to whole body shrimp meat extract by 75%. The allergen bound IgE in 28 of 34 (82%) sera from shrimp-sensitive individuals.

      Pen a 1 compries 312 amino acid residues and 2.9% carbohydrate and has a pI of 5.2. It is referred to as Pen i 1 if isolated from a different species of shrimp, P.indicus.

      Endoproteinase Lys-C studies of Pen a 1 resulted in protein sequencing of a 21-residue peptide that demonstrated significant homology (60 to 85%) with tropomyosin from various species, consistent with the conclusion. The greatest holology occurred in region 129 to 149: 72 to 87% with fruit fly tropomysin, and 60 to 62% with tropomyosin from various mammalian species. The higher homology seen with Drosophila tropomyosin can be cinstrued as bein g indicative of the phylogenic cinnection between shrimp and insects. The  amino acid sequence of the 21 residue peptide is

            V-L-E-N-R-S-L-S-D-E-E-R-M-D-A-L-E-N-Q-L-K.      

      Shanti et al. also reported that sequenced tryptic digests of  Pen i 1 were similar to fruitfly tropomyosin, and that two tryptically derived peptide sequences from shrimp tropomysin bound shrimp-specific IgE. These were regions 50 to 60 and region 153 to 161: 50-66 is

                            M-Q-Q-L-E-N-D-L-D-Q-E-S-L-L-K

and 153-161 is

                                          F-L-A-E-E-A-D-R-K.

Both peptides 50 to 66 and 153 to 161 inhibited binding of SA-II-specific IgE to shrimp tropomyosin and 50% inhibition was attained at 100 pmol/ml for both peptides. Other tryptically derived peptides (some less than 2 kDa in molecular weight) inhibited IgE binding to a lesser extent, but these peptides may not have been free of minor IgE-binding components.

      Corresponding regions of tropomyosins from different vertebrates showed little cross-reactivity in region 50 to66, but demonstrated significant allergenic cross-reactivity with tropomyosins from mammalian species in region 153 to 160: seven of nine amino acids for chicken, rabbitm and humans, and six of nine for rat tropomyosin. Fruit fly tropomyosin was identical to the SA-II allergen in region 153 to 161. Many tropomyosins have homologyin the 155 to 161 region; the authors (Shanti et al.) suggested that lack of homology in residues 153 (Leu) and 154 (Ala) between other tropomyosis and shrimp tropomysins implies that they may be crucial for IgE binding.

      The amino acid composition of shrimp allergens Pen a 1, antigen II, and SA-II is similar (listed here!). This further indicates that these three allergens are the same protein, shrimp tropomyosin, although both antigen II and Pen a 1 have associated carbohydrate moieties.

  d. Met e 1

      Leung et al. produced a recombinant shrimp allergen from a cDNA library of the greaseyback shrimp, Metapenaeus enis. The allergen has 281 amino acid residues, is similar in amino acid composition to Pen i 1 and Pen a 1, and has a molecular weight of 34 kDa in SDS-PAGE. In immunoblotting studies, the recombinant allergen bound IgE in serum samples from all eight individuals in the study with histories of anaphylactic reactions to shrimp. Leung et al. confirmed the observations of other groups inentifying the 34-kDa allergen as shrimp tropomyosin. They also found that the recombinant shrimp allergen Met e 1 possessed an IgE-binding sequence identical to the 50 to 66 region of Shanti et al. showed above, and another small IgE-binding sequence of F-L-A-E-E-A-D-R-K, similar to the 153 to 161 region.

2. Shrimp: Minor allergens

a. Transfer RNA

      A minor allegenic tRNA moiety boiled shrimp (P. indicus) has been described. The "purified" RNA allergen possessed 11% of tis dry weight as amino acids. After enzyme treatment, 84% of the amino acids were lost, but allergenicity was retained. Approximately 1 μg shrimp RNA caused 89% inhibition of a solid-phase shrimp RNA RAST. However, Nagpal et al. only used one patient's sera in their studies, and so their results may not reflect normal clinical reactivity. It is possible that the allergenicity was due to RNA-associated proteins/peptides, as the RNA was not totally barren of amino acid residues. The RNA allergen was not analyzed in skin tests, so its divalent binding abtility was not assessed. This remains the only documented example of a nucleic acid from food implicated in inducing an IgE response.

  b. Dose Response

      Daul et al. found that six subjects reacted positively in a total of seven double blind challenges in 30 subjects with shrimp hypersensutivity. Four positive reactions were to a dose of four shrimp equivalents (one shrimp equivalent is approximately 8 mg, or the amount of protein extract obtained from a standard 4-g medium-sixed shrimp) and three positive reactions occurred to a dose of 16 shrimp equia\valents. It appears to elicit an anaphylactic reation in sensitive individuals.

      Oropharyngeal pruritus and occasional subjective throat-pharyngeal swelling was related by most of the individuals experiencing positive challenges at a lower dose of shrimp than that inducing their objective positive symptoms. In a group of subjects having a history consistent with immediate type I hypersensitivity reactions to shrimp, only atopic patients reported anaphylaxis after ingestion: 30 nonatopic patients reported generalized pruritus as their only symptom.

3. Crab

      Snow crab has been shown to causen allergic sensitization in occupational settings. Heat-labile and -stable allergens have been found in snow crab extract, and snow crab-specific IgE bound more to boiled snow crab than to raw crab.

      The most prominent bands in SDS-PAGE gels were 37-42 kDa in crab cooking water and in extracts of cooked crab meat. Immunoblotting of these SDS-PAGE-separated proteins showed that the majority of snow crab-allergenic  serum samples displayed IgE binding to the 37-to-42-kDa bands, but a makority also demonstrated heavy radiostaining to bands at or near 14 kDa (Hefle, Bush, Cartier, Malo, Lehrer, personal communication).

4. Lobster

      The IgE-binding ability of distinct spiny lobster precipotins resolved in drossed immunoelectrophoresis (CIE) was demonstrated in CRIE using 14 crustacea-sensitive sera. Thirteen crustacea-allergic serum sample reacted in CRIE to these precipitins. Spiny lobstr extract contained four IgE-binding precipitins: antigens 8 (positive in ten sera) and 13 (positive in five sera) are the major allergens, giving the most radiostaining. Antigens 3 and 6 gave weak radiostaining in eight and two sera, respectively.

5. Crawfish (Crayfiah)

     In the study discussed above, crawfiah precipitins were also evaluated for their ability to bind crustacea-specific IgE. Six crawfish antigens produced positive rediostaining using CRIE. Antigen 11 was the main allergenic component (positive in nine sera); antigen 12 (also positive in nine sera) may also be a major allergen, but it was situated under the antigen 11 arc and, therefore, could have been an artfact of coprecipitation. Antigens 6 (positive in one sera), 8 (positive in six), 10 (positive in two), and 13 (positive in seven) exhibited rediostaining to varying degrees.