IgE Reactivity of Blue Swimmer Crab (Portunus pelagicus) Tropomyosin, Por p 1, and Other Allergens; Cross-Reactivity with Black Tiger Prawn and Effects of Heating

Abstract

Shellfish allergy is a major cause of food-induced anaphylaxis, but the allergens are not well characterized. This study examined the effects of heating on blue swimmer crab (Portunus pelagicus) allergens in comparison with those of black tiger prawn (Penaeus monodon) by testing reactivity with shellfish-allergic subjects' serum IgE. Cooked extracts of both species showed markedly increased IgE reactivity by ELISA and immunoblotting, and clinical relevance of IgE reactivity was confirmed by basophil activation tests. Inhibition IgE ELISA and immunoblotting demonstrated cross-reactivity between the crab and prawn extracts, predominantly due to tropomyosin, but crab-specific IgE-reactivity was also observed. The major blue swimmer crab allergen tropomyosin, Por p 1, was cloned and sequenced, showing strong homology with tropomyosin of other crustacean species but also sequence variation within known and predicted linear IgE epitopes. These findings will advance more reliable diagnosis and management of potentially severe food allergy due to crustaceans.

Figure 1. SDS-PAGE analysis of shellfish extracts.

4–12% SDS-PAGE of whole shellfish extracts stained with Coomassie brilliant blue. M, MW markers; RC, raw blue swimmer crab; CC, cooked blue swimmer crab; RP, raw black tiger prawn; CP, cooked black tiger prawn.

Figure 2. ELISA for serum IgE reactivity to shellfish extracts.

ELISA for serum IgE reactivity to raw blue swimmer crab (RC), cooked blue swimmer crab (CC), raw black tiger prawn (RP) and cooked black tiger prawn (CP) for 24 shellfish-allergic subjects. The cut-off of two standard deviations above mean reactivity of 7 non-atopic subjects to each of the extracts is indicated by the dotted line (0.56 for RC, 0.49 for CC, 0.29 for RP and 0.75 for CP).

Figure 3. Immunoblotting for serum IgE reactivity against shellfish extracts.

Sera from 12 shellfish-allergic subjects (5–24) and one non-atopic (NA) subject were tested for IgE reactivity to shellfish extract proteins separated by 4–12% SDS-PAGE. A. RC, B. RP, C. CC, D. CP.

Figure 4. Tropomyosin sequence alignment.

Alignment of TM sequences with Portunus pelagicus (blue swimmer crab) TM, Por p 1 (Genbank accession number JX874982.1), as reference using NCBI Protein BLAST. Species include Penaeus monodon (NCBI protein database accession number: A1KYZ2.1), Portunus trituberculatus (ABL89183.1), Portunus sanguinolentus (ABS12234.1), Charybdis feriatus (Q9N2R3.1) and Homarus americanus (AAC48288.1). Sequences that correlate with known linear epitopes of Penaeus aztecus TM (Pen a 1) – are boxed. Predicted linear IgE epitopes based on studies with Penaeus monodon TM (Pen m 1) are shaded grey.

Figure 5. Characterization of recombinant blue swimmer crab tropomyosin, rPor p 1.

A. 4–12% SDS-PAGE of recombinant blue swimmer crab TM, rPor p 1 stained with Coomassie brilliant blue. M, MW markers. B. Sera from 12 shellfish-allergic subjects (5–24) and one non-atopic (NA) subject were tested for IgE reactivity to rPor p 1 using direct IgE immunoblotting.

Figure 6. In vitro basophil activation by shellfish extracts and rPen m 1.

A. Representative dot plots showing gating of viable basophils (7AAD⁻, high IgE positive). B. Representative dot plots (subject 19) showing analysis of activated basophils (up-regulation of cell surface CD63) for negative and positive controls, and CC (10 µg/mL). C. Dose-dependent activation of basophils from shellfish-allergic subjects (7, 8, 19, 22, 24) by shellfish extracts. D. Dose-dependent activation of basophils from shellfish-allergic subjects (7, 8, 19, 22, 24) by rPen m 1.

Figure 7. Inhibition IgE immunoblotting with shellfish extracts.

Inhibition of serum IgE reactivity to CC (A) and CP (B) by shellfish extracts for subjects 5 (i), 19 (ii), 21 (iii) and 24 (iv). Lanes 1: No inhibitor, 2: 4 µg/ml RC, 3: 20 µg/ml RC, 4: 100 µg/ml RC, 5: 4 µg/ml CC, 6: 20 µg/ml CC, 7: 100 µg/ml CC, 8: 4 µg/ml RP, 9: 20 µg/ml RP, 10: 100 µg/ml RP, 11: 4 µg/ml CP, 12: 20 µg/ml CP, 13: 100 µg/ml CP, 14: 1.2 µg/ml rPen m 1.

Figure 8. Inhibition IgE immunoblotting with recombinant tropomyosin preparations.

Inhibition of serum IgE reactivity to CC (A) and CP (B) by rPor p 1 and rPen m 1 using subjects 24 (lanes 1–7) and 19 (lanes 8–14). Lanes 1: No inhibitor, subject 24, 2: 0.048 µg/ml rPor p 1, 3: 0.24 µg/ml rPor p 1, 4: 1.2 µg/ml rPor p 1, 5: 0.048 µg/ml rPen m 1, 6: 0.24 µg/ml rPen m 1, 7: 1.2 µg/ml rPen m 1, 8: No inhibitor, subject 19, 9: 0.048 µg/ml rPor p 1, 10: 0.24 µg/ml rPor p 1, 11: 1.2 µg/ml rPor p 1, 12: 0.048 µg/ml rPen m 1, 13: 0.24 µg/ml rPen m 1, 14: 1.2 µg/ml rPen m 1.