Background: Shellfish allergy is one of the major causes of life-threatening allergic reactions to food. The shrimp species Pandalus borealis is the commercially most important coldwater shrimp species, and its protein extract is commonly used in shrimp allergy diagnostics. However, the DNA sequence of its major allergen, tropomyosin, designated Pan b 1, was not previously described. Our aim was to identify the cDNA sequence of Pan b 1 and to generate a recombinant protein with similar structure and allergenicity as the natural protein. Methods:P. borealis shrimps were caught in the Oslofjord (Norway). cDNA from Pan b 1 was generated, an N-terminal histidine tag was added, and the protein was expressed in Escherichia coli. The recombinant Pan b 1 was characterized by structural and IgE-binding studies and investigated further with basophil activation tests (BATs) and skin prick tests (SPTs) on Norwegian shrimp-allergic individuals. Results: The open reading frame encoded 284 amino acids that shared 97–100% identity with other shrimp tropomyosins. Mass spectroscopy of natural Pan b 1 confirmed the protein’s molecular mass and indicated the absence of posttranslational modifications. Circular dichroism spectroscopy revealed virtually identical spectra between recombinant and natural Pan b 1, which together with native PAGE and size exclusion chromatography results indicated a similar structure. Furthermore, immunoblot and ELISA studies as well as BATs and SPTs showed equivalent results of recombinant and natural Pan b 1. Conclusion: A recombinant tropomyosin from P. borealis was generated that can be used in diagnostics and further studies on tropomyosin allergenicity and specific immunotherapy.

Background: Epitope mapping of an allergen is generally done by IgE-binding assays with short synthetic peptides, but this provides little information about which domains are responsible for IgE receptor crosslinking on effector cells. Our aim was to map the immunodominant regions of shrimp tropomyosin by both IgE-binding and IgE-receptor crosslinking studies. Methods: Five overlapping fragments covering Pandalus borealis tropomyosin were cloned, expressed in Escherichia coli and characterized by circular dichroism spectroscopy, native PAGE and bis(sulfosuccinimidyl) suberate-crosslinking. IgE binding was detected by Western blot, indirect ELISA and inhibition ELISA, and IgE receptor crosslinking was investigated by basophil activation test and skin prick test with Norwegian shrimp allergic adults. Results: The N- and C-terminal fragments of tropomyosin showed the highest amount of secondary structure. Western blot studies showed preferential binding to the terminal fragments, while indirect and inhibition ELISA studies showed binding to all fragments, but with individual variations. Basophil CD63 expression was upregulated by all fragments at high concentrations (1 µg/ml) and showed individual variations comparable to ELISA results. A mixture of the fragments with equal molar ratios induced comparably strong CD63 activation as for tropomyosin. Skin prick test studies showed positive responses to the terminal and middle fragments and increased responses to the fragment mixture compared to whole tropomyosin. Conclusions: The terminal and middle fragments of tropomyosin had the highest IgE reactivity, but overall no clear immunodominant region was observed in this study. These results correlated well with previous studies with short peptides. Dividing shrimp tropomyosin into five fragments did not reduce the allergenicity of the protein.

BackgroundThe present study investigated to what extent shrimp allergic individuals were IgE-sensitized to anisakis, crab and house dust mite and whether tropomyosin was responsible for IgE cross-reactivity.Methods29 Individuals with self reported shrimp allergy were recruited by advertisements in local and national news-papers in Norway. Anamnesis was taken, skin prick tests (SPT) were performed and positive responders to shrimp were studied further with basophile activation test (BAT), ImmunoCAP analyses and western blotting.ResultsOf the 29 persons studied, 10 (34%) had positive SPT against shrimp and house dust mite, 9 (31%) against shrimp tropomyosin and 3 (10%) against anisakis. Individuals with positive SPT to shrimp all showed positive basophilic responses to house dust mite, while 43% responded to shrimp, 25% to anisakis and 36% to crab in BAT. Moreover, SPT, BAT as well as ImmunoCAP analyses showed a positive correlation of IgE-reactivity between anisakis and shrimp, house dust mite and crab. Immunoblot studies indicated that these responses are not completely explained by cross-reactivity towards tropomyosin.ConclusionsThe current study indicates a positive correlation between IgE-mediated reactions to shrimp, anisakis, house dust mite and crab, which may not be completely explained by cross-reactivity against tropomyosin.

BackgroundFibroleukin or fibrinogen-like protein-2 is a immunomodulatory protein that was described to bind to Fcgamma-receptor-IIb and III. In the present study the effects of a fusion protein consisting of fibroleukin and the major allergen of shrimp, tropomyosin, on human basophilic responses were investigated in vitro.MethodsThe fusion molecule was generated by molecular cloning and expressed in E. coli. Receptor binding studies were performed by immunoblot, ELISA, and flow cytometry. Activation of basophils was studied by basophil activation test (BAT) with blood from shrimp allergic individuals.ResultsTropomyosin and the C-terminal part of fibroleukin were fused by a short flexible linker consisting of the amino acids RADAAP. The fusion protein bound to the human Fcgamma-receptor-IIb in immunoblot and ELISA and binding of the fusion protein to human B-cells was shown by flow-cytometry. Shortening of the allergen into a peptide covering one-fifth of whole tropomyosin increased the binding to B-cells. Futhermore, a decrease in the activation of basophils to shrimp tropomyosin was observed in presence of the fusion protein.ConclusionsHere we describe a novel fusion protein based on fibroleukin and shrimp tropomyosin that may have tolerizing effects on basophils and B-cells in shrimp allergic individuals.