Lopata, A. L. | bioRxiv Science

bioRxiv2020

Novel allergen discovery through comprehensive de novo transcriptomic analyses of 5 shrimp species

Shellfish allergy affects up to 2% of the worlds population and persists for life in most patients. The diagnosis of a shellfish allergy, in particular shrimp, is however often challenging due to the similarity of allergenic proteins in other invertebrates. Despite the clinical importance, the complete allergen repertoire of allergy-causing shrimps remains unclear. Here we mine the complete transcriptome of five frequently consumed shrimp species to identify and compare allergens with all known allergen sources. The transcriptomes were assembled de novo from raw RNA-Seq data of the whiteleg shrimp (Litopenaeus vannamei), black tiger shrimp (Penaeus monodon), banana shrimp (Fenneropenaeus merguiensis), king shrimp (Melicertus latisulcatus), and endeavour shrimp (Metapenaeus endeavouri). Trinity was used to assemble the transcriptome, and Transrate and BUSCO applied to verify the assembly. Blast search with the two major allergen databases, WHO/IUIS Allergen Nomenclature and AllergenOnline, successfully identified all seven known crustacean allergens. Salmon was utilised to measure their relative abundance, demonstrating sarcoplasmic calcium-binding protein, arginine kinase and myosin light chain as highly abundant allergens. In addition, the analyses revealed up to 40 unreported allergens in different shrimp species, including heat shock protein (HSP), alpha-tubulin, chymotrypsin, cyclophilin, beta-enolase, aldolase A, and glyceraldehyde-3-phosphate dehydrogenase (G3PD). Multiple sequence alignment, conducted in Jalview 2.1 with Clustal Omega, demonstrated high homology with allergens from other invertebrates including mites and cockroaches. This first transcriptomic analyses of allergens in a major food source provides a valuable genomic resource for investigating shellfish allergens, comparing invertebrate allergens and developing improved diagnostics and novel immunotherapeutics for food allergy.

bioinformatics↗

bioRxiv2020

Effect of structural stability on endolysosomal degradation and T-cell reactivity of major shrimp allergen tropomyosin

BackgroundTropomyosins are highly conserved proteins, an attribute that forms the molecular basis for their IgE antibody cross-reactivity. Despite structural similarities, their allergenicity varies greatly between ingested and inhaled invertebrate sources. In this study, we investigated the relationship between the structural stability of different tropomyosins, their endolysosomal degradation patterns and T-cell reactivity. MethodsWe investigated the differences between four tropomyosins - the major shrimp allergen Pen m 1 and the minor allergens Der p 10 (dust mite), Bla g 7 (cockroach) and Ani s 3 (fish parasite) - in terms of IgE binding, structural stability, endolysosomal degradation and subsequent peptide generation, and T-cell cross-reactivity in a BALB/c murine model. ResultsDespite their conserved primary structure and consequent IgE co-reactivity, the invertebrate tropomyosins displayed different protein stabilities. Pen m 1 and Ani s 3, but not Der p 10 and Bla g 7 elicited differential melting temperatures that were pH-dependent. Endolysosomal experiments demonstrated differential degradation, as a function of stability, generating different peptide repertoires. Pen m 1 T-cell clones, with specificity for sequences highly conserved in all four tropomyosins, did not proliferate with Der p 10, Bla g 7 and Ani s 3, indicating that these peptides were not naturally produced for other invertebrate tropomyosins. ConclusionsOur data suggest that, although invertebrate tropomyosins exhibit a high degree of IgE cross-reactivity due to conserved B-cell epitopes, they do not necessarily share identical cross-reactive T-cell epitopes. This is likely due to differential endolysosomal processing as a function of different structural stabilities.

immunology↗