Cloning and characterization of tropomyosin from the mite Chortoglyphus arcuatus.

Abstract

Tropomyosin is a pan-allergen that shares a high homology among species. It is involved in cross-reactivity among mites, crustaceans, mollusks and insects. The objectives were to express and purify recombinant tropomyosin from the storage mite Chortoglyphus arcuatus, and to investigate the homology and cross-reactivity with tropomyosin from other invertebrates. Recombinant C. arcuatus tropomyosin (r-Cho a 10) was selected from a library by screening with a pool of patient sera. r-Cho a 10 (UniProt: H2DFL1) was sequenced, expressed in Escherichia coli and purified by ion exchange and affinity chromatography. Polyclonal anti-tropomyosin antibodies were produced in mice. IgE recognition of r-Cho a 10 was checked by immunoblot. Immunoblot inhibition assays were used to identify the native tropomyosin in the complete extract from C. arcuatus and study cross-reactivity between r-Cho a 10 and Der p 10. Identification of tropomyosin in other allergenic sources was performed by immunoblot. r-Cho a 10 showed a high homology (54-96%) with other tropomyosins from different allergenic sources. IgE recognition was observed using a pool of sera from sensitized individuals. Tropomyosins from different extracts were identified not only in the whole C. arcuatus extract but also in Dermatophagoides pteronyssinus, shrimp, mussel, cockroach and Anisakis extracts with polyclonal α-Cho a 10. r-Cho a 10 completely inhibited the recognition of Der p 10. Recombinant C. arcuatus tropomyosin maintained its capacity to recognize IgE. r-Cho a 10 was used to prove cross-reactivity among tropomyosins from other invertebrate species, including mites. This is the first C. arcuatus allergen included in the WHO/IUIS (World Health Organization/International Union of Immunological Societies) Allergen Nomenclature database.