We identified the cysteine protease Amb a 11 as a new major allergen from ragweed pollen. Given the similar physicochemical properties shared by the 2 major allergens, we hypothesize that part of the allergenic activity previously ascribed to Amb a 1 is rather borne by Amb a 11.
Background: We describe the production in Escherichia coli as a recombinant protein of clinical grade wild-type Bet v 1a (rBet v 1a), to be used as a candidate vaccine against birch pollen allergy. Methods: This recombinant protein was purified by hydrophobic interaction and ion exchange chromatography and characterized by SDS-PAGE, immunoprint and circular dichroism in parallel with natural Bet v 1 (nBet v 1) purified from a birch pollen extract. We also compared rBet v 1 and nBet v 1 for their capacity to induce histamine release from basophils and to stimulate T lymphocyte proliferation. Results: rBet v 1a appears in SDS-PAGE as an 18-kDa monomeric protein, whereas purified nBet v 1 comprises a mixture of isoforms (resolving as three distinct bands and six spots after 1-dimensional and 2-dimensional electrophoresis, respectively). Both recombinant and natural purified Bet v 1 molecules are recognized by IgE from birch pollen-allergic patients as well as anti-Bet v 1 murine monoclonal antibodies, suggesting that the recombinant protein is correctly folded in a native configuration. Circular dichroism analysis confirmed that the two Bet v 1 molecules exhibit similar 3-dimensional structures, even if rBet v 1a appears more compact and stable in thermodenaturation/renaturation experiments. Both rBet v 1 and nBet v 1 induce the degranulation of sensitized basophils and proliferation of Bet v 1-specific T lymphocytes in a similar manner. Conclusions: On the basis of these structural and biological properties, rBet v 1a is a valid candidate vaccine against birch pollen allergy, currently evaluated in humans.
Allergy to the short ragweed (Ambrosia artemisiifolia) pollen is a major health problem. The ragweed allergen repertoire has been recently expanded with the identification of Amb a 11, a new major allergen belonging to the cysteine protease family. To better characterize Amb a 11, a recombinant proform of the molecule with a preserved active site was produced in Escherichia coli, refolded, and processed in vitro into a mature enzyme. The enzymatic activity is revealed by maturation following an autocatalytic processing resulting in the cleavage of both N-and C-terminal propeptides. The 2.05-Å resolution crystal structure of pro-Amb a 11 shows an overall typical C1A cysteine protease fold with a network of molecular interactions between the N-terminal propeptide and the catalytic triad of the enzyme. The allergenicity of Amb a 11 was confirmed in a murine sensitization model, resulting in airway inflammation, production of serum IgEs, and induction of Th2 immune responses. Of note, inflammatory responses were higher with the mature form, demonstrating that the cysteine protease activity critically contributes to the allergenicity of the molecule. Collectively, our results clearly demonstrate that Amb a 11 is a bona fide cysteine protease exhibiting a strong allergenicity. As such, it should be considered as an important molecule for diagnosis and immunotherapy of ragweed pollen allergy.
Background: Recombinant allergens with a native conformation represent an alternative to natural extracts for immunotherapy and diagnostic purposes. Methods: We produced the Der p 2 mite allergen in Pichia pastoris and Escherichia coli. After purification by cation exchange chromatography, recombinant molecules were compared to their natural counterpart based upon structural (disulfide bonds, secondary structure, thermal stability) and immunological properties (antibody reactivity, basophil and T cell activation, tolerance induction in a murine sublingual immunotherapy model). Results: The Der p 2.0101 isoform was confirmed to be prevalent in Dermatophagoides pteronyssinus extracts. It was then produced as a secreted molecule in P. pastoris or refolded from E. coli inclusion bodies. The yeast-expressed rDer p 2 molecule exhibits a natural-like disulfide bridge distribution and secondary structure, whereas the E. coli-derived rDer p 2 presents some heterogeneity in cysteine bonds and a lower stability following thermal stress. The two recombinant as well as natural Der p 2 molecules exhibit comparable IgE recognition and activate basophil and CD4+ T cells. Sublingual immunotherapy of nDer p 2- sensitized mice using either one of the rDer p 2 molecules efficiently decreases airway hyperresponsiveness as well as Th2 responses. Conclusions: Natural and recombinant Der p 2 molecules produced in P. pastoris and E. coli exhibit comparable immunological properties despite distinct structural features. Natural-like cysteine pairing is a critical parameter to identify stable, well-folded and homogenous proteins appropriate for immunotherapy and diagnostic purposes.
BackgroundHouse dust mites (HDMs) such as Dermatophagoides farinae and D. pteronyssinus represent major causes of perennial allergy. HDM proteomes are currently poorly characterized, with information mostly restricted to allergens. As of today, 33 distinct allergen groups have been identified for these 2 mite species, with groups 1 and 2 established as major allergens. Given the multiplicity of IgE-reactive mite proteins, potential additional allergens have likely been overlooked.ObjectiveTo perform a comprehensive characterization of the transcriptomes, proteomes and allergomes of D. farinae and D. pteronyssinus in order to identify novel allergens.MethodsTranscriptomes were analyzed by RNA sequencing and de novo assembly. Comprehensive mass spectrometry-based analyses proteomes were combined with two-dimensional IgE reactivity profiling.ResultsTranscripts from D. farinae and D. pteronyssinus were assembled, translated into protein sequences and used to populate derived sequence databases in order to inform immunoproteomic analyses. A total of 527 and 157 proteins were identified by bottom-up MS analyses in aqueous extracts from purified HDM bodies and fecal pellets, respectively. Based on high sequence similarities (>71% identity), we also identified 2 partial and 11 complete putative sequences of currently undisclosed D. pteronyssinus counterparts of D. farinae registered allergens. Immunoprofiling on 2D-gels revealed the presence of unknown 23 kDa IgE reactive proteins in both species. Following expression of non-glycosylated recombinant forms of these molecules, we confirm that these new allergens react with serum IgEs from 42% (8/19) of HDM-allergic individuals.ConclusionsUsing combined transcriptome and immunoproteome approaches, we provide a comprehensive characterization of D. farinae and D. pteronyssinus allergomes. We expanded the known allergen repertoire for D. pteronyssinus and identified two novel HDM allergens, now officially referred by the International Union of Immunological Societies (IUIS) Nomenclature Subcommittee as Der f 36 and Der p 36.
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