Identification of the cysteine protease Amb a 11 as a novel major allergen from short ragweed.

BACKGROUND: Allergy to pollen from short ragweed (Ambrosia artemisiifolia) is a serious and expanding health problem in the United States and in Europe. OBJECTIVE: We sought to investigate the presence of undescribed allergens in ragweed pollen. METHODS: Ragweed pollen proteins were submitted to high-resolution gel electrophoresis and tested for IgE reactivity by using sera from 92 American or European donors with ragweed allergy. Pollen transcriptome sequencing, mass spectrometry (MS), and recombinant DNA technologies were applied to characterize new IgE-binding proteins. RESULTS: High-resolution IgE immunoblotting experiments revealed that 50 (54%) of 92 patients with ragweed allergy were sensitized to a 37-kDa allergen distinct from Amb a 1. The full-length cDNA sequence for this molecule was obtained by means of PCR cloning after MS sequencing of the protein combined with ragweed pollen RNA sequencing. The purified allergen, termed Amb a 11, was fully characterized by MS and confirmed to react with IgEs from 66% of patients. This molecule is a 262-amino-acid thiol protease of the papain family expressed as a combination of isoforms and glycoforms after proteolytic removal of N- and C-terminal propeptides from a proform. Three-dimensional modeling revealed a high structural homology with known cysteine proteases, including the mite Der p 1 allergen. The protease activity of Amb a 11, as well as its capacity to activate basophils from patients with ragweed allergy, were confirmed. The production of a nonglycosylated recombinant form of Amb a 11 in Escherichia coli established that glycosylation is not required for IgE binding. CONCLUSION: 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.

Surprising complexity of the Asf1 histone chaperone-Rad53 kinase interaction.

The histone chaperone Asf1 and the checkpoint kinase Rad53 are found in a complex in budding yeast cells in the absence of genotoxic stress. Our data suggest that this complex involves at least three interaction sites. One site involves the H3-binding surface of Asf11 with an as yet undefined surface of Rad53. A second site is formed by the Rad53-FHA1 domain binding to Asf1-T(270) phosphorylated by casein kinase II. The third site involves the C-terminal 21 amino acids of Rad53 bound to the conserved Asf1 N-terminal domain. The structure of this site showed that the Rad53 C-terminus binds Asf1 in a remarkably similar manner to peptides derived from the histone cochaperones HirA and CAF-I. We call this binding motif, (R/K)R(I/A/V) (L/P), the AIP box for Asf1-Interacting Protein box. Furthermore, C-terminal Rad53-F(820) binds the same pocket of Asf1 as does histone H4-F(100). Thus Rad53 competes with histones H3-H4 and cochaperones HirA/CAF-I for binding to Asf1. Rad53 is phosphorylated and activated upon genotoxic stress. The Asf1-Rad53 complex dissociated when cells were treated with hydroxyurea but not methyl-methane-sulfonate, suggesting a regulation of the complex as a function of the stress. We identified a rad53 mutation that destabilized the Asf1-Rad53 complex and increased the viability of rad9 and rad24 mutants in conditions of genotoxic stress, suggesting that complex stability impacts the DNA damage response.

Expression and characterization of natural-like recombinant Der p 2 for sublingual immunotherapy.

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.

Recombinant fusion proteins assembling Der p 1 and Der p 2 allergens from Dermatophagoides pteronyssinus.

BACKGROUND: Fusion proteins assembling multiple allergens can be engineered by recombinant DNA technologies in order to produce tools for diagnostic and immunotherapeutic purposes. Herein, we developed and characterized chimeras assembling Der p 1 and Der p 2 allergens as potential candidate vaccines against house dust mite allergy. METHODS: Fusion proteins encompassing Der p 2 with either mature or proDer p 1 were expressed in Escherichia coli or Pichia pastoris. Forms with mutation in Der p 1 catalytic site were also engineered. Purified chimeras were characterized by immunoblotting, circular dichroism, disulfide bond mapping, basophil and T lymphocyte stimulation assays. RESULTS: Four fusion proteins were expressed in E. coli as inclusion bodies, whereas only chimeras comprising proDer p 1 were obtained in yeast. All such hybrids formed polymers and aggregates, and yeast-expressed chimeras were unstable. Circular dichroism analysis performed after refolding of bacteria expressed chimeras encompassing mature Der p 1 confirmed partial folding, consistent with the occurrence of both correct and inappropriate intramolecular disulfide bonds. All fusion molecules were recognized by Der p 1- and Der p 2-specific human IgEs, monoclonal and polyclonal antibodies. Fusion proteins activate basophils from mite-allergic patients and trigger the proliferation of specific CD4+ T cells, albeit to a lower level when compared to individual allergens. CONCLUSIONS: Production of multiple Der p 1-Der p 2 fusion proteins exhibiting partial folding and proper antigenic properties has been achieved. Nonetheless, significant solubility and stability issues currently limit the application of such chimeras for immunotherapy or diagnostic.

High throughput screening of mixed-mode sorbents and optimisation using pre-packed lab-scale columns for the purification of the recombinant allergen rBet v 1a.

Mixed-mode chromatography was investigated for the purification of the recombinant allergen rBet v 1a expressed in Escherichia coli (E. coli) and used as an active principle for specific immunotherapy (SIT) treatment against birch pollen allergy. The screening of micro-volumes of three mixed-mode sorbents established that rBet v 1a could be captured without any pre-treatment of the crude feedstock on HEA or PPA HyperCel sorbents equilibrated in "physiological-like" conditions. On a mini-column pre-packed with PPA HyperCel sorbent, rBet v 1a was recovered at pH 4, partially separated from a major methionine Bet v 1 contaminant and purified approximately 9-fold in a single step (85% purity).

Mass spectrometric investigation of molecular variability of grass pollen group 1 allergens.

Natural grass pollen allergens exhibit a wide variety of isoforms. Precise characterization of such microheterogeneity is essential to improve diagnosis and design appropriate immunotherapies. Moreover, standardization of allergen vaccine production is a prerequisite for product safety and efficiency. Both qualitative and quantitative analytical methods are thus required to monitor and control the huge natural variability of pollens, as well as final product quality. A proteomic approach has been set up to investigate in depth the structural variability of five group 1 allergens originating from distinct grass species (Ant o 1, Dac g 1, Lol p 1, Phl p 1, and Poa p 1). Whereas group 1 is the most conserved grass pollen allergen, great variations were shown between the various isoforms found in these five species using mass spectrometry, with many amino acid exchanges, as well as variations in proline hydroxylation level and in main N-glycan motifs. The presence of O-linked pentose residues was also demonstrated, with up to three consecutive units on the first hydroxyproline of Ant o 1. In addition, species-specific peptides were identified that might be used for product authentication or individual allergen quantification. Lastly, natural or process-induced modifications (deamidation, oxidation, glycation) were evidenced, which might constitute useful indicators of product degradation.

Structural basis for the interaction of Asf1 with histone H3 and its functional implications.

Asf1 is a conserved histone chaperone implicated in nucleosome assembly, transcriptional silencing, and the cellular response to DNA damage. We solved the NMR solution structure of the N-terminal functional domain of the human Asf1a isoform, and we identified by NMR chemical shift mapping a surface of Asf1a that binds the C-terminal helix of histone H3. This binding surface forms a highly conserved hydrophobic groove surrounded by charged residues. Mutations within this binding site decreased the affinity of Asf1a for the histone H3/H4 complex in vitro, and the same mutations in the homologous yeast protein led to transcriptional silencing defects, DNA damage sensitivity, and thermosensitive growth. We have thus obtained direct experimental evidence of the mode of binding between a histone and one of its chaperones and genetic data suggesting that this interaction is important in both the DNA damage response and transcriptional silencing.