Abstract
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.
Highlights
Exposure to pollen from the short ragweed (Ambrosia artemisiifolia) is a major cause of severe type I allergy
Cleavage of both propeptides was confirmed by mass spectrometry analysis with up to three additional amino acid residues observed in N or C termini when compared with the natural molecule nAmb
As of today 10 short ragweed pollen allergens have been officially registered by International Union of Immunological Societies [6], we recently reported the identification of seven novel candidate allergens following an extensive characterization of the short ragweed pollen transcriptome and proteome [26]
Summary
Recombinant Protein Expression in E. coli—The cDNA encoding pro-rAmb a 11 preceded by a N-terminal 21-residuelong His tag (MGSSHHHHHHSSGLVPRGSHM) was cloned in the pET-15b vector (Merck Millipore, Molsheim, France), and the resulting plasmid was used to transform BL21(DE3) E. coli (Agilent, Les Ulis, France). Crystal structures were determined by performing molecular replacements with Phaser [16] and using either the monomer of the vignain from Ricinus communis (Protein Data Bank code 1S4V) [17] or the propapain mutant from Carica papaya (Protein Data Bank code 3TNX) [18] as search models for the medium and high resolution data sets, respectively Both pro-rAmb a 11 models were refined with non-crystallographic symmetry restraints and TLS (translation/libration/screw motion) using Buster 2.10 [19]. Measurements of Airway Inflammation and Immune Responses in Amb a 11-sensitized Mice—For sensitization, mice were immunized intraperitoneally (i.p.) on days 0 and 14 with g of either pro-rAmb a or rAmb a 11 activated with 20 mM L-cysteine (Sigma), subsequently inhibited or not with 10 M E64, adsorbed on 2 mg of aluminum hydroxide (Life Technologies), and administered in 100 l of Dulbecco’s PBS. Statistical analyses of data on the inhibition of protease activity by E64 were done using the non-parametric Mann-Whitney t test. p values less than 0.05 were considered as significant: *, p Ͻ 0.05; **, p Ͻ 0.01; and ***, p Ͻ 0.001
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