Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jun 17;291(25):13076-87.
doi: 10.1074/jbc.M115.702001. Epub 2016 Apr 19.

Structural and Functional Characterization of the Major Allergen Amb a 11 from Short Ragweed Pollen

Rachel Groeme  1 , Sabi Airouche  1 , David Kopečný  2 , Judith Jaekel  1 , Martin Savko  3 , Nathalie Berjont  1 , Laetitia Bussieres  1 , Maxime Le Mignon  1 , Franck Jagic  4 , Petra Zieglmayer  5 , Véronique Baron-Bodo  1 , Véronique Bordas-Le Floch  1 , Laurent Mascarell  1 , Pierre Briozzo  4 , Philippe Moingeon  6
Affiliations

Structural and Functional Characterization of the Major Allergen Amb a 11 from Short Ragweed Pollen

Rachel Groeme et al. J Biol Chem. .

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.

Keywords: allergen; cysteine protease; immunotherapy; protein processing; ragweed; structure-function.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
In vitro maturation of pro-Amb a 11. A, schematic representation of pro-rAmb a 11, pro-rAmb a 11 C155S, and pro-rAmb a 11 ΔCT molecules with His tag shown in green, N-terminal propeptide (N-term prop) in magenta, mature domain in black, and C-terminal propeptide (C-term prop) in blue. aa, amino acids. B, SDS-PAGE analysis of pro-rAmb a 11 before (lane 1) and after activation at pH 5.0 (lane 2), pro-rAmb a 11 C155S (lane 3), and pro-rAmb a 11 ΔCT (lane 4) after dialysis at pH 5.0. C, MALDI-TOF MS analysis of cleavage sites after maturation of pro-rAmb a 11. Cleavage sites are indicated with arrows. D, SRCD spectra of pro-rAmb a 11, pro-rAmb a 11 ΔCT, and rAmb a 11.
FIGURE 2.
FIGURE 2.
Protease activity of Amb a 11. A, protease activity of rAmb a 11 was monitored with (+) and without (−) l-cysteine preactivation. B, relative activity profiles under various pH conditions using casein (▴) or Boc-VLK-AMC (■) substrates. C, protease activities were measured in the presence of different protease inhibitors specific for either cysteine (E64), cysteine and serine (leupeptin), serine (AEBSF and PMSF), metallo- (bestatin), and aspartic (pepstatin A) proteases. D, protease activities of rAmb a 11 (left panel) and nDer p 1 (right panel) in the presence of either N- (N-ter prop) or C-terminal propeptides (C-ter prop) of Amb a 11 or the N-terminal propeptide of Der p 1. Results are expressed as counts per second/microamperes (CPS/μAmps).
FIGURE 3.
FIGURE 3.
Overall fold of pro-Amb a 11. A, superposition of the pro-rAmb a 11 high resolution and medium resolution structures (ribbon representations) viewed along the 2-fold non-crystallographic symmetry axis. In the high resolution structure, monomer A (left) and monomer B (right) are shown in green and orange with the N-terminal propeptide in cyan and magenta, respectively. Dotted lines connect residues that delineate a segment for which no clear density was observed. The medium resolution structure is shown in gray with N and C termini labeled. The C-terminal propeptide (monomer A, medium resolution) is red. B, high resolution structure of the pro-rAmb a 11 dimer. The 2-fold non-crystallographic symmetry axis is indicated as a black ellipsis. The catalytic triad is labeled in purple-blue with the Cys-155 shown in spheres and His-289 and Asn-310 in sticks. The N and C termini of the protein and helices α1–α5 of the N-terminal propeptide are labeled.
FIGURE 4.
FIGURE 4.
Sequence and structure comparison of pro-Amb a 11 and other allergenic cysteine proteases. A, sequence alignment of pro-Amb a 11 (V5LU01), pro-Der p 1 (P08176), and propapain (P00784) with Clustal Omega. Conserved residues are labeled in yellow, and semi-conserved residues are in gray. The first amino acid of the different mature forms is highlighted in blue. Each of the residues of the catalytic triad (i.e. Cys-155, His-289, and Asn-310) is identified with a star. A triangle indicates the Ser-99 residue of the N-terminal Amb a 11 propeptide. B, superposition of the crystal structures of pro-rAmb a 11 (cyan and green as in Fig. 3), pro-Der p 1 (red; Protein Data Bank code 1XKG), and propapain (yellow; Protein Data Bank code 3TNX). The N- and C termini of pro-rAmb a 11 and helices α1–α5 of its N-terminal propeptide are labeled.
FIGURE 5.
FIGURE 5.
Network of hydrogen bonds between catalytic site and N-terminal propeptide. Residues are shown in ball-and-sticks with residues of the N-terminal propeptide in cyan. The magenta sphere represents a water molecule. Catalytic residues are pink with hydrogen bonds (maximal accepted length, 3.2 Å) shown as black dotted lines.
FIGURE 6.
FIGURE 6.
Murine model of sensitization to Amb a 11. A, study design. B, AHR was assessed by whole body plethysmography at day 25 and expressed as enhanced pause (Penh) index values when using a 50 mg/ml dose of methacholine. C, percentages of eosinophils and ILC2s evaluated in BALs by flow cytometry. D, serum IgE and IgG1 antibody responses assessed by ELISA. E, levels of IL-5 and IL-13 were assessed in culture supernatants of lung and spleen cells after in vitro stimulation with pro-rAmb a 11 (10 μg/ml) by using a multiplex cytokine quantification assay. Background levels of cytokines produced by non-activated cells were subtracted. Results are expressed as mean values ±S.E. (error bars) with n = 6 mice per group. Statistical differences between groups were assessed using the non-parametric Kruskal-Wallis test with subsequent Dunnett's multiple analyses when comparing treated mice with naive mice. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
FIGURE 7.
FIGURE 7.
Impact of the inhibition of protease activity on Amb a 11 allergenicity. A, AHR was assessed by whole body plethysmography at day 25 in the presence of a 50 mg/ml dose of methacholine and expressed as enhanced pause (Penh) index values. B, percentages of eosinophils and ILC2s evaluated in BALs by flow cytometry. C, levels of IL-5 and IL-13 were assessed by using a multiplex cytokine quantification assay in culture supernatants of lung and spleen cells after in vitro stimulation with pro-rAmb a 11 (10 μg/ml). Background levels of cytokines produced by non-activated cells were subtracted. Results are expressed as mean values ± S.E. (error bars) with n = 6 mice per group. Statistical differences between rAmb a 11 and E64-inhibited rAmb a 11 were assessed using the non-parametric Mann-Whitney t test. *, p < 0.05; **, p < 0.01.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Arbes S. J. Jr., Gergen P. J., Elliott L., and Zeldin D. C. (2005) Prevalences of positive skin test responses to 10 common allergens in the US population: results from the third National Health and Nutrition Examination Survey. J. Allergy Clin. Immunol. 116, 377–383 - PubMed
    1. D'Amato G., Cecchi L., Bonini S., Nunes C., Annesi-Maesano I., Behrendt H., Liccardi G., Popov T., and van Cauwenberge P. (2007) Allergenic pollen and pollen allergy in Europe. Allergy 62, 976–990 - PubMed
    1. Burbach G. J., Heinzerling L. M., Röhnelt C., Bergmann K. C., Behrendt H., Zuberbier T., and GA(2)LEN study (2009) Ragweed sensitization in Europe—GA(2)LEN study suggests increasing prevalence. Allergy 64, 664–665 - PubMed
    1. Oswalt M. L., and Marshall G. D. (2008) Ragweed as an example of worldwide allergen expansion. Allergy Asthma Clin. Immunol. 4, 130–135 - PMC - PubMed
    1. Smith M., Cecchi L., Skjøth C. A., Karrer G., and Šikoparija B. (2013) Common ragweed: a threat to environmental health in Europe. Environ. Int. 61, 115–126 - PubMed

Publication types

MeSH terms

LinkOut - more resources