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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 15, 2002 as doi:10.1096/fj.02-0472fje. |
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Institute of Genetics and General Biology, University of Salzburg, A-5020 Salzburg, Austria;
* Institute of Pathophysiology, University of Vienna, A-1090 Vienna, Austria;
Institute of Chemistry, Agricultural University, A-1190 Vienna, Austria; and
Department of Immunopathology, Sanquin Research at CLB, 1066 CX Amsterdam, The Netherlands
2Correspondence: Institute of Genetics and General Biology, University of Salzburg, Hellbrunnerstr. 34, A-5020 Salzburg, Austria. E-mail: fatima.ferreira{at}sbg.ac.at
SPECIFIC AIMS
Mugwort is widely spread throughout the European temperate climate zone, North America, and parts of Asia and is one of the main causes of hay fever in late summer and autumn. Yet no information about the complete molecular structure of any mugwort pollen allergen has been reported so far. The primary aim of this study was to perform molecular and immunological characterization of Art v 1, the major allergen of mugwort pollen. Art v 1 is recognized by > 90% of mugwort pollen-sensitized patients. The cDNA clone coding for Art v 1 was isolated and used to produce the recombinant allergen in Escherichia coli. For a detailed characterization of Art v 1, the structural and immunological features of recombinant Art v 1 (rArt v 1) purified from E. coli were compared with those of the natural protein (nArt v 1) purified from mugwort pollen.
PRINCIPAL FINDINGS
1. Art v 1 is a secreted glycoprotein
Sequence analysis revealed that Art v 1 is a secreted protein consisting of two domains: 1) a cysteine-rich NH2 terminus with high sequence similarities to antifungal proteins belonging to the plant defensin family and 2) a carboxyl-terminal (amino acids 56 to 108) proline-rich domain containing several (Ser/Ala)(Pro)2–4 extensin-like repeats (Fig. 1
A). Structural analysis showed that nArt v 1 has two types of post-translational modifications: proline hydroxylation and O-glycosylation of hydroxyproline residues. In addition, our results demonstrated that the Art v 1 sugars represent a new type of plant O-glycan composed of 3 galactoses and 9–16 arabinoses linked to a hydroxyproline residue in the carboxyl-terminal proline-rich domain. Based on the homology of the amino-terminal region of Art v 1 to plant defensins, we generated 3-dimensional structural models of the Art v 1 defensin domain (Fig. 1B
) using the horse chestnut defensin (Ah-AMP1, 1bk8) as template. Plant defensins adopt a compact globular fold known as cysteine-stabilized alpha/beta motif. Using the ProFIT program to calculate fold recognition, the carboxyl-terminal part was predicted to adopt an extended left-helical structure similar to that of collagen. The complete 3D model of Art v 1 resembles a characteristic "head and tail" structure (Fig. 1D
). This extended type of structure predicted for the proline-rich domain is suited to accommodate large sugar chains and fits to our findings showing that Art v 1 contains O-glycans of 
3500–5200 Da attached to hydroxyproline residues.
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2. Post-translational modifications play a role in IgE recognition of Art v 1
IgE binding properties of natural and recombinant Art v 1 were evaluated in immunoblots, RAST, and ELISA inhibition experiments. Two subgroups of patients were identified in immunoblots and RAST (Fig. 2
A): one with similar IgE recognition of nArt v 1 and rArt v 1 and another with significantly lower or no reactivity to rArt v 1. In ELISA inhibition, rArt v 1 caused only a partial inhibition (30%) of IgE binding to nArt v 1 (Fig. 2B
). These results suggest the involvement of post-translational modifications, either directly or indirectly, in the formation of the IgE binding epitopes of Art v 1. No inhibition of binding to either natural (Fig. 2B
) or rArt v 1 (Fig. 2C
) was observed on preincubation of sera with reduced and alkylated rArt v 1, indicating that the defensin domain also plays a role in IgE recognition of the allergen.
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3. Post-translational modifications do not influence T cell recognition of Art v 1
T cell proliferation in response to natural and rArt v 1 was assessed in peripheral blood mononuclear cells (PBMC) and in allergen-specific T cell lines (TCL) derived from mugwort pollen-allergic patients. In PBMC, comparable primary in vitro responses were induced with natural and rArt v 1 (Fig. 2D
). Allergen-specific TCL were established with natural or rArt v 1 as initial stimulus. Both antigens induced comparable proliferative responses in the TCL obtained with natural or rArt v 1 (Fig. 2E
).
CONCLUSIONS AND SIGNIFICANCE
It is widely accepted that recombinant allergens are promising tools for diagnosis and therapy of type I allergy. As a first step to establish a recombinant-based diagnostic and therapy approach for weed pollen allergy (Fig. 3
), we present here a detailed characterization of Art v 1, the major allergen of mugwort pollen. Our results clearly demonstrate even though rArt v 1 produced in E. coli lacks all post-translational modifications shown to be present in nArt v 1, both protein preparations share common epitopes recognized by human IgE and T lymphocytes. In principle, rArt v 1 produced in E. coli cannot be considered a suitable reagent for diagnosis of weed pollen allergies because only 30–50% of Art v 1-allergic patients recognize this molecule. Plant-based expression systems might be the choice for production of glycosylated Art v 1 for diagnostic purposes (Fig. 3)
. However, its IgE and T cell activation properties make nonglycosylated rArt v 1 a good starting template for engineering of hypoallergenic forms to be used in safer forms of specific immunotherapy (SIT) (Fig. 3)
. Genetically engineered allergens with reduced IgE binding epitopes (hypoallergens) and preserved structural motifs necessary for T cell recognition (T cell epitopes) combine the advantage of low risk of IgE-mediated side effects with the possibility of administration of the allergen at higher doses, which increases the efficacy of SIT. Based on the results presented here, hypoallergenic forms of Art v 1 for SIT could be engineered by targeting the disulfide bonds that stabilize the defensin domain and by production of the nonglycosylated mutant protein in E. coli (Fig. 3)
.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0472fje; to cite this article, use FASEB J. (November 15, 2002) 10.1096/fj.02-0472fje 
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  B. Jahn-Schmid, P. Sirven, V. Leb, W. F. Pickl, G. F. Fischer, G. Gadermaier, M. Egger, C. Ebner, F. Ferreira, B. Maillere, et al. Characterization of HLA Class II/Peptide-TCR Interactions of the Immunodominant T Cell Epitope in Art v 1, the Major Mugwort Pollen Allergen J. Immunol., September 1, 2008; 181(5): 3636 - 3642. [Abstract] [Full Text] [PDF]
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-helix, respectively. B) Calculated model of the defensin domain of Art v 1 using the Ah-AMP1 (PDB 1bk8) defensin as template. Two views representing a rotation of 180° around the vertical (Z) axis showing the ribbon representation of secondary structure elements with ß-strands in yellow and the 
