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      Pectate Lyase Pollen Allergens: Sensitization Profiles and Cross-Reactivity Pattern

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          Abstract

          Background

          Pollen released by allergenic members of the botanically unrelated families of Asteraceae and Cupressaceae represent potent elicitors of respiratory allergies in regions where these plants are present. As main allergen sources the Asteraceae species ragweed and mugwort, as well as the Cupressaceae species, cypress, mountain cedar, and Japanese cedar have been identified. The major allergens of all species belong to the pectate lyase enzyme family. Thus, we thought to investigate cross-reactivity pattern as well as sensitization capacities of pectate lyase pollen allergens in cohorts from distinct geographic regions.

          Methods

          The clinically relevant pectate lyase pollen allergens Amb a 1, Art v 6, Cup a 1, Jun a 1, and Cry j 1 were purified from aqueous pollen extracts, and patients´ sensitization pattern of cohorts from Austria, Canada, Italy, and Japan were determined by IgE ELISA and cross-inhibition experiments. Moreover, we performed microarray experiments and established a mouse model of sensitization.

          Results

          In ELISA and ELISA inhibition experiments specific sensitization pattern were discovered for each geographic region, which reflected the natural allergen exposure of the patients. We found significant cross-reactivity within Asteraceae and Cupressaceae pectate lyase pollen allergens, which was however limited between the orders. Animal experiments showed that immunization with Asteraceae allergens mainly induced antibodies reactive within the order, the same was observed for the Cupressaceae allergens. Cross-reactivity between orders was minimal. Moreover, Amb a 1, Art v 6, and Cry j 1 showed in general higher immunogenicity.

          Conclusion

          We could cluster pectate lyase allergens in four categories, Amb a 1, Art v 6, Cup a 1/Jun a 1, and Cry j 1, respectively, at which each category has the potential to sensitize predisposed individuals. The sensitization pattern of different cohorts correlated with pollen exposure, which should be considered for future allergy diagnosis and therapy.

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          Most cited references24

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          The spectrum of allergens in ragweed and mugwort pollen.

          Ragweed and mugwort are important allergenic weeds belonging to the Asteraceae or Compositae plant family. Pollen of mugwort is one of the main causes of allergic reactions in late summer and autumn in Europe and affects about 10-14% of the patients suffering from pollinosis. Ragweed pollen represents the major source of allergenic protein in the United States, with a prevalence of about 50% in atopic individuals. In Europe, ragweed allergy is now rapidly increasing particularly in certain areas in France, Italy, Austria, Hungary, Croatia, and Bulgaria. Amb a 1 and Art v 1, the major allergens of ragweed and mugwort, respectively, are unrelated proteins. Amb a 1 is an acidic 38-kDa nonglycosylated protein. The natural protein undergoes proteolysis during purification and is cleaved into a 26-kDa alpha chain, which associates noncovalently with the beta chain of 12 kDa. The two-chain form seems to be immunologically indistinguishable from the full-length molecule. Art v 1 is a basic glycoprotein comprising two domains: an N-terminal cysteine-rich, defensin-like domain and a C-terminal proline/hydroxyproline-rich module. The proline/hydroxyproline-rich domain was recently shown to contain two types of glycosylation: (1) a large hydroxyproline-linked arabinogalactan composed of a short beta1,6-galactan core substituted by a variable number (5-28) of alpha-arabinofuranose residues forming branched side chains with 5-, 2,5-, 3,5-, and 2,3,5-substituted arabinoses, and (2) single and adjacent beta-arabinofuranoses linked to hydroxyproline. As described for other pollen, ragweed and mugwort pollen also contain the pan-allergen profilin and calcium-binding proteins, which are responsible for extensive cross-reactivity among pollen-sensitized patients. Copyright (c) 2005 S. Karger AG, Basel.
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            Development of a functional in vitro assay as a novel tool for the standardization of allergen extracts in the human system.

            Biochemical and immunochemical methods used for batch control of allergen extracts rely on the binding of IgE molecules to allergens. They do not measure the ability of a protein to induce type I allergic reactions. Therefore, a biological assay was established that is based on the cellular mechanisms of allergies in order to assess the cross-linking capacity of allergens. Rat basophilic leukaemia cells were transfected with cDNA coding for the human high affinity IgE receptor chains. The surface expression of the IgE-binding alpha-chain was detected by FACS analysis and the functional integration of the 'humanized' receptors into the signal transduction cascade was addressed by intracellular calcium mobilization. Mediator release was measured in response to human IgE and a variety of cross-linking allergen preparations. Several clones were obtained that were able to bind allergen-specific human IgE. The results of the biological assay were compared with those obtained by immunochemical methods. The biological assay was used to determine the potency of allergen extracts, including highly diluted products that cannot be analysed by conventional methods. A stable 'humanized' basophil cell line was established that will be a useful tool for the standardization and batch control of allergen extracts. Because of its high sensitivity, it can also be used to detect minute quantities of potentially allergenic proteins, e.g. in processed foods. In addition, the test may support the development of novel allergy vaccines, such as recombinant hypoallergenic molecules.
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              Art v 1, the major allergen of mugwort pollen, is a modular glycoprotein with a defensin-like and a hydroxyproline-rich domain.

              In late summer, pollen grains originating from Compositae weeds (e.g., mugwort, ragweed) are a major source of allergens worldwide. Here, we report the isolation of a cDNA clone coding for Art v 1, the major allergen of mugwort pollen. Sequence analysis showed that Art v 1 is a secreted allergen with an N-terminal cysteine-rich domain homologous to plant defensins and a C-terminal proline-rich region containing several (Ser/Ala)(Pro)2-4 repeats. Structural analysis showed that some of the proline residues in the C-terminal domain of Art v 1 are posttranslationally modified by hydroxylation and O-glycosylation. The O-glycans are composed of 3 galactoses and 9-16 arabinoses linked to a hydroxyproline and represent a new type of plant O-glycan. A 3-D structural model of Art v 1 was generated showing a characteristic "head and tail" structure. Evaluation of the antibody binding properties of natural and recombinant Art v 1 produced in Escherichia coli revealed the involvement of the defensin fold and posttranslational modifications in the formation of epitopes recognized by IgE antibodies from allergic patients. However, posttranslational modifications did not influence T-cell recognition. Thus, recombinant nonglycosylated Art v 1 is a good starting template for engineering hypoallergenic vaccines for weed-pollen therapy.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                15 May 2015
                2015
                : 10
                : 5
                : e0120038
                Affiliations
                [1 ]Christian Doppler Laboratory for Allergy Diagnosis and Therapy, Department of Molecular Biology, University of Salzburg, Salzburg, Austria
                [2 ]Associated Centers for Molecular Allergology, Rome, Italy
                [3 ]Centro di Allergologia Molecolare, IDI-IRCCS, Rome, Italy
                [4 ]Department of Molecular Biology, University of Salzburg, Salzburg, Austria
                [5 ]Allergieambulatorium am Reumanplatz, Vienna, Austria
                [6 ]Department of Otolaryngology, Kyorin University, School of Medicine, Tokyo, Japan
                [7 ]Atopy (Allergy) Research Center, Juntendo University, Graduate School of Medicine, Tokyo, Japan
                [8 ]Stallergenes S.A., Antony, France
                [9 ]ZAUM, Center for Allergy and Environment, Munich, Germany
                Mie University Graduate School of Medicine, JAPAN
                Author notes

                Competing Interests: MH, GG, MW, and FF are inventors of the patent “Polysaccharide Lyases”, EP2729568 (A1). AD is an employee of Stallergenes SA. FF received funding from Biomay AG. This does not alter the adherence to PLOS ONE policies on sharing data and materials

                Conceived and designed the experiments: AM HB M. Wallner FF. Performed the experiments: UP MH M. Wolf MLB GG RW PB M. Wallner. Analyzed the data: MH M. Wallner AM PB UP RW GG M. Wolf. Contributed reagents/materials/analysis tools: AM MLB CR CE HY TT AD. Wrote the paper: UP M. Wolf M. Wallner FF.

                Article
                PONE-D-14-42633
                10.1371/journal.pone.0120038
                4433284
                25978036
                6242b601-47e0-49f2-a565-2f27f734d678
                Copyright @ 2015

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 14 October 2014
                : 18 January 2015
                Page count
                Figures: 8, Tables: 0, Pages: 19
                Funding
                This work was supported by CK-CARE AG Individual Project 2009-02, Davos, Switzerland; Biomay AG, Vienna Austria; Christian Doppler Research Association, Vienna, Austria. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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