Isoforms of Bet v 1, the Major Birch Pollen Allergen, Analyzed by Liquid Chromatography, Mass Spectrometry, and cDNA Cloning

Swoboda, Ines; Jilek, Alexander; Ferreira, Fátima; Engel, Edwin; Hoffmann‐Sommergruber, Karin; Scheiner, Otto; Kraft, Dietrich; Breiteneder, Heimo; Pittenauer, Ernst; Schmid, E.; Vicente, Óscar; Heberle‐Bors, Erwin; Ahorn, Horst; Breitenbach, Michael

doi:10.1074/jbc.270.6.2607

Cited by 191 publications

(162 citation statements)

References 30 publications

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“…2B). Only few PR-10 proteins, such as AmPR-10 from the Chinese medicinal plant Astragalus mongholicus (Yan et al, 2008), Bet v 1 from birch (Swoboda et al, 1995;Koistinen et al, 2002), and SPE-16 from the Mexican potato Pachyrrizus erosus (Wu et al, 2003), have been found to form dimers in solution. However, the functional importance of dimerization in PR-10 proteins has not been clarified.…”

Section: Discussionmentioning

confidence: 99%

The RNA Hydrolysis and the Cytokinin Binding Activities of PR-10 Proteins Are Differently Performed by Two Isoforms of the Pru p 1 Peach Major Allergen and Are Possibly Functionally Related

et al. 2009

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PR-10 proteins are a family of pathogenesis-related (PR) allergenic proteins playing multifunctional roles. The peach (Prunus persica) major allergen, Pru p 1.01, and its isoform, Pru p 1.06D, were found highly expressed in the fruit skin at the pit hardening stage, when fruits transiently lose their susceptibility to the fungal pathogen Monilinia spp. To investigate the possible role of the two Pru p 1 isoforms in plant defense, the recombinant proteins were expressed in Escherichia coli and purified. Light scattering experiments and circular dichroism spectroscopy showed that both proteins are monomers in solution with secondary structures typical of PR-10 proteins. Even though the proteins do not display direct antimicrobial activity, they both act as RNases, a function possibly related to defense. The RNase activity is different for the two proteins, and only that of Pru p 1.01 is affected in the presence of the cytokinin zeatin, suggesting a physiological correlation between Pru p 1.01 ligand binding and enzymatic activity. The binding of zeatin to Pru p 1.01 was evaluated using isothermal titration calorimetry, which provided information on the stoichiometry and on the thermodynamic parameters of the interaction. The structural architecture of Pru p 1.01 and Pru p 1.06D was obtained by homology modeling, and the differences in the binding pockets, possibly accounting for the observed difference in binding activity, were evaluated.

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Section: Discussionmentioning

confidence: 99%

The RNA Hydrolysis and the Cytokinin Binding Activities of PR-10 Proteins Are Differently Performed by Two Isoforms of the Pru p 1 Peach Major Allergen and Are Possibly Functionally Related

et al. 2009

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“…Natural pollen extracts represent allergen mixtures that also contain isoallergenic variants of certain allergens (43). Therefore, we were interested to investigate whether rabbit anti-rBet v 1 fragment antisera can also inhibit IgE binding of allergic patients to birch pollen extract and to pollen extracts from botanically related trees (alder, hornbeam, and oak) containing Bet v 1-homologous allergens.…”

Section: Rbet V 1 Fragment-induced Rabbit Abs Inhibit Ige Binding Of mentioning

confidence: 99%

T Cell Epitope-Containing Hypoallergenic Recombinant Fragments of the Major Birch Pollen Allergen, Bet v 1, Induce Blocking Antibodies

Vrtala

Akdiş²,

Budak³

et al. 2000

The Journal of Immunology

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Allergen-specific immunotherapy represents one of the few curative approaches toward type I allergy. Up to 25% of allergic patients are sensitized against the major birch pollen allergen, Bet v 1. By genetic engineering we produced two recombinant (r) Bet v 1 fragments comprising aa 1–74 and aa 75–160 of Bet v 1, which, due to a loss of their native-like fold, failed to bind IgE Abs and had reduced allergenic activity. Here we show that both fragments covering the full Bet v 1 sequence induced human lymphoproliferative responses similar to rBet v 1 wild type. The C-terminal rBet v 1 fragment induced higher lymphoproliferative responses than the N-terminal fragment and represented a Th1-stimulating segment with high IFN-γ production, whereas the N-terminal fragment induced higher IL-4, IL-5, and IL-13 secretion. Immunization of mice and rabbits with rBet v 1 fragments induced IgG Abs, which cross-reacted with complete Bet v 1 and Bet v 1-related plant allergens and strongly inhibited the IgE binding of allergic patients to these allergens. Thus, our results demonstrate that hypoallergenic T cell epitope-containing rBet v 1 fragments, despite lacking IgE epitopes, can induce Abs in vivo that prevent the IgE binding of allergic patients to the wild-type allergen. The overall demonstration of the immunogenic features of the hypoallergenic rBet v 1 fragments will now enable clinical studies for safer and more efficient specific immunotherapy.

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“…Review of the various cloning attempts shows that IgE immunoscreening of expression cDNA libraries prepared from the allergen source yielded recombinant allergens which most closely mimicked their natural counterparts in terms of antibody-binding capacity and ability lo stimulate specific T cells as well as to elicit allergic effector mechanisms (6,7). DNA-based techniques, such as polymerase chain reaction amplification, frequently lead to the isolation of cDNAs coding for allergen isovariants with reduced or eliminated IgE-binding capacity and diminished biologic activity (8)(9)(10). While the first approach, i.e., IgE immunoscreening, delivered recombinant allergens that are extremely useful for antibody-based in vitro, as well as in vivo, diagnosis, the DNA-based approach allowed the identification of allergen variants with greatly reduced IgEbinding capacity and anaphylactic activity.…”

Section: Strategies For Obtaining Recombinant Allergensmentioning

confidence: 99%

Recombinant allergens

Valenta

Vrtala

Laffer

et al. 1998

Allergy

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A great variety of recombinant plant, mite, mold, mammal, and insect allergens have been expressed in heterologous hosts (e.g., Escherichia coli), their cDNA being used as a template. The number of biologically active recombinant allergens available for experimental, diagnostic, and therapeutic purposes is increasing tremendously. Recombinant allergens have proven to be valuable tools to investigate T‐cell and B‐cell recognition of allergens as well as to study mechanisms of specific IgE regulation. The immunologic equivalence of many relevant recombinant allergens with their natural counterparts has been demonstrated, and the three‐dimensional structures of several recombinant allergens have been described recently. As a result of extensive cross‐reactivities among the relevant allergens, it appears that the number of epitopes needed for diagnosis and specific immunotherapy is less diverse than originally anticipated and might be soon covered by recombinant molecules. Recombinant allergens have been used for successful in vitro, as well as in vivo, allergy diagnosis, and work is in progress to produce recombinant allergen derivatives with reduced anaphylactic potential to improve current forms of immunotherapy.

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Isoforms of Bet v 1, the Major Birch Pollen Allergen, Analyzed by Liquid Chromatography, Mass Spectrometry, and cDNA Cloning

Cited by 191 publications

References 30 publications

The RNA Hydrolysis and the Cytokinin Binding Activities of PR-10 Proteins Are Differently Performed by Two Isoforms of the Pru p 1 Peach Major Allergen and Are Possibly Functionally Related

The RNA Hydrolysis and the Cytokinin Binding Activities of PR-10 Proteins Are Differently Performed by Two Isoforms of the Pru p 1 Peach Major Allergen and Are Possibly Functionally Related

T Cell Epitope-Containing Hypoallergenic Recombinant Fragments of the Major Birch Pollen Allergen, Bet v 1, Induce Blocking Antibodies

Recombinant allergens

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