Structure of the functional domain of the major grass-pollen allergen Phlp 5b

Rajashankar, Kanagalaghatta R.; Bufe, Albrecht; Weber, Wolfgang; Eschenburg, Susanne; Lindner, Buko; Betzel, Christian

doi:10.1107/s0907444902007254

Cited by 30 publications

(24 citation statements)

References 29 publications

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“…This is unexpected as the homologous Der p 5 protein is predicted to have coiled-coil helical structure (10). Helical bundle structures have also been observed in major timothy grass pollen allergens Phl p 5b (35) and Phl p 6 (36). These allergens, however, form a right-handed four-helical up-and-down bundle topology instead of the three-helical bundle structure in Blo t 5.…”

Section: Discussionmentioning

confidence: 99%

“…These allergens, however, form a right-handed four-helical up-and-down bundle topology instead of the three-helical bundle structure in Blo t 5. While Blo t 5 is monomeric in solution, Phl p 5b is reported to form a dimer stabilized by one intermolecular disulfide bridge (35). Attempt to predict the biological function of Blo t 5 through the search of homologous structures using the DALI server (37) has also failed.…”

Section: Discussionmentioning

confidence: 99%

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Nuclear Magnetic Resonance Structure and IgE Epitopes of Blo t 5, a Major Dust Mite Allergen

Chan¹,

Ong²,

Gao³

et al. 2008

The Journal of Immunology

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A high incidence of sensitization to Blomia tropicalis, the predominant house dust mite species in tropical regions, is strongly associated with allergic diseases in Singapore, Malaysia, and Brazil. IgE binding to the group 5 allergen, Blo t 5, is found to be the most prevalent among all B. tropicalis allergens. The NMR structure of Blo t 5 determined represents a novel helical bundle structure consisting of three antiparallel α-helices. Based on the structure and sequence alignment with other known group 5 dust mite allergens, surface-exposed charged residues have been identified for site-directed mutagenesis and IgE binding assays. Four charged residues, Glu76, Asp81, Glu86, and Glu91 at around the turn region connecting helices α2 and α3 have been identified to be involved in the IgE binding. Using overlapping peptides, we have confirmed that these charged residues are located on a major putative linear IgE epitope of Blo t 5 from residues 76–91 comprising the sequence ELKRTDLNILERFNYE. Triple and quadruple mutants have been generated and found to exhibit significantly lower IgE binding and reduced responses in skin prick tests. The mutants induced similar PBMC proliferation as the wild-type protein but with reduced Th2:Th1 cytokines ratio. Mass screening on a quadruple mutant showed a 40% reduction in IgE binding in 35 of 42 sera of atopic individuals. Findings in this study further stressed the importance of surface-charged residues on IgE binding and have implications in the cross-reactivity and use of Blo t 5 mutants as a hypoallergen for immunotherapy.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Nuclear Magnetic Resonance Structure and IgE Epitopes of Blo t 5, a Major Dust Mite Allergen

Chan¹,

Ong²,

Gao³

et al. 2008

The Journal of Immunology

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“…These lysines were exchanged for alanines in a mutant allergen. Interpretation of the crystal structure published for the wildtype allergen [19] indicated that 2 out of 12 lysines were relevant for the stability of the molecule by forming intramolecular salt bridges and allowed to predict that substitution of the 10 external lysines by alanines should not affect the conformation of the molecule. This notion was confirmed by the finding that in CD spectroscopy, no differences in the 2-dimensional structures of wildtype and mutant allergen could be detected.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, this C-terminal part of the molecule exhibits allergenic and biologic activity comparable with the holoprotein [18]. When analyzing the surface of the 3-dimensional structure of Phl 5b C terminus, our model allergen [19], it was striking that 10 out of the 12 lysines were located on the surface of the allergen and only 2 were found in the inner core of the molecule and were involved in the formation of stabilizing salt bridges.…”

Section: Introductionmentioning

confidence: 99%

Lysine as a Critical Amino Acid for IgE Binding in Phl p 5b C Terminus

Gehlhar

Rajashankar²,

Hofmann³

et al. 2006

Int Arch Allergy Immunol

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Background: Allergens induce the formation of specific immunoglobulin (Ig)E and harbor at least two IgE-binding regions (epitopes) to facilitate crosslinking of basophilic or mast-cell-bound specific IgE antibodies. Studies mapping linear epitopes have shown that these regions often contain charged or hydrophobic amino acids. Nevertheless, these studies are hampered by limited significance due to the often conformational nature of IgE epitopes. This prompted us to study the role of lysines in the context of an intact 3-dimensional model. Methods: Major allergen Phl p 5b from timothy grass bears 12 lysines in its C-terminal half. Using site-directed mutagenesis, we substituted all 10 surface-exposed lysines by alanines. Results: Although structural integrity of the lysine-deficient mutant was not altered, IgE-binding capacity measured by ELISA inhibition tests and crosslinking activity in ex vivo basophil stimulation and in vivo skin prick tests were significantly diminished. Interestingly, binding of specific IgG antibodies was considerably less reduced by loss of lysines. Conclusion: Lysine is an important amino acid for IgE binding in more than one epitope of major grass pollen allergen Phl p 5b C terminus. Allergenicity, but not IgG binding of the molecule, is substantially diminished by single amino acid substitutions without structural integrity being hampered.

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“…The occurrence of Bet v 1-dimers in pollen extracts as well as in recombinant allergen preparations has previously been observed in SDS-PAGE gels and Western blots (9 -13). Furthermore, many important allergens are known to appear as homodimers or -oligomers in nature, e.g., Phl p 1 (14), Phl p 5b (15), Phl p 7 (16), and Phl p 11 (17) from grass pollen; Fel d 1 from cat dander (18); parvalbumin from cod (19); the panallergen tropomyosin (20,21); Api m 4 from bee venom (22); Equ c 1 from horse (23,24); Sol i II from fire ant (25); Ara h 1 (26,27) and Ara h 2 (28) from peanut; ABA-1 from Ascaris (helminth) (29,30); bovine ␤-lactoglobulin (31-34) and bovine dander allergen (35); BGP-2 from Bermuda grass pollen (36); or Ves v 5 from wasp venom (37).…”

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confidence: 99%

Dimerization of the Major Birch Pollen Allergen Bet v 1 Is Important for its In Vivo IgE-Cross-Linking Potential in Mice

Schöll¹,

Kalkura

Shedziankova

et al. 2005

The Journal of Immunology

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In type I allergy, the cross-linking of membrane IgE on B lymphocytes and of cytophilic IgE on effector cells by their respective allergens are key events. For cross-linking two IgE molecules, allergens need at least two epitopes. On large molecules, these could be different epitopes in a multivalent, or identical epitopes in a symmetrical, fashion. However, the availability of epitopes may be limited on small allergens such as Bet v 1, the major birch pollen allergen. The present work analyzes whether dimerization is required for the cross-linking capacity of this allergen. In immunoblots, murine monoclonal and polyclonal human Bet v 1-specific Abs detected, besides a Bet v 1 monomer of 17 kDa, a dimer of 34 kDa. In dynamic light scattering, Bet v 1 appeared as dimers and even multimers, but a single condition could be defined where it behaved exclusively monomerically. Small-angle x-ray scattering of the monomeric and dimeric samples resulted in diagrams agreeing with the calculated models. Circular dichroism measurements indicated that the structure of Bet v 1 was preserved under monomeric conditions. Skin tests in Bet v 1-allergic mice were positive with Bet v 1 dimer, but remained negative using the monomer. Furthermore, in contrast to dimeric Bet v 1, the monomer was less capable of activating murine memory B cells for IgE production in vivo. Our data indicate that the presentation of two identical epitopes by dimerized allergens is a precondition for cross-linking of IgE on mast cells and B lymphocytes.

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Structure of the functional domain of the major grass-pollen allergen Phlp 5b

Cited by 30 publications

References 29 publications

Nuclear Magnetic Resonance Structure and IgE Epitopes of Blo t 5, a Major Dust Mite Allergen

Nuclear Magnetic Resonance Structure and IgE Epitopes of Blo t 5, a Major Dust Mite Allergen

Lysine as a Critical Amino Acid for IgE Binding in Phl p 5b C Terminus

Dimerization of the Major Birch Pollen Allergen Bet v 1 Is Important for its In Vivo IgE-Cross-Linking Potential in Mice

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