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Crystal structure of Pla l 1 reveals both structural similarity and allergenic divergence within the Ole e 1–like protein family

2016; Elsevier BV; Volume: 140; Issue: 1 Linguagem: Inglês

10.1016/j.jaci.2016.10.035

1097-6825

Teresa Stemeseder, Regina Freier, Sabrina Wildner, Julian E. Fuchs, Peter Briza, Roland Lang, Eva Batanero, Jonas Lidholm, Klaus R. Liedl, Paloma Campo, Thomas Hawranek, Mayte Villalba, Hans Brandstetter, Fátima Ferreira, Gabriele Gadermaier,

Plant Reproductive Biology

Knowledge of structural and immunological features of allergens is important for understanding IgE sensitization and disease eliciting mechanisms of allergenic molecules. Despite the fact that Ole e 1–like proteins are driving type I allergies within several pollen sources,1Villalba M. Rodriguez R. Batanero E. The spectrum of olive pollen allergens: from structures to diagnosis and treatment.Methods. 2014; 66: 44-54Crossref PubMed Scopus (56) Google Scholar, 2Gadermaier G. Eichhorn S. Vejvar E. Weilnbock L. Lang R. Briza P. et al.Plantago lanceolata: an important trigger of summer pollinosis with limited IgE cross-reactivity.J Allergy Clin Immunol. 2014; 134: 472-475Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar there is currently no information regarding their tertiary structure. Ole e 1–like proteins are characterized by 3 conserved disulfide bonds and the [EQT]-G-X-V-Y-C-D-[TNP]-C-R consensus pattern while their biological function is unknown. To date, 14 allergenic members of this protein family, all originating from pollen, have been reported (www.allergen.org). They exhibit varying degrees of sequence identity, typically high among Oleaceae species (>82%) but medium/low between botanically distant plants (25% to 60%). This prompted us to solve the crystal structure of Pla l 1,3Calabozo B. Barber D. Polo F. Purification and characterization of the main allergen of Plantago lanceolata pollen, Pla l 1.Clin Exp Allergy. 2001; 31: 322-330Crossref PubMed Scopus (37) Google Scholar an Ole e 1–like allergen of English plantain pollen and compare its immunological properties to homologous allergens from pollen of olive, ash, grass, chenopod, and Russian thistle. Recombinant Pla l 1.0101 was expressed as soluble nontagged protein in Escherichia coli Rosetta-gami B(DE3)pLysS to enable disulfide bond formation, and the molecule was purified by cation exchange and size exclusion chromatography. We were able to obtain a nonglycosylated homogenous batch (Fig 1, A and B) with correct molecular mass and established disulfide bonds. Pla l is thermostable (Tm = 72°C) and able to partially refold on heating (Fig 1, C). Fourier transform infrared spectroscopy predicted 40% β-strands and no α-helix (Fig 1, D). Detailed materials and methods describing physicochemical, structural, and immunological characterization can be found online. Within this work, the first structure of an Ole e 1–like protein was solved by crystallography, revealing a 7-stranded β-barrel with 4 pronounced loop regions. The proposed glycosylation site of Asn107 is surface located within loop 4. Intramolecular disulfide bonds were found between β1b and β6 strands, as well as between β2 and β5, and the C-terminus and loop C-terminal of β2, thus forming a closed branched loop (Fig 1, E). One extended loop forms a small cavity with β5 and β7b of approximately 300 Å,3Calabozo B. Barber D. Polo F. Purification and characterization of the main allergen of Plantago lanceolata pollen, Pla l 1.Clin Exp Allergy. 2001; 31: 322-330Crossref PubMed Scopus (37) Google Scholar flanked by hydrophobic residues in the protein core and polar residues pointing outward, potentially serving as a ligand binding site. In the crystal structure, His21 and Glu88 were coordinating 1 zinc ion together with Asp45 and Asp73 of the neighboring monomer. Thus, extended long chains in the presence of zinc might be possible, though our preparation is found to be monomeric in dynamic light scattering. Molecular dynamics simulations allowed identification of 5 regions with high degrees of flexibility (see Fig E1 in this article's Online Repository at www.jacionline.org). Coordinates and structure factors were deposited at Protein Data Bank with ID code 4Z8W. Further details on the structure and molecular dynamics simulation are provided in Figs E2-E4 and Video E1 (see this article's Online Repository at www.jacionline.org). A homology model of Ole e 1 was pursued using 3 different modelling approaches (see Fig E5 in this article's Online Repository at www.jacionline.org). Ole e 1 and homologs share the same β-barrel fold, stabilized by 3 disulfide bonds. There are, however, differences, especially in the long connection between β5 and β7, as some homologs lack residues in this region (Fig 2, A and B). Surface models also showed considerable differences in hydrophobicity between distantly related members especially in those flexible loop regions (see Fig E6 in this article's Online Repository at www.jacionline.org). A Kunitz-type trypsin inhibitor function could not be confirmed due to structural dissimilarity and was also experimentally ruled out as Pla l 1 was completely digested by trypsin within 0.5 hours. Despite a low primary sequence identity of 14.9%, highest structural similarity was obtained for a cell wall surface anchor protein (3PHS) of Streptococcus agalactiae (Fig 2, C). These pilin structures encompass an IgG-like fold and can adhere to host tissues and modulate immune responses. Pla l 1 and Ole e 1 were previously found to be localized in mature pollen tissue and frequently associated with the endoplasmic reticulum.4Castro A.J. Alche J.D. Calabozo B. Rodriguez-Garcia M.I. Polo F. Pla 1 1 and Ole e 1 pollen allergens share common epitopes and similar ultrastructural localization.J Investig Allergol Clin Immunol. 2007; 17: 41-47PubMed Google Scholar To investigate their immunological properties, purified Pla l 1, Ole e 1, Fra e 1, Phl p 11, Che a 1, and Sal k 5 (see Table E1 in this article's Online Repository at www.jacionline.org) were tested with sera of 27 Austrian Pla l 1–sensitized and 19 Spanish Ole e 1–sensitized patients. All patients presented with rhinitis or rhinoconjunctivitis to plantain or olive pollen; demographic and further clinical details are found in Table E2 (see this article's Online Repository at www.jacionline.org). In ELISA, the samples from Austrian patients sensitized to Pla l 1 also demonstrated IgE reactivity to Ole e 1 (44.4%), Fra e 1 (40.7%), Phl p 11 (37.0%), Che a 1 (14.8%), and Sal k 5 (7.4%) (Fig 2, D). Preincubation of sera with any of the Ole e 1–like homologs did not result in substantial IgE inhibition to Pla l 1, with mean inhibition values of 4.3% to 6.0% (Fig 2, E). This suggests that Pla l 1–mediated plantain allergy represents an independent allergy that is not a result of cross-reactivity with ash or grass, even though cosensitization to these sources is frequently observed. The patients studied here were mostly multisensitized to pollen, mite, and cat allergens, which is in line with previous findings that monosensitization to plantain is rare.2Gadermaier G. Eichhorn S. Vejvar E. Weilnbock L. Lang R. Briza P. et al.Plantago lanceolata: an important trigger of summer pollinosis with limited IgE cross-reactivity.J Allergy Clin Immunol. 2014; 134: 472-475Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar On a speculative basis, plantain allergy might preferentially manifest in patients with an already established "allergic background." All Spanish patients reacted to Ole e 1 as well as to the highly homologous Fra e 1, showing correlating values in ELISA (r = 0.951) and substantial IgE cross-reactivity, as expected.5Imhof K. Probst E. Seifert B. Regenass S. Schmid-Grendelmeier P. Ash pollen allergy: reliable detection of sensitization on the basis of IgE to Ole e 1.Allergo J Int. 2014; 23: 78-83Crossref PubMed Scopus (15) Google Scholar In this cohort, Spanish patients who are allergic to olive pollen did not react to Pla l 1, and sensitization frequencies to Phl p 11, Che a 1, and Sal k 5 were 26.3%, 26.3%, and 21.1%, respectively (Fig 2, F). Sal k 5 showed low IgE cross-reactivity with Ole e 1, with a mean inhibition value of 13.2%. Little or no IgE cross-reactivity was observed between Ole e 1 and the homologous allergens from plantain, grass, and chenopod (Fig 2, G). In comparison to the Austrian cohort, Spanish patients showed a less diverse IgE profile and preferentially reacted to other olive allergens as well as grass pollen. In both cohorts, Ole e 1 and Pla l 1 showed no IgE cross-reactivity with each other, suggesting functionally different epitopes. Especially the fact that Pla l 1 lacks the C-terminal region previously shown to be an important epitope of Ole e 1 may explain this finding.6Gonzalez E.M. Villalba M. Quiralte J. Batanero E. Roncal F. Albar J.P. et al.Analysis of IgE and IgG B-cell immunodominant regions of Ole e 1, the main allergen from olive pollen.Mol Immunol. 2006; 43: 570-578Crossref PubMed Scopus (27) Google Scholar However, also epitopes in other regions may be highly antibody specific because a homolog from birch with 29% sequence identity to Ole e 1 entirely lacks allergenic activity.7Marazuela E.G. Hajek R. Villalba M. Barber D. Breiteneder H. Rodriguez R. et al.A non-allergenic Ole e 1–like protein from birch pollen as a tool to design hypoallergenic vaccine candidates.Mol Immunol. 2012; 50: 83-90Crossref PubMed Scopus (14) Google Scholar Structural similarity is a prerequisite for cross-reactivity of discontinuous B-cell epitopes. However, allergens of the Ole e 1–like protein family are apparently not ubiquitously cross-reactive except for closely related members.4Castro A.J. Alche J.D. Calabozo B. Rodriguez-Garcia M.I. Polo F. Pla 1 1 and Ole e 1 pollen allergens share common epitopes and similar ultrastructural localization.J Investig Allergol Clin Immunol. 2007; 17: 41-47PubMed Google Scholar, 8Castro L. Mas S. Barderas R. Colas C. Garcia-Selles J. Barber D. et al.Sal k 5, a member of the widespread Ole e 1–like protein family, is a new allergen of Russian thistle (Salsola kali) pollen.Int Arch Allergy Immunol. 2014; 163: 142-153Crossref PubMed Scopus (14) Google Scholar, 9Barderas R. Purohit A. Papanikolaou I. Rodriguez R. Pauli G. Villalba M. Cloning, expression, and clinical significance of the major allergen from ash pollen, Fra e 1.J Allergy Clin Immunol. 2005; 115: 351-357Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Notably, the highest level of sequence divergence and gaps could now be localized in loop regions, whereas residues involved in β-strands of the core structure were more conserved. Therefore, we suggest that Ole e 1–like loop regions could constitute antibody binding sites that are highly specific for each allergen. In silico predictions frequently determine loops as candidates, and Pla l 1 residues 45 to 47 and 105 to 113 are suggested as discontinuous epitopes using software predictions. Both epitopes are located in loop regions with low sequence identity to other homologs. In summary, the 3-dimensional structure of Pla l 1 was solved by crystallography, thus unravelling the Ole e 1–like protein fold. Detailed characterization led to the conclusion that family members share the same core structure, whereas loop regions can be heterogeneous. Thus, limited antibody cross-reactivity between distantly related members is explained, which is useful for highly specific diagnosis toward the pollen allergen source. https://www.jacionline.org/cms/asset/cdde3474-fc09-4106-ae02-95c4ce8783fe/mmc1.mp4Loading ... Download .mp4 (14.31 MB) Help with .mp4 files Video E1 Download .docx (.07 MB) Help with docx files Online Repository text Download .pdf (.13 MB) Help with pdf files Table E1 Download .pdf (1.02 MB) Help with pdf files Table E2 Download .pdf (.31 MB) Help with pdf files Fig E1 Download .pdf (.59 MB) Help with pdf files Fig E2 Download .pdf (1.2 MB) Help with pdf files Fig E3 Download .pdf (.26 MB) Help with pdf files Fig E4 Download .pdf (.59 MB) Help with pdf files Fig E5 Download .pdf (.93 MB) Help with pdf files Fig E6