Plantago lanceolata: An important trigger of summer pollinosis with limited IgE cross- reactivity Gabriele Gadermaier, PhD, Stephanie Eichhorn, MSc, Eva.

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Plantago lanceolata: An important trigger of summer pollinosis with limited IgE cross- reactivity Gabriele Gadermaier, PhD, Stephanie Eichhorn, MSc, Eva Vejvar, MSc, Lisa Weilnböck, MSc, Roland Lang, PhD, Peter Briza, PhD, Christian G. Huber, PhD, Fatima Ferreira, PhD, Thomas Hawranek, MD Journal of Allergy and Clinical Immunology Volume 134, Issue 2, Pages 472-475.e5 (August 2014) DOI: 10.1016/j.jaci.2014.02.016 Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 IgE sensitization and cross-inhibition. A, Patients' IgE levels toward 8 allergen extracts were determined in ELISAs, and sensitization frequencies are shown at the top. B, Patients' sera were preincubated with pollen extracts, and remaining IgE reactivity to plantain extract was evaluated. Individual IgE levels and inhibitions are shown as symbols, mean values are shown, and the dotted line represents the cutoff value. Journal of Allergy and Clinical Immunology 2014 134, 472-475.e5DOI: (10.1016/j.jaci.2014.02.016) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Identification of IgE-reactive proteins in Plantago lanceolata pollen. A, Sensitization to plantain extract using 26 patients' sera in a 1-dimensional immunoblot. B and C, Coomassie staining (Fig 2, B) and immunoblotting (Fig 2, C) of 2-dimensional separated plantain extract. D, Mass spectrometry–based analysis of trypsin-digested spots and protein identification. E, Sensitization frequency to identified plantain pollen allergens. BC, Buffer control; NHS, nonatopic human serum. Journal of Allergy and Clinical Immunology 2014 134, 472-475.e5DOI: (10.1016/j.jaci.2014.02.016) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Amino acid sequence alignment of Pla l 1 and investigated OLPs (numbering according to Pla l 1 sequence). The mature sequences of Pla l 1.0101 (UniProt P82242), Pla l 1.0102 (UniProt P82242), and Pla l 1.0103 (UniProt P82242) from plantain (Plantago lanceolata); Lol p 11 (UniProt Q7M1X5) from rye grass (Lolium perenne); Phl p 11 (UniProt Q8H6L7) from timothy grass (Phleum pratense); Fra e 1.0101 (UniProt Q7XAV4), Fra e 1.0102 (UniProt Q5EXJ6), and Fra 3 1.0103 (UniProt Q6U740) from ash (Fraxinus excelsior); Ole e 1.0101 (UniProt P19963), Ole e 1.0102 (GenBank X76395), Ole e 1.0103 (GenBank X76396), Ole e 1.0104 (GenBank X76397), Ole e 1.0105 (UniProt P19963), Ole e 1.0106 (UniProt P19963), and Ole e 1.0107 (UniProt P19963) from olive (Olea europaea); and BB18 (UniProt O49813) from birch (Betula verrucosa) were aligned by using Clustal Omega on EMBL-EBI, and identity scores are shown in percentages. Amino acids identical to Pla l 1.0101 are highlighted in blue, and similar residues are shown in light blue. Residues corresponding to the Ole e 1 consensus pattern [EQT]-G-X-V-Y-C-D-[TNP]-C-R 1 are underlined and shown as boldface and violet. Potential glycosylation sites of Pla l 1 (N107), Lol p 11 (N24), Phl p 11 (N24), and Ole e 1 (N111) are indicated by red boldface letters. IgE-binding epitopes of Ole e 1 2 are boxed, and relevant residues are highlighted in black boldface letters. % ID, Percentage of amino acid identity; pI, isoelectric point. Information given in the figure and the legend from Sigrist et alE1 and Gonzalez et al.E2 Journal of Allergy and Clinical Immunology 2014 134, 472-475.e5DOI: (10.1016/j.jaci.2014.02.016) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Immunoblot inhibition to English plantain pollen extract. Selected patients' sera cross-reactive with all tested extracts (patients 8 and 17), cross-reactive with grass extracts (patients 18 and 21), and without cross-reactivity (patients 4 and 24) were preincubated with pollen extracts from English plantain (Plantago lanceolata), timothy grass (Phleum pratense), rye grass (Lolium perenne), ash (Fraxinus excelsior), olive (Olea europaea), and birch (Betula verrucosa). Sera without inhibitor (n.i.) were used as positive controls. Journal of Allergy and Clinical Immunology 2014 134, 472-475.e5DOI: (10.1016/j.jaci.2014.02.016) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions