Identification and gene cloning of a new major allergen Cha o 3 from Chamaecyparis obtusa (Japanese cypress) pollen Toshihiro Osada, MSc, Takafumi Harada,

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Identification and gene cloning of a new major allergen Cha o 3 from Chamaecyparis obtusa (Japanese cypress) pollen Toshihiro Osada, MSc, Takafumi Harada, MSc, Naomasa Asaka, MSc, Tomonori Haruma, MSc, Kohsuke Kino, PhD, Eiji Sasaki, PhD, Mitsuhiro Okano, MD, PhD, Akira Yamada, PhD, Teruhiro Utsugi, PhD Journal of Allergy and Clinical Immunology Volume 138, Issue 3, Pages 911-913.e7 (September 2016) DOI: 10.1016/j.jaci.2016.03.026 Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 SDS-PAGE analysis and correlation between ImmunoCAP values and Cha o allergen–specific IgE. A, Cha o 3: purified Cha o 3 under nonreducing conditions, Cha o 1: purified Cha o 1 under nonreducing conditions. B, Purified Cha o 3 under reducing conditions. C, Purified recombinant Cha o 2. Because only a limited amount of Cha o 2 was obtained from the cypress extract, recombinant Cha o 2 was prepared. Correlation between ImmunoCAP values and Cha o 3–specific IgE binding measured by ELISA (D) or Cha o 1–specific IgE (E). Statistical analysis was performed using the Pearson correlation coefficient test. Lines indicate 95% probability ellipses. rCha o 2, recombinant Cha o 2. Journal of Allergy and Clinical Immunology 2016 138, 911-913.e7DOI: (10.1016/j.jaci.2016.03.026) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Proliferative response of PBMCs. PBMCs were stimulated with antigen (10 μg/mL) for 3 days. After incubation for an additional 16 hours with [3H]-thymidine, [3H]-thymidine incorporation was measured. Statistical significance versus Cha o 3 was evaluated using Dunnett test (*P < .01). The complementary DNA sequence of Cha o 3 was deposited in the GenBank sequence database under accession number HV942721. rCha o 2, recombinant Cha o 2; SBP, Sugi basic protein. Journal of Allergy and Clinical Immunology 2016 138, 911-913.e7DOI: (10.1016/j.jaci.2016.03.026) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Cha o 3 induces basophil activation in patients with Japanese cypress pollinosis. A, Whole blood of V043 was stimulated with antigen (0.1 μg/mL) or anti-human IgE (10 μg/mL) as a positive control for 15 minutes at 37°C. Whole blood of V016 (B) or V037 (C) was stimulated with each allergen (1 ng/mL). CRTH2, Chemoattractant receptor-homologous molecule on TH2 cells; SBP, Sugi basic protein. Journal of Allergy and Clinical Immunology 2016 138, 911-913.e7DOI: (10.1016/j.jaci.2016.03.026) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Sequence of cloned cDNA of Cha o 3 and deduced amino acid sequence. Domains to which primers were applied for PCR cloning are shown by arrows (black arrows, first primers; blue arrows, second primers; red arrows, primers for 5′-RACE; green arrow, primer for 3′-RACE). Arrow direction indicates forward or reverse primers. The blue box represents the predicted signal peptide. Peptides that were identified by amino acid sequence analysis are shown in red boxes. Several independently isolated cDNA clones identified polymorphisms at nucleotides 88 (C or A), 264 (A or C), 681 (C or T), 772 (G or A), 792 (A or G), 870 (C or T), 895 (G or A), and 1029 (G or A). Nearly half the mutations encoded the same amino acids, except nucleotides 88 (Pro or Thr), 772 (Val or Ile), and 895 (Val or Ile). Because of these polymorphisms, the most frequent cDNA clone is discussed in this report. Journal of Allergy and Clinical Immunology 2016 138, 911-913.e7DOI: (10.1016/j.jaci.2016.03.026) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 A, Predicted Cha o 3 protein. B, ClustalW multiple sequence alignment for homology analysis. The predicted cellulase domain of Cha o 3 (from Pro70 to Gly353) was compared with the cellulases of Medicago truncatula (accession no. KEH20742), Theobroma cacao (accession no. XP_007044872), and Arabidopsis thaliana (accession no. NP_172772). Shaded areas indicate consistent amino acid sequence compared with Cha o 3. Journal of Allergy and Clinical Immunology 2016 138, 911-913.e7DOI: (10.1016/j.jaci.2016.03.026) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 IL-5 production by PBMCs collected from patients with Japanese cedar and cypress pollinosis with or without Japanese cedar–specific immunotherapy. PBMCs were stimulated with 10 μg/mL of the indicated allergens for 72 hours. The IL-5 content of the supernatant was determined by ELISA. Statistical analyses were performed using the 2-tailed Student t test (*P < .01). Journal of Allergy and Clinical Immunology 2016 138, 911-913.e7DOI: (10.1016/j.jaci.2016.03.026) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions