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Der f 34, a Novel Major House Dust Mite Allergen Belonging to a Highly Conserved Rid/YjgF/YER057c/UK114 Family of Imine Deaminases*

  • Kareem Gamal ElRamlawy
    Footnotes
    Affiliations
    From the Hiroshima Research Center for Healthy Aging (HiHA), Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan,

    the Department of Zoology, Faculty of Science, Minia University, Minia 61519, Egypt,
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  • Takashi Fujimura
    Correspondence
    To whom correspondence may be addressed: Dept. of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan. Tel.: 81-82-424-4434; Fax: 81-82-424-7867;.
    Footnotes
    Affiliations
    From the Hiroshima Research Center for Healthy Aging (HiHA), Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan,
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  • Koji Baba
    Affiliations
    From the Hiroshima Research Center for Healthy Aging (HiHA), Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan,
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  • Ji Won Kim
    Affiliations
    From the Hiroshima Research Center for Healthy Aging (HiHA), Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan,
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  • Chika Kawamoto
    Affiliations
    From the Hiroshima Research Center for Healthy Aging (HiHA), Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan,
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  • Toshihide Isobe
    Affiliations
    From the Hiroshima Research Center for Healthy Aging (HiHA), Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan,
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  • Takuya Abe
    Affiliations
    From the Hiroshima Research Center for Healthy Aging (HiHA), Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan,
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  • Kelsey Hodge-Hanson
    Affiliations
    the Department of Microbiology, University of Georgia, Athens, Georgia 30602,
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  • Diana M. Downs
    Affiliations
    the Department of Microbiology, University of Georgia, Athens, Georgia 30602,
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  • Inas Hussein Refaat
    Affiliations
    the Department of Zoology, Faculty of Science, Minia University, Minia 61519, Egypt,
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  • Diaa Beshr Al-Azhary
    Affiliations
    the Department of Zoology, Faculty of Science, Minia University, Minia 61519, Egypt,
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  • Tsunehiro Aki
    Affiliations
    From the Hiroshima Research Center for Healthy Aging (HiHA), Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan,
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  • Yoshiko Asaoku
    Affiliations
    the Takanobashi Central Hospital, Hiroshima, Hiroshima 730-0042,
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  • Takaharu Hayashi
    Affiliations
    the Takanobashi Central Hospital, Hiroshima, Hiroshima 730-0042,
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  • Takashi Katsutani
    Affiliations
    the Katsutani Ogasawara Clinic, Hatsukaichi, Hiroshima 738-0042,
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  • Shinji Tsuboi
    Affiliations
    the Tsuboi Clinic, Ohtake, Hiroshima, 739-0613, and
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  • Kazuhisa Ono
    Affiliations
    From the Hiroshima Research Center for Healthy Aging (HiHA), Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan,

    the Department of Food Sciences and Biotechnology, Faculty of Life Sciences, Hiroshima Institute of Technology, Hiroshima, Hiroshima 731-5193, Japan
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  • Seiji Kawamoto
    Correspondence
    To whom correspondence may be addressed: Dept. of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan. Tel.: 81-82-424-7753; Fax: 81-82-424-7867;.
    Affiliations
    From the Hiroshima Research Center for Healthy Aging (HiHA), Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan,
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  • Author Footnotes
    * This work was supported by a Grant-in-Aid for Scientific Research (B), JSPS KAKENHI Grant 14360209 (to K. O. and S. K.), and National Institutes of Health Grant GMO95837 (to D. M. D.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
    1 Both authors contributed equally to this work.
    2 Supported by the Ministry of higher education (MoHE), Egypt.
Open AccessPublished:August 18, 2016DOI:https://doi.org/10.1074/jbc.M116.728006
      The high prevalence of house dust mite (HDM) allergy is a growing health problem worldwide, and the characterization of clinically important HDM allergens is a prerequisite for the development of diagnostic and therapeutic strategies. Here, we report a novel HDM allergen that belongs structurally to the highly conserved Rid/YjgF/YER057c/UK114 family (Rid family) with imine deaminase activity. Isolated HDM cDNA, named der f 34, encodes 128 amino acids homologous to Rid-like proteins. This new protein belongs to the Rid family and has seven conserved residues involved in enamine/imine deaminase activity. Indeed, we demonstrated that purified Der f 34 had imine deaminase activity that preferentially acted on leucine and methionine. Native Der f 34 showed a high IgE binding frequency as revealed by two-dimensional immunoblotting (62.5%) or ELISA (68%), which was comparable with those of a major HDM allergen Der f 2 (77.5 and 79%, respectively). We also found that Der f 34 showed cross-reactivity with another prominent indoor allergen source, Aspergillus fumigatus. This is the first report showing that the Rid family imine deaminase represents an additional important pan-allergen that is conserved across organisms.

      Introduction

      Asthma is a serious global health problem that significantly reduces quality of life. In a cross-sectional World Health Organization survey of 178,215 individuals from 70 countries conducted in 2002–2003, it was estimated that 4.3% of adults (range, 0.2–21.0%) had been diagnosed with asthma by a medical doctor (
      • To T.
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      Global asthma prevalence in adults: findings from the cross-sectional world health survey.
      ). Inhaled airborne allergens cause allergic inflammation via the cross-linking of allergen-specific IgEs bound on high affinity IgE receptor (FceRI) located on the surface of airway or lung mast cells, causing the secretion of inflammatory chemical mediators (e.g. histamine and leukotrienes) from activated mast cells. House dust mites (HDMs)
      The abbreviations used are: HDM, house dust mite; AIT, allergen-specific immunotherapy; nDer f 34, native Der f 34; rDer f 34, recombinant Der f 34 without tag; rGST-Der f 34, rDer f 34 with GST tag; rTF-Der f 34, rDer f 34 as a trigger factor fusion protein; rTF, recombinant trigger factor; pAb, polyclonal antibody; ANOVA, analysis of variance; SNK, Student-Newman-Keuls.
      and fungi are major sources of airborne allergens and trigger asthmatic attacks in allergic patients concomitant with air pollutants (
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      ).
      Allergen-specific immunotherapy (AIT) is the only curative treatment for allergic asthma that changes the natural course of an allergy (
      • Fujimura T.
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      Antigen-specific immunotherapy against allergic rhinitis: the state of the art.
      ). AIT induces allergen-specific tolerance by administering increasing doses of causative allergens subcutaneously or sublingually (
      • Jutel M.
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      ). The precise mechanisms underlying AIT remain to be fully elucidated, but the involvement of regulatory T cells and B cells or the induction of T cell anergy is suggested to be a key mechanism for an efficient AIT. AIT induces antigen-specific immunomodulatory cells or the anergic response of T cells specific to major allergens included in an allergen extract as an AIT vaccine. Therefore, the standardization of AIT vaccines using important major allergens and the characterization of major component allergens in an allergen source are important issues for the development of an effective AIT (
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      Spectrum of allergens for Japanese cedar pollinosis and impact of component-resolved diagnosis on allergen-specific immunotherapy.
      ,
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      ).
      An HDM extract commonly used for AIT of HDM-allergic rhinitis and asthma is standardized by its concentrations of major allergens, Der f 1 and 2 or Der p 1 and 2. Only Der f 1/2 and Der p 1/2 are used for the standardization of a vaccine for AIT of HDM. On the other hand, many allergens have been identified and characterized from HDM, including Dermatophagoides farinae (American HDM) and Dermatophagoides pteronyssinus (European HDM), as Der f/Der p allergens or mite allergen gene (Mag) allergens, such as Der f 6 and Mag3 (
      • Thomas W.R.
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      Cloning and expression of Der f 6, a serine protease allergen from the house dust mite, Dermatophagoides farinae.
      • Kawamoto S.
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      ). Among the allergens, 28 from D. farinae have been formally registered in a database for systematic allergen nomenclature approved by the World Health Organization and the International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-committee. These allergens have been identified and characterized from mite body, mite feces, or a cDNA library of mite bodies. Der f 1 (cysteine protease), Der f 2 (MD2-like lipid-binding domain protein), and Der f 23 (peritrophin-like protein) are serodominant allergens in D. farinae (
      • Thomas W.R.
      Hierarchy and molecular properties of house dust mite allergens.
      ,
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      Crystal structures of mite allergens Der f 1 and Der p 1 reveal differences in surface-exposed residues that may influence antibody binding.
      ,
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      Lipopolysaccharide binding of the mite allergen Der f 2.
      • Weghofer M.
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      • Pauli G.
      • Horak F.
      • Keller W.
      • et al.
      Identification of Der p 23, a peritrophin-like protein, as a new major Dermatophagoides pteronyssinus allergen associated with the peritrophic matrix of mite fecal pellets.
      ). Serine proteases from D. farinae, including Der f 3 (trypsin), Der f 6 (chymotrypsin), and Der f 9 (collagenase), act on mucosal epithelial cells to induce chemokine production that triggers the migration of inflammatory cells (
      • Kawamoto S.
      • Mizuguchi Y.
      • Morimoto K.
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      Cloning and sequencing of the Dermatophagoides pteronyssinus group III allergen, Der p III.
      • Nishiyama C.
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      ,
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      ,
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      ). Tropomyosin as Der f 10 and gelsolin as Der f 16 are also important HDM allergens as pan-allergens (
      • Aki T.
      • Kodama T.
      • Fujikawa A.
      • Miura K.
      • Shigeta S.
      • Wada T.
      • Jyo T.
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      ,
      • Kawamoto S.
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      • Shigeta S.
      • Ono K.
      Der f 16: a novel gelsolin-related molecule identified as an allergen from the house dust mite, Dermatophagoides farinae.
      ).
      We previously reported that subcutaneous AIT using an allergen fraction prepared from D. farinae feces extract (HM2 fraction) significantly reduced symptom-medication scores for bronchial symptoms without any severe adverse events, such as asthmatic attacks or anaphylaxis (
      • Kawamoto S.
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      [Desensitization immunotherapy on patients with mite-positive bronchial asthma using purified mite feces antigen fractions].
      ). Interestingly, although the HM2 fraction has the highest allergenicity and tolerability, it does not contain major HDM allergens Der f 1 and Der f 2. This suggests that new HDM allergens, other than Der f 1 and Der f 2, are responsible for the immunotherapeutic effects of the HM2 fraction. These findings prompted us to characterize the component allergens in HM2 fraction, which should contain important uncharacterized allergens applicable to AIT.
      In this study, we identified a novel allergen, Der f 34, belonging to the Rid/YjgF/YER057c/UK114 family (Rid family) with imine deaminase activity from HM2 fraction. The Rid family protein is broadly conserved from prokaryotes to mammals with their high homology of amino acid sequences (
      • Niehaus T.D.
      • Gerdes S.
      • Hodge-Hanson K.
      • Zhukov A.
      • Cooper A.J.
      • ElBadawi-Sidhu M.
      • Fiehn O.
      • Downs D.M.
      • Hanson A.D.
      Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114 (Rid) protein family.
      ). We further demonstrated that native Der f 34 showed a high IgE binding frequency comparable with that of a major HDM allergen Der f 2 by two-dimensional allergenome analysis and ELISA. Furthermore, our ELISA inhibition analysis showed that Der f 34 shared its immunoglobulin-binding epitopes with those of Aspergillus fumigatus. Taken together, these results indicate that Der f 34 could be a novel and important cross-reactive allergen for HDM allergy.

      Discussion

      To achieve a more effective AIT for HDM allergy, it is important to characterize component allergens from the fraction of HDM that has the most potent allergenicity and tolerability by subcutaneous AIT. We characterized a novel allergen named Der f 34 as an imine deaminase belonging to the Rid family. This is the first report to show the IgE binding capacity of a Rid family protein in allergic patients. Der f 34 showed that imine deaminase activity involved in amino acid metabolisms cooperated with amino acid oxygenase or hydrogenase. Imine deaminase catalyzes the hydrolysis of imine to produce ammonia and ketone (
      • Lambrecht J.A.
      • Flynn J.M.
      • Downs D.M.
      Conserved YjgF protein family deaminates reactive enamine/imine intermediates of pyridoxal 5′-phosphate (PLP)-dependent enzyme reactions.
      ). Rid family proteins are highly conserved in various species, including prokaryotes and eukaryotes, and their sequences for the UPF0076 signature pattern and residues for enamine/imine deaminase activity are highly conserved (Fig. 1, A and B) (
      • Niehaus T.D.
      • Gerdes S.
      • Hodge-Hanson K.
      • Zhukov A.
      • Cooper A.J.
      • ElBadawi-Sidhu M.
      • Fiehn O.
      • Downs D.M.
      • Hanson A.D.
      Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114 (Rid) protein family.
      ). Due to sequence similarities among members of the Rid family, Der f 34 is a candidate for a novel pan-allergen in various allergen sources. We elucidated that Der f 34 shares its immunoglobulin-binding epitopes with those from Aspergillus species (Fig. 4B). This strongly suggests that Der f 34-specific IgE can bind with allergens that share their immunoglobulin-binding epitopes from other allergenic sources, because binding between epitopes and antigen-binding regions of immunoglobulins (Fab region) is crucial for antigen recognition of immunoglobulin, and this binding is independent of the immunoglobulin isotype (Fc region). Although we found its cross-reactivity only in Aspergillus species, it seems to cross-react with other allergens in the Rid family or with enamine/imine deaminases. Elucidation of the cross-reactivity of Der f 34 with other homologous allergens in various allergen sources may also be important for the precise diagnosis of HDM allergy.
      High doses (100 and 300 μg/ml) of crude extract from A. fumigatus showed statistically significant inhibition of binding between Der f 34 and Der f 34-specific IgG, although an excess amount of a nonspecific negative control protein (rTF) failed to block the binding. This clearly indicates that the observed competition with rTF-Der f 34 as well as with A. fumigatus extracts is not a nonspecific binding. The present data also indicate that the nonspecific binding kinetics of rTF are distinct from those seen in rTF-Der f 34, D. farinae feces extract, and A. fumigatus extract (Fig. 4B). Our estimation from the inhibition assay suggests that the content of rDer f 34-cross-reactive allergens in A. fumigatus is only 0.001% in the crude extract. One possible reason for the low allergen content is that those cross-reactive allergens in A. fumigatus might be lost during the extraction procedures, because the Rid proteins are small molecules (<20 kDa), albeit the inhibition is statistically significant compared with that of A. alternata and rTF as a negative control. To gain further insight into the cross-reactivity, experiments using purified native A. fumigatus Rid proteins and Rid protein-deficient A. fumigatus extract (prepared by immunodepletion with anti-Der f 34 pAb or made from rid gene disruptants) are needed.
      We characterized Der f 34 as a candidate for a major allergen available to develop a more efficient AIT vaccine. UK114 protein in the Rid family was reported to induce Th2-skewing responses in murine splenocyte and to induce high IL4 with low IFN-γ production by costimulation with concanavalin A (
      • Panerai A.E.
      • Sacerdote P.
      • Bianchi M.
      • Nicoletti F.
      • Manfredi B.
      • Gaspani L.
      • Bartorelli A.
      • Ceciliani F.
      • Ronchi S.
      Chronic administration of UK-114, a multifunctional emerging protein, modulates the Th1/Th2 cytokine pattern and experimental autoimmune diseases.
      ). We analyzed the potential for such an immunological skewing property of rDer f 34 produced by the COS-7 (monkey kidney fibroblast) expression system, which is a lipopolysaccharide-negative protein expression system, by culturing with murine splenocytes. However, we could not conclude that Der f 34 possesses the capacity to skew the Th2 response in mice (data not shown). It will be interesting to elucidate whether Der f 34 has the potency to skew the Th1/Th2 response in humans. Interestingly, UK114 protein was also identified in humans as a tumor-associated antigen (NCBI GenBankTM accession number P80601; 47.4% identical to and 75.2% similar to Der f 34), and human monoclonal antibody against the UK114 antigen showed complement-dependent cytotoxicity to tumor cell lines but not to normal cells, except for a small fraction of hepatocytes of fetal origin (
      • Ceciliani F.
      • Faotto L.
      • Negri A.
      • Colombo I.
      • Berra B.
      • Bartorelli A.
      • Ronchi S.
      The primary structure of UK114 tumor antigen.
      ,
      • Funaro A.
      • Horenstein A.L.
      • Ghisolfi G.
      • Bussolati B.
      • Bartorelli A.
      • Bussolati G.
      Identification of a 220-kDa membrane tumor-associated antigen by human anti-UK114 monoclonal antibodies selected from the immunoglobulin repertoire of a cancer patient.
      ). The UK114 protein also inhibits the growth of mammary carcinomas induced in Sprague-Dawley rats, and the authors of that report speculated that the UK114 antigen may be involved in antigen presentation and tumor immunosurveillance (
      • Racca S.
      • Di Carlo F.
      • Bartorelli A.
      • Bussolati B.
      • Bussolati G.
      Growth inhibition of DMBA-induced rat mammary carcinomas by UK 114.
      ). We tested cross-reactivity between rTF-Der f 34 and human recombinant GST-tagged UK114 but failed to find significant cross-reactivity as assessed by an ELISA inhibition assay and immunoblotting using anti-Der f 34 pAb (data not shown). One possible reason for this poor cross-reactivity is that UK114 was GST-tagged recombinant protein produced by the wheat germ cell-free protein expression system, whose conformation could be different from its natural counterparts. We could not clarify the cross-reactivity between native Der f 34 and native UK114 nor whether anti-Der f 34 antibody was involved in the progression or clinical condition of tumors or other human diseases. Further analysis is needed to clarify the cross-reactivity between Der f 34 and human UK114 protein. However, if native UK114 is cross-reactive antigen with Der f 34, it may possible that Der f 34-specific antibody might be involved in the mechanisms underlying the inverse association between an allergy and specific types of cancer (
      • Rittmeyer D.
      • Lorentz A.
      Relationship between allergy and cancer: an overview.
      ). These reports implied that Der f 34 is capable of immunomodulation and may be involved in the therapeutic mechanisms of AIT.
      Native Der f 34 had a high IgE binding capacity, comparable with that of Der f 2 (62.5 and 68% for Der f 34, 77.5 and 79% for Der f 2 by two-dimensional immunoblotting and ELISA, respectively; Fig. 4A) (data not shown), and Der f 34 cross-reacted with fungal allergens (A. fumigatus; Fig. 4B). These data strongly suggested that Der f 34 is an important cross-reactive allergen for HDM and other allergen sources and that the Rid family will be a new category for pan-allergens, such as profilins and nonspecific lipid transfer protein families (
      • Hauser M.
      • Roulias A.
      • Ferreira F.
      • Egger M.
      Panallergens and their impact on the allergic patient.
      ). Allergen sources that digested amino acid in their metabolic pathways may contain enamine/imine deaminase in the Rid family homologous to Der f 34. Therefore, we consider that Der f 34 is also a clinically important major allergen for HDM-allergic rhinitis and asthma.
      In this paper, we identified and characterized a novel mite imine deaminase, named der f 34, from HDM as a pan-allergen for the first time. Our results suggested that Rid family proteins are important as novel pan-allergens for the cross-reactivity among various allergen sources. It will be important to elucidate in the near future the contribution of Der f 34 to AIT using HDM extract.

      Author Contributions

      K. G. E., T. F., T. Abe, K. H., J. W. K., K. B., C. K., and T. I. performed experiments, including production of recombinant fusion proteins; Y. A., T. H., T. K., and S. T. contributed to obtaining plasma from HDM patients and healthy volunteers as well as the clinical data for them; K. G. E., T. F., and S. K. analyzed experimental data; T. F., T. Aki, K. O., and S. K. conceived, arranged, and supervised the study; K. H.-H., D. M. D., I. H. R., and D. B. A. analyzed experimental data and reviewed the manuscript before submission; T. F. and S. K. wrote the paper and revised the manuscript; K. G. E., T. F., and S. K. have agreed to be accountable for all aspects of the work and in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; S. K. has given final approval of the version of the manuscript to be published.

      Acknowledgments

      The cDNA sequence for der f 34 (originally named mag133) has been registered in the DNA Data Bank Japan (DDBJ) with accession number LC120618 as Dermatophagoides farinae MAG133 mRNA for imine deaminase. The sequence of Der f 34 has been submitted for registration in a database for the systematic allergen nomenclature approved by the World Health Organization and the International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-committee as the newly registered D. farinae allergen Der f 34.

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