Identification of a 53 kDa protein, as a new high molecular weight allergen from Fraxinusexcelsior (Ash) pollen



Fraxinus excelsior (Ash) is a common tree and is important cause of winter–spring pollinosis in many temperate regions in the world. In this study, a high molecular weight allergen from ash pollen was identified.


In all, 20 individuals allergic to ash participated in the study. Characterization and immunoreactivity of ash pollen proteins was performed using sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE), two-dimensional (2D) gel electrophoresis and immunoblotting.


Immunoglobulin E (IgE)-binding proteins with apparent molecular mass ranging from 9 to 110 kDa were detected in ash pollen extract. Serum IgE of 7 (35%) patients reacted with a 53-kDa protein band. Analysis of 2D immunoblots showed several IgE-binding proteins. Moreover, mass spectrometry analysis indicated that the 53-kDa allergen was homologous to calreticulin.


We defined a novel allergen from F. excelsior pollen with a molecular weight of about 53 kDa. This allergen could be considered as an important high molecular weight allergenic protein for further studies on cross-reactivity and development of diagnostic and therapeutic approaches.

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Fig. 1
Fig. 2



Two-dimensional electrophoresis


Bovine serum albumin


Comassie brilliant blue




Endoplasmic reticulum


High molecular weight


Immunology, Asthma and Allergy Research Institute


Isoelectric focusing


Immobilized pH gradient


International protein index


High-sensitivity liquid diaminobenzidine


Matrix assisted laserdesorption/ionization


Molecular weight


Polyvinylidene fluoride


SDS-polyacrylamide gel electrophoresis


Tris-buffered saline


Time of flight


  1. 1.

    Poncet P, Senechal H, Clement G, Purohit A, Sutra JP, Desvaux FX, et al. Evaluation of ash pollen sensitization pattern using proteomic approach with individual sera from allergic patients. Allergy. 2010;65:571–80.

    CAS  Article  Google Scholar 

  2. 2.

    Wallander E. Systematics of Fraxinus (Oleaceae) and evolution of dioecy. Plant Syst Evol. 2008;273:25–49.

    Article  Google Scholar 

  3. 3.

    Lee HS, Park SH, Wallander E, Chang C‑S. A flavonoid survey of Fraxinus (Oleaceae) in eastern Asia, and the overlooked species Fraxinus hopeiensis T. Tang in northern China. Biochem Syst Ecol. 2012;41:150–6.

    CAS  Article  Google Scholar 

  4. 4.

    Albert B, Morand-Prieur M‑E, Brachet S, Gouyon P‑H, Frascaria-Lacoste N, Raquin C. Sex expression and reproductive biology in a tree species, Fraxinus excelsior L. C R Biol. 2013;336:479–85.

    Article  Google Scholar 

  5. 5.

    García B, Lizaso M, Moreno C, Rodríguez R, Villalba M, Ledesma A, et al. 5 Oleaceae-induced pollinosis in an area with exposure to olive and ash trees. J Investig Allergol Clin Immunol. 2011;21:34.

    PubMed  Google Scholar 

  6. 6.

    Gassner M, Schmid-Grendelmeier P, Clot B. Ash pollen allergy and aerobiology. Allergo J Int. 2019;28:289–98.

    Article  Google Scholar 

  7. 7.

    Majd A, Shoushtari MS, Moin M, Nejad MM, Zandieh F, Bayat P, et al. Pollen database registry in Iran. Iran J Allergy Asthma Immunol. 2018;17:91–2.

    Google Scholar 

  8. 8.

    Barderas R, Purohit A, Papanikolaou I, Rodríguez 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;115:351–7.

    CAS  Article  Google Scholar 

  9. 9.

    Hemmer W, Focke M, Wantke F, Götz M, Jarisch R, Jäger S, et al. Ash (Fraxinus excelsior)-pollen allergy in central Europe: specific role of pollen panallergens and the major allergen of ash pollen, Fra e 1. Allergy. 2000;55:923–30.

    CAS  Article  Google Scholar 

  10. 10.

    Hrabina M, Purohit A, Oster J‑P, Papanikolaou I, Jain K, Pascal P, et al. Standardization of an ash (Fraxinus excelsior) pollen allergen extract. Int Arch Allergy Immunol. 2007;142:11–8.

    Article  Google Scholar 

  11. 11.

    Imhof 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;23:78–83.

    Article  Google Scholar 

  12. 12.

    Niederberger V, Purohit A, Oster JP, Spitzauer S, Valenta R, Pauli G. The allergen profile of ash (Fraxinus excelsior) pollen: cross-reactivity with allergens from various plant species. Clin Exp Allergy. 2002;32:933–41.

    CAS  Article  Google Scholar 

  13. 13.

    Robledo-Retana T, Zenteno E, Agundis-Mata M, Pereyra-Morales M, Calderón-Segura M, Calderón-Ezquerro M. Detection of immunogens from Fraxinus spp. pollen grains. Aerobiologia. 2015;31:403–10.

    Article  Google Scholar 

  14. 14.

    Mas S, Torres M, Garrido-Arandia M, Salamanca G, Castro L, Barral P, et al. Ash pollen immunoproteomics: identification, immunologic characterization, and sequencing of 6 new allergens. J Allergy Clin Immunol. 2014;133:923–6.

    CAS  Article  Google Scholar 

  15. 15.

    Sharif Shoushtari M, Majd A, Pourpak Z, Shahali Y, Moin M, Eslami MB. Differential allergenicity of mature and immature pollen grains in Shasta daisy (Chrysanthemum maximum Ramond). Iran J Allergy Asthma Immunol. 2013;12:99–106.

    PubMed  Google Scholar 

  16. 16.

    Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54.

    CAS  Article  Google Scholar 

  17. 17.

    Assarehzadegan MA, Sankian M, Jabbari F, Tehrani M, Varasteh A. Expression of the recombinant major allergen of Salsola kali pollen (Sal k 1) and comparison with its low-immunoglobulin E‑binding mutant. Allergol Int. 2010;59:213–22.

    CAS  Article  Google Scholar 

  18. 18.

    Ghosal K, Saha B, Bhattacharya SG. Clinical and immuno-proteomic approach on Lantana camara pollen allergy—a major health hazard. Allergy Asthma Clin Immunol. 2016;12:33.

    Article  Google Scholar 

  19. 19.

    Assarehzadegan M‑A, Khodadadi A, Amini A, Shakurnia A‑H, Marashi SS, Ali-Sadeghi H, et al. Immunochemical characterization of Prosopis juliflora pollen allergens and evaluation of cross-reactivity pattern with the most allergenic pollens in tropical areas. Iran J Allergy Asthma Immunol. 2014;4:74–82.

    Google Scholar 

  20. 20.

    Assarehzadegan MA, Sankian M, Jabbari F, Tehrani M, Falak R, Varasteh A. Identification of methionine synthase (Sal k 3), as a novel allergen of Salsola kali pollen. Mol Biol Rep. 2011;38:6–73.

    Article  Google Scholar 

  21. 21.

    Mousavi F, Majd A, Shahali Y, Ghahremaninejad F, Shoormasti RS, Pourpak Z. Immunoproteomics of tree of heaven (Ailanthus atltissima) pollen allergens. J Proteomics. 2017;154:94–101.

    CAS  Article  Google Scholar 

  22. 22.

    Assarehzadegan MA, Shakurnia A, Amini A. The most common aeroallergens in a tropical region in Southwestern Iran. World Allergy Organ J. 2013;6:7.

    Article  Google Scholar 

  23. 23.

    Fereidouni M, Hossini RF, Azad FJ, Assarehzadegan M‑A, Varasteh A. Skin prick test reactivity to common aeroallergens among allergic rhinitis patients in Iran. Allergol Immunopathol (Madr). 2009;37:73–9.

    Article  Google Scholar 

  24. 24.

    Barderas R, Purohit A, Rodriguez R, Pauli G, Villalba M. Isolation of the main allergen Fra e 1 from ash (Fraxinus excelsior) pollen: comparison of the natural and recombinant forms. Ann Allergy Asthma Immunol. 2006;96:557–63.

    CAS  Article  Google Scholar 

  25. 25.

    Roth M. The C1q-receptor-calreticulin-contributes to the pathogenesis of asthma. J Respir Med Lung Dis. 2016;1:1002.

    Google Scholar 

  26. 26.

    Abolhassani M, Roux KH. cDNA cloning, expression and characterization of an allergenic 60s ribosomal protein of almond (Prunus dulcis). Iran J Allergy Asthma Immunol. 2009;8:77–84.

    CAS  PubMed  Google Scholar 

  27. 27.

    Radauer C, Bublin M, Wagner S, Mari A, Breiteneder H. Allergens are distributed into few protein families and possess a restricted number of biochemical functions. J Allergy Clin Immunol. 2008;121:847–852.e7.

    CAS  Article  Google Scholar 

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This study was supported by Immunology, Asthma and Allergy Research Institute (IAARI), Children’s Medical Center, Tehran University of Medical Sciences (grant # 93-03-40-27007).

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Corresponding author

Correspondence to Gholam Ali Kardar PhD.

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Conflict of interest

M. Sharif Shoushtari, A. Majd, M.-A. Assarehzadegan, S. Fanuel, M. Moin, T. Nejadsattari, R. S. Shoormasti,M. Badalzadeh, S. Tajik, M.R. Fazlollahi, B. Tayebi, Z. Pourpak and G.A. Kardar declare that they have no competing interests.

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Sharif Shoushtari, M., Majd, A., Assarehzadegan, M. et al. Identification of a 53 kDa protein, as a new high molecular weight allergen from Fraxinusexcelsior (Ash) pollen. Allergo J Int (2020).

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  • Ash pollen
  • Allergen
  • 2D electrophoresis
  • IgE immunoblotting
  • Calreticulin