Purification and molecular characterization of phospholipase, antigen 5 and hyaluronidases from the venom of the Asian hornet (Vespa velutina)

Autoři: Rafael I. Monsalve aff001;  Ruth Gutiérrez aff001;  Ilka Hoof aff002;  Manuel Lombardero aff001
Působiště autorů: CMC Research and Development, ALK-Abelló S.A., Madrid, Spain aff001;  Global Research, ALK-Abelló A/S, Hørsholm, Denmark aff002
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0225672


The aim of this study was to purify potential allergenic components of Vespa velutina venom, the yellow legged Asian Hornet, and perform a preliminary characterization of the purified proteins. Starting from the whole venom of V.velutina, several chromatographic steps allowed to purify the phospholipase (named Vesp v 1), as well as the antigen 5 (Vesp v 5, the only allergenic component described as such so far). The two hyaluronidase isoforms found (Vesp v 2A and Vesp v 2B) cannot be separated from each other, but they are partially purified and characterized. Purity of the isolated proteins in shown by SDSPAGE, as well as by the results of the N-terminal sequencing. This characterization and nLC-MS/MS data provide most of the sequence for Vesp v 1 and Vesp v 5 (72 and 84% coverage, respectively), confirming that the whole sequences of the isolated natural components match with the data available in public transcriptomic databases. It is of particular interest that Vesp v 1 is a glycosylated phospholipase, a fact that had only described so far for the corresponding allergen components of Dolichovespula maculata and Solenopsis invicta. The availability of the complete sequences of Vespa velutina components permits comparison with homologous sequences from other Hymenoptera. These data demonstrate the higher similarity among the species of the genera Vespa and Vespula, in comparison to Polistes species, as it is especially observed with the hyaluronidases isoforms: the isoform Vesp v 2A only exists in the former genera, and not in Polistes; in addition, the most abundant isoform (Vesp v 2B) exhibits 93% sequence identity with the Ves v 2 isoform of Vespula vulgaris. Finally, the isolated components might be useful for improving the diagnosis of patients that could be allergic to stings of this invasive Asian hornet, as it has been the case of an improved diagnosis and treatment of other Hymenoptera-sensitized patients.

Klíčová slova:

Allergens – Multiple alignment calculation – Protein sequencing – Sequence alignment – Sequence databases – Transcriptome analysis – Venoms – Phospholipases


1. Castro L, Pagola-Carte S. Vespa velutina Lepeletier, 1836 (Hymenoptera: Vespidae), collected in the Iberian Peninsula. Heteropterus Rev Entomol 2010;10(2):193–6.

2. Leza M, Herrera C, Marques A, Roca P, Sastre-Serra J, Pons DG. The impact of the invasive species Vespa velutina on honeybees: A new approach based on oxidative stress. Sci Total Environ 2019 Jul 1;689:709–15. doi: 10.1016/j.scitotenv.2019.06.511 31280152

3. Milanesio D, Saccani M, Maggiora R, Laurino D, Porporato M. Recent upgrades of the harmonic radar for the tracking of the Asian yellow-legged hornet. Ecol Evol 2017 Jul;7(13):4599–606. doi: 10.1002/ece3.3053 28690790

4. Villemant C, Zuccon D, Rome Q, Muller F, Poinar GO Jr., Justine JL. Can parasites halt the invader? Mermithid nematodes parasitizing the yellow-legged Asian hornet in France. PeerJ 2015;3:e947. doi: 10.7717/peerj.947 26038716

5. Arca M, Papachristoforou A, Mougel F, Rortais A, Monceau K, Bonnard O, et al. Defensive behaviour of Apis mellifera against Vespa velutina in France: testing whether European honeybees can develop an effective collective defence against a new predator. Behav Processes 2014 Jul;106:122–9. doi: 10.1016/j.beproc.2014.05.002 24857979

6. Chugo S, Lizaso MT, Alvarez MJ, Arroabaren E, Lizarza S, Tabar AI. Vespa velutina nigritorax: A New Causative Agent in Anaphylaxis. J Investig Allergol Clin Immunol 2015;25(3):231–2. 26182696

7. Liu Z, Chen S, Zhou Y, Xie C, Zhu B, Zhu H, et al. Deciphering the venomic transcriptome of killer-wasp Vespa velutina. Sci Rep 2015;5:9454. doi: 10.1038/srep09454 25896434

8. Schiener M, Graessel A, Ollert M, Schmidt-Weber CB, Blank S. Allergen-specific immunotherapy of Hymenoptera venom allergy—also a matter of diagnosis. Hum Vaccin Immunother 2017 Oct 3;13(10):2467–81. doi: 10.1080/21645515.2017.1334745 28604163

9. Antolin-Amerigo D, Ruiz-Leon B, Boni E, Alfaya-Arias T, Alvarez-Mon M, Barbarroja-Escudero J, et al. Component-resolved diagnosis in Hymenoptera allergy. Allergol Immunopathol 2017 Jul 21;46(3):253–62.

10. Monsalve RI, Vega A, Marques L, Miranda A, Fernandez J, Soriano V, et al. Component-resolved diagnosis of vespid venom-allergic individuals: phospholipases and antigen 5s are necessary to identify Vespula or Polistes sensitization. Allergy 2012 Apr;67(4):528–36. doi: 10.1111/j.1398-9995.2011.02781.x 22229815

11. King TP, Hoffman D, Lowenstein H, Marsh DG, Platts-Mills TA, Thomas W. Allergen nomenclature. WHO/IUIS Allergen Nomenclature Subcommittee. Int Arch Allergy Immunol 1994;105(3):224–33. doi: 10.1159/000236761 7920024

12. Pomes A, Davies JM, Gadermaier G, Hilger C, Holzhauser T, Lidholm J, et al. WHO/IUIS Allergen Nomenclature: Providing a common language. Mol Immunol 2018 Aug;100:3–13. doi: 10.1016/j.molimm.2018.03.003 29625844

13. King TP, Kochoumian L, Joslyn A. Wasp venom proteins: phospholipase A1 and B. Arch Biochem Biophys 1984;230(1):1–12. doi: 10.1016/0003-9861(84)90080-8 6712224

14. King TP, Lu G, González M, Qian NF, Soldatova L. Yellow jacket venom allergens, hyaluronidase and phospholipase: sequence similarity and antigenic cross-reactivity with their hornet and wasp homologs and possible implications for clinical allergy. Journal of Allergy and Clinical Immunology 1996;98:588–600. doi: 10.1016/s0091-6749(96)70093-3 8828537

15. Morrissey JH. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 1981;117(2):307–10. doi: 10.1016/0003-2697(81)90783-1 6172996

16. Habermann E, Hardt KL. A sensitive and specific plate test for the quantitation of phospholipases. Anal Biochem 1972;50(1):163–73. doi: 10.1016/0003-2697(72)90495-2 4342994

17. Richman PG, Baer H. A convenient plate assay for the quantitation of hyaluronidase in Hymenoptera venoms. Anal Biochem 1980;109(2):376–81. doi: 10.1016/0003-2697(80)90663-6 7224162

18. Speicher KD, Gorman N, Speicher DW. N-terminal sequence analysis of proteins and peptides. Curr Protoc Protein Sci 2009 Aug;Chapter 11:Unit11.

19. Kall L, Canterbury JD, Weston J, Noble WS, MacCoss MJ. Semi-supervised learning for peptide identification from shotgun proteomics datasets. Nat Methods 2007 Nov;4(11):923–5. doi: 10.1038/nmeth1113 17952086

20. Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, et al. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc 2013 Aug;8(8):1494–512. doi: 10.1038/nprot.2013.084 23845962

21. Artimo P, Jonnalagedda M, Arnold K, Baratin D, Csardi G, de CE, et al. ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res 2012 Jul;40(Web Server issue):W597–W603. doi: 10.1093/nar/gks400 22661580

22. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990 Oct 5;215(3):403–10. doi: 10.1016/S0022-2836(05)80360-2 2231712

23. Kolarich D, Loos A, Leonard R, Mach L, Marzban G, Hemmer W, et al. A proteomic study of the major allergens from yellow jacket venoms. Proteomics 2007;7(10):1615–23. doi: 10.1002/pmic.200600800 17443842

24. Skov LK, Seppala U, Coen JJ, Crickmore N, King TP, Monsalve R, et al. Structure of recombinant Ves v 2 at 2.0 Angstrom resolution: structural analysis of an allergenic hyaluronidase from wasp venom. Acta Crystallogr D Biol Crystallogr 2006;62(Pt 6):595–604. doi: 10.1107/S0907444906010687 16699186

25. Soldatova L, Kochoumian L, King TP. Sequence similarity of a hornet (D. maculata) venom allergen phospholipase A1 with mammalian lipases. FEBS Lett 1993;320(2):145–9. doi: 10.1016/0014-5793(93)80080-e 8458431

26. Hoffman DR, Sakell RH, Schmidt M. Sol i 1, the phospholipase allergen of imported fire ant venom. J Allergy Clin Immunol 2005 Mar;115(3):611–6. doi: 10.1016/j.jaci.2004.11.020 15753912

27. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994 Nov 11;22(22):4673–80. doi: 10.1093/nar/22.22.4673 7984417

28. Perez-Riverol A, Lasa AM, Dos Santos-Pinto JRA, Palma MS. Insect venom phospholipases A1 and A2: Roles in the envenoming process and allergy. Insect Biochem Mol Biol 2019 Feb;105:10–24. doi: 10.1016/j.ibmb.2018.12.011 30582958

29. Erwin EA, Custis NJ, Satinover SM, Perzanowski MS, Woodfolk JA, Crane J, et al. Quantitative measurement of IgE antibodies to purified allergens using streptavidin linked to a high-capacity solid phase. J Allergy Clin Immunol 2005 May;115(5):1029–35. doi: 10.1016/j.jaci.2004.12.1131 15867862

30. Goldberg A, Shefler I, Panasoff J, Paitan Y, Confino-Cohen R. Immunotherapy with commercial venoms is efficacious for anaphylactic reactions to Vespa orientalis stings. Int Arch Allergy Immunol 2013;161(2):174–80. doi: 10.1159/000345139 23363701

31. Kosnik M, Korosec P, Silar M, Music E, Erzen R. Wasp venom is appropriate for immunotherapy of patients with allergic reaction to the European hornet sting. Croat Med J 2002;43(1):25–7. 11828554

32. Macchia D, Cortellini G, Mauro M, Meucci E, Quercia O, Manfredi M, et al. Vespa crabro immunotherapy versus Vespula-venom immunotherapy in Vespa crabro allergy: a comparison study in field re-stings. World Allergy Organ J 2018;11(1):3. doi: 10.1186/s40413-018-0183-6 29441146

Článek vyšel v časopise


2020 Číslo 1
Nejčtenější tento týden