Hyaluronidase From The Venom Of The Social Wasp Polybia Paulista (hymenoptera, Vespidae): Cloning, Structural Modeling, Purification, And Immunological Analysis

In this study, we describe the cDNA cloning, sequencing, and 3-D structure of the allergen hyaluronidase from Polybia paulista venom (Pp-Hyal). Using a proteomic approach, the native form of Pp-Hyal was purified to homogeneity and used to produce a Pp-specific polyclonal antibody. The results revealed that Pp-Hyal can be classified as a glycosyl hydrolase and that the full-length Pp-Hyal cDNA (1315 bp; GI: 302201582) is similar (80-90%) to hyaluronidase from the venoms of endemic Northern wasp species. The isolated mature protein is comprised of 338 amino acids, with a theoretical pI of 8.77 and a molecular mass of 39,648.8 Da versus a pI of 8.13 and 43,277.0 Da indicated by MS. The Pp-Hyal 3D-structural model revealed a central core (α/β)7 barrel, two sulfide bonds (Cys 19-308 and Cys 185-197), and three putative glycosylation sites (Asn79, Asn187, and Asn325), two of which are also found in the rVes v 2 protein. Based on the model, residues Ser299, Asp107, and Glu109 interact with the substrate and potential epitopes (five conformational and seven linear) located at surface-exposed regions of the structure. Purified native Pp-Hyal showed high similarity (97%) with hyaluronidase from Polistes annularis venom (Q9U6V9). Immunoblotting analysis confirmed the specificity of the Pp-Hyal-specific antibody as it recognized the Pp-Hyal protein in both the purified fraction and P. paulista crude venom. No reaction was observed with the venoms of Apis mellifera, Solenopsis invicta, Agelaia pallipes pallipes, and Polistes lanio lanio, with the exception of immune cross-reactivity with venoms of the genus Polybia (sericea and ignobilis). Our results demonstrate cross-reactivity only between wasp venoms from the genus Polybia. The absence of cross-reactivity between the venoms of wasps and bees observed here is important because it allows identification of the insect responsible for sensitization, or at least of the phylogenetically closest insect, in order to facilitate effective immunotherapy in allergic patients. © 2013 Elsevier Ltd.647080Al-Ghouleh, A., Johal, R., Sharquie, I.K., Emara, M., Harrington, H., Shakib, F., Ghaemmaghami, A.M., The glycosylation pattern of common allergens: the recognition and uptake of Der p 1 by epithelial and dendritic cells is carbohydrate dependent (2012) PLoS ONE, 7 (3), pp. e33929Barbaro, K.C., Knysak, I., Martins, R., Hogan, C., Winkel, K., Enzymatic characterization, antigenic cross-reactivity and neutralization of dermonecrotic activity of five loxosceles spider venoms of medical importance in the Americas (2005) Toxicon, 45, pp. 489-499Cevallos, M.A., Navarro-Duque, C., Varela-Julia, M., Alagon, A.C., Molecular mass determination and assay of venom hyaluronidases by sodium dodecyl sulfate-polyacrilamide gel electrophoresis (1992) Toxicon, 30, pp. 925-930Cherr, G.N., Meyers, S.A., Yudin, A.I., Van de Voort, C.A., Myles, D.G., Primakoff, P., Overstreet, J.W., The PH-20 protein in Cynomolgus macaque spermatozoa: identification of two different forms exhibiting hyaluronidase activity (1996) Dev. Biol., 175, pp. 142-153Cramer, J.A., Bailey, L.C., Bailey, C.A., Miller, R.T., Kinetic and mechanistic studies with bovine testicular hyaluronidase (1994) Biochim. Biophys. Acta B, 1200, pp. 315-321Csóka, A.B., Scherer, S.W., Stern, R., Expression analysis of six paralogous human hyaluronidase genes clustered on chromosomes 3p21 and 7q31 (1999) Genomics, 60, pp. 356-361Delano, W.L., (2002) The PyMOL Molecular Graphics System DeLano Scientific, , Palo Alto, CaliforniaEberlein, B., Krischan, L., Darsow, U., Ollert, M., Ring, J., Double positivity to bee and wasp venom: improved diagnostic procedure by recombinant allergen-based IgE testing and basophil activation test including data about cross-reactive carbohydrate determinants (2012) J. Allergy Clin. Immunol., 130, pp. 155-161Fiszer-Szafarz, B., Hyaluronidase polymorphism detected by polyacrylamide gel electrophoresis. Application to hyaluronidases from bacteria, slime molds, bee and snake venoms, bovine testes, rat liver lysosomes, and human serum (1984) Anal. Biochem., 143, pp. 76-81Fiszer-Szafarz, B., Szafarz, D., Vannier, P., Polymorphism of hyaluronidase in serum from man, various mouse strains and other vertebrate species revealed by electrophoresis (1990) Biol. Cell., 68, pp. 95-100Gmachl, M., Kreil, G., Bee venom hyaluronidase is homologous to a membrane protein of mammalian sperm (1993) Proc. Natl. Acad. Sci. U. S. A., 90, pp. 3569-3573Hemmer, W., Cross-reactivity to honeybee and wasp venom (2008) Hautarzt, 59, pp. 194-199Henrissat, B., Bairoch, A., Updating the sequence-based classification of glycosyl hidrolases (1996) Biochem. J., 316, pp. 695-696Hotez, P., Cappello, M., Hawdon, J., Beckers, C., Sakanari, J., Hyaluronidases of the gastrointestinal invasive nematodes Ancylostoma conium and Anisakissimplex: possible functions in the pathogenesis of human zoonoses (1994) J. Infect. Dis., 170, pp. 918-926Jin, C., Hantusch, B., Hemmer, W., Stadlmann, J., Altmann, F., Affinity of IgE and IgG against cross-reactive carbohydrate determinants on plant and insect glycoproteins (2008) J. Allergy Clin. Immunol., 121, pp. 185-190Jin, C., Focke, M., Léonard, R., Jarisch, R., Altmann, F., Hemmer, W., Reassessing the role of hyaluronidase in yellow jacket venom allergy (2010) J. Allergy Clin. Immunol., 125, pp. 184-190e1Kemeny, D.M., Dalton, N., Lawrence, A.J., Pearce, F.L., Vernon, C.A., The purification and characterization of hyaluronidase from the venom of the honey bee, Apis mellijera (1984) Eur. J. Biochem., 139, pp. 217-233Kemparaju, K., Girish, K.S., Snake venom hyaluronidase: a therapeutic target (2006) Cell Biochem. Funct., 24, pp. 7-12Kolarich, D., Altmann, F., N-glycan analysis by matrix-assisted laser desorption/ionization mass spectrometry of electrophoretically separated nonmammalian proteins: application to peanut allergen Ara h 1 and olive pollen allergen Ole e 1 (2000) Anal. Biochem., 285, pp. 64-75Kolarich, D., Léonard, R., Hemmer, W., Altman, F., The N-glycans of yellow jacket venom hyaluronidases and the protein sequence of its major isoform in Vespula vulgaris (2005) FEBS J., 272, pp. 5182-5190Kreil, G., Hyaluronidases - a group of neglected enzymes (1995) Protein Sci., 4, pp. 1666-1669Kubelka, V., Altmann, F., März, L., The asparagine-linked carbohydrate of honeybee venom hyaluronidase (1995) Glycoconj. J., 12, pp. 77-83Kulkarni-Kale, U., Bhosle, S., Kolaskar, A.S., CEP: a conformational epitope prediction server (2005) Nucleic Acids Res., 33, pp. 168-171Laskowski, R.A., MacArthur, M.W., Moss, D.S., Thornton, J.M., PROCHECK: a program to check the stereochemical quality of protein structures (1993) J. Appl. Cryst., 26, pp. 238-291Laurent, T.C., The Biology of Hyaluronan (1989) Ciba Foundation Symp. 143, pp. 1-298. , John Wiley & Sons, New YorkLong-Rowe, K.O., Burnett, J.W., Characteristics of hyaluronidase and hemolytic activity in fishing tentacle nematocyst venom of Chrysaora quinquecirrha (1994) Toxicon, 32, pp. 165-174Lu, G., Kochoumian, L., King, T.P., Sequence identity and antigenic crossreactivity of hite face hornet venom allergen, also a hyaluronidase, with other proteins (1995) J. Biol. Chem., 270, pp. 4457-4465Markovic-Housley, Z., Miglierini, G., Soldatova, L., Rizkallah, P.J., Muller, U., Schirmer, T., Crystal structure of hyaluronidase, a major allergen of bee venom (2000) Structure, 8, pp. 1025-1035McConkey, B.J., Sobolev, V., Edelman, M., Quantification of protein surfaces, volumes and atom-atom contacts using a constrained Voronoi procedure (2002) Bioinformatics, 18, pp. 1365-1373Meyer, K., Hyaluronidases (1971) The Enzymes, 5, pp. 307-320. , Academic Press, New YorkPalma, M.S., Insect venom peptides (2006) Handbook of Biologically Active Peptides, pp. 389-396. , Academic Press, Burlington, A.J. Kastin (Ed.)Pinto, J.R., Santos, L.D., Arcuri, H.A., Dias, N.B., Palma, M.S., Proteomic characterization of the hyaluronidase (E.C. 3.2.1.35) from the venom of the social wasp Polybia paulista (2012) Protein Pep. Lett., 19, pp. 624-634Senff-Ribeiro, A., Henrique Da Silva, P., Chaim, O.M., Gremski, L.H., Paludo, K.S., Bertoni Da Silveira, R., Gremski, W., Veiga, S.S., Biotechnological applications of brown spider (Loxosceles genus) venom toxins (2008) Biotechnol. Adv., 26, pp. 210-218Sanchez, R., Sali, A., Advances in comparative protein-structure modeling (1997) Curr. Opin. Struct. Biol., 7, pp. 206-214Santos, L.D., Santos, K.S., Pinto, J.R., Dias, N.B., Souza, B.M., Santos, M.F., Perales, J., Palma, M.S., Profiling the proteome of the venom from the social wasp Polybia paulista: a clue to understand the envenoming mechanism (2010) J. Proteome Res., 9, pp. 3867-3877Sedmak, J.J., Grossberg, S.E., A rapid, sensitive and versatile assay for protein using Coomassie brilliant blue G250 (1977) Anal. Biochem., 79, pp. 544-552Seo, J., Lee, K.J., Post-translational modifications and their biological functions: proteomic analysis and systematic approaches (2004) J. Biochem. Mol. Biol., 37, pp. 35-44Silva, G.P., Brochetto-Braga, M.R., Ruberti, M., Ternero, M.L., Gobbi, N., A comparative study of protein and enzymatic activity in venoms of some common wasps (hymenoptera: vespidae) from São Paulo State (2004) Sociobiology, 44, pp. 271-282Skov, L.K., Seppala, U., Coen, J.J., Crickmore, N., King, T.P., Monsalve, R., Kastrup, J.S., Gajhede, M., Structure of recombinant Ves v 2 at 2.0 angstrom resolution: structural analysis of an allergenic hyaluronidase from wasp venom (2006) Acta Crystallogr. D. Biol. Crystallogr., 62, pp. 595-604Steinberg, T.H., Pretty On Top, K., Berggren, K.N., Kemper, C., Jones, L., Diwu, Z., Haugland, R.P., Patton, W.F., Rapid and simple single nanogram detection of glycoproteins in polyacrylamide gels and on electroblots (2001) Proteomics, 7, pp. 841-855Stern, R., Jedrzejas, M.J., Hyaluronidases: their genomics, structures, and mechanisms of action (2006) Chem. Rev., 106, pp. 818-839Takagaki, K., Nakamura, T., Izumi, J., Saitoh, H., Endo, M., Kojima, K., Kato, I., Majima, M., Characterization of hydrolysis and transglycosylation by testicular hyaluronidase using ion-spray mass spectroscopy (1994) Biochemistry, 33, pp. 6503-6507Wright, R.P., Elgert, K.D., Campbell, B.J., Barret, J.T., Hyaluronidase and esterase activities of the venom of the poisonous brown recluse spider (1973) Arch. Biochem. Biophys., 159, pp. 415-42

Standard methods for <em>Apis mellifera</em> venom research.

Honey bees have a sting which allows them to inject venomous substances into the body of an opponent or attacker. As the sting originates from a modified ovipositor, it only occurs in the female insect, and this is a defining feature of the bee species that belong to a subclade of the Hymenoptera called Aculeata. There is considerable interest in bee venom research, primarily because of an important subset of the human population who will develop a sometimes life threatening allergic response after a bee sting. However, the use of honey bee venom goes much further, with alleged healing properties in ancient therapies and recent research. The present paper aims to standardize selected methods for honey bee venom research. It covers different methods of venom collection, characterization and storage. Much attention was also addressed to the determination of the biological activity of the venom and its use in the context of biomedical research, more specifically venom allergy. Finally, the procedure for the assignment of new venom allergens has been presented. Las abejas meliferas tienen un aguijon que les permite inyectar sustancias venenosas en el cuerpo de un oponente o atacante. El aguijon es un ovipositor modificado que solo se manifiesta en el insecto hembra, siendo este una caracteristica que define a las especies de abejas que pertenecen al subclado de himenopteros llamada Aculeata. Hay un interes considerable en la investigacion del veneno de abeja, principalmente debido a que un porcentaje importante de la poblacion humana desarrollara una respuesta alergica - a veces mortal - a la picadura de abeja. Sin embargo, el uso del veneno de la abeja melifera abarca mucho mas, con presuntas propiedades curativas en terapias antiguas e investigaciones recientes. El presente trabajo tiene como objetivo estandarizar metodos seleccionados para la investigacion del veneno de las abejas meliferas. Cubre diferentes metodos de recoleccion, caracterizacion y almacenamiento de veneno. Tambien se presto mucha atencion a la determinacion de la actividad biologica del veneno y su uso en el contexto de la investigacion biomedica, mas especificamente la alergia al veneno. Finalmente, se ha presentado el procedimiento para la asignacion de nuevos alergenos de veneno