Cloning, structural modelling and characterization of VesT2s, a wasp venom hyaluronidase (HAase) from Vespa tropica
© The Author(s). 2016
Received: 20 April 2016
Accepted: 29 September 2016
Published: 22 October 2016
Wasp venom is a complex mixture containing proteins, enzymes and small molecules, including some of the most dangerous allergens. The greater banded wasp (Vespa tropica) is well-known for its lethal venom, whose one of the major components is a hyaluronidase (HAase). It is believed that the high protein proportion and activity of this enzyme is responsible for the venom potency.
In the present study, cDNA cloning, sequencing and 3D-structure of Vespa tropica venom HAase were described. Anti-native HAase antibody was used for neutralization assay.
Two isoforms, VesT2a and VesT2b, were classified as members of the glycosidase hydrolase 56 family with high similarity (42–97 %) to the allergen venom HAase. VesT2a gene contained 1486 nucleotide residues encoding 357 amino acids whereas the VesT2b isoform consisted of 1411 residues encoding 356 amino acids. The mature VesT2a and VesT2b are similar in mass and pI after prediction. They are 39119.73 Da/pI 8.91 and 39571.5 Da/pI 9.38, respectively. Two catalytic residues in VesT2a, Asp107 and Glu109 were substituted in VesT2b by Asn, thus impeding enzymatic activity. The 3D-structure of the VesT2s isoform consisted of a central core (α/β)7 barrel and two disulfide bridges. The five putative glycosylation sites (Asn79, Asn99, Asn127, Asn187 and Asn325) of VesT2a and the three glycosylation sites (Asn1, Asn66 and Asn81) in VesT2b were predicted. An allergenic property significantly depends on the number of putative N-glycosylation sites. The anti-native HAase serum specifically recognized to venom HAase was able to neutralize toxicity of V. tropica venom. The ratio of venom antiserum was 1:12.
The wasp venom allergy is known to cause life-threatening and fatal IgE-mediated anaphylactic reactions in allergic individuals. Structural analysis was a helpful tool for prediction of allergenic properties including their cross reactivity among the vespid HAase.
Vespidae venom consists of complex mixtures of enzymes, proteins, peptides and small molecules responsible for many of the non-allergic and mild allergic reactions – such as local pain, inflammation and itching – as well as moderate and serious allergic reactions – such as anaphylaxis, and delayed hypersensitivity – including systemic toxic reactions, coagulopathy, acute renal failure and hepatotoxicity [1, 2]. Wasp venom contains many biological active compounds [3, 4]. The major allergens are phospholipase A1, hyaluronidase (HAase) and antigen 5 [5–8].
Venom HAase is an enzyme that hydrolyses hyaluronic acid (HA), one of the primary components of the extracellular matrix of vertebrates, which facilitates venom toxin diffusion into the tissue and blood circulation of the prey [9, 10]. HAase mainly acts as a “spreading factor” to enhance venom action. It has been identified in the venom of animals including snakes, bees, scorpions, fish, spiders, ants, wasps, caterpillars etc. [11–16]. Clinical studies have demonstrated that HAase is an “allergic factor” due to its ability to initiate pathogenic reactions in the majority of venom allergic patients [17–19]. It is also able to induce several anaphylactic IgE-mediated reactions in humans and has been suggested to be involved in the difficulties in the clinical diagnosis of venom allergic individuals [20–22]. The wasp venom HAase belongs to the hyaluronate glycanohydrolase family (EC 126.96.36.199), which degrades hyaluronic acid (HA) [23, 24]. Wasp venom HAase is responsible for the cross-reactivity of wasp and bee venom sera in patients as well [2, 25].
The greater banded wasp (Vespa tropica) is mostly distributed in the forest throughout Indochina peninsula including Thailand. It has a body length of up to 5 cm and its nest is usually found underground . V. tropica is among the most venomous known insects. The lethal dose of its pure venom in experimental animals (LD50 of approximately 2.8 mg/kg in mice) is more potent than that of V. affinis venom [26, 27]. The potency of V. tropica venom has been reported to nearly stop the end plate potentials of Drosophila larvae in nerve-muscle preparation in response to treatment with this venom . HAase was reported to be a major protein in V. tropica venom, where it is found by 2.5-fold the proportion observed in V. affinis venom . The understanding of HAase in terms of biochemical and structural characterization of these wasps is important for the development of new tools for treating multiple stings and for diagnosis and therapy of allergic reactions caused by this venom. Therefore, the present study aimed to characterize HAase isoforms in the venom of V. tropica by analyzing its sequence and 3D modelling.
The wasps were collected from Siang Sao Village, Sri Songkram district, Nakorn Panom Province, northeastern Thailand . The worker wasps were immediately shocked on ice. The venom reservoirs were removed from the sting apparatus by removing them from the bodies with forceps and squeezing. The droplets of venom and specimens of V. tropica were collected in a 1.5-mL microcentrifuge tube and then keep at −80 °C until use.
RT-PCR and rapid amplification of cDNA ends (5′ and 3′ RACE)
Primer design of gene-specific primers and PCR product size
Product size (bp)
Full nucleotide sequence active form
F4 GCCAGACTTTTCATGGAGGA (GSP1 for active)
R3 (7) ATCAGGGGTCAGTTCACGTC (GSP1 for active)
Adaptor primer (AP)
5′GGCCACGCGTCGACTAGTAC (T) 16
(GSP for cDNA synthesis of 3′ RACE system)
R4 (8) CGTCGGTCTCGGTAAGAAAA
Abridged universal amplification primer (AUAP)
R5 (9) GTTCTCGTGCATCGCTGTAA
VesT2a (F) NcoI
VesT2a (R) XhoI
Full nucleotide sequence inactive form
(GSP for RT-PCR inactive form)
R1 CATCTTGTCGTTCTCGCTCA (GSP for RT-PCR inactive form)
F2 CTTCGGCGTCTATTTCAAGG (GSP for RT-PCR inactive form)
R2CCGCTAAGACAGTGGGGATA (GSP for inactive form)
Adaptor primer (AP)
5′GGCCACGCGTCGACTAGTAC (T) 16
(GSP for cDNA synthesis of 3′ RACE system)
R2 (1) CATCTTGTCGTTCTCGCTCA (GSP for RT-PCR inactive form)
Abridged universal amplification primer (AUAP)
R1 (2) CCGCTAAGACAGTGGGGATA (GSP for inactive form)
Sequence analysis and structure modelling
The basic characterizations of the gene and protein sequences were analyzed using NCBI (http://www.ncbi.nlm.nih.gov/Database/index.html) and the basic local alignment search tool BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). The phylogenic tree was created using CLUSTAL-X software analysis using the Neighbour-Joining method . The three-dimensional models were created using the SWISS-MODEL program, the automated protein homology modelling template at ExPASY (Switzerland) and a template search with the Alignment Mode program from the protein database (http://swissmodel.expasy.org/) [31, 32]. The model was elucidated as a PDB file, and the structure was previewed and analyzed using Swiss-Pdb Viewer Deep View v4 software (http://www.expasy.org/). The molecular mass and isoelectric points were computed using the Compute pI/MW tool of ExPASy Bioinformatics (http://web.expasy.org/compute_pi/). The N-glycosylation sites were predicted using the CBS prediction severs (http://www.cbs.dtu.dk/services/NetNGlyc/) and compared with other wasp and bee venom HAases.
Zymographic HAase activity assay
The V. tropica venom HAase activity was detected using 10 % SDS-PAGE containing hyaluronic acid as a substrate. Proteins were separated at 15 mA. The gel was incubated in 3 % Triton X-100 for 1 h with agitation in order to remove SDS and then transferred to the HAase assay buffer (0.15 M NaCl in 0.1 M formate buffer), rinsed twice with assay buffer, and then incubated on a rotating shaker for 16 h at 37 °C. The gels were rinsed twice with distilled water and stained in 0.5 % Alcian blue solution for 1 h. The destain was performed with 7 % acetic acid that was changed every 1 h until clear bands appeared on a pale blue background .
Turbidity HAase activity assay
The turbidity HAase method followed the one by Pukrittayakamee et al.  with slight modifications. We mixed 0.5 mg/mL HA and buffer containing 0.15 M NaCl to a final volume of 100 μL and incubated for 30 min at 37 °C. The reaction was stopped using 200 μL of 2 % CTAB containing 2.5 % NaOH. The absorbance was measured at 405 nm. The turbidity reducing activity was expressed as the percentage of remaining HA by taking the absorbance of the tube at 100 % in which no enzyme was added. The optimal pH of the venom HAase was determined by changing the buffers of the enzymatic turbidimetric venom HAase activity assay as follows: 0.2 M formate buffer, pH 2–4; 0.2 M acetate buffer, pH 5–6; 0.2 M Tris–HCl buffer, pH 7–10.
Mouse anti-hyaluronidase serum
The HAase band from zymographic gel were cut and frozen at −70 °C overnight, the gel was freeze-dried and ground. Anesthetized mice were subcutaneously immunized with gel swollen in PBS buffer (135 mM NaCl, 1.5 mM KH2PO4, 2.5 mM KCl, and 8 mM Na2HPO4) emulsified with Freud’s complete adjuvant. Mice were four times boosted with the antigen emulsified with incomplete Freund’s adjuvant. After retro-orbital plexus bleeding, blood was kept at 4 °C for 12 h and centrifuged at 10000 × g for antiserum collection.
Proteins were separated by SDS-PAGE and blotted onto a nitrocellulose membrane (Bio-Rad, USA). After being eletrotransferred, the membrane was incubated with 5 % nonfat dry milk for 1 h, anti-HAase antibody for 1 h and goat anti-mouse IgG linked alkaline phosphatase (1:500) for 1 h. The blotted bands were detected by a substrate kit (GE Healthcare, Sweden). The membrane was intensive washed before the next incubation.
Crickets (Gryllus sp.) were abdominally injected with venom pre-incubated with anti-HAase serum 10 min before considered paralyzed. The paralyzed crickets were defined as those that could return from the overturned position.
Sequence and structural modelling analysis of VesT2s
HAase activity of wasp venom VesT2a
The neutralization assay of V. tropica venom against anti-HAase serum in crickets (Gryllus sp.)
V. tropica venom: Anti-HAase serum (μL/μL)
Neutralized crickets/total crickets after injections with V. tropica venom and anti-HAase serum
In this study, we described the identification, biochemistry, bioactivity and structural characteristics of the HAase from the venom of greater banded wasp V. tropica. This study describes the existence of two isoforms of VesT2s, VesT2a and VesT2b. The primary sequence of VesT2a and VesT2b were clearly isoenzymes with 61.52 % similarity but with different molecular masses and pIs of the mature sequence (357 amino acids/39119.73 Da/pI 8.91 and 337 amino acids/39571.53 Da/pI 9.38, respectively). Mass differences were mainly estimated from amino acid variations, including the degree of glycosylation of VesT2s. However, they were classified into the same family of glycoside hydrolase family 56 by sequence similarity. This phenomenon also occurs with HAases in many species, such asVesV2a and VesV2b, the HAase isoenzymes in Vespula vugaris venom. VesV2a and b share 58 % amino acid identity to each other [5, 20].
Rungsa et al.  indicated that the mass of HAase in V. tropica venom was approximately 43 kDa after analysis by denaturing two-dimensional electrophoresis, which was confirmed by peptide mass fingerprinting. However, the mature sequence of HAase in this study, VesT2s, was smaller in size, with approximately 39 kDa. The molecular mass of about 43 kDa of native VesT2s was not surprising, since wasp venom HAase is a glycoprotein whose differences in estimated values of theoretical pI and molecular masses are frequent [9, 38, 39].
The phylogenetic tree demonstrated that VesT2a is found in the same cluster of active HAase from insect venoms. VesT2b is also found in a cluster of inactive HAase from insect venoms [2, 20, 35, 38, 40]. The enzyme function of VesT2s is different because of two catalytic residues in VesT2a, Asp107 and Glu109. Both are substituted by Asn in VesT2b that has no HAase enzymatic activity towards various substrates [20, 35, 41]. The less acidic Asn cannot act as a proton donor as the acidic amino acids, Asp and Glu [36, 37].
Glycosylation sites are the most common post-translational modification of many insect venom proteins as they contribute to biological activity, immunogenicity, and solubility, stability and protease resistance. VesT2s represents one of the strongest conserved hymenoptera venom allergens in wasps, yellow jackets and honeybees [42, 43]. VesT2a is highly similar to VesMa2 (Vespa magnifica HAase) while VesT2b is close to VesV2b (Vespula vugaris HAase b). V. vugaris and V. magnifica also belong to the Vespidae family [20, 35, 40]. Therefore, we presume that the VesT2s isoform might have a similar structure and allergic properties.
N-glycosylation in wasp venom HAase. Asn-Xaa-Ser/Thr residues represent the possible N-glycosylation sites predicted by NetNGlyc 1.0 Server (N-glycosylation in V. vulgaris and V. magnifica HAase was obtained in the experiment in the native form)
A previous study showed the high potency of V. tropica venom (PD50 ~ 3 μg/g body weight of cricket) . Venom HAase, a “spreading factor”, is well-known for its toxin-enhancing activity. Therefore, the anti-HAase serum was produced. The anti-HAase serum shows neutralizing efficiency against crude venom by ratio the ratio of 1:12 (venom:antiserum). Inhibition of HAase activity not only prevents local tissue damage, but also retards the venom toxin diffusion into the tissues and blood circulation, resulting in the delay of fatal outcomes in several cases . HAase activity may play a vital role in allergenicity and toxicity of venoms.
Hymenoptera venom showed cross-reactivity with bee and wasp venoms . The allergic responses to wasp venom are known to cause life-threatening and fatal IgE-mediated anaphylactic reactions in sensitive individuals. The cross reactivity among the hyaluronidase from yellow jacket and bee venom are presumably induced by CCDs, but less often shared by peptide epitopes . Knowledge on the structural determinants responsible for the allergic potency is expected to have important clinical implications.
This work was mainly supported by the Higher Education Research Promotion and National Research University (NRU) Project of Thailand, Office of the Higher Education Commission (CHE), through the Food and Functional Food Research Cluster of Khon Kaen University (KKU). It was also partially supported by the “The Thailand Research Fund – Master Research Granted (TRF–MAG)” year 2008 (MRG-WII515S069), “TRF–CHE jointly funded Research Grant for Mid-Career University Faculty”, fiscal years 2007–2009; and KKU Research Fund, fiscal years 2007–2010.
PR conducted most of the experiments, coordinated the data analysis and drafted the manuscript. PI and SS contributed to bioinformatics analyses. NU conducted Western blotting experiments. SK contributed to the study design and writing of the manuscript. JD performed the molecular analyses and contributed to the writing of the manuscript. RP contributed to writing and editing of the manuscript. SR performed the proteomic study. SD designed the research and the experiments, coordinated the study, wrote and edited the manuscript. All authors read and approved the final manuscript.
The authors declare that there are no competing interests.
Ethics approval and consent to participate
The present study was approved by the Animal Ethics Committee of Khon Kaen University based on the Ethics for Animal Experimentation of the National Research Council of Thailand (reference. 05188.8.131.52/1).
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