Cloning, structural modelling and characterization of VesT2s, a wasp venom hyaluronidase (HAase) from Vespa tropica

 

Prapenpuksiri Rungsa¹, Paroonkorn Incamnoi², Sophida Sukprasert³, Nunthawun Uawonggul⁴, Sompong Klaynongsruang¹, Jureerut Daduang⁵, Rina Patramanon¹, Sittiruk Roytrakul⁶, Sakda Daduange^1  7

 

1 Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand

2 Department of Chemistry, Faculty of Engineering, Rajamangala University of Technology Isan, Khon Kaen Campus, Khon Kaen, Thailand

3 Chulabhorn International College of Medicine, Thammasat University (Rangsit Campus), Pathumthani, Thailand

4 Division of Chemistry, Faculty of Science, Nakhon Phanom University, Nakhon Phanom, Thailand

5 Department of Clinical Chemistry, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand

6 Genome Institute, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency (NSTDA), Pathumthani, Thailand

7 Division of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand

 

ABSTRACT

 

Background:

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.

 

Methods:

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.

 

Results:

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.

 

Conclusions:

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.

 

Keyword: Wasp venom; Vespa tropica; Hyaluronidase (HAase)

 

Received: April 20, 2016.

Accepted: September 29, 2016.

 

Correspondence: sakdad@kku.ac.th

 

Authors' contributions

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.

 

Competing interests

The authors declare that there are no competing interests.