Characterisation of the human tetraspanin protein Tspan2

The tetraspanins are a superfamily of integral membrane proteins characterised by 4 ‘crosses’ of the plasma membrane. Although they have been implicated in a variety of key cellular processes and diseases, it has been challenging to characterise their precise functions. Specific antibodies that recognise the native molecule, and the soluble large extracellular loop (EC2) of tetraspanins, are important tools in tetraspanin research. Such tools can be exploited not only in the study of the cancer metastatic properties of the tetraspanins, but also in the biology multinucleated giant cell (MNGC) and bacterial pathogenicity and spread from cell to cell. To characterise the tetraspanin Tspan2 protein, one of the less well-characterised members of tetraspanins, we have followed a novel strategy to generate monoclonal antibodies (mAbs) in which BALB/c mice were immunised with mouse myeloma NS0 cells expressing the full-length Tspan2-GFP. In order to facilitate a rapid and efficient screening for hybridomas secreting the desired monoclonal antibody (mAb), we have expressed the soluble EC2 as a GST fusion protein in a bacterial expression system, for use in a high-throughput ELISA screening. This was followed by testing the hybridoma supernatants against the native Tspan2 expressed on the cell surface by flow cytometry. The recombinant EC2 developed in this work is biologically active in a variety of functional assays, and its identity has been validated by western blotting (WB) and mass spectrometry. The generated ED5 mAb bound to the surface of Tspan2-GFP transfected NS0 and other cell lines that are known to express high levels of Tspan2, indicating that ED5 mAb is able to recognise the native form of Tspan2 protein. ED5 mAb also showed strong binding against the soluble Tspan2EC2-GST in ELISA and the denatured Tspan2 protein in western blotting and immunohistochemistry. Furthermore, we tested the effect of anti-Tspan2 mAb on the adhesion of several human cancer cell lines and the data showed a significant decrease in the adhesion ability for cells pre-treated with the antibody. This suggests a role for Tspan2 in cancer cell adhesion. Tspan2-GFP is mainly localised on the cell membrane, and co-localised with PDI on the ER and with LAMP-1 in endocytic vesicles. Also, the internalisation of the cell surface Tspan2 occurred very rapidly and was inhibited by up to 50% using the dynamin-inhibitor agent Dynasore. This indicates that Tspan2 might be localised on the endosomes and multivesicular bodies (MVBs) and potentially contributes to the extracellular vesicle (EV) biogenesis and secretion. To test this, we quantified the EVs secreted from cells overexpressing Tspan2 or cells treated with siRNA against Tspan2, and the data showed a significant increase or decrease in the EV number, respectively. This suggests a role for Tspan2 in EV production. Anti-Tspan2, the soluble EC2, overexpression and downregulation of Tspan2 showed a role in human lung epithelial giant cell (HLEGC) formation induced by Burkholderia thailandensis CDC272, and the infection by the same bacteria. HLEGC formation and the bacterial uptake and intracellular replication increased with cells overexpressing Tspan2 and decreased with cells treated with Tspan2 siRNA, Tspan2EC2-GST or anti-Tspan2 mAb. In the future, anti-Tspan2 mAb would be extremely useful to perform further functional studies to characterise Tspan2 in health and disease.