The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

Koo, Chek Ziu; Harrison, Neale; Noy, Peter J.; Szyroka, Justyna; Matthews, Alexandra L.; Hsia, Hung En; M�ller, Stephan A.; T�shaus, Johanna; Goulding, Joelle; Willis, Katie; Apicella, Clara; Cragoe, Bethany; Davis, Edward; Keles, Murat; Malinova, Antonia; McFarlane, Thomas A.; Morrison, Philip R.; Nguyen, Hanh T.H.; Sykes, Michael C.; Ahmed, Haroon; Di Maio, Alessandro; Seipold, Lisa; Saftig, Paul; Cull, Eleanor; Pliotas, Christos; Rubinstein, Eric; Poulter, Natalie S.; Briddon, Stephen J.; Holliday, Nicholas D.; Lichtenthaler, Stefan F.; Tomlinson, Michael G.

Abstract

© 2020 Koo et al. A disintegrin and metalloprotease 10 (ADAM10) is a transmembrane protein essential for embryonic development, and its dysregulation underlies disorders such as cancer, Alzheimer's disease, and inflammation. ADAM10 is a "molecular scissor" that proteolytically cleaves the extracellular region from >100 substrates, including Notch, amyloid precursor protein, cadherins, growth factors, and chemokines. ADAM10 has been recently proposed to function as six distinct scissors with different substrates, depending on its association with one of six regulatory tetraspanins, termed TspanC8s. However, it remains unclear to what degree ADAM10 function critically depends on a TspanC8 partner, and a lack of monoclonal antibodies specific for most TspanC8s has hindered investigation of this question. To address this knowledge gap, here we designed an immunogen to generate the first monoclonal antibodies targeting Tspan15, a model TspanC8. The immunogen was created in an ADAM10-knockout mouse cell line stably overexpressing human Tspan15, because we hypothesized that expression in this cell line would expose epitopes that are normally blocked by ADAM10. Following immunization of mice, this immunogen strategy generated four Tspan15 antibodies. Using these antibodies, we show that endogenous Tspan15 and ADAM10 co-localize on the cell surface, that ADAM10 is the principal Tspan15-interacting protein, that endogenous Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Furthermore, two of the four antibodies impaired ADAM10/Tspan15 activity. These findings suggest that Tspan15 directly interacts with ADAM10 in a functional scissor complex.

Citation

Koo, C. Z., Harrison, N., Noy, P. J., Szyroka, J., Matthews, A. L., Hsia, H. E., Müller, S. A., Tüshaus, J., Goulding, J., Willis, K., Apicella, C., Cragoe, B., Davis, E., Keles, M., Malinova, A., McFarlane, T. A., Morrison, P. R., Nguyen, H. T., Sykes, M. C., Ahmed, H., …Tomlinson, M. G. (2020). The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex. Journal of Biological Chemistry, 295(36), 12822-12839. https://doi.org/10.1074/jbc.RA120.012601

• Journal Article Type: Article
• Acceptance Date: Feb 28, 2020
• Online Publication Date: Feb 28, 2020
• Publication Date: Sep 4, 2020
• Deposit Date: Feb 29, 2020
• Publicly Available Date: Mar 4, 2020
• Journal: The Journal of biological chemistry
• Print ISSN: 0021-9258
• Electronic ISSN: 1083-351X
• Publisher: American Society for Biochemistry and Molecular Biology
• Peer Reviewed: Peer Reviewed
• Volume: 295
• Issue: 36
• Pages: 12822-12839
• DOI: https://doi.org/10.1074/jbc.RA120.012601
• Keywords: tetraspanin, a disintegrin and metalloprotease (ADAM), metalloproteinase, ADAM10, Tspan15, Tspan14, membrane protein, shedding, monoclonal antibody, molecular cell biology
• Public URL: https://nottingham-repository.worktribe.com/output/4064649
• Publisher URL: https://www.jbc.org/content/early/2020/02/28/jbc.RA120.012601
• Additional Information: This research was originally published in the Journal of Biological Chemistry. Chek Ziu Koo1,2*, Neale Harrison1*, Peter J. Noy1*, Justyna Szyroka1, Alexandra L. Matthews1, Hung-En Hsia3, Stephan A. Müller3, Johanna Tüshaus3, Joelle Goulding2,4, Katie Willis1, Clara Apicella1, Bethany Cragoe1, Edward Davis1, Murat Keles1, Antonia Malinova1, Thomas A. McFarlane1, Philip R. Morrison1, Hanh T.H. Nguyen1, Michael C. Sykes1, Haroon Ahmed1, Alessandro Di Maio1, Lisa Seipold5, Paul Saftig5, Eleanor Cull1, Christos Pliotas6, Eric Rubinstein7, Natalie S. Poulter2,8, Stephen J. Briddon2,4, Nicholas D. Holliday4, Stefan F. Lichtenthaler3 and Michael G. Tomlinson1. The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex . J Biol Chem. Year; Vol:pp-pp. © the authors