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

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Authors

  • Chek Ziu Koo
  • Justyna Szyroka
  • Alexandra L Matthews
  • Hung-En Hsia
  • Stephan A Müller
  • Johanna Tüshaus
  • Joelle Goulding
  • Katie Willis
  • Bethany Cragoe
  • Edward Davis
  • Murat Keles
  • Antonia Malinova
  • Thomas A McFarlane
  • Philip R Morrison
  • Hanh T H Nguyen
  • Michael C Sykes
  • Haroon Ahmed
  • Alessandro Di Maio
  • Lisa Seipold
  • Paul Saftig
  • Christos Pliotas
  • Eric Rubinstein
  • Stephen J Briddon
  • Nicholas D Holliday
  • Stefan F Lichtenthaler
  • Michael Tomlinson

Abstract

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.

Bibliographic note

Funding Information: This work was funded by a British Heart Foundation Ph.D. Studentship Grant FS/18/9/33388 and COMPARE (to C. Z. K.), Biotechnology and Biological Sciences Research Council Project Grant BB/P00783X/1 (to N. H.), British Heart Foundation Project Grant PG/13/92/30587 (to P. J. N.), Biotechnol-ogy and Biological Sciences Research Council Ph.D. Studentships (to J. S. and A. L. M.), and a Biochemical Society Summer Vacation Studentship (to H. T. H. N.). This work was also supported by the Deutsche Forschungsge-meinschaft (German Research Foundation) within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy, project ID 390857198) and by the BMBF through CLINSPECT-M (to S. F. L.). Further support was provided by Deutsche Forschungsgemeninschaft Grant DFG-SFB877-A3 (to P. S.). The authors declare that they have no conflicts of interest with the contents of this article.

Details

Original languageEnglish
Pages (from-to)12822-12839
Number of pages18
JournalThe Journal of biological chemistry
Volume295
Issue number36
Early online date28 Feb 2020
Publication statusPublished - 4 Sep 2020

Keywords

  • tetraspanin, a disintegrin and metalloprotease (ADAM), metalloproteinase, ADAM10, Tspan15, Tspan14, membrane protein, shedding, monoclonal antibody, molecular cell biology, ADAM, a disintegrin and metalloprotease