Tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

Abstract

1A disintegrin and metalloprotease 10 (ADAM10) is essential for embryonic development and 2 impacts on diseases such as cancer, Alzheimer's and inflammatory diseases. ADAM10 is a 3 'molecular scissor' that proteolytically cleaves the extracellular region from over 100 substrates, 4 including Notch, amyloid precursor protein, cadherins, growth factors and chemokines. 5 ADAM10 was recently proposed to function as six distinct scissors with different substrates, 6 depending on its association with one of six regulatory tetraspanins, termed TspanC8s. 7 However, it remains unclear to what degree ADAM10 function is critically dependent on a 8 TspanC8 partner. To address this, we generated the first monoclonal antibodies to Tspan15 as a 9 model TspanC8. These were used to show that ADAM10 is the principal Tspan15-interacting 10 protein, that Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a 11 synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Together these findings 12 suggest that ADAM10 exists as an intimate ADAM10/TspanC8 scissor complex. 13 14 15A disintegrin and metalloproteinase 10 (ADAM10) is a ubiquitously-expressed transmembrane 16 protein which acts as a 'molecular scissor' by cleaving the extracellular region from over 100 17 substrates, a process termed ectodomain shedding (Lichtenthaler et al. 2018). ADAM10 is 18 essential for embryonic development by activating Notch proteins which determine cell fate. 19 Other substrates include cadherin adhesion molecules, amyloid precursor protein, and 20 transmembrane growth factors and chemokines. As such, ADAM10 is important for health and 21 in diseases such as cancer, Alzheimer's and inflammatory diseases (Wetzel et al. 2017). 22 The tetraspanins are a superfamily of 33 transmembrane proteins in mammals which interact 23 with specific transmembrane partner proteins and regulate their intracellular trafficking, lateral 24 mobility and clustering at the cell surface (Termini and Gillette 2017; van Deventer et al. 2017). 25 42 Jouannet et al. 2016; Noy et al. 2016; Seipold et al. 2018). Tspan15 is also upregulated, and is a 43 marker of poor prognosis, in certain cancers (Zhang et al. 2018; Hiroshima et al. 2019; 44 Sidahmed-Adrar et al. 2019) and promotes cancer progression in a mouse model (Zhang et al. 45 2018). The aims of this study were to generate the first Tspan15 mAbs and to test three 46 hypotheses that would support the theory that Tspan15 and ADAM10 exist together as a scissor 47 complex: first, that ADAM10 is the principal Tspan15-interacting protein; second, that Tspan15 48 expression requires ADAM10; and third, that covalently linking Tspan15 and ADAM10 together 49 as a single fusion protein yields a functional scissor. 50 51 Results 52Generation of Tspan15 mAbs 53 The majority of anti-tetraspanin mAbs have epitopes within the large extracellular loop (LEL). 54 However, it has been traditionally difficult to make mAbs to many tetraspanins due to lack of 55 efficacy of recombinant LELs a...

TspanC8 tetraspanins differentially regulate ADAM10 endocytosis and half-life

“…The mAb directed to human ADAM10 (11G2), CD9 (TS9), CD81 (TS81), CD63 (TS63), and Tspan5 (TS5-2, TS5-3, and TS5-1r) were generated in our laboratory (Charrin et al, 2001; Saint-Pol et al, 2017a; Arduise et al, 2008). The mAbs to Tspan15 (5F4 (IgG2b) and 5D4 (IgG1)) are described in a separate publication (Koo et al, 2019 Preprint ). Labelling of antibodies with DyLight 650 or 550 N-Hydroxysuccinimide Esters was performed according to the manufacturer’s instructions (Thermo Fisher Scientific).…”

miR-27-3p Enhances the Sensitivity of Triple-Negative Breast Cancer Cells to the Antitumor Agent Olaparib by Targeting PSEN-1, the Catalytic Subunit of Γ-Secretase

“…In cancerous cells, the Notch pathway is activated via a two-step cleavage process in the presence of ligand-binding ( 21 ). ADAMs (a disintegrin and metalloproteinase), including ADAM17 and ADAM10, mediate the first cleavage of Notch, and γ-secretase mediates the second cleavage of Notch; finally, the notch intracellular domain (NICD) is released and translocated to the nucleus to mediate the transcription of certain genes related to drug resistance ( 22 , 23 ). These genes often encode cellular pro-survival, anti-apoptotic, and epithelial-mesenchymal transition-related factors ( 24 , 25 ).…”