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

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 investigating 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.

Analysis of the Conditions That Affect the Selective Processing of Endogenous Notch1 by ADAM10 and ADAM17

“…One potential explanation for this could be that the Notch1 receptor exists in complexes in the cell membrane so that it is available for processing by ADAM10 but inaccessible to ADAM17. Evidence in the literature suggests that the TspanC8 family of tetraspanins, which have been shown to be required for the proper maturation and trafficking of ADAM10, might be candidate ADAM10-Notch1 complex partners [ 68 , 69 , 70 , 71 ]. In fact, work conducted in Drosophila suggests that silencing TspanC8 family members results in decreased Notch activity [ 69 ].…”

“…Our data showing that the exposed S2 site is a preferred ADAM17 cleavage site provides a plausible explanation for this finding. On the other hand, the genetic and cell biological studies that point to ADAM10 as the relevant protease in ligand-dependent Notch signaling could be explained by a growing literature that suggests that ADAM10, gamma-secretase, tetraspanins, and perhaps even Notch may be found together in membrane complexes [ 69 , 70 , 71 , 72 ]. It is also possible that the conformational change of the NRR induced by ligand binding is smaller than following treatment with EDTA, which could conceivably only provide access to the S2 cleavage site to ADAM10, but not to ADAM17.…”

The roles of tetraspanins in bacterial infections

“…In fact, S. aureus secretes an α‐toxin that interacts with and activates the metalloprotease ADAM10, which has been shown to be a receptor for bacteria α‐toxin (Figure 2J) (Virreira Winter, Zychlinsky, & Bardoel, 2016). Consequently, ADAM10 cleaves cadherins present on epithelial and endothelial cells, thus disrupting the barrier and leading to tissue damage and spread of the bacteria (Figure 2J) (Koo et al, 2020).…”

Tspan18 is a novel regulator of thrombo-inflammation

“…We have proposed a 'six scissor' hypothesis, which postulates that ADAM10 is not one scissor, but six different scissors with different substrates depending on which of the six regulatory TspanC8s it is associated with [20][21][22]. Substantial recent evidence supports this hypothesis [24][25][26][27][28][29][30][31][32].…”