New insights into the tetraspanin Tspan5 using novel monoclonal antibodies

J Biol Chem. 2017 Jun 9;292(23):9551-9566. doi: 10.1074/jbc.M116.765669. Epub 2017 Apr 20.


Tspan5 is a member of a subgroup of tetraspanins referred to as TspanC8. These tetraspanins directly interact with the metalloprotease ADAM10, regulate its exit from the endoplasmic reticulum and subsequent trafficking, and differentially regulate its ability to cleave various substrates and activate Notch signaling. The study of Tspan5 has been limited by the lack of good antibodies. This study provides new insights into Tspan5 using new monoclonal antibodies (mAbs), including two mAbs recognizing both Tspan5 and the highly similar tetraspanin Tspan17. Using these mAbs, we show that endogenous Tspan5 associates with ADAM10 in human cell lines and in mouse tissues where it is the most abundant, such as the brain, the lung, the kidney, or the intestine. We also uncover two TspanC8-specific motifs in the large extracellular domain of Tspan5 that are important for ADAM10 interaction and exit from the endoplasmic reticulum. One of the anti-Tspan5 mAbs does not recognize Tspan5 associated with ADAM10, providing a convenient way to measure the fraction of Tspan5 not associated with ADAM10. This fraction is minor in the cell lines tested, and it increases upon transfection of cells with TspanC8 tetraspanins such as Tspan15 or Tspan33 that inhibit Notch signaling. Finally, two antibodies inhibit ligand-induced Notch signaling, and this effect is stronger in cells depleted of the TspanC8 tetraspanin Tspan14, further indicating that Tspan5 and Tspan14 can compensate for each other in Notch signaling.

Keywords: ADAM; ADAM10; Notch pathway; Tspan5; intracellular trafficking; metalloprotease; monoclonal antibody; tetraspanin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADAM10 Protein / genetics
  • ADAM10 Protein / immunology
  • ADAM10 Protein / metabolism
  • Amino Acid Motifs
  • Amyloid Precursor Protein Secretases / genetics
  • Amyloid Precursor Protein Secretases / immunology
  • Amyloid Precursor Protein Secretases / metabolism
  • Animals
  • Antibodies, Monoclonal, Murine-Derived / chemistry*
  • Antibodies, Monoclonal, Murine-Derived / immunology
  • Cell Line, Tumor
  • Endoplasmic Reticulum / genetics
  • Endoplasmic Reticulum / immunology
  • Endoplasmic Reticulum / metabolism*
  • Humans
  • Membrane Proteins / genetics
  • Membrane Proteins / immunology
  • Membrane Proteins / metabolism
  • Mice
  • Mice, Knockout
  • Protein Domains
  • Receptors, Notch / genetics
  • Receptors, Notch / immunology
  • Receptors, Notch / metabolism
  • Signal Transduction / physiology*
  • Tetraspanins / genetics
  • Tetraspanins / immunology
  • Tetraspanins / metabolism*


  • Antibodies, Monoclonal, Murine-Derived
  • Membrane Proteins
  • Receptors, Notch
  • TSPAN5 protein, human
  • Tetraspanins
  • Tspan5 protein, mouse
  • Amyloid Precursor Protein Secretases
  • ADAM10 Protein
  • ADAM10 protein, human
  • Adam10 protein, mouse