Saint-Pol Julien, Billard Martine, Dornier Emmanuel, Eschenbrenner Etienne, Danglot Lydia, Boucheix Claude, Charrin Stéphanie, Rubinstein Eric
From Inserm, U935, F-94807 Villejuif.
the Université Paris-Sud, Institut André Lwoff, F-94807 Villejuif.
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.
Tspan5是四跨膜蛋白亚组TspanC8的成员。这些四跨膜蛋白直接与金属蛋白酶ADAM10相互作用，调节其从内质网的输出及随后的运输，并差异调节其切割各种底物和激活Notch信号的能力。对Tspan5的研究因缺乏优质抗体而受到限制。本研究使用新的单克隆抗体（mAb）对Tspan5有了新的认识，其中包括两种能识别Tspan5和高度相似的四跨膜蛋白Tspan17的单克隆抗体。利用这些单克隆抗体，我们发现内源性Tspan5在人类细胞系和小鼠组织（如大脑、肺、肾脏或肠道，Tspan5在这些组织中最为丰富）中与ADAM10相关联。我们还在Tspan5的大细胞外结构域中发现了两个TspanC8特异性基序，它们对于ADAM10相互作用和从内质网输出很重要。一种抗Tspan5单克隆抗体不能识别与ADAM10相关的Tspan5，这为测量未与ADAM10相关的Tspan5比例提供了一种便捷方法。在测试的细胞系中，这一比例较小，在用抑制Notch信号的TspanC8四跨膜蛋白（如Tspan15或Tspan33）转染细胞后，该比例会增加。最后，两种抗体抑制配体诱导的Notch信号，并且在缺乏TspanC8四跨膜蛋白Tspan14的细胞中这种效应更强，这进一步表明Tspan5和Tspan14在Notch信号中可以相互补偿。
