The disintegrin metalloprotease TACE, also known as ADAM17, is an essential regulator of diverse pathways that include development, immunity, inflammation and cancer progression. It cleaves an array of more than 90 different substrates such as cytokines, growth factors and adhesion molecules. Even though TACE has been extensively studied, molecular mechanisms that govern cleavage of some substrates have still not been elucidated. One of such substrates is a transmembrane glycoprotein EpCAM, which was identified as a tumour-associated antigen due to its high expression level in rapidly growing epithelial tumours. Cleavage with TACE is the first step in a signalling pathway that leads to oncogene expression. In vivo studies in combination with mass spectrometry have shown that TACE cleaves EpCAM at two sites, which are located at the interface of the two subunits in a dimer - the native oligomeric state of EpCAM. The cleavage sites are thereby inaccessible to TACE. To better understand mechanisms by which TACE is able to cleave EpCAM we designed various full length and extracellular constructs of TACE and expressed them in a Sf9 insect cell line. We optimized the expression and purification methods for the construct containing the whole wild-type ectodomain (TACE-EX) and performed the first in vitro cleavage of different EpCAM ectodomain constructs. We were able to show that TACE is able to recognize and cleave EpCAM in vitro at proposed cleavage sites and that dimerization of EpCAM obstructs cleavage because of steric hindrance. This is also the first experimental proof of the hypothesis that EpCAM has to be in a monomeric state to be cleaved.
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