Regulated Intramembrane Proteolysis and Degradation of Murine Epithelial Cell Adhesion Molecule mEpCAM

Abstract

Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is highly and frequently expressed in carcinomas and (cancer-)stem cells, and which plays an important role in the regulation of stem cell pluripotency. We show here that murine EpCAM (mEpCAM) is subject to regulated intramembrane proteolysis in various cells including embryonic stem cells and teratocarcinomas. As shown with ectopically expressed EpCAM variants, cleavages occur at α-, β-, γ-, and ε-sites to generate soluble ectodomains, soluble Aβ-like-, and intracellular fragments termed mEpEX, mEp-β, and mEpICD, respectively. Proteolytic sites in the extracellular part of mEpCAM were mapped using mass spectrometry and represent cleavages at the α- and β-sites by metalloproteases and the b-secretase BACE1, respectively. Resulting C-terminal fragments (CTF) are further processed to soluble Aβ-like fragments mEp-β and cytoplasmic mEpICD variants by the g-secretase complex. Noteworthy, cytoplasmic mEpICD fragments were subject to efficient degradation in a proteasome-dependent manner. In addition the γ-secretase complex dependent cleavage of EpCAM CTF liberates different EpICDs with different stabilities towards proteasomal degradation. Generation of CTF and EpICD fragments and the degradation of hEpICD via the proteasome were similarly demonstrated for the human EpCAM ortholog. Additional EpCAM orthologs have been unequivocally identified in silico in 52 species. Sequence comparisons across species disclosed highest homology of BACE1 cleavage sites and in presenilin-dependent γ-cleavage sites, whereas strongest heterogeneity was observed in metalloprotease cleavage sites. In summary, EpCAM is a highly conserved protein present in fishes, amphibians, reptiles, birds, marsupials, and placental mammals, and is subject to shedding, γ-secretase-dependent regulated intramembrane proteolysis, and proteasome-mediated degradation.

Cleavage and Cell Adhesion Properties of Human Epithelial Cell Adhesion Molecule (HEPCAM)

“…Regulated intramembrane proteolysis of mouse Epcam at pH 4.5 resulted in complete cleavage of full-length protein, which was not the case at pH 7 (31). Although cleavage at pH 4.5 appeared total, cleavage at pH 7 affected ϳ20% of molecules, which is in line with results presented here for human EpCAM.…”

“…Studies of cleavage of murine Epcam demonstrated variable cleavages and degradation of the protein (31). To delineate exact cleavage sites and study their availability, we applied biochemical and mass spectrometry analysis on recombinant HEPCAM variants in combination with structure-based approaches.…”

Crystal structure and its bearing towards an understanding of key biological functions of EpCAM

“…3b), and several sites within the TM region including the g 2 -and g 1 -sites Ala270-Val271 and Val273-Val274, respectively (Fig. 4c) 47 . The a-cleavage site within the RCD would imply that the protease active site is at least 5 nm from the cell surface as inferred from the EpEX dimensions or is a result of proteolytic action of a protease at the surface of a neighbouring cell or even a soluble protease.…”

“…While the a-site appears accessible in the cis-dimer, the b-cleavage within bC suggests a dynamic equilibrium between EpCAM monomer and cis-dimer (discussed above) since in the cis-dimer this cleavage site is inaccessible. It is tempting to speculate that the b-cleavage is triggered by a local drop of pH since this both destabilizes the EpEX cis-dimer and at the same time enhances the activity of the BACE1 protease involved in the cleavage 47,48 . However, both cleavages within the CD as identified using murine EpCAM seem to be incompatible with the model where the formation of trans-intercellular contacts triggers the signalling-associated cleavage.…”

A high-content screen for small-molecule regulators of epithelial cell-adhesion molecule (EpCAM) cleavage yields a robust inhibitor

“…These assays were performed as previously described (50,68,69). Briefly, the cells were treated with 20 M compound or inhibitor overnight in assay buffer (150 mM sodium citrate, pH 6.4, 10 M ZnCl 2 , protease inhibitor).…”