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.
Highlights
Regulated intramembrane proteolysis (RIP) is an important means of regulation for a growing number of transmembrane proteins [1,2]
Small amounts of the C-terminal fragment mCTF-yellow fluorescent protein (YFP) were present at the initial time point, which might reflect the overall status of murine EpCAM (mEpCAM) cleavage at the time of membrane isolation
Regulated intramembrane proteolysis of EpCAM was first described in human carcinoma cells and after ectopic expression in Human embryonic kidney 293 cells (HEK293) cells [10]
Summary
Regulated intramembrane proteolysis (RIP) is an important means of regulation for a growing number of transmembrane proteins [1,2]. Through the function of various sheddases and the multiprotein c-secretase complex comprised of minimally presenilin, APH-1, PEN-2, and nicastrin, RIP substrates are sequentially cleaved to release an extracellular ectodomain and an intracellular domain (ICD). Apart from an ectodomain and an ICD, processing of APP results in the formation of a small peptides corresponding to the remainder of the extracellular domain after shedding and parts of the transmembrane domain [3]. One of these peptides, termed b-amyloid (Ab), can misfold and form plaques in brains of Alzheimer’s disease patients. The biological roles of RIP are several-fold and include the generation of soluble extracellular domains with ligand activity, formation of Ab-like peptides, release and nuclear translocation of ICDs with transcriptional capacity, and protein degradation [1,11]
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