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

<div><p>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 <i>in silico</i> 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.</p></div

Determination of the sheddase cleavage amino acid sequence in mEpEX.

<p>(<b>A</b>) Schematic representation of mEpCAM-TF containing a TEV protease recognition site and a Flag-Tag in the mEpEX domain 42 amino acids before the predicted transmembrane domain. After cleavage by sheddases, the largest part of mEpEX can be removed through digestion with TEV protease and the resulting small fragment immunoprecipitated using Flag-specific antibodies. (<b>B</b>) Representative mass spectrometry spectrum of HEK293, NIH3T3, and mF9 cells stably expressing mEpCAM-TF and of vector control HEK293 cells as a control. Four major peak species are indicated. (<b>C</b>) Tabular overview of sheddase cleavage sites within mEpEX as determined upon mass spectrometric analysis and alignment to potential molecular weights. Calculated and determined masses are given in Dalton including error and charge of each peptide. (<b>D</b>) Representative mass spectrometry spectrum of HEK293 cells stably expressing mEpCAM-TF after treatment with DMSO, the metalloprotease protease inhibitor TAPI-1, and the phorbol ester PMA. (<b>E</b>) Representative mass spectrometry spectrum of HEK293 cells stably expressing mEpCAM-TF after treatment with DMSO, the BACE1 protease inhibitor C3, and after transient transfection of BACE1 expression plasmid. (<b>F</b>) Sequence alignment of murine and human EpCAM (top), and murine EpCAM and murine Trop-2 (bottom). metalloprotease protease cleavage (a-secretase) and BACE1 cleavage sites (b-secretase) are indicated.</p

Cleavage of murine EpCAM in membrane assays.

<p>HEK293 cells were stably transfected with full-length mEpCAM in fusion with YFP (EpCAM-YFP). (<b>A</b>) Schematic representation of cleavage processes resulting in the generation of soluble EpEX, CTF-YFP, and intracellular EpICD-YFP fragments. (<b>B–C</b>) Membranes of stable transfectants were isolated and either kept at 0°C (0 h) or incubated at 37°C in reaction buffer for the indicated time points. Thereafter, pellets and supernatant were collected upon differential centrifugation. Pellets (<b>B</b>) and supernatants (<b>C</b>) of membrane assays were separated in a 10% SDS-PAGE and probed with mEpICD- and YFP-specific antibodies. (<b>D</b>) Embryonic stem cell line E14TG2a and teratocarcinoma cells mF9 were treated with DMSO (control) or the γ-secretase inhibitor DAPT before being subjected to a membrane assay. The total fraction of the membrane assay was separated in a 10% SDS-PAGE, and probed with a YFP-specific antibody. Treatment with DAPT resulted in the accumulation of CTF-YFP and in the inhibition of mEpICD-YFP formation. Protein bands corresponding to mEpCAM-YFP, CFT-YFP, and mEpICD-YFP are indicated in each immunoblot. Shown are the representative results of three independent experiments.</p

Schematic representation of EpCAM presenilin-dependent regulated intramembrane proteolysis (PS-RIP) and endocytosis.

<p>Murine EpCAM (mEpCAM) is cleaved at the plasma membrane to release soluble EpEX (smEpEX). The resulting C-terminal fragment (mCTF) is a substrate for γ-secretase, which cleaves mCTF to generate soluble, extracellular mEp-β fragments (γ-cleavage) and intracellular mEpICD fragments, which are prone to proteasomal degradation. Additionally, mEpICD can be endocytosed and processed either by BACE1 in acidic intracellular compartments (endosome) and/or by acidic hydrolases in lysosomes.</p

Sequence conservation of cleavage sites in orthologs of EpCAM found in fishes, amphibians, birds, to placental mammals.

<p>Amino acid sequences of 52 orthologs of human EpCAM were aligned using ClustallW and sequence conservation of each amino acid was calculated (maximum score 11. Shown are the mean conservation score throughout all orthologs (mean) and conservation scores of single amino acids ranging positions p⁻³ to p⁺³ around determined cleavage sites of metalloproteases (<b>A</b>), BACE1 (<b>B</b>), γ-cleavage of γ-secretase (<b>C</b>), and ε-cleavage of γ-secretase (<b>D</b>).</p

Cleavage of endogeneous mEpCAM.

<p>Proteolytic cleavage of mEpCAM was addressed in mF9 (<b>A</b>) and E14TG2a (<b>B</b>) cells using membrane assays at pH 7 and pH 4. Membranes of mF9 and E14TG2a cells were incubated for 0 h and 24 h at 37°C and EpCAM fragments were detected in immunoblots using a mEpICD-specific antibody in combination with an HRP-conjugated secondary antibody. Inhibition of the γ-secretase complex was achieved upon treatment with DAPT where indicated. Shown are the representative results of three independent experiments.</p

Cleavage and proteasomal degradation of human EpCAM.

<p>HEK293 cells were stably transfected with hEpCAM-YFP and used to determine cleavage products of hEpCAM in membrane assays. Membranes of stable transfectants were isolated and either kept at 0°C (0 h) or incubated at 37°C in reaction buffer for the indicated time points. Thereafter, pellets and supernatant were collected upon differential centrifugation. Pellets (<b>A</b>) and supernatants (<b>B</b>) of membrane assays were separated in a 10% SDS-PAGE and probed with hEpICD- and YFP-specific antibodies. (<b>C</b>) HEK293 hEpCAM-YFP transfectants were treated with DMSO (control) or the γ-secretase inhibitor DAPT before being subjected to a membrane assay. The total fraction of the membrane assay was separated in a 10% SDS-PAGE, and probed with a YFP-specific antibody. Treatment with DAPT resulted in the accumulation of CTF-YFP and in the inhibition of hEpICD-YFP formation. (<b>D</b>) HEK293 human Myc-CTF-TF-YFP transfectants were treated with DMSO (control), the γ-secretase inhibitor DAPT, the proteasome inhibitor β-lacto-lactocystin (β-Lac), or combination of both. Thereafter, whole cell lysates were separated in a 10% SDS-PAGE, and probed with a YFP-specific antibody. Treatment with β-lacto-lactocystin resulted in an accumulation of hEpICD-YFP. Similar loading of protein lysates was visualised upon staining of tubulin on the same blots. Protein bands corresponding to human Myc-CTF-TF-YFP and mEpICD-YFP are indicated in each immunoblot. Shown are the representative results of three independent experiments. (<b>E</b>) YFP fluorescence was analysed in dependency of the treatment of HEK293 human Myc-CTF-TF-YFP transfectants. DMSO treatment served as a reference and values were normalised to one. Shown are the mean values with standard deviations from three independent experiments. (<b>F</b>) HEK293 cells stably expressing hEpCAM-YFP were transiently transfected with expression plasmids for luciferase (Luc) as a control or BACE1 (BACE1). After 24 hours, supernatants were removed and cells treated with the indicated inhibitors of BACE1 (C3), γ-secretase (DAPT) or combinations thereof. After additional 24 hours, supernatants were collected and hEpEX was immunoprecipitated and visualised upon immunoblotting with specific antibodies. Shown are two representative results with exposure times (1 s and 10 s). Over-expression of BACE1 and equal protein loading were verified upon immunobloting (lower left and right panel, respectively).</p

Determination of the γ-secretase cleavage amino acid sequence in mEpCAM.

<p>(<b>A</b>) Schematic representation of Myc-CTF-YFP containing an N-terminal c-Myc-tag, a Flag-Tag and a TEV protease recognition site as well as YFP C-terminally of mEpICD. After cleavage by g-secretase, the Ab-like fragment can be isolated upon immunoprecipitation with c-Myc-specific antibodies. The YFP moiety is removed through digestion with TEV protease and the resulting small peptide isolated upon immunoprecipitation with Flag-specific antibodies. (<b>B</b>) Representative mass spectrometry spectrum of HEK293, NIH3T3, and mF9 cells stably expressing Myc-CTF-TF-YFP after immunoprecipitation of supernatants with c-Myc-specific antibodies. Two major peak species representing g-cleavages are indicated. (<b>C</b>) Tabular overview of γ-secretase cleavage sites within mEpCAM as determined upon mass spectrometric analysis and alignment to potential molecular weights. Calculated and determined masses are given in Dalton including error of each peptide. (<b>D</b>) Representative mass spectrometry spectrum of HEK293 cells stably expressing mEpCAM-TF after immunoprecipitation with c-Myc-specific antibodies and treatment with DMSO and the γ-secretase inhibitor DAPT. (<b>E</b>) Representative mass spectrometry spectrum of HEK293, mF9, and NIH3T3 stably expressing Myc-CTF-TF-YFP after TEV digestion and immunoprecipitation with Flag-specific antibodies. Four major peaks representing ε-cleavages are indicated. (<b>F</b>) Representative mass spectrometry spectrum of HEK293 cells stably expressing mEpCAM-TF after TEV digestion, immunoprecipitation with Flag-specific antibodies, and treatment with DMSO and the γ-secretase inhibitor DAPT. (<b>G</b>) Sequence alignment of murine and human EpCAM (top), and murine EpCAM and murine Trop-2 (bottom). γ-Secretase cleavages at g-position and e-position are indicated.</p