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Regulated intramembrane proteolysis and degradation of murine epithelial cell adhesion molecule mEpCAM.

Hachmeister M, Bobowski KD, Hogl S, Dislich B, Fukumori A, Eggert C, Mack B, Kremling H, Sarrach S, Coscia F, Zimmermann W, Steiner H, Lichtenthaler SF, Gires O - PLoS ONE (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, Head and Neck Surgery, Ludwig-Maximilians-University, Munich, Germany.

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.

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Cleavage of endogeneous mEpCAM.Proteolytic cleavage of mEpCAM was addressed in mF9 (A) and E14TG2a (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.
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pone-0071836-g004: Cleavage of endogeneous mEpCAM.Proteolytic cleavage of mEpCAM was addressed in mF9 (A) and E14TG2a (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.

Mentions: Cleavage of endogeneous mEpCAM was addressed in mF9 and E14TG2a cell lines with membrane assays. Since metalloproteases and BACE1 differ in their pH optimum, membrane assays were performed at pH7 and pH4. At pH7 and in the absence of any inhibitor, no mEpCAM fragment was detectable except for full-length mEpCAM. Inhibition of the γ-secretase complex with DAPT resulted in the accumulation of endogeneous mCTF after 24 h in teratocarcinoma (mF9) and ES cells (E14TG2α) (Figure 4A and B, left panels). Owing to the abovementioned shedding of mEpCAM by BACE1, the same experiments were performed at pH = 4, which represents the pH optimum of BACE1 [38]. In this acidic environment, endogeneous mEpCAM was quantitatively cleaved to generate mCTF, while mEpICD was not detectable possibly due to the inappropriate pH for the enzymatic activity of γ-secretase and owing to the very small size of the resulting protein (Figure 4A and B, right panels). Hence, endogeneous mEpCAM is subject to proteolytic cleavage but detection of mEpICD was technically not feasible under the assay conditions. Detection of ICDs generated through presenilin-dependent RIP is usually a very difficult task due to rapid and efficient degradation of ICDs after release into the intracellular space [27], [28], [29]. Detection of human EpICD was highly inefficient and achieved only upon immunoprecipitation of large amounts of protein lysate in the milligram range. This led the authors to the notion that hEpICD is a very small protein, whose biochemical properties hamper thorough detection and to the assumption of a potential degradation of hEpICD through the proteasome [10].


Regulated intramembrane proteolysis and degradation of murine epithelial cell adhesion molecule mEpCAM.

Hachmeister M, Bobowski KD, Hogl S, Dislich B, Fukumori A, Eggert C, Mack B, Kremling H, Sarrach S, Coscia F, Zimmermann W, Steiner H, Lichtenthaler SF, Gires O - PLoS ONE (2013)

Cleavage of endogeneous mEpCAM.Proteolytic cleavage of mEpCAM was addressed in mF9 (A) and E14TG2a (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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756971&req=5

pone-0071836-g004: Cleavage of endogeneous mEpCAM.Proteolytic cleavage of mEpCAM was addressed in mF9 (A) and E14TG2a (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.
Mentions: Cleavage of endogeneous mEpCAM was addressed in mF9 and E14TG2a cell lines with membrane assays. Since metalloproteases and BACE1 differ in their pH optimum, membrane assays were performed at pH7 and pH4. At pH7 and in the absence of any inhibitor, no mEpCAM fragment was detectable except for full-length mEpCAM. Inhibition of the γ-secretase complex with DAPT resulted in the accumulation of endogeneous mCTF after 24 h in teratocarcinoma (mF9) and ES cells (E14TG2α) (Figure 4A and B, left panels). Owing to the abovementioned shedding of mEpCAM by BACE1, the same experiments were performed at pH = 4, which represents the pH optimum of BACE1 [38]. In this acidic environment, endogeneous mEpCAM was quantitatively cleaved to generate mCTF, while mEpICD was not detectable possibly due to the inappropriate pH for the enzymatic activity of γ-secretase and owing to the very small size of the resulting protein (Figure 4A and B, right panels). Hence, endogeneous mEpCAM is subject to proteolytic cleavage but detection of mEpICD was technically not feasible under the assay conditions. Detection of ICDs generated through presenilin-dependent RIP is usually a very difficult task due to rapid and efficient degradation of ICDs after release into the intracellular space [27], [28], [29]. Detection of human EpICD was highly inefficient and achieved only upon immunoprecipitation of large amounts of protein lysate in the milligram range. This led the authors to the notion that hEpICD is a very small protein, whose biochemical properties hamper thorough detection and to the assumption of a potential degradation of hEpICD through the proteasome [10].

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, Head and Neck Surgery, Ludwig-Maximilians-University, Munich, Germany.

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.

Show MeSH
Related in: MedlinePlus