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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 murine EpCAM in membrane assays.HEK293 cells were stably transfected with full-length mEpCAM in fusion with YFP (EpCAM-YFP). (A) Schematic representation of cleavage processes resulting in the generation of soluble EpEX, CTF-YFP, and intracellular EpICD-YFP fragments. (B–C) 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) and supernatants (C) of membrane assays were separated in a 10% SDS-PAGE and probed with mEpICD- and YFP-specific antibodies. (D) 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.
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pone-0071836-g001: Cleavage of murine EpCAM in membrane assays.HEK293 cells were stably transfected with full-length mEpCAM in fusion with YFP (EpCAM-YFP). (A) Schematic representation of cleavage processes resulting in the generation of soluble EpEX, CTF-YFP, and intracellular EpICD-YFP fragments. (B–C) 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) and supernatants (C) of membrane assays were separated in a 10% SDS-PAGE and probed with mEpICD- and YFP-specific antibodies. (D) 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.

Mentions: Regulated intramembrane proteolysis (RIP) of human EpCAM was reported in carcinoma and HEK293 cells [10]. Similarly to these previous reports, the murine ortholog of EpCAM (mEpCAM) was fused to yellow fluorescent protein (YFP) to increase the size of cleavage products and to facilitate their detection (see schematic view in Figure 1A). Cleavage and functionality of hEpCAM-YFP/hEpICD-YFP were demonstrated in earlier approaches in vitro and in vivo[10]. Cleavage was investigated in isolated membranes of stable HEK293 transfectants expressing full-length mEpCAM-YFP as described before [34]. Isolated membranes of these cells were incubated for 0 h to 22 h in a time course at 37°C to allow for cleavage to occur. Thereafter, membranous and soluble fractions were harvested separately via differential centrifugation and the presence of cleaved variants of mEpCAM determined in immunoblot experiments with mEpICD- and YFP-specific antibodies. Three distinct proteins were detected with mEpICD- and YFP-specific antibodies in particulate fractions of membrane-based assays (Figure 1B). Apparent molecular masses were calculated using the Chemidoc XRS+ imaging system and corresponded to the predicted molecular mass for mEpCAM-YFP (predicted: 62.55 kDa; apparent: 66.7 kDa), CTF-YFP (predicted: 34.5–37 kDa; apparent: 34.9 kDa), and mEpICD-YFP (predicted: 31 kDa; apparent: 29.9 kDa). These molecular weights refer to fusions with YFP, hence 25 kDa must be subtracted to determine actual EpCAM fragment sizes. Only small amounts of the C-terminal fragment mCTF-YFP were present at the initial time point, which might reflect the overall status of mEpCAM cleavage at the time of membrane isolation. At this time point, a major mCTF fragment with an approximate molecular weight of 34.9 kDa represented the dominant mCTF band. Two additional bands of weaker intensity and with molecular weights of 37 kDa and 40 kDa were detected using mEpICD-specific antibodies (Figure 1B). Upon time, these two proteins disappeared and after 2.5 h, the level of the 34.9 kDa mCTF-YFP strongly increased and remained stable over the observation time of 22 h (Figure 1B). At later time points, we observed the appearance of a smaller mEpICD-reactive protein, which corresponded to mEpICD-YFP, in comparably small amounts (Figure 1B). All three major protein species were identified with mEpICD and YFP-specific antibodies. In supernatants of membrane assays, two mEpICD and YFP-reactive proteins were detected, which corresponded to the 34.9 kDa mCTF-YFP and mEpICD-YFP (Figure 1C). The ratio of mCTF-YFP to mEpICD-YFP was inversed in supernatants versus pellets, in line with the notion that mEpICD is release from membrane-bound mCTF-YFP as a soluble protein. Residual levels of mEpCAM-YFP and mCTF-YFP occasionally seen in supernatants and of mEpICD-YFP in pellets might represent minor cross-contaminations of subcellular fractions.


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 murine EpCAM in membrane assays.HEK293 cells were stably transfected with full-length mEpCAM in fusion with YFP (EpCAM-YFP). (A) Schematic representation of cleavage processes resulting in the generation of soluble EpEX, CTF-YFP, and intracellular EpICD-YFP fragments. (B–C) 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) and supernatants (C) of membrane assays were separated in a 10% SDS-PAGE and probed with mEpICD- and YFP-specific antibodies. (D) 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756971&req=5

pone-0071836-g001: Cleavage of murine EpCAM in membrane assays.HEK293 cells were stably transfected with full-length mEpCAM in fusion with YFP (EpCAM-YFP). (A) Schematic representation of cleavage processes resulting in the generation of soluble EpEX, CTF-YFP, and intracellular EpICD-YFP fragments. (B–C) 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) and supernatants (C) of membrane assays were separated in a 10% SDS-PAGE and probed with mEpICD- and YFP-specific antibodies. (D) 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.
Mentions: Regulated intramembrane proteolysis (RIP) of human EpCAM was reported in carcinoma and HEK293 cells [10]. Similarly to these previous reports, the murine ortholog of EpCAM (mEpCAM) was fused to yellow fluorescent protein (YFP) to increase the size of cleavage products and to facilitate their detection (see schematic view in Figure 1A). Cleavage and functionality of hEpCAM-YFP/hEpICD-YFP were demonstrated in earlier approaches in vitro and in vivo[10]. Cleavage was investigated in isolated membranes of stable HEK293 transfectants expressing full-length mEpCAM-YFP as described before [34]. Isolated membranes of these cells were incubated for 0 h to 22 h in a time course at 37°C to allow for cleavage to occur. Thereafter, membranous and soluble fractions were harvested separately via differential centrifugation and the presence of cleaved variants of mEpCAM determined in immunoblot experiments with mEpICD- and YFP-specific antibodies. Three distinct proteins were detected with mEpICD- and YFP-specific antibodies in particulate fractions of membrane-based assays (Figure 1B). Apparent molecular masses were calculated using the Chemidoc XRS+ imaging system and corresponded to the predicted molecular mass for mEpCAM-YFP (predicted: 62.55 kDa; apparent: 66.7 kDa), CTF-YFP (predicted: 34.5–37 kDa; apparent: 34.9 kDa), and mEpICD-YFP (predicted: 31 kDa; apparent: 29.9 kDa). These molecular weights refer to fusions with YFP, hence 25 kDa must be subtracted to determine actual EpCAM fragment sizes. Only small amounts of the C-terminal fragment mCTF-YFP were present at the initial time point, which might reflect the overall status of mEpCAM cleavage at the time of membrane isolation. At this time point, a major mCTF fragment with an approximate molecular weight of 34.9 kDa represented the dominant mCTF band. Two additional bands of weaker intensity and with molecular weights of 37 kDa and 40 kDa were detected using mEpICD-specific antibodies (Figure 1B). Upon time, these two proteins disappeared and after 2.5 h, the level of the 34.9 kDa mCTF-YFP strongly increased and remained stable over the observation time of 22 h (Figure 1B). At later time points, we observed the appearance of a smaller mEpICD-reactive protein, which corresponded to mEpICD-YFP, in comparably small amounts (Figure 1B). All three major protein species were identified with mEpICD and YFP-specific antibodies. In supernatants of membrane assays, two mEpICD and YFP-reactive proteins were detected, which corresponded to the 34.9 kDa mCTF-YFP and mEpICD-YFP (Figure 1C). The ratio of mCTF-YFP to mEpICD-YFP was inversed in supernatants versus pellets, in line with the notion that mEpICD is release from membrane-bound mCTF-YFP as a soluble protein. Residual levels of mEpCAM-YFP and mCTF-YFP occasionally seen in supernatants and of mEpICD-YFP in pellets might represent minor cross-contaminations of subcellular fractions.

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