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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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Sequence conservation of cleavage sites in orthologs of EpCAM found in fishes, amphibians, birds, to placental mammals.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 (A), BACE1 (B), γ-cleavage of γ-secretase (C), and ε-cleavage of γ-secretase (D).
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pone-0071836-g007: Sequence conservation of cleavage sites in orthologs of EpCAM found in fishes, amphibians, birds, to placental mammals.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 (A), BACE1 (B), γ-cleavage of γ-secretase (C), and ε-cleavage of γ-secretase (D).

Mentions: In 52 out of 61 sequences of potential orthologs, all exons encoding the mature EpCAM protein could be identified and were further considered to study the conservation of EpCAM cleavage sites (selected species are marked in bold letters in Table S2). It must be noted that exon 1, which encodes the signal peptide and part of the first glutamine within the mature protein, was identified in orthologs using SignalP 4.0 and disclosed from the following comparison (Figure S1). The conservation of cleavage sites within EpCAM across the 52 orthologs was assessed using the ClustalW algorithm with translated proteins sequences. Conservation coefficients were automatically calculated for each individual amino acid in the complete sequence and reached a value of 11 in case of a 100% identity throughout all species. The mean value of the conservation coefficient (CC) of the complete protein sequence of EpCAM across all 52 species was 5.86 and, thus, disclosed an intermediate 53% conservation of EpCAM in all orthologs (Figure 7). Cleavage sites were analysed in form of six amino acids from position P−3 to P+3 centred on the defined cleavage. The metalloproteinase cleavage sequence 227FHS*KKM232 in the extracellular domain of EpCAM (cleavage site *) displayed very low conservation below the protein average (CC 1–4) (Figure 7A), whereas the BACE1 cleavage site 249LIY*YVD254 was highly conserved (Figure 7B). Except for leucine in position P−3, all amino acids in the vicinity of the defined BACE1 cleavage site were characterised by higher-than-average conservation coefficient of 9 to 11 and both tyrosines, in between which cleavage occurs, have a conservation coefficient of 11, representing an almost perfect conservation throughout all species (Figure 7B). Cleavage sites of γ-secretase, which give rise to mEp-β fragments (γ-cleavage) also displayed very high sequence homology and a conservation coefficient of 9 to 11 (Figure 7C). In contrast, the conservation of cleavage sites of γ-secretase, which result in the generation of mEpICD fragments (e-cleavage), was more heterogeneous (CC 4–9). Cleavage ε2 was slightly more conserved in the leucine residue (CC 9) and cleavage ε4 was less conserved than the entire amino acid sequence of full-length EpCAM (CC 4–5) (Figure 7D).


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)

Sequence conservation of cleavage sites in orthologs of EpCAM found in fishes, amphibians, birds, to placental mammals.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 (A), BACE1 (B), γ-cleavage of γ-secretase (C), and ε-cleavage of γ-secretase (D).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0071836-g007: Sequence conservation of cleavage sites in orthologs of EpCAM found in fishes, amphibians, birds, to placental mammals.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 (A), BACE1 (B), γ-cleavage of γ-secretase (C), and ε-cleavage of γ-secretase (D).
Mentions: In 52 out of 61 sequences of potential orthologs, all exons encoding the mature EpCAM protein could be identified and were further considered to study the conservation of EpCAM cleavage sites (selected species are marked in bold letters in Table S2). It must be noted that exon 1, which encodes the signal peptide and part of the first glutamine within the mature protein, was identified in orthologs using SignalP 4.0 and disclosed from the following comparison (Figure S1). The conservation of cleavage sites within EpCAM across the 52 orthologs was assessed using the ClustalW algorithm with translated proteins sequences. Conservation coefficients were automatically calculated for each individual amino acid in the complete sequence and reached a value of 11 in case of a 100% identity throughout all species. The mean value of the conservation coefficient (CC) of the complete protein sequence of EpCAM across all 52 species was 5.86 and, thus, disclosed an intermediate 53% conservation of EpCAM in all orthologs (Figure 7). Cleavage sites were analysed in form of six amino acids from position P−3 to P+3 centred on the defined cleavage. The metalloproteinase cleavage sequence 227FHS*KKM232 in the extracellular domain of EpCAM (cleavage site *) displayed very low conservation below the protein average (CC 1–4) (Figure 7A), whereas the BACE1 cleavage site 249LIY*YVD254 was highly conserved (Figure 7B). Except for leucine in position P−3, all amino acids in the vicinity of the defined BACE1 cleavage site were characterised by higher-than-average conservation coefficient of 9 to 11 and both tyrosines, in between which cleavage occurs, have a conservation coefficient of 11, representing an almost perfect conservation throughout all species (Figure 7B). Cleavage sites of γ-secretase, which give rise to mEp-β fragments (γ-cleavage) also displayed very high sequence homology and a conservation coefficient of 9 to 11 (Figure 7C). In contrast, the conservation of cleavage sites of γ-secretase, which result in the generation of mEpICD fragments (e-cleavage), was more heterogeneous (CC 4–9). Cleavage ε2 was slightly more conserved in the leucine residue (CC 9) and cleavage ε4 was less conserved than the entire amino acid sequence of full-length EpCAM (CC 4–5) (Figure 7D).

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