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. 2018 Sep 5;8(1):13269.
doi: 10.1038/s41598-018-31482-7.

EpCAM Homo-Oligomerization Is Not the Basis for Its Role in Cell-Cell Adhesion

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Free PMC article

EpCAM Homo-Oligomerization Is Not the Basis for Its Role in Cell-Cell Adhesion

Aljaž Gaber et al. Sci Rep. .
Free PMC article

Abstract

Cell-surface tumor marker EpCAM plays a key role in proliferation, differentiation and adhesion processes in stem and epithelial cells. It is established as a cell-cell adhesion molecule, forming intercellular interactions through homophilic association. However, the mechanism by which such interactions arise has not yet been fully elucidated. Here, we first show that EpCAM monomers do not associate into oligomers that would resemble an inter-cellular homo-oligomer, capable of mediating cell-cell adhesion, by using SAXS, XL-MS and bead aggregation assays. Second, we also show that EpCAM forms stable dimers on the surface of a cell with pre-formed cell-cell contacts using FLIM-FRET; however, no inter-cellular homo-oligomers were detectable. Thus, our study provides clear evidence that EpCAM indeed does not function as a homophilic cell adhesion molecule and therefore calls for a significant revision of its role in both normal and cancerous tissues. In the light of this, we strongly support the previously suggested name Epithelial Cell Activating Molecule instead of the Epithelial Cell Adhesion Molecule.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
EpCAM’s function in cell-cell adhesion and signalling. (a) EpCAM mediated adhesion. EpCAM is depicted as a shape outline. Subunits in cis-dimers are colored cyan and magenta, transmembrane regions are depicted in paler colors. Cell membrane is shown in grey. (b) EpCAM signalling via RIP. EpCAM is depicted as a shape outline. Structures of ADAM’s catalytic domain, γ-secretase, β-catenin are presented as ribbons (PDB: 1bkc, 5a63 and 2z6h, respectively). The portion ADAM beside the catalytic domain, FHL2 and Lef1, whose 3D structures are not known are depicted as shapes with sizes corresponding to their mass.
Figure 2
Figure 2
SAXS analysis shows no evidence of oligomerization. (a) Scaled SAXS profiles of ngEpEX at different concentrations in the range from 0.5 mg/ml (17.5 µM) to 26.2 mg/ml (919.4 µM) presented and overlaid in the same plot. (b) MW and Rg values calculated from SAXS profiles. Dotted lines represent values calculated for dimer structure 57 kDa and 24 Å. Predicted area of tetramer values are depicted with a grey rectangle and the hypothetical tetramer values at 114 kDa and 54 Å are marked with black triangles. (c) Three orientations of EpEX dimer structure docked in the ab initio shape (grey envelope) reconstructed from the merged SAXS profile. Subunits in the dimer are depicted as cyan and magenta ribbons.
Figure 3
Figure 3
XL-MS analysis of EpCAM oligomerization. (a) SDS-PAGE analysis of cross-linking experiment. Black triangle at the top indicates increasing molar ratio of crosslinker (DSS) vs protein. White rectangles denote excised areas, which were analysed with MS. Lanes were cropped and greyscaled for clarity. Full-length gel is presented in Supplementary Fig. 3. (b) Cross-links identified in MS analysis. Columns represent corresponding areas of origin, EX and FL stand for identification in ngEpEX or ngEpFL XL experiment, respectively. (c,d) Shortest SASD for each matched cross-link in monomer model and dimer structure. In dimer structure, only one of each ambiguous inter-dimer cross-links is represented. Subunits are depicted as shape outlines. Subunits in cis-dimers are colored cyan and magenta, with membrane proximal and membrane distal parts depicted in grey and yellow, respectively. (e) Modelling of tetramers based on cross-links, non-accessible in dimer. Proposed trans-tetrameric adhesive unit model was taken from literature (Pavšič, 2014). Jwalk results are presented as three clusters, generated from 25 best scoring random tetramer models, clustered at 10 Å. DisVis results represent average ligand occupancy at default cut-off, as outputted by the web server.
Figure 4
Figure 4
Bead Aggregation Assays. (a) Schematic representation of analysed proteins. Subunits in EpEX and EpFL are depicted as in Fig. 1. Glycosylation is depicted as grey sticks. E-CadEX dimer is green and Fc-dimer is orange and yellow. (b) Representative bead aggregation images after image analysis with ImageJ. Bead aggregation is seen as clustering of black spots. (c) Comparison of Aggregation ratios. Values represent mean values of 15 independent images with s.d., ***p < 0.001, one-way ANOVA test with Bonferroni post hoc analysis.
Figure 5
Figure 5
FLIM-FRET (HEK 293 T). (a) Analysed combinations of fluorescently tagged EpCAM proteins. sfGFP (G) fluorescence is colored cyan and mCherry (R) fluorescence is colored magenta. The same color scheme applies to schematic representations on the left. White line represents 20 µm. FLIM color scale is the same in all presented FLIM measurements. (b) Representative fluorescence lifetime decays for each analysed combination. (c) Mean lifetimes with s.d. of all analysed combinations. For each combination, results obtained in HEK 293 T cells are presented on the left and results obtained in HCT8EpCAM− on the right, ***p < 0.001, one-way ANOVA test with Bonferroni post hoc analysis, compared to mean values of negative controls. Color scheme is the same as in figure b.

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