Fig 1: Mapping the AGR2-binding site on EpCAM using hydrogen-deuterium exchange mass spectrometry. The indicated wt or mutated (Y251A) versions of EpCAM were incubated for 60 min at room temperature with buffer only or AGR2 protein at a molar ratio of AGR2/EpCAM of 4:1. The proteins were then deuterated by dilution into deuterated buffer then incubated over a time course of up to 3600 s followed by reduction, acidification, pepsinization, and separation of fragments using mass spectrometry as indicated in the methods. The deuterium exchange rates of individual EpCAM peptides (supplemental Fig. S7) is summarized using the HDX exchange plots highlighting % deuteration as a function of amino acid position. The numbering of amino acids in the deuteration plots ranges from 1–241, in which the EpCAM used was from amino acids 24–265 creating a 24-amino acid stagger. A, wt-EpCAM deuteration alone or with AGR2 after 600 s; B, wt-EpCAM deuteration alone or with AGR2 after 3600 s; C, EpCAMY251A deuteration alone or with AGR2 after 3600 s; D, a summary of the key regions in EpCAM whose deuteration is suppressed by AGR2 (in red) based on EpCAM PDB (4MZV), and E, a table summarizing the key peptic peptides derived from wt or mutated EpCAM and how their deuteration changes as a function of Y251A mutation without or after stable AGR2 binding. In D, the detergent decyl-beta-d-maltopyranoside decylmaltoside (PDB 4MZV) is included in the image to highlight the proximity of the detergent binding domain to the stable AGR2-binding site. The green highlights the location of the TLIYY motif in the ß-strand residing at the extreme C terminus of the recombinant EpCAM protein. In E, the yellow highlights the raw deuteration data of the overlapping TLIYY motif containing peptide that reveal no changes in deuteration after the 60 min preincubation allowing the complex to form between AGR2 and EpCAM. The pale green highlights the wt or mutated EpCAM peptic peptides identified that exhibit suppressed deuteration in the presence of AGR2 protein.
Fig 2: Expected Outcomes(A) Yield of a single assay. Shown are day 12 organoids distributed into 24 wells.(B) Single 24-well of the same assay.(C) High efficiency of tubule formation, shown are day 12 organoids.(D) Confocal images of immunohistological stainings on sections of day 12 organoids (derived from the MANZ-2-2 iPSC line) showing WT1+ podocytes, EPCAM+ or HNF1B+ tubular epithelia, LRP2+ or CUBN+ proximal tubules, and CHD1+ distal tubules and connecting/collecting duct epithelia. Scale bars, 0.5 cm (B); 1 mm (C); 100 µm (D).
Fig 3: EpCAM as a candidate AGR2 client protein. A, Homology between EpCAM and its paralogue TACD2 as aligned using Clustal Omega. Both proteins were identified using ScanProsite (supplemental Table S1) and harbor the TLIYY motif implicated as an AGR2 linear peptide docking site. B, Secondary structure of EpCAM which consists of a N-domain (ND, green), Thyroglobulin type-1 domain (TY, blue) and C-domain (CD, dark pink) which altogether make up for extracellular domain (EpEX), transmembrane domain (TM, gray), intracellular domain (EpIC, yellow), and the amino acids from 247–251 containing the sequence TLIYY. C, Three-dimensional cartoon representation of extracellular part of human EpCAM (PDB code: 4MZ) highlighting the AGR2 linear peptide motif at amino acid position Thr247 to Tyr251 9 (gray). Color coding is the same as in (B). D, Schematic representation of his-tagged EpCAM protein sequence highlighting the extracellular domain (EpEX), the TEV cleavage site, and the TLIYY motif. E, Coomassie Blue staining of purified His-EpCAM showing the major band at 32 kDa under reducing SDS-PAGE condition. F–G, Solid-phase binding assay to measure AGR2 binding to EpCAM protein. Increasing amounts of EpCAM were immobilized on the surface of a microtiter plate (0–1 µg). AGR2 (0–1 µg) was titrated in the mobile phase and AGR2 binding to immobilized EpCAM was quantified using AGR2 specific antibody. The binding of AGR2 is plotted as the extent of protein-protein complex formation as RLU as a function of increasing protein in the mobile phase. H, DNA sequencing chromatogram traces of EpCAMY251A. I, Coomassie Blue staining of purified His- EpCAMY251A mutant protein showing the major band at 32 kDa under denaturing SDS-PAGE. J, His-EpCAM or His-EpCAMY251A (1 µg) was immobilized onto the well surface of a microtiter plate as in (F) His-AGR2 WT (0–1 µg) was titrated in the mobile phase. AGR2 binding to immobilized EpCAM was quantified using AGR2 specific antibody. The binding is plotted as the extent of protein-protein complex formation as RLU as a function of increasing protein in the mobile phase.
Fig 4: Expression of tumor markers in 2D (A) and 3D (B) MCF-7 cell cultures by IFNa- 2b (104 U/mL), microphotography, hematoxylin/eosin, 400× magnification: a) CK expression, MCF-7 control, b) Expression of CK, MCF-7 + IFNa-2b, c) EpCAM expression, MCF-7 control, d) EpCAM, MCF-7 + IFNa-2b expression, e) Vim expression, MCF-7 control, f) Vim expression, MCF-7 + IFNa-2b.
Fig 5: Effects of Y251A mutation on EpCAM localization in cells. A–C, Fluorescently labeled versions of AGR2 (mCHERRY) and EpCAM (EGFP) with the signal peptides were generated and expression validated in cells using immunoblotting (B and C). B, The immunoblotting of mCHERRY and mCHERRY-AGR2 transfected cells highlights the expression of mCHERRY alone (lane 1) and mCHERRY-AGR2 (lane 2). Blots were incubated with an anti-mCHERRY antibody. The arrow marks the location of full-length mCHERRY-AGR2 and the asterisk marks the location of mCHERRY. We noticed the reproducible small molecular mass “cleavage” or synthesis products when mCHERRY was transfected into cells. C, The immunoblotting of EGFP and EGFP-EpCAM transfected cells highlights the expression of EGFP alone (lane 1) and EGFP-EpCAM (lane 2). Blots were incubated with a GFP antibody. The arrow marks the location of full-length EGFP-EpCAM and the asterisk mark the location of EGFP. The EGFP was not subjected to the production of smaller molecular mass adducts as was the mCHERRY protein. Fluorescent microscopy was used to measure the relative localization of the following proteins; D, mCHERRY-AGR2 E, EGFP-EpCAM F, EGFP-EpCAMY251A G, mCHERRY and H, EGFP. I and J, The impact of cotransfection of mCHERRY-AGR2 and EGFP-EpCAM or EGFP-EpCAMY251A on their respective localizations. Representative images of the wild-type and mutant EpCAM, as well as AGR2, are highlighted in both panels. The arrow in J highlights AGR2 mislocalization to the plasma membrane periphery in EpCAM mutant cotransfections that mirrors the mutant EpCAM mislocalization to the nuclear membrane in the same cells.
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