EpCAM is a tumor and stem cell marker involved in nuclear signaling via proteolytic cleavage. A hydrophobic pocket was observed in the crystal structure of EpCAM, into which the crystallization additive N-decyl-β-D-maltopyranoside was bound. The structure could represent a binding site for a physiological hydrophobic ligand with similar chemical properties, i.e. a longer hydrophobic chain. Preliminary in silico research suggested the palmitate chain as a possible ligand. The purpose of our work was to investigate hydrophobic ligand binding in vitro using 11-(danzylamino)undecanoic acid (DAUDA) as ligand. After the expression of the protein in the unmutated and mutated (closed pocket) form and purification by nickel chromatography and gel filtration, we performed binding tests by measuring DAUDA fluorescence and EpEX tyrosine fluorescence analysis, namely without and in the presence of palmitate as a competitor. The first set of experiments shows that DAUDA binds weakly to the protein and that palmitate displaces it. The results also show that the binding of DAUDA does not depend on the oligomeric state of EpEX. Binding on the mutated form of EpCAM was not detected in any case, which indicates that binding occurs in the hydrophobic pocket or in the adjacent area. A change in tyrosine fluorescence of EpEX was observed when palmitate was present, but it cannot be unequivocally attributed to the binding of this ligand. The results are a good basis for further experiments, which could eventually lead to the identification of a physiologically relevant EpCAM ligand.
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