The epithelial cell adhesion molecule (EpCAM) is a homodimeric transmembrane glycoprotein, involved in cell signalling pathways. Physiologically present higher expression levels of EpCAM on the surface of stem cells and lower on the surface of differentiated cells indicate its role in the tissue development and regeneration of the organism. The collapse in the balance of the cleavage abundance is a sign for the tumorigenic transformation of cells. Higher levels of EpCAM expression are typical for many types of adenocarcinoma and metastatic cells and therefore relevant as a tumor marker in the prognostics for some types of cancers. The difference in EpCAM expression levels in normal and cancer cells implies its potential role as an immunotherapeutic target. The extracellular domain of EpCAM (EpEX) is subjected to proteolytic cleavages mediated by proteolytical enzymes, called sheddases. Such cleavages result in a release of a soluble EpEX into the matrix. The sequence of following cleavages leads to the release of an intracellular domain (EpIC), which is further integrated in the signalling processes of β-catenin pathway. The increased extent of cleavages ultimately leads to more abundant transcription of genes, correlated to cell migration and proliferation.
The cleavages by sheddases are presumably regulated by yet unidentified regulators/interaction partners. One of the approaches for studying the effect of such regulators is by the selective mutagenesis of EpEX surface amino acids and the analysis of the effect those mutantions have on the extend of the cleavages through diminished or abolished interaction with the particular regulator. The results of one of such analysis imply that the cleavage rate of some mutants by proteases significantly differs from that of the wild type. Secondary structure analysis using CD spectrometry would assure that increased rate of the cleavage is not a result of a misfolded protein due to the introduced mutations but a biologically relevant consequence of the absence of an important cleavage/RIP regulator.
This work is combined of a short review on current approaches addressing the identification of key cleavages during the shedding process and of the cloning of DNA constructs of EpCAM mutants for further analysis of proper folding. Mutations were introduced into the DNA sequence using the method of the site-directed mutagenesis Quik-Change which is based on the mutant primers design and the polymerase chain reaction. We increased its efficiency by separating primers into two separated reaction mixtures. The prepared DNA constructs will be further used for the detailed in vitro analysis of the EpCAM cleavage.
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