-GlcNAcylation and Regulation of Galectin-3 in Extraembryonic Endoderm Differentiation.

The regulation of proteins through the addition and removal of -linked β--acetylglucosamine (-GlcNAc) plays a role in many signaling events, specifically in stem cell pluripotency and the regulation of differentiation. However, these post-translational modifications have not been explored in extraembryonic endoderm (XEN) differentiation. Of the plethora of proteins regulated through -GlcNAc, we explored galectin-3 as a candidate protein known to have various intracellular and extracellular functions. Based on other studies, we predicted a reduction in global -GlcNAcylation levels and a distinct galectin expression profile in XEN cells relative to embryonic stem (ES) cells. By conducting dot blot analysis, XEN cells had decreased levels of global -GlcNAc than ES cells, which reflected a disbalance in the expression of genes encoding -GlcNAc cycle enzymes. Immunoassays (Western blot and ELISA) revealed that although XEN cells (low -GlcNAc) had lower concentrations of both intracellular and extracellular galectin-3 than ES cells (high -GlcNAc), the relative secretion of galectin-3 was significantly increased by XEN cells. Inducing ES cells toward XEN in the presence of an -GlcNAcase inhibitor was not sufficient to inhibit XEN differentiation. However, global -GlcNAcylation was found to decrease in differentiated cells and the extracellular localization of galectin-3 accompanies these changes. Inhibiting global -GlcNAcylation status does not, however, impact pluripotency and the ability of ES cells to differentiate to the XEN lineage.