Elements of the Endomucin Extracellular Domain Essential for VEGF-Induced VEGFR2 Activity.

Endomucin (EMCN) is the type I transmembrane glycoprotein, mucin-like component of the endothelial cell glycocalyx. We have previously shown that EMCN is necessary for vascular endothelial growth factor (VEGF)-induced VEGF receptor 2 (VEGFR2) internalization and downstream signaling. To explore the structural components of EMCN that are necessary for its function and the molecular mechanism of EMCN in VEGF-induced endothelial functions, we generated a series of mouse EMCN truncation mutants and examined their ability to rescue VEGF-induced endothelial functions in human primary endothelial cells (EC) in which endogenous EMCN had been knocked down using siRNA. Expression of the mouse full-length EMCN (FL EMCN) and the extracellular domain truncation mutants increment 21-81 EMCN and increment 21-121 EMCN, but not the shortest mutant increment 21-161 EMCN, successfully rescued the VEGF-induced EC migration, tube formation, and proliferation. increment 21-161 EMCN failed to interact with VEGFR2 and did not facilitate VEGFR2 internalization. Deletion of COSMC (C1GalT1C1) revealed that the abundant mucin-typeO-glycans were not required for its VEGFR2-related functions. Mutation of the twoN-glycosylation sites on increment 21-121 EMCN abolished its interaction with VEGFR2 and its function in VEGFR2 internalization. These results reveal increment 21-121 EMCN as the minimal extracellular domain sufficient for VEGFR2-mediated endothelial function and demonstrate an important role forN-glycosylation in VEGFR2 interaction, internalization, and angiogenic activity.