Abstract
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 ∆21-81 EMCN and ∆21-121 EMCN, but not the shortest mutant ∆21-161 EMCN, successfully rescued the VEGF-induced EC migration, tube formation, and proliferation. ∆21-161 EMCN failed to interact with VEGFR2 and did not facilitate VEGFR2 internalization. Deletion of COSMC (C1GalT1C1) revealed that the abundant mucin-type O-glycans were not required for its VEGFR2-related functions. Mutation of the two N-glycosylation sites on ∆21-121 EMCN abolished its interaction with VEGFR2 and its function in VEGFR2 internalization. These results reveal ∆21-121 EMCN as the minimal extracellular domain sufficient for VEGFR2-mediated endothelial function and demonstrate an important role for N-glycosylation in VEGFR2 interaction, internalization, and angiogenic activity.
Highlights
Angiogenesis, the process by which new blood vessels arise from existing venules through budding and sprouting, plays an essential role in growth and development, wound healing, and repairing as well as in numerous physiological conditions [1]
COSMC (C1GALT1C1) is the unique and sole chaperone of EMCN-VEGF receptor 2 (VEGFR2) interaction, we examined how the role O-glycosylation in the extracellular domain (ECD) is involved in EMCN
O-glycosylation did not impact the interaction of EMCN and VEGFR2; n = 3 (E,F) To examine VEGFR2 internalization, CRIPSR control Human retinal endothelial cells (HRECs) or COSMC knockout HRECs were stimulated with vascular endothelial growth factor A (VEGF) or BSA and VEGF2 tracked with an antibody that recognizes the VEGFR2 extracellular domain (ECD)
Summary
Angiogenesis, the process by which new blood vessels arise from existing venules through budding and sprouting, plays an essential role in growth and development, wound healing, and repairing as well as in numerous physiological conditions [1]. While several factors are involved in the regulation of new blood vessel growth, vascular endothelial growth factor A (VEGF) has been shown to play a central role. VEGFR2 co-receptors [4], like neuropilin (NRP) and heparan sulfate proteoglycans (HSPGs), as well as integrins and endoglin [5] Of all these associated proteins, the role of neuropilin in VEGFR2 signaling has been the best characterized. VEGF signaling is tightly regulated at all of these levels, including by receptor expression, by interactions with co-receptors or auxiliary proteins, and by the rate of internalization. Heavily glycosylated proteins with extended conformations, are important components of the glycocalyx [24] and have been shown to be involved in signal transduction by interacting with growth factor receptors or extracellular domain-mediated ligand binding [25,26]. We characterized the contribution of O-glycans and N-glycans on EMCN’s ECD in VEGF-induced endothelium functions
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