Glycosylation and glycan interactions can serve as extracellular machinery facilitating clathrin-independent endocytosis.

In contrast to clathrin-mediated endocytosis (CME) which is well characterized and understood, little is known about the regulation and machinery underlying clathrin-independent endocytosis (CIE). There is also a wide variation in the requirements each individual CIE cargo has for its internalization. Recent studies have shown that CIE is affected by glycosylation and glycan interactions. We briefly review these studies and explore how these studies mesh with one another. We then discuss what this sensitivity to glycan interactions could indicate for the regulation of CIE. We address the spectrum of responses CIE has been shown to have with respect to changes in glycan interactions and attempt to reconcile disparate observations onto a shared conceptual landscape. We focus on the mechanisms by which cells can alter the glycan interactions at the plasma membrane and propose that glycosylation and glycan interactions could provide cells with a tool box with which cells can manipulate CIE. Altered glycosylation is often associated with a number of diseases and we discuss how under different disease settings, glycosylation-based modulation of CIE could play a role in disease progression.

Distinct cargo-specific response landscapes underpin the complex and nuanced role of galectin-glycan interactions in clathrin-independent endocytosis.

Clathrin-independent endocytosis (CIE) is a form of endocytosis that lacks a defined cytoplasmic machinery. Here, we asked whether glycan interactions, acting from the outside, could be a part of that endocytic machinery. We show that the perturbation of global cellular patterns of protein glycosylation by modulation of metabolic flux affects CIE. Interestingly, these changes in glycosylation had cargo-specific effects. For example, in HeLa cells, GlcNAc treatment, which increases glycan branching, increased major histocompatibility complex class I (MHCI) internalization but inhibited CIE of the glycoprotein CD59 molecule (CD59). The effects of knocking down the expression of galectin 3, a carbohydrate-binding protein and an important player in galectin-glycan interactions, were also cargo-specific and stimulated CD59 uptake. By contrast, inhibition of all galectin-glycan interactions by lactose inhibited CIE of both MHCI and CD59. None of these treatments affected clathrin-mediated endocytosis, implying that glycosylation changes specifically affect CIE. We also found that the galectin lattice tailors membrane fluidity and cell spreading. Furthermore, changes in membrane dynamics mediated by the galectin lattice affected macropinocytosis, an altered form of CIE, in HT1080 cells. Our results suggest that glycans play an important and nuanced role in CIE, with each cargo being affected uniquely by alterations in galectin and glycan profiles and their interactions. We conclude that galectin-driven effects exist on a continuum from stimulatory to inhibitory, with distinct CIE cargo proteins having unique response landscapes and with different cell types starting at different positions on these conceptual landscapes.

Microdomain Formation Controls Spatiotemporal Dynamics of Cell-Surface Glycoproteins.

The effect of galectin-mediated microdomain formation on the spatiotemporal dynamics of glycosylated membrane proteins in human microvascular endothelial cells (HMEC-1) was studied qualitatively and quantitatively by high-resolution fluorescence microscopy and artificially mimicked by metabolic glycoprotein engineering. Two types of membrane proteins, sialic acid-bearing proteins (SABPs) and mucin-type proteins (MTPs), were investigated. For visualization they were metabolically labeled with azido sugars and then coupled to a cyclooctyne-conjugated fluorescent dye by click chemistry. Both spatial (diffusion) and temporal (residence time) dynamics of SABPs and MTPs on the membrane were investigated after treatment with exogenous galectin-1 or -3. Strong effects of galectin-mediated lattice formation were observed for MTPs (decreased spatial mobility), but not for SABPs. Lattice formation also strongly decreased the turnover of MTPs (increased residence time on the cell membrane). The effects of galectin-mediated crosslinking was accurately mimicked by streptavidin-mediated crosslinking of biotin-tagged glycoproteins and verified by single-molecule tracking. This technique allows the induction of crosslinking of membrane proteins under precisely controlled conditions, thereby influencing membrane residence time and the spatial dynamics of glycans on the cell membrane in a controlled way.

Galectin-lattice sustains function of cationic amino acid transporter and insulin secretion of pancreatic ß cells.

Maintenance of cell surface residency and function of glycoproteins by lectins are essential for regulating cellular functions. Galectins are ß-galactoside-binding lectins and form a galectin-lattice, which regulates stability, clustering, membrane sub-domain localization and endocytosis of plasmalemmal glycoproteins. We have previously reported that galectin-2 (Gal-2) forms a complex with cationic amino acid transporter 3 (CAT3) in pancreatic ß cells, although the biological significance of the molecular interaction between Gal-2 and CAT3 has not been elucidated. In this study, we demonstrated that the structure of N-glycan of CAT3 was either tetra- or tri-antennary branch structure carrying ß-galactosides, which works as galectin-ligands. Indeed, CAT3 bound to Gal-2 using ß-galactoside epitope. Moreover, the disruption of the glycan-mediated bindings between galectins and CAT3 significantly reduced cell surface expression levels of CAT3. The reduced cell surface residency of CAT3 attenuated the cellular arginine uptake activities and subsequently reduced nitric oxide production, and thus impaired the arginine-stimulated insulin secretion of pancreatic ß cells. These results indicate that galectin-lattice stabilizes CAT3 by preventing endocytosis to sustain the arginine-stimulated insulin secretion of pancreatic ß cells. This provides a novel cell biological insight into the endocrinological mechanism of nutrition metabolism and homeostasis.