Abstract
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.
Highlights
Clathrin-independent endocytosis (CIE) is a form of endocytosis that lacks a defined cytoplasmic machinery
Similar to the effect of lactose, swainsonine treatment leads to a significant inhibition of CD59 internalization (Fig. 2E) and no significant effect on major histocompatibility complex class I (MHCI) (Fig. 2D) or transferrin internalization (Fig. 2F). These results further indicate that the internalization of CIE cargo can be mediated via galectin– glycan interactions, whereas clathrin-mediated endocytosis (CME) is unaffected
We found that galectin 3 pulled down along with MHCI and that GlcNAc treatment led to an increase in the amount of galectin 3 pulled down (Fig. 3D)
Summary
Cell Biology and Physiology Center, NHLBI, National Institutes of Health, Bethesda, Maryland 20892. All galectins are synthesized in the cytoplasm and are secreted by nonclassical secretory mechanisms [17] They are known to play roles both intracellularly (such as regulating RNA splicing [18]) and extracellularly (regulating cell adhesion and signaling [19]). In the CIE of the epidermal growth factor receptor, the galectin– glycan interactions have been demonstrated to inhibit internalization via galectin lattice–mediated cell-surface sequestration [25,26,27,28,29,30]. Glycan branching has been shown to modulate cellsurface levels of glucose transporter 2 by altering cell-surface sequestration of the receptor [31, 32]
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