Galectin‐4‐Regulated Delivery of Glycoproteins to the Brush Border Membrane of Enterocyte‐Like Cells

Abstract

We have previously reported that silencing of galectin-4 expression in polarized HT-29 cells perturbed apical biosynthetic trafficking and resulted in a phenotype similar to the inhibitor of glycosylation, 1-benzyl-2-acetamido-2-deoxy-b-D-galactopyranoside (GalNAca-O-bn). We now present evidence of a lipid raft-based galectin-4-dependent mechanism of apical delivery of glycoproteins in these cells. First, galectin-4 recruits the apical glycoproteins in detergent-resistant membranes (DRMs) because these glycoproteins were depleted in DRMs isolated from galectin-4-knockdown (KD) HT-29 5M12 cells. DRM-associated glycoproteins were identified as ligands for galectin-4. Structural analysis showed that DRMs were markedly enriched in a series of complex N-glycans in comparison to detergent-soluble membranes. Second, in galectin-4-KD cells, the apical glycoproteins still exit the Golgi but accumulated inside the cells, showing that their recruitment within lipid rafts and their apical trafficking required the delivery of galectin-4 at a post-Golgi level. This lectin that is synthesized on free cytoplasmic ribosomes is externalized from HT-29 cells mostly in the apical medium and follows an apical endocytic-recycling pathway that is required for the apical biosynthetic pathway. Together, our data show that the pattern of N-glycosylation of glycoproteins serves as a recognition signal for endocytosed galectin-4, which drives the raft-dependent apical pathway of glycoproteins in enterocyte-like HT-29 cells.Key words: brush border membrane, DRMs, enterocytelike cells, galectin-4, glycosylation A characteristic feature of enterocyte polarization is the formation of distinct apical and basolateral membranes separated by tight junctions. The apical membrane faces the lumen and consists of a brush border of microvilli containing enzymes and transporters for intestinal digestion. This asymmetric structure originates from polarized vesicular trafficking to apical or basolateral membrane domains.

Galectin-3 regulates MUC1 and EGFR cellular distribution and EGFR downstream pathways in pancreatic cancer cells

“…Galectin-3 promotes MUC1 and EGFR endocytosis J Merlin et al Stechly et al, 2009). In the case of MUC1, galectin-3 can interact either with O-glycans branched on the TR domain (Yu et al, 2007) or with an N-glycan branched on Asn 36 of the C-terminal domain (Ramasamy et al, 2007).…”

The Role of Glycans in Apical Sorting of Proteins in Polarized Epithelial Cells

“…A group of small lectins with one or two carbohydrate binding domains are called galectins. Although galectins are synthesized in the cytoplasm and are devoid of known signals for membrane translocation, several members of this family of proteins have been shown to be of the outmost importance for epithelial cell differentiation and glycoprotein sorting mechanisms (88)(89)(90)(91)(92). The galectins are to a variable extent able to cross intracellular membranes or the plasma membrane to gain access to the lumen of intracellular compartments like endosomes, or to the outside of the cell, where (in both cases) the glycans of glycoproteins and glycolipids are localized.…”

“…The effect on sorting seems to be that apical transport requires galectin-3 mediated glycoprotein clustering to be efficient (94). In polarized epithelial HT-29 cells, galectin-4 recruits N-linked glycoproteins to detergent resistant membrane fractions and further to the apical membrane (89). Knock-down of galectin-4 did not inhibit Golgi exit of glycoproteins, but reduced their surface appearance.…”

Novel Role for Galectin-8 Protein as Mediator of Coagulation Factor V Endocytosis by Megakaryocytes

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