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

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-β- d -galactopyranoside (GalNAcα- 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.     

Lactobacillus casei NY1301 Increases the Adhesion of Lactobacillus gasseri NY0509 to Human Intestinal Caco-2 Cells

In the presence of Lactobacillus casei NY1301, the adhesion of Lactobacillus gasseri NY0509 to cultured human intestinal Caco-2 cells was significantly increased (P<0.01). In contrast, L. gasseri NY0509 did not affect the adhesion of L. casei NY1301. A heat-stable cell component of L. casei NY1301 was involved in this increase of adhesion. These results suggest that a combination of these strains may have synergistic effects of adhesion to human intestinal mucosa.     

Regulation of Intestinal Epithelial Brush Border Na+/H+ Exchanger Isoforms, NHE2 and NHE3, in C2bbe Cells

Until recently, studies to characterize the intestinal epithelial Na⁺/H⁺ exchangers had to be done in nonepithelial, mutated fibroblasts. In these cells, detection of any Na⁺/H⁺ exchange activity requires prior acid loading. Furthermore, most of these experiments used intracellular pH changes to measure NHE activity. Because changes in pH _i only approximate Na⁺/H⁺ exchange activity, and may be confounded by alterations in buffering capacity and/or non-NHE contributions to pH regulation, we have used ²²[Na] unidirectional apical to cell uptake to measure activities specific to NHE2 or NHE3. Furthermore, we performed these measurements under basal, nonacid-stimulated conditions to avoid bias from this nonphysiological experimental precondition. Both brush border NHEs, when expressed in the well-differentiated, intestinal villuslike Caco-2 subclone, C2bbe (C2), localize to the C2 apical domain and are regulated by second messengers in the same way they are regulated in vivo. Increases in intracellular calcium and cAMP inhibit both isoforms, while phorbol ester affects only NHE3. NHE2 inhibition by cAMP and Ca⁺⁺ involves changes to both K _Na and V _max . In contrast, the same two second messengers inhibit NHE3 by a decrease in V _max exclusively. Phorbol ester activation of protein kinase C alters both V _max and K _Na of NHE3, suggesting a multilevel regulatory mechanism. We conclude that NHE2 and NHE3, in epithelial cells, are basally active and are differentially regulated by signal transduction pathways. Received: 28 January 1999/Revised: 18 May 1999