Galectin-4 and sulfatides in apical membrane trafficking in enterocyte-like cells.
Bottom Line: Moreover, galectin-4 depletion altered the DRM association characteristics of apical proteins.Sulfatides with long chain-hydroxylated fatty acids, which were also enriched in DRMs, were identified as high-affinity ligands for galectin-4.Together, our data propose that interaction between galectin-4 and sulfatides plays a functional role in the clustering of lipid rafts for apical delivery.
Affiliation: Unité INSERM 560, 59045 Lille Cedex, France.
We have previously reported that 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (GalNAc alpha-O-bn), an inhibitor of glycosylation, perturbed apical biosynthetic trafficking in polarized HT-29 cells suggesting an involvement of a lectin-based mechanism. Here, we have identified galectin-4 as one of the major components of detergent-resistant membranes (DRMs) isolated from HT-29 5M12 cells. Galectin-4 was also found in post-Golgi carrier vesicles. The functional role of galectin-4 in polarized trafficking in HT-29 5M12 cells was studied by using a retrovirus-mediated RNA interference. In galectin-4-depleted HT-29 5M12 cells apical membrane markers accumulated intracellularly. In contrast, basolateral membrane markers were not affected. Moreover, galectin-4 depletion altered the DRM association characteristics of apical proteins. Sulfatides with long chain-hydroxylated fatty acids, which were also enriched in DRMs, were identified as high-affinity ligands for galectin-4. Together, our data propose that interaction between galectin-4 and sulfatides plays a functional role in the clustering of lipid rafts for apical delivery.
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Mentions: To determine whether galectin-4 depletion affected the organization of DRMs in HT-29 5M12 cells, we tested the detergent insolubility at 4 and 37°C of glycoproteins at the apical membrane on living cells. After Triton X-100 treatment of control cells at 37°C, apical staining of CEA and MUC1 and to some extent DPP-IV was still seen. In galectin-4-KD cells, CEA, MUC1, and DPP-IV were no longer detected after Triton X-100 extraction at 37°C (Fig. 7). We also examined the surface delivery of tsO45 VSVG, using basolateral and apical versions of this protein. The apical delivery was inhibited in the galectin-4-KD cells, whereas the basolateral VSV-G protein was transported normally to the basolateral membrane (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200407073/DC1).