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 gain further insight into the role of these glycosphingolipid–galectin-4 complexes in detergent insolubility of raft microdomains, DRMs were first isolated from a total membrane preparation of control and GalNAcα-O-bn–treated cells using 1% Triton X-100 at 4°C. These DRMs, insoluble at 4°C, were then incubated at 37°C and both the soluble and the insoluble material (DRMs being detergent insoluble at 37°C) were examined by HPTLC (Fig. 5). The results showed the presence of galactosylceramides in all DRM fractions of control and GalNAcα-O-bn–treated cells, whereas sulfatides were only present in the DRM fraction detergent insoluble at 37°C in control cells.