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Galectin-4 and sulfatides in apical membrane trafficking in enterocyte-like cells.

Delacour D, Gouyer V, Zanetta JP, Drobecq H, Leteurtre E, Grard G, Moreau-Hannedouche O, Maes E, Pons A, André S, Le Bivic A, Gabius HJ, Manninen A, Simons K, Huet G - J. Cell Biol. (2005)

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.

View Article: PubMed Central - PubMed

Affiliation: Unité INSERM 560, 59045 Lille Cedex, France.

ABSTRACT
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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Apical glycoproteins are no longer associated with DRMs in galectin-4-KD HT-29 5M12 cells. Confocal microscopy with antibodies directed against MUC1, CEA, and DPP-IV, on empty-RVH-1-virus–infected cells or galectin-4-KD cells, after cell treatment with Triton X-100 at 4°C or 37°C. xz sections were shown.
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fig7: Apical glycoproteins are no longer associated with DRMs in galectin-4-KD HT-29 5M12 cells. Confocal microscopy with antibodies directed against MUC1, CEA, and DPP-IV, on empty-RVH-1-virus–infected cells or galectin-4-KD cells, after cell treatment with Triton X-100 at 4°C or 37°C. xz sections were shown.

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).


Galectin-4 and sulfatides in apical membrane trafficking in enterocyte-like cells.

Delacour D, Gouyer V, Zanetta JP, Drobecq H, Leteurtre E, Grard G, Moreau-Hannedouche O, Maes E, Pons A, André S, Le Bivic A, Gabius HJ, Manninen A, Simons K, Huet G - J. Cell Biol. (2005)

Apical glycoproteins are no longer associated with DRMs in galectin-4-KD HT-29 5M12 cells. Confocal microscopy with antibodies directed against MUC1, CEA, and DPP-IV, on empty-RVH-1-virus–infected cells or galectin-4-KD cells, after cell treatment with Triton X-100 at 4°C or 37°C. xz sections were shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171948&req=5

fig7: Apical glycoproteins are no longer associated with DRMs in galectin-4-KD HT-29 5M12 cells. Confocal microscopy with antibodies directed against MUC1, CEA, and DPP-IV, on empty-RVH-1-virus–infected cells or galectin-4-KD cells, after cell treatment with Triton X-100 at 4°C or 37°C. xz sections were shown.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Unité INSERM 560, 59045 Lille Cedex, France.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus