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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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Galectin-4 is no longer bound to sulfatides under GalNAcα-O-bn treatment. (A) Co-immunoprecipitation of galectin-4 complexes and HPTLC analysis of glycolipid ligands. Co-immunoprecipitation was performed from the same quantity of control and GalNAcα-O-bn–treated (14 d and 18 h) cells. Plates were iodine stained and scanned. The material migrating as two bands of galactosylceramides (Cer and Cer*) and the trailing corresponding to sulfatides (Sulf and Sulf*) were recovered from the HPTLC plate, submitted to acid-catalyzed methanolysis and analyzed by GC-MS. Cer, sphinganine and nonhydroxylated fatty acids; Cer*, sphinganine and 2-hydroxylated fatty acids; Sulf and Sulf*, sphingosine, sphinganine, phytosphingosine and 6-hydroxy-sphingosine, and 2-hydroxylated fatty acids from 16 to 28 carbon atoms. (B) Overlay experiments on the four fractions corresponding to the galactosylceramides and sulfatides purified from human sciatic nerve. Fractions were identified by their eluting methanol percent. Their glycosphingolipids were visualized by orcinol staining and analyzed for binding to the NH2-terminal CRD and the COOH-terminal CRD of galectin-4. Control without lectin is presented. arrows show the glycolipids which bind the NH2-terminal or COOH-terminal domain of galectin-4.
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fig4: Galectin-4 is no longer bound to sulfatides under GalNAcα-O-bn treatment. (A) Co-immunoprecipitation of galectin-4 complexes and HPTLC analysis of glycolipid ligands. Co-immunoprecipitation was performed from the same quantity of control and GalNAcα-O-bn–treated (14 d and 18 h) cells. Plates were iodine stained and scanned. The material migrating as two bands of galactosylceramides (Cer and Cer*) and the trailing corresponding to sulfatides (Sulf and Sulf*) were recovered from the HPTLC plate, submitted to acid-catalyzed methanolysis and analyzed by GC-MS. Cer, sphinganine and nonhydroxylated fatty acids; Cer*, sphinganine and 2-hydroxylated fatty acids; Sulf and Sulf*, sphingosine, sphinganine, phytosphingosine and 6-hydroxy-sphingosine, and 2-hydroxylated fatty acids from 16 to 28 carbon atoms. (B) Overlay experiments on the four fractions corresponding to the galactosylceramides and sulfatides purified from human sciatic nerve. Fractions were identified by their eluting methanol percent. Their glycosphingolipids were visualized by orcinol staining and analyzed for binding to the NH2-terminal CRD and the COOH-terminal CRD of galectin-4. Control without lectin is presented. arrows show the glycolipids which bind the NH2-terminal or COOH-terminal domain of galectin-4.

Mentions: Galectin-4 immunoprecipitates were analyzed by HPTLC (Fig. 4 A), GC-MS, and MALDI-TOF mass spectrometry. In control cells, galactosyl-ceramides and sulfatides were identified as major constituents. Gangliosides were not detected. The fatty acid composition showed a high content of 2-hydroxylated FAMEs with chain length of 18 or 22 to 26 carbon atoms (>50%; 24:0).


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)

Galectin-4 is no longer bound to sulfatides under GalNAcα-O-bn treatment. (A) Co-immunoprecipitation of galectin-4 complexes and HPTLC analysis of glycolipid ligands. Co-immunoprecipitation was performed from the same quantity of control and GalNAcα-O-bn–treated (14 d and 18 h) cells. Plates were iodine stained and scanned. The material migrating as two bands of galactosylceramides (Cer and Cer*) and the trailing corresponding to sulfatides (Sulf and Sulf*) were recovered from the HPTLC plate, submitted to acid-catalyzed methanolysis and analyzed by GC-MS. Cer, sphinganine and nonhydroxylated fatty acids; Cer*, sphinganine and 2-hydroxylated fatty acids; Sulf and Sulf*, sphingosine, sphinganine, phytosphingosine and 6-hydroxy-sphingosine, and 2-hydroxylated fatty acids from 16 to 28 carbon atoms. (B) Overlay experiments on the four fractions corresponding to the galactosylceramides and sulfatides purified from human sciatic nerve. Fractions were identified by their eluting methanol percent. Their glycosphingolipids were visualized by orcinol staining and analyzed for binding to the NH2-terminal CRD and the COOH-terminal CRD of galectin-4. Control without lectin is presented. arrows show the glycolipids which bind the NH2-terminal or COOH-terminal domain of galectin-4.
© Copyright Policy
Related In: Results  -  Collection

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fig4: Galectin-4 is no longer bound to sulfatides under GalNAcα-O-bn treatment. (A) Co-immunoprecipitation of galectin-4 complexes and HPTLC analysis of glycolipid ligands. Co-immunoprecipitation was performed from the same quantity of control and GalNAcα-O-bn–treated (14 d and 18 h) cells. Plates were iodine stained and scanned. The material migrating as two bands of galactosylceramides (Cer and Cer*) and the trailing corresponding to sulfatides (Sulf and Sulf*) were recovered from the HPTLC plate, submitted to acid-catalyzed methanolysis and analyzed by GC-MS. Cer, sphinganine and nonhydroxylated fatty acids; Cer*, sphinganine and 2-hydroxylated fatty acids; Sulf and Sulf*, sphingosine, sphinganine, phytosphingosine and 6-hydroxy-sphingosine, and 2-hydroxylated fatty acids from 16 to 28 carbon atoms. (B) Overlay experiments on the four fractions corresponding to the galactosylceramides and sulfatides purified from human sciatic nerve. Fractions were identified by their eluting methanol percent. Their glycosphingolipids were visualized by orcinol staining and analyzed for binding to the NH2-terminal CRD and the COOH-terminal CRD of galectin-4. Control without lectin is presented. arrows show the glycolipids which bind the NH2-terminal or COOH-terminal domain of galectin-4.
Mentions: Galectin-4 immunoprecipitates were analyzed by HPTLC (Fig. 4 A), GC-MS, and MALDI-TOF mass spectrometry. In control cells, galactosyl-ceramides and sulfatides were identified as major constituents. Gangliosides were not detected. The fatty acid composition showed a high content of 2-hydroxylated FAMEs with chain length of 18 or 22 to 26 carbon atoms (>50%; 24:0).

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