Galectin-4 and sulfatides in apical membrane trafficking in enterocyte-like cells - PubMed (original) (raw)

. 2005 May 9;169(3):491-501.

doi: 10.1083/jcb.200407073.

Valérie Gouyer, Jean-Pierre Zanetta, Hervé Drobecq, Emmanuelle Leteurtre, Georges Grard, Odile Moreau-Hannedouche, Emmanuel Maes, Alexandre Pons, Sabine André, André Le Bivic, Hans Joachim Gabius, Aki Manninen, Kai Simons, Guillemette Huet

Affiliations

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

Delphine Delacour et al. J Cell Biol. 2005.

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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Figures

Figure 1.

Figure 1.

Analysis of proteins contained in DRMs of control and GalNAcα-_O_-bn–treated HT-29 cells. 2-D patterns were obtained using 300 μg of DRM proteins isolated from control and GalNAcα-_O_-bn–treated (14 d) cells. Each protein spot was numbered and submitted to mass spectrometry analysis in MALDI-TOF mode. Spot number 32 was identified as galectin-4 (arrows).

Figure 2.

Figure 2.

GalNAcα- O -bn decreases the apical localization of galectin-4. (A) Western blot of culture media, cytosol and membrane fractions of control and GalNAcα-_O_-bn–treated (14 d) cells after permeabilization with saponin, using anti–galectin-4 antibody. (B) Confocal microscopy with an anti–galectin-4 antibody on permeabilized or unpermeabilized control and GalNAcα-_O_-bn–treated (14 d) cells. Apical and xz sections are shown. Bars, 22 μm.

Figure 3.

Figure 3.

Galectin-4 is associated with DRMs of post-Golgi carrier vesicles. (A) Immunogold labeling of nascent carrier vesicles isolated from HT-29 5M12 cells. Galectin-4 (arrowhead) was labeled with 18-nm gold particles and DPP-IV (arrow) by 12-nm gold. Bars, 156 nm. In the vesicle preparation, 30% of the vesicles were labeled with the anti–DPP-IV antibody. 20% of these DPP-IV–positive vesicles were also labeled with galectin-4. (B) Western blotting of galectin-4 in carrier vesicles. Trypsin digestion of galectin-4 in untreated or Triton X-100–treated carrier vesicles is shown. (C) Detergent extractability of galectin-4, DPP-IV, and annexins XIIIb and XIIIa in carrier vesicles from HT-29 5M12 cells. Detergent extracts, i.e., Triton X-100 soluble (S), insoluble at 4°C but soluble at 37°C (P1), and insoluble at 37°C (P2), were analyzed by Western blotting.

Figure 4.

Figure 4.

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.

Figure 5.

Figure 5.

Sulfatides are found in DRMs which are detergent insoluble at 37°C DRMs were isolated from a total membrane fraction of control and GalNAcα-_O_-bn–treated (14 d) cells. DRMs were further warmed at 37°C and both the soluble and insoluble material were collected and examined by HPTLC.

Figure 6.

Figure 6.

KD of galectin-4 expression in HT-29 5M12 cells induces mistargeting of apical proteins. (A) Western blot analysis of galectin-4 in empty-RVH-1-virus–infected cells or galectin-4-KD cells. (B) Confocal microscopy with antibodies directed against apical (MUC1, CEA, DPP-IV) and basolateral (E-cadherin) proteins, on empty-RVH-1-virus–infected cells or galectin-4 KD cells. xz sections are shown.

Figure 7.

Figure 7.

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.

Figure 8.

Figure 8.

Apical and basolateral delivery of DPP-IV in control and galectin-4-KD cells. (A) Cells were pulse labeled for 30 min, and newly synthesized proteins were chased for 4 or 6 h and biotinylated from the apical or basolateral side. Aliquots from the immunoprecipitations (total) and streptavidin precipitations (cell surface) are shown. Ap, apical; Bl, basolateral. ○, DDP-IV precursor; •, mature DPP-IV. (B) Apical and total DPP-IV signals were quantitated in control and in galectin-4-KD cells. The apical to total ratio in control cells was set to 100% and apical transport efficiency in galectin-4-KD cells is shown relative to this value. Data are means ± SD.

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