Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171948&req=5

fig3: 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.

Mentions: We then proceeded to analyze whether galectin-4 was present in membrane vesicles released from perforated HT-29 cells (Wandinger-Ness et al., 1990). Immunoelectron microscopy demonstrated that galectin-4 was in carrier vesicles containing DPP-IV (Fig. 3 A).


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: 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.
Mentions: We then proceeded to analyze whether galectin-4 was present in membrane vesicles released from perforated HT-29 cells (Wandinger-Ness et al., 1990). Immunoelectron microscopy demonstrated that galectin-4 was in carrier vesicles containing DPP-IV (Fig. 3 A).

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