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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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Related in: MedlinePlus

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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fig8: 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.

Mentions: To evaluate the impact of galectin-4 depletion on membrane trafficking to the apical or basolateral surfaces, the surface delivery of DPP-IV was studied using cell surface biotinylation. Chase times (4 and 6 h) were selected according to a previous study of the raft association of DPP-IV along its biosynthetic pathway in this cell type (Delacour et al., 2003). DPP-IV became detergent insoluble after 4 h. At this time, the maturation of the protein precursor into the mature glycosylated form was nearly complete and only the mature form was detergent insoluble. In galectin-4-KD cells, we observed a fourfold inhibition of DPP-IV delivery to the apical membrane (Fig. 8). Significant missorting of DPP-IV to the basolateral surface was not observed.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: 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.
Mentions: To evaluate the impact of galectin-4 depletion on membrane trafficking to the apical or basolateral surfaces, the surface delivery of DPP-IV was studied using cell surface biotinylation. Chase times (4 and 6 h) were selected according to a previous study of the raft association of DPP-IV along its biosynthetic pathway in this cell type (Delacour et al., 2003). DPP-IV became detergent insoluble after 4 h. At this time, the maturation of the protein precursor into the mature glycosylated form was nearly complete and only the mature form was detergent insoluble. In galectin-4-KD cells, we observed a fourfold inhibition of DPP-IV delivery to the apical membrane (Fig. 8). Significant missorting of DPP-IV to the basolateral surface was not observed.

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