To identify proteins possibly associated with lipid rafts in HT-29 5M12 we used 2-dimensional (2-D) electrophoresis and matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometry analysis of detergent-resistant membrane fractions (DRMs).
Galectin-4 is a major component of DRMs in HT-29 5M12 cells
The 2-D gel electrophoresis pattern of DRMs is shown in , and the identity of corresponding proteins in . Besides flotillin-1, an ubiquitous marker of DRMs, the proteins identified could be divided into eight groups: (1) G proteins (G(i) α1,2,3, G11, GTPase-activating protein for Rab6); (2) proteins of SNARE machinery (NSF, SNAP23); (3) proteins of vesicular structures (Rab22a, annexin II); (4) chaperone proteins (heat-shock protein 90 (hsp90), BiP, endoplasmin, hsp73); (5) ionic pumps (V-ATPase, voltage-dependent anion channel 2); (6) membrane cytoskeleton and intermediate filament-associated proteins (αII-spectrin, α4-actinin, myosin light chain, periplakin, mitofilin, cytokeratins 8, 18, 19, and 20); (7) apical membrane glycoproteins dipeptidylpeptidase-IV (DPP-IV), carcinoembryonic antigen (CEA), nonspecific cross-reacting antigen, 5′-nucleotidase, CD59); and (8) a member of the galectin family of lectins, galectin-4. Proteins of the three latter groups were the major proteins of the DRMs.
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 (more ...)
Identification of common protein components in DRMs from control and GalNAcα-O-benzyl–treated HT-29 5M12 cells
GalNAcα-O-bn decreases the amount of galectin-4 associated with DRMs
Prompted by the hypothesis of a role for glycans in apical transport, we analyzed whether galectin-4 was present in DRMs in GalNAcα-O-bn–treated cells. The 2-D gel electrophoresis protein pattern of DRMs of GalNAcα-O-bn–treated cells was similar to that of control cells (). Marked qualitative and/or quantitative changes were observed for a number of proteins. Interestingly, the levels of galectin-4 in DRMs were strongly decreased in GalNAcα-O-bn–treated cells, pointing to an effect of GalNAcα-O-bn on the interaction of galectin-4 with raft-associated compounds.
GalNAcα-O-bn modifies the cellular distribution of galectin-4
The distribution of galectin-4 in control and in GalNAcα-O-bn–treated cells was studied by analyzing the culture medium, the cytosolic and the total membrane fractions from saponin-permeabilized cells for the presence of galectin-4 ( A). In control cells, galectin-4 was found in the membrane fraction, whereas in GalNAcα-O-bn–treated cells, galectin-4 was found in the soluble cytosolic fraction. Neither the control cells nor the GalNAcα-O-bn–treated cells secreted detectable levels of galectin-4 to the culture medium.
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 (more ...)
Confocal microscopy on permeabilized cells showed that galectin-4 was distributed throughout the cytoplasm, but mostly in the subapical region ( B). Furthermore, immunostaining on nonpermeabilized cells revealed the presence of galectin-4 at the extracellular surface of the apical membrane. This extracellular galectin-4 staining disappeared if cells were treated with trypsin from the outside (unpublished data). In permeabilized GalNAcα-O-bn–treated cells accumulation of galectin-4 in the subapical region was no longer observed ( B). Moreover, a clear decrease in the extracellular fraction of galectin-4 at the apical membrane was observed in GalNAcα-O-bn–treated, nonpermeabilized cells ( B).
Galectin-4 is associated with DRMs in post-Golgi carrier vesicle preparations
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 ( A).
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, (more ...)
To determine whether galectin-4 was localized on the lumenal or cytoplasmic side of these vesicles, we subjected the vesicle preparations to trypsin. Galectin-4 was not sensitive to trypsin, suggesting that the lectin was localized on the lumenal side of post-Golgi vesicles ( B). Galectin-4 in these preparations became susceptible to trypsin upon addition of detergent ( B).
Galectin-4 has been previously described as an intestinal brush border protein with potential functions as a raft stabilizer/organizer characterized by insolubility in Triton X-100 at 37°C (“super-rafts”; Danielsen and van Deurs, 1997
; Braccia et al., 2003
). We therefore examined the DRM association of galectin-4 in isolated post-Golgi vesicles of HT-29 5M12 cells using the two-step procedure described by Braccia et al. (2003)
. Vesicle preparations were treated by 1% Triton X-100 at 4°C, fractionated into supernatant (S) and pellet; the pellet was resuspended into Triton X-100, incubated at 37°C and then further fractionated into supernatant (P1) and pellet (P2). Galectin-4 was only present in P2, i.e., the detergent-insoluble fraction at 37°C ( C). DPP-IV was similarly detected only in the Triton X-100–insoluble fraction at 37°C. Annexin XIIIb and XIIIa were partially Triton X-100 soluble at 4°C but their DRM fraction remained insoluble at 37°C (particularly for annexin XIIIb).
GalNAcα-O-bn treatment decreases the amount of glycosphingolipids in DRMs of HT-29 5M12 cells
We then investigated the effect of GalNAcα-O-bn on the lipid composition of the DRMs using a quantitative analysis by high performance thin layer chromatography (HPTLC) and gas chromatography mass spectrometry (GC-MS) in reference to the total membrane fraction. Results in control cells demonstrated enrichment of glycosphingolipids (from 12.16% in total membrane preparation to 34.90% in total DRM preparation) and cholesterol (from 22.78 to 35.13% in DRMs). Sphingomyelin was barely detectable. Among the glycosphingolipids, galactosyl-ceramides were the major class (51.18%), followed by sulfatides (33.05%), GM3 (12.98%), and GM1 (2.79%; ). Fatty-acid methyl esters (FAMEs) analysis revealed that DRMs were enriched in hydroxylated fatty acids (all hydroxylated in position 2 with a predominance of the C24:0 chain; 11.36% vs. 0.32% in total membranes).
Lipid analysis of total membranes and DRMs
When DRMs were prepared from cells treated with GalNAcα-O-bn the amount of cholesterol increased by twofold (54.56% vs. 35.13% in control cells), whereas the amount of glycosphingolipids was decreased by twofold (16.50% vs. 34.90% in control cells; ). FAMEs analysis showed a decrease in hydroxylated C24:0 (7.58% vs. 11.36% in control cells), with a simultaneous increase in mono-unsaturated FAMEs (C16:1 and C18:1). These changes after GalNAcα-O-bn treatment prompted us to look for galactosylated glycosphingolipids as ligands for galectin-4.
Identification of glycosphingolipids as carbohydrate ligands of galectin-4
Galectin-4 immunoprecipitates were analyzed by HPTLC ( 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).
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- (more ...)
When similar experiments were performed on cells treated with GalNAcα-O-bn for 12 d, the quantity of material recovered was extremely low (<1% of the control cells) and not detectable by HPTLC, indicating a dramatic inhibition in the formation of glycosphingolipid–galectin-4 complexes ( A). A short time exposure to GalNAcα-O-bn (18 h) was also used and trace amounts of less polar sulfatides were recovered (~10% of the control).
To gain further insight into possible glycosphingolipids–galectin-4 interactions, we used human glycosphingolipid preparations from sciatic nerve, for overlay experiments with the recombinant NH2- or COOH-terminal domains of galectin-4. Each of these domains contains one of the two carbohydrate recognition domains (CRDs) in galectin-4. A strong binding of both the NH2-terminal and COOH-terminal domain of galectin-4 was observed to the more polar fraction of sulfatides, i.e., those substituted by 2-hydroxylated fatty acids ( B). In contrast, the less polar sulfatides did not show any binding to the domains of galectin-4. In addition, a weak binding to the more polar galactosyl-ceramides was observed with the COOH-terminal domain of galectin-4. The addition of galactose (0.3 M) and lactose (0.1 M) during the incubation did not significantly reduce the binding of the two galectin-4 domains to the glycosphingolipids. No binding to gangliosides was observed (unpublished data).
The human sciatic nerve galectin-4 ligands were further characterized by GC-MS and MALDI-TOF mass spectrometry. Results showed that these ligands corresponded to sulfatides and galactosylceramides showing a similar composition as those identified after co-immunoprecipitation from HT-29 5M12 cells with the anti–galectin-4 antibody. By NMR spectroscopy, we demonstrated that galectin-4 ligands were β-galactosylceramides and 3-sulfated-β-galactosylceramide both containing 2-hydroxylated fatty acids (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200407073/DC1
Sulfatides are found in DRMs, which are detergent insoluble at 37°C
To gain further insight into the role of these glycosphingolipid–galectin-4 complexes in detergent insolubility of raft microdomains, DRMs were first isolated from a total membrane preparation of control and GalNAcα-O-bn–treated cells using 1% Triton X-100 at 4°C. These DRMs, insoluble at 4°C, were then incubated at 37°C and both the soluble and the insoluble material (DRMs being detergent insoluble at 37°C) were examined by HPTLC (). The results showed the presence of galactosylceramides in all DRM fractions of control and GalNAcα-O-bn–treated cells, whereas sulfatides were only present in the DRM fraction detergent insoluble at 37°C in control cells.
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 (more ...)
Inhibition of galectin-4 expression abrogates apical targeting
To analyze whether galectin-4 had a role in the delivery of apical proteins, we depleted galectin-4 expression in HT-29 5M12 cells by RNA interference (RNAi). Galectin-4 RNAi constructs and controls are described in Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200407073/DC1
The level of galectin-4 mRNA depletion was analyzed by quantitative RT-PCR and Western blotting. The galectin-4 mRNA was reduced by 80% in the galectin-4-knockdown (KD) cell population and correlated well with the observed strong reduction (~80%) of the galectin-4 protein levels ( A).
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 (more ...)
We then analyzed the cellular localization of DPP-IV and two other apical markers, CEA, a glycosylphosphatidylinositol-anchored protein, and mucin (MUC1), a transmembrane protein, in the galectin-4-KD cells. In addition, localization of a basolateral marker, E-cadherin, was also studied. In the galectin-4-KD cells, the amount of DPP-IV, CEA, and MUC1 delivered to the apical membrane was significantly decreased ( B). Nevertheless, we did not observe mistargeting of DPP-IV, CEA, and MUC1 to the basolateral membrane upon galectin-4 depletion. Instead, we saw intracellular accumulation of these glycoproteins ( B). Costaining experiments with different organellar markers in galectin-4-KD cells showed partial colocalization of the cargo with lysosome-associated membrane protein 2 (not depicted). E-Cadherin localization was not altered in galectin-4-KD cells ( B). These results indicated that galectin-4 is required for efficient apical delivery of glycoproteins.
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 (). 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
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 (more ...)
Inhibition of galectin-4 expression decreases apical delivery of newly synthesized DPP-IV
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 (). Significant missorting of DPP-IV to the basolateral surface was not observed.
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 (more ...)