2Department of Medical Biochemistry, Göteborg University, P.O. Box 440, 405 30 Gothenburg, Sweden, and 3Cell Biology and Biophysics Programme, EMBL, Meyerhofstrasse 1, 69017 Heidelberg, Germany
Received on December 27, 2000; revised on March 20, 2001; accepted on March 20, 2001.
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Abstract |
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Key words: ammonium chloride/bafilomycin/glycoprotein/glycosylation/Golgi apparatus
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Introduction |
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We observed that a disruption of the pH gradient over the secretory pathway by ammonium chloride (NH4Cl) treatment of cultured cells changed the overall glycosylation of gel-forming mucins. One possible explanation for this observation was that the disrupted pH gradient caused a relocalization of glycosyltransferases. This idea was studied by neutralizing the Golgi pH gradient and monitoring the localization of glycosyltransferases in the Golgi apparatus. Two pH-disrupting agents were used: NH4Cl, causing its effect via ammonia diffusing into the cell, and bafilomycin A1, an inhibitor of the H+-K+-ATPase present both in the secretory and endocytotic pathways (Bowman et al., 1988; Nelson and Tai, 1989
). As glycosyltransferase markers we examined the O-glycosylation relevant polypeptide:N-acetylgalactosaminyltransferase 2 (GalNAc-T2), the N-glycosylation relevant N-acetylglucosaminyltransferase I (NAGT I) and the classical trans-Golgi/TGN marker ß1,4 galactosyltransferase 1 (Gal-T1). NAGT I and Gal-T1 normally reside mainly in the medial/trans-Golgi and the trans-Golgi/TGN, respectively (Nilsson et al., 1993
), whereas GalNAc-T2 is found throughout the Golgi stack with some preference for the trans cisternae (Röttger et al., 1998
).
Indirect immunofluorescence microscopy, immuno-electron microscopy (EM) and subcellular fractionation revealed that the glycosyltransferases were mislocalized. These results could suggest that a pH gradient over the secretory pathway is somehow involved in the proper localization of glycosyltransferases over the Golgi apparatus. When the O-glycans from purified gel-forming mucins of NH4Cl treated LS 174T cells were released and structurally characterized, altered O-glycosylation was observed.
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Results |
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Disruption of the pH gradient results in displacement of glycosyltransferases to endosomal compartments or the cell surface
To study if the pH gradient over the secretory pathway was essential for the localization of glycosyltransferases, HeLa cells stable expressing a VSV-G-tagged GalNAc-T2 were used for indirect immunofluorescence microscopy. Endogenous and VSV-G-tagged GalNAc-T2 are normally found throughout the Golgi stack with a slight preference for the trans cisternae (Röttger et al., 1998). Cells were treated with NH4Cl for 40 h or with bafilomycin A1 for 12 h prior to fixation. Cells were permeabilized by detergent or kept nonpermeabilized, and GalNAc-T2 was monitored by indirect immunofluorescence. Nonpermeabilized control cells (Figure 1A) revealed no cell surface staining of GalNAc-T2, and this transferase was localized to the juxtanuclear Golgi apparatus as shown by the permeabilized cells (Figure 1B). Similarly, in nonpermeabilized NH4Cl treated cells no surface staining was observed (Figure 1C). Instead a low but significant autofluorescence from enlarged endosomes, not due to antibody staining, could be detected (counterstaining not shown). Such osmotic swelling of endosomes on NH4Cl treatment has been observed before, including HeLa cells (Cover et al., 1991
, 1992) and could contribute to the effects observed. In permeabilized NH4Cl-treated cells, GalNAc-T2 showed a partial relocalization from the Golgi stack to punctuate compartments reminiscent of endosomal structures (Figure 1D). In bafilomycin A1treated nonpermeabilized cells, staining for GalNAc-T2 revealed cell surface expression (Figure 1E). This was also observed in permeabilized cells, showing surface as well as Golgi staining (Figure 1F). Surface expression was also demonstrated in living cells incubated with antibodies at 4°C followed by fixation (not shown).
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Relocalization of GalNAc-T2 and Gal-T1 from Golgi stacks was further evaluated by quantitative immunostaining of the electron micrographs. Fifteen of the Golgi stacks identified for each cell category were labeled with the polyclonal antibody against VSV-G-tagged GalNAc-T2, and 15 with a polyclonal against endogenous Gal-T1. The labeling intensity against GalNAc-T2 was for control cells (Figure 4A) 156 gold particles per µm2, for NH4Cl-treated cells (Figure 4C) 123 µm2 (79% of control), and for bafilomycin A1treated cells (Figure 4E) 104 µm2 (67%). These results are in accordance with the light microscopy in Figure 1, suggesting that the GalNAc-T2 was only partly relocalized from the Golgi stack and/or that the relocation was compensated by production of new enzymes. For Gal-T1 the corresponding values were 152 µm2 for control cells (Figure 4B), 76 µm2 (50%) for NH4Cl-treated cells (Figure 4D), and 26 µm2 (17%) for bafilomycin A1treated cells (Figure 4F). Thus, these findings suggest a more complete relocalization from the Golgi stack of Gal-T1 compared to GalNAc-T2, as was also suggested by Figure 3.
Quantification of GalNAc-T2 over endosomal compartments was also performed by immuno-EM. Fifteen individual cells, expressing the VSV-G-tagged enzyme construct, were randomly selected, and the average number of gold particles over endosomal compartments per cell was calculated. This was for control cells (Figure 6A) 0.2 cell1, for NH4Cl-treated cells (Figure 6B) 26 cell1, and for bafilomycin A1treated cells (Figure 6C) 3.8 cell1. Background values found in cells (N = 15) not expressing the construct were 0.0 cell1, 2.5 cell1, and 0.3 cell1, respectively. These results support the conclusion from Figure 5 that the GalNAc-T2 is relocalized to endosomal vesicles on NH4Cl treatment. It also suggests a minor relocalization to endosomes, in addition to the cell surface relocalization, on bafilomycin A1 treatment. Dilation of endosomes was evident in the NH4Cl-treated cells (Figure 6B) and, as discussed, assumed to be an effect of NH4+ as described for HeLa cells (Cover et al., 1991, 1992).
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Possible effects of NH4Cl on the Golgi stack structure in LS 174T cells were investigated by EM. Fifteen Golgi stacks were identified in control cells (Figure 8A) and in NH4Cl-treated cells (Figure 8B), respectively. The average number of cisternae per stack was 3.6 and 3.7, the cisternal widths 16.4 nm and 17.3 nm, and the average stack areas 0.045 µm2 and 0.047 µm2, respectively. Interestingly, no growth of the Golgi apparatus was found in these cells, rather the opposite, with on average 2.3 Golgi stacks per cell in the control cells and 1.3 in the NH4Cl-treated cells. This suggests that the glycosyltransferase relocalization is probably not secondary to a growth of the Golgi as could have been assumed from the HeLa cells.
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Disruption of the pH gradient by NH4Cl caused a decreased O-glycosylation of mucins
To examine if the observed glycosyltransferase relocalization had any effect on O-glycosylation of proteins, we examined O-glycosylated mucins from the LS 174T cells. To obtain pure mucins in a quantity allowing a detailed analysis of glycan structures, the guanidinium chlorideinsoluble mucins of the LS 174T cells, known to contain the MUC2 mucin (Axelsson et al., 1998), were purified. The mucins were washed in guanidinium chloride, solubilized by thiol reduction, and purified by three rounds of isopycnic density gradient ultracentrifugation, two in 4 M and one in 0.2 M guanidinium chloride. The mucin peaks from control and NH4Cl-treated cells were found at 1.45 g/ml and 1.40 g/ml, respectively, on density gradient ultracentrifugation in 4 M guanidinium chloride. The differences in 0.2 M guanidinium chloride were smaller with peaks at 1.51 g/ml and 1.49 g/ml, respectively. These observations suggest that mucins formed under NH4Cl treatment had reduced glycan content, because carbohydrates have higher density than proteins.
To analyze the purified mucins further, these were subjected to monosaccharide and amino acid compositional analyses (Table I). As no blood group Atype determinants were found by the gas chromatographymass spectrometry (GC-MS) analyses, all N-acetylgalactosamine (GalNAc) was assumed to be attached directly to the peptide core. All monosaccharides, except for mannose, showed decreased amounts relative GalNAc on NH4Cl treatment. As mannose is added cotranslationally in the initial step of N-glycan biosynthesis, one should not expect this to be affected. Instead of decreasing, the relative amount of mannose was increased, suggesting that the N-glycans might be less processed after NH4Cl treatment and consequently contain more mannose. The amount of monosaccharides per GalNAc gives an estimation of the mean oligosaccharide length. This was decreased from 4.1 to 2.7 (from 4.0 to 2.5 if mannose is excluded) after NH4Cl treatment. The number of potential O-glycosylation sites utilized is given from the relative amount of GalNAc per serine and threonine. In the control mucins about 59% of the serines/threonines were substituted and about 51% in the NH4Cl-treated cells. Thus, the value for the NH4Cl-treated cells was somewhat lower, suggesting that the number of sites utilized might be a little lower. However, a slightly higher content of serine relative to threonine was observed in the NH4Cl-treated cells, possibly due to the presence of contaminating proteins. If these extra serines are excluded, rather similar substitution levels are obtained for control and treated cells.
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Discussion |
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The three enzymes showed different redistribution patterns on the Golgi pH neutralization, something that maybe could be expected as they have different normal steady-state localization. GalNAc-T2 is found throughout the Golgi stack with a somewhat higher level in the trans cisternae (Röttger et al., 1998), NAGT I resides mostly in the medial/trans cisternae (Nilsson et al., 1993
), and Gal-T1 in the trans cisternae and the TGN (Nilsson et al., 1993
). Because Gal-T1 is largely localized to the TGN, which is more acidic than the cisternae, it may be predicted that Gal-T1 is more sensitive to pH gradient disruption than the two other enzymes. The present results suggest that this is the case; Gal-T1 disappeared to a great extent from the Golgi on treatment with the two substances, whereas the Golgi density of the two other transferases appeared to be less affected. In contrast to the two other transferases, Gal-T1 showed a decreased total antigenicity after NH4Cl treatment. This may be due to the fact that Gal-T1, in addition to being a membrane-bound protein, also occurs in a cleaved secreted form. The conversion from a membrane-bound to a soluble protein probably occurs in the late secretory pathway, distal to the main Gal-T1 localization in the trans cisternae and TGN (Strous, 1986
). It is therefore reasonable to believe that displacement of Gal-T1 in a distal direction could lead to an increased conversion into the soluble form and loss from the cell. The decreased level of Gal-T1 on treatment with the two substances may therefore reflect an increased secretion, secondary to distal relocalization.
NH4Cl and bafilomycin A1 gave different redistribution patterns of both GalNAc-T2 and NAGT I. This could be due to bafilomycin A1 being more efficient than NH4Cl in neutralizing Golgi pH, as was indicated by the more complete relocalization of Gal-T1 by bafilomycin A1 than by NH4Cl (Figure 4). Another explanation might be that the enzymes are endocytosed in the presence of NH4Cl but not in the presence of bafilomycin A1, assuming that the primary effect of both substances is a relocalization to the cell surface. That bafilomycin A1 inhibited endocytosis of both GalNAc-T2 and NAGT I was demonstrated by late endosomal staining after washing out the drug from the cells (not shown).
Why, then, does pH have an effect on glycosyltransferase distribution? Clues to this come from the notion of a complete absence of sequence similarities between the different enzymes. The enzymes have a common domain structure with an amino terminal cytoplasmic tail followed by a transmembrane region, a luminal stem region, and a terminating catalytic domain. In general, the first three domains are responsible for proper steady-state distribution, whereas in some cases the transmembrane domain or the luminal stalk is sufficient for correct cisternal localization (for review see Colley, 1997). However, even if the sequences differ completely between enzymes, a given enzyme appears highly conserved when compared between different species. Such a high selective pressure to conserve the primary structure is consistent with the enzymes being sensitive to differences in pH, for example. What role or function would such sensitivity to the pH then play? It is possible that the enzyme undergoes a conformational alteration at a particular pH and that this is necessary for a proper preservation of the enzyme in the cisternae to which it is destined (Füllekrug and Nilsson, 1998
; Weiss and Nilsson, 2000).
The importance of maintaining a pH gradient over the secretory pathway was further supported by the observed changes in mucin O-glycosylation on NH4Cl treatment. The altered localization of a core 2 ß1,6N-acetylglucosaminyltransferase was shown to cause a decreased synthesis of branched O-glycans, showing that relocalization of glycosyltransferases can cause an altered glycosylation (Skrincosky et al., 1997). Although we have analyzed the mislocalization of only a few glycosyltransferases, the general glycosylation alterations observed in the mucins indirectly suggest that other transferases were displaced as well. Many of the more distal O-glycosylation-relevant transferases are not yet characterized, making studies of potential relocalization of these enzymes impossible. Some specific connections between the glycosyltransferase relocalization and glycosylation alterations observed could be suggested. On NH4Cl treatment, the GalNAc-T2 density over the Golgi apparatuses was not substantially changed, whereas the density of Gal-T1 was reduced. This could be in accordance with the number of serines/threonines utilized by GalNAc-transferases being essentially unchanged (Table I), whereas the number of structures containing Gal that could have been attached by the Gal-T1 was reduced (structures 4.17.1 and s5.1s6.1, Figure 10). It is, however, important to bear in mind that both these enzyme activities are carried out by enzyme families containing several members (Clausen and Bennett, 1996
; Almeida et al., 1997
) and that the role of GalNAc-T2 and Gal-T1 in the mucin biosynthesis of LS 174T cells has not been proven. An alternative explanation for the observed changes in mucin glycosylation could be that glycosyltransferases have different enzyme activity pH optima, giving decreased activity on pH neutralization. Such an explanation does not contradict that the transferases are redistributed on elevated pH.
Bafilomycin A1 could not be used in the mucin glycosylation experiments, because longer incubation times were toxic to the cells. When mucins were produced in preparative scale for oligosaccharide analysis, pH manipulation with NH4Cl had to be extended over 10 days due to the slow turnover of mucins in the LS 174T cells (up to 6 days, Sheehan et al., 1996). The slow turnover was illustrated by analysis of cells cultured in NH4Cl for only 3 days, where differences in O-glycosylation between control and treated cells were smaller than after 10 days (not shown). This was presumably due to residing mucin molecules produced before the NH4Cl treatment and stored in the secretory granules for long periods of time.
O-glycosylation is necessary for the gel-forming properties of polymerizing mucins and thus for the function of the vitally essential mucus barrier. The results presented here suggest that pathological changes of the Golgi pH gradient might influence mucin glycosylation and consequently the mucus quality. This might be relevant in the pathogenesis of several mucus related disorders. In cystic fibrosis, the pH of the Golgi apparatus has been proposed to be elevated due to lack of Cl counterions, caused by the defective Cl transport (Barasch et al., 1991; Lukacs et al., 1992
). Later results have, however, contradicted elevated Golgi pH in this disorder (Seksek et al., 1996
). Instead, a lowered pH of the Golgi apparatus might be suggested, as an HCO3 transport has been suggested to be defective (Poulsen et al., 1994
). If the latter is correct, one may speculate that the decreased pH results in a relocalization of glycosyltransferases in a proximal direction, causing increased glycosylation. The direct relation between Helicobacter pylori infection and the epithelial damage in gastritis and peptic ulcers is poorly understood. It is known that H. pylori produces large amounts of ammonia due to a urease. Gastric mucus ammonia concentration around 25 mM has been reported from infected patients (Thomsen et al., 1989
). An intracellular ammonia concentration of 25 mM was also found in H. pyloriinfected culture cells in medium containing physiological urea concentrations (Mégraud et al., 1992
). Because these levels of ammonia can cause glycosyltransferase relocalization and changes in mucin O-glycosylation, as shown in this study, it is possible that H. pylori infection could cause a decreased mucus glycosylation due to the ammonia produced. The defective glycosylation might result in a defective mucus barrier, allowing the hydrochloric acid of the stomach lumen to reach and injure the epithelial cells.
In conclusion, we have suggested the relocalization of glycosyltransferases when the secretory pathway pH gradient is disrupted. This may suggest that pH has a role in maintaining the steady state distribution of Golgi resident glycosyltransferases and that this could have direct consequences for proper glycosylation of secreted proteins.
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Materials and methods |
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Fluorescence measurement and calculation of intracellular pH
The effect of NH4Cl or bafilomycin A1 on intracellular pH was measured using dual-emission ratiometric fluorescence imaging. In all experiments, cells were grown for 23 days on glass coverslips. Cells were rinsed three times with a Tyrodes buffered solution and loaded with BCECF-AM (Molecular Probes, OR) for 45 min at room temperature. The coverslips were rinsed in Tyrodes and attached to the bottom of a chamber (about 1 ml) using silicone grease, and mounted on a microscope equipped for epifluorescence (Axiovert 100 TV, Zeiss, Germany). Excitation light (xenon arc lamp) was passed through one of two computer-controlled differential interference filters (440 or 490 nm ± 20 nm) mounted in a wheel (Ion Optix Corporation, MA). The light was passed through a 440/490 ± 20 nm dichroic mirror and a 100x Zeiss plan-neofluor oi-immersion objective lens. Fluorescent light was passed back through the dichroic mirror (510 ± 40 nm) and a 520-nm-long pass barrier filter to reduce background fluorescence. The emitted fluorescence was measured by a cooled video camera (KAPPA Messtechnik, Gleichen, Germany). Cells were excited for a 250-ms period at each excitation wavelength, and the emitted fluorescence analyzed using a computer-based system (Bildanalys, Stockholm, Sweden). Background values for each excitation wavelength were obtained from cell-free areas of the coverslip. Individual cells were identified using the image of fluorescence during excitation at 440 nm, and the cytosolic area was marked. Three pairs (490 nm and 440 nm) of values were obtained in succession for each defined area, at intervals of 250 ms, and an average was obtained. The experiment was calibrated in vitro with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (James-Kracke, 1992). For each pair of averaged values, the ratio of the fluorescence intensity at 490 nm to the fluorescence intensity at 440 nm was calculated. Background values were subtracted from the experimental values before the ratios were calculated.
In NH4Cl experiments, 25 mM was added to the cells 24 h prior to the experiment and was present in the loading and initial experimental buffer. The fluorescence intensity was measured over a 15-min period, cells were washed three times in NH4Cl-free solution, and after 15 min in this solution the fluorescence intensity was measured again, prior to an in vitro calibration. The pH values for each cell before and after NH4Cl removal were compared. As the effect of bafilomycin A1 removal turned out to be slow, experiments with this drug instead compared pH values from two separate sets of experiments, one from nontreated cells and one from cells treated with 300 nM bafilomycin A1 for 6 h.
Indirect immunofluorescence microscopy
HeLa cells, stable expressing VSV-G-tagged GalNAc-T2 or myc-tagged NAGT I, were seeded onto glass coverslips and fixed at about 75% confluence. NH4Cl (25 mM) was added 40 h prior to fixation and bafilomycin A1 (300 nM) 12 h prior to fixation. Cells were fixed for 20 min in 3% paraformaldehyde in phosphate buffered saline (PBS), washed 3 x 5 min in PBS, incubated for 10 min in 50 mM NH4Cl to quench aldehyde groups, washed 3 x 5 min in PBS, permeabilized (if denoted) for 4 min in 0.1% Triton X-100 in PBS, and washed 3 x 5 min in PBS. After blocking 2 x 5 min in PBS-gelatin (PBS with 0.2% teleostean gelatin, Sigma) and washing 3 x 5 min in PBS, coverslips were incubated for 20 min in anti-VSV-G-tag (1:100) or anti-myc-Tag (1:25) rabbit polyclonal antiserum or anti-Gal-T1 monoclonal (1:100) and in some cases 2C2 monoclonal (1:50) in PBS-gelatin, washed three times in PBS-gelatin and twice in PBS, all over 5 min, and incubated for 20 min in anti-rabbitfluorescein isothiocyanate (FITC) (1:100) and/or anti-mouse-Cy3 (1:750). After washing in PBS and finally in water, coverslips were mounted onto slides using Mowiol mounting medium (Rodriguez and Deinhardt, 1960) containing 0.1 g/ml DABCO (Sigma). The whole process was carried out at room temperature. Microscopy was performed in a Axiovert 100 TV microscope (Zeiss), and images were captured using a Hamamatsu RGB CCD camera controlled by the Open Lab software 2.0 (Improvision, Coventry, UK).
Immunoelectron microscopy and quantitation
HeLa cells, stable expressing VSV-G-tagged GalNAc-T2, were kept as controls or were treated with NH4Cl (25 mM for 40 h) or with bafilomycin A1 (300 nM for 12 h), prior to preparation for immuno-EM as described (Moolenaar et al., 1997). The anti-VSV-G-tag and anti-GalT1 polyclonals were used at 1:100 and 1:50, respectively, and secondary gold-conjugated anti-rabbit antibodies were used at 1:100. LS 174T cells, nontreated or treated with NH4Cl (25 mM for 40 h), were prepared similarly, but only structural information was obtained. Grids were viewed in a Zeiss EM10 at 80 kV. Golgi stacks were defined as membrane structures or parts thereof, where three cisternae or more overlap along more than 0.2 µm. Golgi stacks were selected at random, and, when found, all stacks within the same cell were identified and included. The boundaries of Golgi stacks were defined as the utmost cisternal membranes, and the stack areas were measured. Cisternal width (average from all available stacks) was defined as the average stack width/2n-1, were n is the number of cisternae, that is, the cisternal lumens and the spaces between the cisternae were assumed to have the same width. For quantification of endosomal labeling, HeLa cells with an intact shape and an area of at least 75 µm2 were selected randomly. Individual cells were regarded to express the VSV-G-GalNAc-T2 when at least 10 gold particles associated to membrane structures could be found. Endosomes were defined as electron-dense vesicle-shaped structures with an area of at least 0.005 µm2.
Subcellular fractionation and western blotting
NH4Cl (25 mM) was added to 70% confluent cells (1080 cm2 HeLa or 540 cm2 LS 174T); after 40 h cells (and nontreated controls) were washed twice in ice-cold PBS and harvested (cell scraper) at 4°C. Cells were pelleted at 200 x g for 5 min, resuspended, and equilibrated in K-Hop buffer (130 mM KCl, 25 mM TrisHCl, pH 7.5) at 4°C, pelleted as above, and resuspended, using a micropipette, in 1 ml/g cells of K-Hop with 0.1% dimethyl sulfoxide (DMSO), 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml antipain, 1 mM benzamidine-HCl, and 40 µg/ml phenylmethylsulfonyl fluoride. Cells were homogenized by passage through syringe needles (5 x 22 G/0.7 mm, 5 x 25 G/0.5 mm, 3 x 27 G/0.4 mm). The homogenate was centrifuged for 5 min at 1000 x g, and 1.3 ml of the obtained postnuclear supernatant was layered on top of a linear 10 ml 525% (w/v) Nycodenz (Nycomed, Oslo, Norway) gradient in K-Hop, with a 0.66-ml 40% Nycodenz bottom cushion. Ultracentrifugation was performed in a Beckman SW40 (swinging bucket) rotor, at 28,500 r.p.m. for 60 min (HeLa) or 40 min (LS 174T). Nine 1.33-ml fractions were recovered from the top to the bottom and diluted 1:1 with K-Hop prior to pelleting for 1 h at 45,000 r.p.m. in a Beckman TLA45 rotor, using a Beckman tabletop ultracentrifuge. Pellets were redissolved in 50 mM TrisHCl, pH 6.8, 20% glycerol, 10 mM dithiothreitol (DTT), 95°C, 5 min, and subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) (10 µl per lane). The separation gel was 12% polyacrylamide, 0.1% SDS, 375 mM TrisHCl, pH 8.8, and the stacking gel 4.5% polyacrylamide, 0.1% SDS, 126 mM TrisHCl, pH 6.8. After gel electrophoresis (100 mM Tris, 768 mM glycine, 0.2% SDS as anode and cathode buffer), proteins were western blotted onto nitrocellulose (Hybond-C Extra, Amersham) membranes for 1 h at 1 mA/cm2 using a semi-dry blotter (Semi-Phor, Hoefer, San Francisco, CA) and 20 mM Tris, 50 mM glycine, 20% methanol as transfer buffer. Membranes were blocked for 1 h at room temperature in 10% nonfat milk and 0.1% Tween-20 in PBS, and incubated for 1 h at room temperature in anti-calnexin (1:20,000) or anti-VSV-G (1:2000) or anti-Gal-T1 (1:1000) or anti-GalNAc-T2 (1:4) antibody in PBS with 2% nonfat milk and 0.02% Tween-20. Membranes were washed six times in 0.1% Tween-20 in PBS, the first two times in the presence of 10% nonfat milk. Second incubation in peroxidase-conjugated anti-rabbit (1:2500) or anti-mouse (1:2500) antibody was for 1 h at room temperature in PBS with 2% nonfat milk and 0.02% Tween-20. The blots were washed six times as above, and developed using the ECL reagent (Amersham).
Purification of insoluble mucins from LS174T cells
Cells were cultured in four 1000-cm2 roller bottles for 10 days with daily medium changes, one bottle in the presence of 25 mM NH4Cl except for the first 24 h. Cells were extracted in 20 ml guanidinium chloride per bottle, and the insoluble lysate components were washed, solubilized by reduction of disulfide bonds, and alkylated to stabilize obtained cysteine groups as described elsewhere (Carlstedt et al., 1993; Axelsson et al., 1998
). The mucins were purified by three rounds of isopycnic CsCl density gradient ultracentrifugation (Carlstedt et al., 1983
), two with 4 M guanidinium chloride and one with 0.2 M guanidinium chloride. After recovering the gradients into fractions from the bottom up, the mucin peaks were identified by periodic acidSchiff slot blot. After the last ultracentrifugation, the samples were dialyzed six times against water, lyophilized, and dissolved in water.
Release, fractionation, and analysis of oligosaccharides from the purified mucins
The monosaccharide composition of the purified mucins was analyzed after hydrolysis (Karlsson and Hansson, 1995), and the amino acid composition was determined using an Alpha Plus amino acid analyzer (Pharmacia). Released neutral and sialylated oligosaccharides from approximately 7 mg mucins of nontreated and 1.5 mg mucins of NH4Cl-treated cells were isolated, permethylated, and analyzed by GC and GC-MS as described (Karlsson et al., 1995
, 1997). The sequence and linkage positions of the structures were interpreted from the obtained mass recorded on GC-MS. The hexoses observed in the mass spectra were assumed to be galactoses (Gal), the N-acetylhexosamines to be N-acetylglucosamines (GlcNAc), deoxyhexoses to be fucoses (Fuc), and N-acetylhexosaminitols to be N-acetylgalactosaminitols (GalNAcol), as these were the monosaccharides found by sugar composition analysis of the released oligosaccharides.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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