Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins

Magnus A.B. Axelsson2, Niclas G. Karlsson2, Daniella M. Steel2, Joke Ouwendijk3, Tommy Nilsson3 and Gunnar C. Hansson1,2

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.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Addition of the weak base ammonium chloride (NH4Cl) or the proton pump inhibitor bafilomycin A1 to cultured HeLa and LS 174T cells effectively neutralized the pH gradient of the secretory pathway. This resulted in relocalization of the three studied glycosyltransferases, N-acetylgalactosaminyltransferase 2, ß1,2 N-acetylglucosaminyltransferase I, and ß1,4 galactosyltransferase 1, normally localized to the Golgi stack, the medial/trans-Golgi and the trans-Golgi/TGN, respectively. Indirect immunofluorescence microscopy, immunoelectron microscopy, and subcellular fractionation of the tagged or native glycosyltransferases showed that NH4Cl caused a relocalization of the enzymes mainly to vesicles of endosomal type, whereas bafilomycin A1 gave mainly cell surface staining. The general morphology of the endoplasmic reticulum and Golgi apparatus was retained as judged from immunofluorescence and electron microscopy studies. When the O-glycans on the guanidinium chloride insoluble gel-forming mucins from the LS 174T cells were analyzed by gas chromatography–mass spectrometry after neutralization of the secretory pathway pH by NH4Cl over 10 days shorter O-glycans were observed. However, no decrease in the number of oligosaccharide chains was indicated. Together, the results suggest that pH is a contributing factor for proper steady-state distribution of glycosyltransferases over the Golgi apparatus and that altered pH may cause alterations in glycosylation possibly due to a relocalization of glycosyltransferases.

Key words: ammonium chloride/bafilomycin/glycoprotein/glycosylation/Golgi apparatus


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Glycosylation is the major posttranslational modification of most extracellular and cell surface membrane proteins. The individual glycosyltransferases performing this multistep process show a discrete gradient-like distribution along the secretory pathway (Nilsson et al., 1993Go; Rabouille et al., 1995Go; Röttger et al., 1998Go). This presumably allows enzymes to act in a sequential manner on the maturing glycoprotein. Glycans are attached via either an N- or an O-linkage. The latter form is often referred to as mucin type glycosylation, because this is abundant in mucins, highly glycosylated glycoproteins with the O-linked glycans concentrated in mucin domains (Gendler and Spicer, 1995Go). The largest mucins are the gel-forming ones, which serve as a barrier between the external and internal milieu on all mucosal surfaces. The O-glycans contribute to the gel-forming properties of mucins and their proteolytic resistance, both necessary for the barrier function.

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., 1988Go; Nelson and Tai, 1989Go). 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., 1993Go), whereas GalNAc-T2 is found throughout the Golgi stack with some preference for the trans cisternae (Röttger et al., 1998Go).

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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
NH4Cl and bafilomycin A1 treatment neutralize the pH of acidic cell compartments
Two agents were used to neutralize the acidic pH of the distal Golgi and secretory vesicles: NH4Cl, mediating its effect through ammonia diffusing into the cell, and bafilomycin A1, inhibiting the ATPase proton pumps of the secretory and endocytotic pathways (Bowman et al., 1988Go; Nelson and Tai, 1989Go). To demonstrate that these drugs had the desired effect in the HeLa cells used, measurements with the 10 µM 2', 7'-bis-(2-carboxyethyl)-5-(6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM) dye over large parts of the cell cytoplasm were performed. These revealed an increase of 0.25 (N = 4) pH units on NH4Cl treatment and 0.22 (N = 5) pH units on treatment with bafilomycin A1. Measurements were also performed on ammonia-treated LS 174T cells, but their tendency to grow in multiple layers and their mucin-filled secretory granulae made light microscopy difficult. However, repeated measurements showed consequently increased pH values in a similar range. Because the pH could only be estimated over large portions of the cell, the increased pH observed suggests that the two drugs caused the expected neutralization of the acidic intracellular compartments of the two cell lines used in this study.

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., 1998Go). 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., 1991Go, 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 A1–treated 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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Fig. 1. Effect of NH4Cl and bafilomycin A1 treatment on GalNAc-T2 localization. Immunofluorescence microscopy of HeLa cells stable expressing VSV-G-tagged GalNAc-T2 kept as nontreated controls (A and B) or treated with 25 mM NH4Cl for 40 h (C and D) or 300 nM bafilomycin A1 for 12 h (E and F). Cells were fixed, permeabilized with 0.1% Triton X-100 (B, D, and F) or kept nonpermeabilized (A, C, and E), and were incubated with anti-VSV-G antibody prior to incubation with a FITC-labeled secondary antibody. Bar, 3 µm.

 
To illustrate that pH is essential for Golgi localization of other transferases, identical experiments were performed in HeLa cells expressing myc-tagged NAGT I, normally residing in the medial/trans cisternae (Nilsson et al., 1993Go). The control cells, as well as the nonpermeabilized NH4Cl cells, showed similar properties as for GalNAc-T2 (Figure 2A–C). In permeabilized NH4Cl-treated cells, some redistribution to punctuate compartments occurred (Figure 2D) but less than observed for the GalNAc-T2 expressing cells. The effect of bafilomycin A1 on NAGT I was similar to that on GalNAc-T2 with a relocalization of NAGT I to the surface, as observed in both nonpermeabilized and permeabilized cells (Figure 2E–F).



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Fig. 2. Effect of NH4Cl and bafilomycin A1 treatment on NAGT I localization. Immunofluorescence microscopy of HeLa cells stable expressing myc-tagged NAGT I analyzed as in Figure 1 using an anti-myc-tag antibody. Panels as in Figure 1. Bar, 3 µm.

 
Endogenous Gal-T1, normally present in the trans cisternae and the trans-Golgi network (TGN) (Nilsson et al., 1993Go), was also studied. Only permeabilized cells are shown as no surface staining could be observed. In control cells, a staining pattern corresponding to a juxtanuclear Golgi was observed (Figure 3A). On pH neutralization by both NH4Cl (Figure 3B) and bafilomycin A1 (Figure 3C), Gal-T1 staining largely disappeared from the Golgi consistent with a redistribution. Even though the signal was relatively weak, Gal-T1 appeared to have redistributed to punctuate compartments in a similar manner as for GalNAc-T2 after treatment with NH4Cl (Figure 3B). No surface staining could be seen after bafilomycin A1 treatment (Figure 3C). As will be discussed, this is probably due to cleavage and secretion of Gal-T1 when it is relocalized in a distal direction along the secretory pathway.



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Fig. 3. Effect of NH4Cl and bafilomycin A1 treatment on Gal-T1 localization. Immunofluorescence microscopy of HeLa cells (the cell line expressing myc-tagged NAGT I) kept as nontreated controls (A) or treated with 25 mM NH4Cl for 40 h (B) or 300 nM bafilomycin A1 for 12 h (C). Cells were fixed, permeabilized with 0.1% Triton X-100, and incubated with an anti-Gal-T1 antibody prior to incubation with Cy3-labeled secondary antibody. Bar, 3 µm.

 
To study if the drugs used had any structural effects on the Golgi stacks, EM was performed. In HeLa cells, expressing the VSV-G tagged GalNAc-T2, 30 Golgi stacks were identified in control cells (Figure 4A–B), in NH4Cl-treated cells (Figure 4C–D) and in bafilomycin A1–treated cells (Figure 4E–F), respectively. The average number of cisternae per stack was 3.29, 3.15, and 3.35; the average cisternal width 17.1 nm, 17.1 nm and 19.5 nm; and the average stack area 0.039 µm2, 0.047 µm2, and 0.060 µm2, respectively. The average number of stacks observed per cell was increased from 1.2 in the control cells to 2.0 in NH4Cl-treated cells and 1.6 in bafilomycin A1–treated cells. Areas with fewer than three Golgi-like cisternae overlapping each other, here not defined as Golgi stacks, were also increased by the drugs. Such areas continuous with complete Golgi stacks were found to increase proportional to the enlargement of the stacks (not shown). Taken together, these data suggest a growth of the Golgi apparatus by both substances, but no changes in the cisternal arrangement or otherwise in the fundamental Golgi architecture. Thus, the glycosyltransferase relocalizations observed seem to represent a primary pH effect on the enzymes, rather than being a phenomenon secondary to disorganization of the Golgi compartment. The early compartments of the secretory pathway were intact in HeLa cells (Figure 5A–B) as shown by antibody staining for PDI (a general marker for the endoplasmic reticulum, ER) (Sitia and Meldolesi, 1992Go) and the expression of a green fluorescent protein (GFP)–tagged KDEL receptor (marker for cis-Golgi network) (Griffiths et al., 1994Go) after bafilomycin A1 treatment.



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Fig. 4. Effect of NH4Cl and bafilomycin A1 treatment on the Golgi structure and on the density of GalNAc-T2 and Gal-T1 over Golgi stacks. Immuno-EM of HeLa cells stable expressing VSV-G-tagged GalNAc-T2 kept as nontreated controls (A and B) or treated with 25 mM NH4Cl for 40 h (C and D) or 300 nM bafilomycin A1 for 12 h (E and F). Ultrathin cryosections were labeled with a polyclonal against VSV-G-tagged GalNAc-T2 (revealed by 15 nm gold) (A, C, and E) or against endogenous Gal-T1 (revealed by 10 nm gold) (B, D, and F). Bar, 200 nm.

 


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Fig. 5. Effect of bafilomycin A1 on the ER (A and B) and identification of compartments to which GalNAc-T2 relocalizes on treatment with NH4Cl (C and D). HeLa cells stable expressing the cis localized KDEL receptor tagged with GFP at its N-terminus were incubated for 8 h with bafilomycin A1 (B). Nontreated control is shown in A. PDI (stained red) was visualized using a rabbit polyclonal antibody. The arrows mark the nuclear membrane. Note that not all cells express the KDEL receptor. HeLa cells stable expressing VSV-G-tagged GalNAc-T2 were treated with 25 mM NH4Cl for 40 h and the transferase was stained with FITC (C). Double staining was performed with the antibody 2C2 against a marker for late endosomes, using a Cy3-labeled secondary antibody (D). Bar, 5 µm (A and B), 3 µm (C and D).

 
To identify the punctuate compartments to which GalNAc-T2 and Gal-T1 redistributed on NH4Cl treatment, counterstaining was performed using different compartment markers. The GalNAc-T2 was found to partly colocalize with the antibody 2C2, revealing that the compartments were late endosomes (Figure 5C–D). No colocalization was found with sec13 (ER exit sites), p53 (ER to Golgi intermediate compartment), or OKT 9 (early endosomes) (not shown). A weak colocalization with COPI was observed (not shown), indicating that some GalNAc-T2 was present in COPI-coated structures.

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 µm–2 (79% of control), and for bafilomycin A1–treated cells (Figure 4E) 104 µm–2 (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 µm–2 for control cells (Figure 4B), 76 µm–2 (50%) for NH4Cl-treated cells (Figure 4D), and 26 µm–2 (17%) for bafilomycin A1–treated 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 cell–1, for NH4Cl-treated cells (Figure 6B) 26 cell–1, and for bafilomycin A1–treated cells (Figure 6C) 3.8 cell–1. Background values found in cells (N = 15) not expressing the construct were 0.0 cell–1, 2.5 cell–1, and 0.3 cell–1, 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., 1991Go, 1992).



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Fig. 6. GalNAc-T2 staining over endosomal compartments. Immuno-EM of HeLa cells stable expressing VSV-G-tagged GalNAc-T2, nontreated (A) or treated with 25 mM NH4Cl for 40 h (B) or 300 nM bafilomycin A1 for 12 h (C). Ultrathin cryosections were labeled with a polyclonal against VSV-G-tagged GalNAc-T2 revealed by 15 nm gold. Bar, 200 nm.

 
The glycosyltransferase relocalization in HeLa cells was also monitored by subcellular fractionations of control and NH4Cl-treated cells, followed by western blots. These were assayed for VSV-G-tagged GalNAc-T2, endogenous Gal-T1, and calnexin, the latter as a marker for the ER (Ou et al., 1993Go). Figure 7 shows western blots of the nine fractions, recovered from the top to the bottom. Calnexin was concentrated in fractions 8–9, and its localization was not affected by the NH4Cl treatment (Figure 7A). GalNAc-T2 was most prominent in fractions 3–5 of control cells, the expected position of the Golgi stack, whereas in the NH4Cl-treated cells GalNAc-T2 was spread mainly over fractions 1–6, consistent with a relocalization (Figure 7B). The Gal-T1 was present mostly in fraction 3 of both the control cells and the NH4Cl-treated cells (Figure 7C), which could reflect its main localization in trans-Golgi/TGN in HeLa cells (Nilsson et al., 1993Go). The enzyme was present in smaller amounts in the NH4Cl-treated cells, consistent with the reduced density already observed by immuno-EM. The increased total antigenicity found for GalNAc-T2 suggests a partial relocalization to endosomes, possibly compensated by production of new enzymes maintaining the density over the Golgi stack. The decreased antigenicity of Gal-T1 might be due to a more complete relocalization from the Golgi stack of this enzyme together with secretion of the enzyme (see Discussion).



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Fig. 7. Subcellular fractionation of HeLa (A–C) and LS 174T (D–F) cells for assay of glycosyltransferase relocalization on NH4Cl treatment. Cells were homogenized in buffered KCl by passage through syringe needles, debris was pelleted, and the obtained postnuclear supernatant was fractionated by ultracentrifugation on a linear Nycodenz gradient. The nine fractions, recovered from the top to the bottom, were subjected to tabletop ultracentrifugation, and the obtained pellets were reduced and analyzed by western blots of SDS–PAGE, assaying calnexin (A and D), GalNAc-T2/VSV-G (B), endogenous GalNAc-T2 (E) and Gal-T1 (C and F), and developed by the ECL method. In addition to images of the western blots, where the upper in every pair is from control cells and the lower from NH4Cl-treated cells, video densitometry measurements are shown. Open circles, control cells; filled-in circles, NH4Cl-treated cells. The difference in calnexin staining between control and treated cells in A, fraction 8, is not significant.

 
To examine if the glycosyltransferase relocalization observed in HeLa cells could be induced also in other cell lines, the mucin-producing cell line LS 174T was studied. These cells could not be readily examined by immunofluorescence microscopy due to their tendency to grow in multiple layers and the presence of mucin-filled secretory granulae. Immuno-EM also failed due to low reactivity with available antibodies against endogenous transferases. A possible relocalization of glycosyltransferases could therefore only be studied by subcellular fractionation. Calnexin, an ER marker, was present mainly in fractions 7–9, and its localization was not affected by the NH4Cl (Figure 7D). Endogenous GalNAc-T2 was found in fractions 5–6 of control cells and in fractions 3–7 of NH4Cl-treated cells, consistent with a relocalization (Figure 7E). The total antigenicity of GalNAc-T2 was also increased. The Gal-T1 was present in fractions 5–7 of the control cells, indicating that this transferase might have another distribution in LS 174T cells than in HeLa cells. Gal-T1 was found in substantial amounts only in fraction 4 of the NH4Cl-treated cells (Figure 7F), implying both a distal relocalization and a decreased amount. Taken together, the subcellular fractionation data from LS 174T cells are essentially in accordance with the observations in HeLa cells, with relocalization and increased amount of GalNAc-T2, and relocalization and a decreased amount of Gal-T1.

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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Fig. 8. Effect of NH4Cl treatment on the Golgi structure in LS 174T cells. Immuno-EM of ultrathin cryosections of control cells (A) and cells treated with 25 mM NH4Cl for 40 h (B). Bar, 200 nm.

 
In summary, the results suggest that the three studied glycosyltransferases were mislocalized as a consequence of neutralizing the acidic pH of the Golgi and the TGN. These results could suggest that the pH gradient over the secretory pathway has a role in maintaining glycosyltransferase localization.

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 chloride–insoluble mucins of the LS 174T cells, known to contain the MUC2 mucin (Axelsson et al., 1998Go), 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 A–type determinants were found by the gas chromatography–mass 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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Table I. Composition of purified insoluble mucins from LS 174T cells treated and nontreated with NH4Cl
 
To analyze the observed glycan alterations in more detail, the neutral and sialylated oligosaccharides released from the purified mucins were separated and quantified by GC (Figure 9). Without knowing the structure in each peak, one immediately observes that the profiles were altered by NH4Cl treatment as that the height (amount) of the larger saccharides (late eluting) were decreased. A general trend of relatively shorter neutral and sialylated oligosaccharides was observed. The structure of the oligosaccharide in each major peak was then analyzed by GC-MS, where the determination of the individual structures was based on the principles for MS fragmentation found earlier (Karlsson et al., 1989Go, 1994; Thomsson et al., 1997Go). The major oligosaccharide structures and the amount of each oligosaccharide relative to the smallest neutral (GalNAcol) and sialic acid–containing (NeuAc-6GalNAcol) oligosaccharide are shown in Figure 10. The relative alterations in amounts observed in NH4Cl-treated cells compared to control cells (diagram in Figure 10) show that all saccharides were decreased in amount, except for the Fuc-Gal-3GalNAcol compound. Analysis of the relative abundance of the individual compounds in relation to known biosynthetic pathways for O-glycans (Brockhausen, 1995Go), suggests that the alterations observed were not due to loss of individual glycosyltransferases, but rather to a more general mechanism, such as glycosyltransferase relocalization.



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Fig. 9. Gas chromatograms of released mucin oligosaccharides. O-linked oligosaccharides were released from purified insoluble mucins (MUC2) of LS 174T cells by ß-elimination and fractionated into neutral, sialylated, and sulfated fractions. Gas chromatograms of permethylated neutral (A and C) and sialylated (B and D) oligosaccharides released from mucins of control (A and B) and NH4Cl-treated (C and D) cells. Designations on the peaks refer to Figure 10.

 


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Fig. 10. Mucin oligosaccharide structures and alterations in their amounts in NH4Cl-treated cells compared to control cells. The oligosaccharides were quantified by GC (Figure 9) and characterized by GC-MS. The columns to the left show the relative amounts of oligosaccharides (compared to GalNAcol or NeuAc-6GAlNAcol) in control cells and NH4Cl-treated cells, respectively. The diagram shows the relative change when NH4Cl-treated cells are compared to control cells. The basis for the interpretation of the oligosaccharide structures is explained in the Materials and methods section.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Three different glycosyltransferases were relocalized when disrupting the pH gradient over the secretory pathway using two different principles: NH4Cl neutralizing the pH of acidic compartments via NH3 buffering, and bafilomycin A1 inhibiting the proton ATPases generating the acidic pH. One possible explanation for the observed effects is that these were secondary to a disruption of the Golgi apparatus. EM and a morphometric evaluation of the Golgi stacks revealed an essentially preserved Golgi architecture. Some quantitative differences in number and size of the Golgi stacks were noticed, however. Treated HeLa cells showed an increased number and size of stacks, whereas LS 174T cells showed a decrease in number and unaffected sizes. These changes were thus in opposite directions in the two cell lines, making it less likely that they caused the relocalization of glycosyltransferases, because these were similar in the two cell lines.

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., 1998Go), NAGT I resides mostly in the medial/trans cisternae (Nilsson et al., 1993Go), and Gal-T1 in the trans cisternae and the TGN (Nilsson et al., 1993Go). 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, 1986Go). 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, 1997Go). 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, 1998Go; 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., 1997Go). 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 4.1–7.1 and s5.1–s6.1, 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, 1996Go; Almeida et al., 1997Go) 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., 1996Go). 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., 1991Go; Lukacs et al., 1992Go). Later results have, however, contradicted elevated Golgi pH in this disorder (Seksek et al., 1996Go). Instead, a lowered pH of the Golgi apparatus might be suggested, as an HCO3 transport has been suggested to be defective (Poulsen et al., 1994Go). 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., 1989Go). An intracellular ammonia concentration of 25 mM was also found in H. pylori–infected culture cells in medium containing physiological urea concentrations (Mégraud et al., 1992Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Antibodies, cell lines, and chemicals
Affinity-purified rabbit polyclonals against the VSV-G- and the myc-tag epitopes (Röttger et al., 1998Go), a rabbit polyclonal antiserum against PDI, the affinity-purified monoclonal 2C2 against late endosomal/early lysosomal structures (gift from Dr. Marsh, MRC, University College London, UK), FITC-conjugated anti-rabbit antibodies (Tago Inc., Burlingame, CA), and Cy3 conjugated anti-mouse antibodies (Jackson Immuno Research Laboratories, West Grove, PA) were used for immunofluorescence. The same polyclonal against VSV-G, a rabbit polyclonal against Gal-T1 (Watzele et al., 1991Go, kindly provided by Dr. E. Berger, Zurich), and gold-conjugated anti-rabbit antibodies (British BioCell, Cardiff, UK) were used for immuno-EM. The anti-VSV-G polyclonal, an affinity-purified antiserum against calnexin (Ou et al., 1993Go), a monoclonal against GalNAc-T2 (6B7) (Mandel et al., 1999Go), and peroxidase-conjugated anti-mouse and anti-rabbit antibodies (Tago) were used for western blot. The purified monoclonal (Y2C4) against the cytoplasmic tail (long form) of Gal-T1, used for immunofluorescence microscopy and western blot, will be described elsewhere. The MUC2-producing colon adenocarcinoma cell line LS 174T (ATCC CL 188) (Asker et al., 1995Go), and monolayer HeLa cells (ATCC CCL 185), stable expressing VSV-G-tagged GalNAc-T2 (Röttger et al., 1998Go), myc-tagged NAGT I (Nilsson et al., 1993Go), and GFP-tagged KDEL receptor, were cultivated as described. Transfected HeLa cells were kept in the presence of 250 or 500 µg/ml geneticin (G-418 sulfate). The bafilomycin A1 was a kind gift from Astra Hässle (Mölndal, Sweden).

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 2–3 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, 1992Go). 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-rabbit–fluorescein 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, 1960Go) 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., 1997Go). 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 Tris–HCl, 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 5–25% (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 Tris–HCl, pH 6.8, 20% glycerol, 10 mM dithiothreitol (DTT), 95°C, 5 min, and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (10 µl per lane). The separation gel was 12% polyacrylamide, 0.1% SDS, 375 mM Tris–HCl, pH 8.8, and the stacking gel 4.5% polyacrylamide, 0.1% SDS, 126 mM Tris–HCl, 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., 1993Go; Axelsson et al., 1998Go). The mucins were purified by three rounds of isopycnic CsCl density gradient ultracentrifugation (Carlstedt et al., 1983Go), 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 acid–Schiff 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, 1995Go), 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., 1995Go, 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.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This work was supported by grants from EU-BioTech (BIO4-CT96-0129) (G.H., T.N.); the Visitors Programme, EMBL, Heidelberg (M.A.); the Swedish Medical Research Council (no. 7461) (G.H.); the IngaBritt and Arne Lundbergs Stiftelse (G.H.); the Swedish Cystic Fibrosis Foundation (M.A., D.S.); the Göteborg Medical Society (Göteborgs Läkaresällskap) (M.A.); the Anna Cederbergs Stiftelse (M.A.); the Johannes and Sonja Magnussons Fond (M.A.); the Glycoconjugates in Biological Systems program sponsored by the Swedish Foundation for Strategic Research (G.H.); and the Human Frontiers Science Program (LTO 482) (J.O.).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
BCECF-AM, 10 µM 2', 7'-bis-(2-carboxyethyl)-5-(6)-carboxyfluorescein, acetoxymethyl ester; EM, electron microscopy; ER, endoplasmic reticulum; FITC, fluorescein isothiocyanate; GalNAcol, N-acetylgalactosaminitol; GalNAc-T2, UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 2; Gal-T1, ß1,4 galactosyltransferase 1; GC, gas chromatography; GFP, green fluorescent protein; GlcNAc, N-acetylglucosamine; K-Hop, 130 mM KCl, 25 mM Tris–HCl, pH 7.5; MS, mass spectrometry; NAGT I, ß1,2 N-acetylglucosaminyltransferase I; NeuAc, N-acetylneuraminic acid (sialic acid); PBS, phosphate buffered saline; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TGN, trans-Golgi network.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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