Glycobiology vol. 11 no. 8 pp. 633–644, 2001
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
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, β1,2 N-acetylglucosaminyltransferase I, and β1,4 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
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., 1993; Rabouille et al., 1995;
1To
whom correspondence should be addressed
© 2001 Oxford University Press
Röttger et al., 1998). 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, 1995). 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., 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 β1,4
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.
Results
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,
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M.A.B. Axelsson et al.
inhibiting the ATPase proton pumps of the secretory and
endocytotic pathways (Bowman et al., 1988; Nelson and Tai,
1989). 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., 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 NH4Cltreated 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).
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., 1993). 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
634
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.
of NAGT I to the surface, as observed in both nonpermeabilized
and permeabilized cells (Figure 2E–F).
Endogenous Gal-T1, normally present in the trans cisternae
and the trans-Golgi network (TGN) (Nilsson et al., 1993), 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.
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
Golgi pH, glycosyltransferase localization, and glycosylation
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.
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, 1992) and the expression of a
green fluorescent protein (GFP)–tagged KDEL receptor
(marker for cis-Golgi network) (Griffiths et al., 1994) after
bafilomycin A1 treatment.
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
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.
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)
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M.A.B. Axelsson et al.
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.
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
636
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).
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., 1993).
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 transGolgi/TGN in HeLa cells (Nilsson et al., 1993). 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).
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
Golgi pH, glycosyltransferase localization, and glycosylation
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).
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.
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., 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 A–type determinants were found
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M.A.B. Axelsson et al.
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.
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., 1989, 1994; Thomsson et al., 1997). 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, 1995), suggests that the
alterations observed were not due to loss of individual glycosyltransferases, but rather to a more general mechanism, such
as glycosyltransferase relocalization.
Discussion
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.
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
638
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., 1998),
NAGT I resides mostly in the medial/trans cisternae (Nilsson
Golgi pH, glycosyltransferase localization, and glycosylation
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.
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).
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M.A.B. Axelsson et al.
Table I. Composition of purified insoluble mucins from LS 174T cells treated
and nontreated with NH4Cl
Relative abundance (mole)
Control cells
Treated cells
GalNAca
1.00
1.00
Fuca
0.46
0.11
GlcNAca
0.91
0.32
Mana
0.05
0.19
Gala
1.68
1.04
Serb
0.33
0.55
Thrb
1.36
1.39
Saccharide lengthc
4.1
2.6
%
glycosylationd
59
51
aFrom
mean of two analyses using high-performance anion exchange
chromatography–pulsed amperometric detection, relative to GalNAc.
bFrom amino acid composition analyses, relative to GalNAc.
cMean length calculated from the amount of monosaccharides per GalNAc.
dCalculated from the amount of GalNAc per serine and threonine.
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.
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
640
Fig. 9. Gas chromatograms of released mucin oligosaccharides. O-linked
oligosaccharides were released from purified insoluble mucins (MUC2) of LS
174T cells by β-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.
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 β1,6N-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
Golgi pH, glycosyltransferase localization, and glycosylation
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.
glycosyltransferases, the general glycosylation alterations
observed in the mucins indirectly suggest that other transferases
were displaced as well. Many of the more distal O-glycosylationrelevant 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, 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. pylori–infected
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.
Materials and methods
Antibodies, cell lines, and chemicals
Affinity-purified rabbit polyclonals against the VSV-G- and
the myc-tag epitopes (Röttger et al., 1998), a rabbit polyclonal
antiserum against PDI, the affinity-purified monoclonal 2C2
against late endosomal/early lysosomal structures (gift from
641
M.A.B. Axelsson et al.
Dr. Marsh, MRC, University College London, UK), FITCconjugated 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., 1991, kindly
provided by Dr. E. Berger, Zurich), and gold-conjugated antirabbit antibodies (British BioCell, Cardiff, UK) were used for
immuno-EM. The anti-VSV-G polyclonal, an affinity-purified
antiserum against calnexin (Ou et al., 1993), a monoclonal
against GalNAc-T2 (6B7) (Mandel et al., 1999), and peroxidaseconjugated 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., 1995), and monolayer HeLa cells (ATCC CCL 185), stable expressing VSV-Gtagged GalNAc-T2 (Röttger et al., 1998), myc-tagged NAGT I
(Nilsson et al., 1993), 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 100× 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.
642
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 × 5 min in PBS,
incubated for 10 min in 50 mM NH4Cl to quench aldehyde
groups, washed 3 × 5 min in PBS, permeabilized (if denoted)
for 4 min in 0.1% Triton X-100 in PBS, and washed 3 × 5 min
in PBS. After blocking 2 × 5 min in PBS-gelatin (PBS with
0.2% teleostean gelatin, Sigma) and washing 3 × 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 PBSgelatin 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, 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 antiVSV-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
Golgi pH, glycosyltransferase localization, and glycosylation
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 vesicleshaped 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 × 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 benzamidineHCl, and 40 µg/ml phenylmethylsulfonyl fluoride. Cells were
homogenized by passage through syringe needles (5 × 22 G/
0.7 mm, 5 × 25 G/0.5 mm, 3 × 27 G/0.4 mm). The homogenate
was centrifuged for 5 min at 1000 × 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., 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 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, 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.
Acknowledgments
This work was supported by grants from EU-BioTech (BIO4CT96-0129) (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
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,
β1,4 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, β1,2 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.
643
M.A.B. Axelsson et al.
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