(Received for publication, March 26, 1996, and in revised form, September 20, 1996)
From the In order to study the subcellular
localization and organization of the enzymes involved in the
glycosylation of the hybrid proteoglycan serglycin, mouse mastocytoma
cells were metabolically labeled with [35S]sulfate or
[3H]glucosamine in the absence or presence of brefeldin
A. This drug is known to induce a disassembly of the proximal part of the Golgi complex, resulting in a redistribution of cis-,
medial-, and trans-Golgi resident enzymes back
to the endoplasmic reticulum, and to block the anterograde transport of
proteins to the trans-Golgi network. Although the total
incorporation of [3H]glucosamine into glycosaminoglycan
chains was reduced to about 25% in brefeldin A-treated cells compared
to control cells, both control cells and cells treated with brefeldin A
synthesized heparin as well as chondroitin sulfate chains. Therefore,
enzymes involved in the biosynthesis of both types of glycosaminoglycan
chains seem to be present proximal to the trans-Golgi
network in these cells. Chondroitin sulfate and heparin synthesized in
cells exposed to brefeldin A were undersulfated, as demonstrated by
ion-exchange chromatography, compositional analyses of disaccharides,
as well as by a lower
[35S]sulfate/[3H]glucosamine ratio compared
to controls. In heparin biosynthesis, both N- and
O-sulfation reactions were impaired, with a larger relative
decrease in 2-O-sulfation than in
6-O-sulfation. Despite undersulfation, the heparin chains
synthesized in the presence of brefeldin A were larger (30 kDa) than
the heparin synthesized by control cells (20 kDa). The reduced
[3H]glucosamine incorporation in brefeldin A-treated
cells was partly due to decreased number of glycosaminoglycan chains
synthesized, but also to the biosynthesis of chondroitin sulfate chains
of smaller molecular size (8 versus 15 kDa in control
cells). Brefeldin A had no effect on the glycosaminoglycan synthesis
when used in a cell-free, microsomal fraction, indicating that
brefeldin A does not interfere directly with the enzymes involved in
the biosynthesis of glycosaminoglycans.
Most mammalian cells synthesize proteoglycans, a special group of
glycoproteins containing a core protein with covalently linked
glycosaminoglycan (GAG)1 side chains (1).
Whereas the majority of proteoglycans are substituted with either
heparan sulfate or chondroitin sulfate, some cells also synthesize
hybrid proteoglycans, in which both types of GAGs are linked to the
same core protein (2-4). The biosynthesis of the GAG chains takes
place during the transport of the core protein from the endoplasmic
reticulum through the Golgi complex. An initial polymerization product
is formed, composed of repeating glucuronic acid (GlcA) and hexosamine
units, N-acetylglucosamine (GlcNAc) in heparan
sulfate/heparin and N-acetylgalactosamine (GalNAc) in
chondroitin sulfate/dermatan sulfate. Subsequent modification involves
sulfate substitution at various positions and may include C5
epimerization of GlcA to iduronic acid (IdceA) units. Based on studies
of heparin biosynthesis, the enzymes responsible for the generation of
a GAG chain appear to be strictly organized and tightly clustered into
one or more enzyme complex(es) (5). Recently it has been suggested that
the enzymes involved in the biosynthesis of chondroitin sulfate are
located to the trans-Golgi network (6), whereas the enzymes
involved in the biosynthesis of heparan sulfate are located in the
proximal part of the Golgi apparatus (7). In both these studies
brefeldin A (BFA) was used as an experimental tool to segregate
biosynthetic processes occurring in the trans-Golgi
network.
BFA is a fungal metabolite that has been shown to induce a disassembly
of the cis-, medial-, and trans-Golgi
subcompartments, followed by a fusion of these subcompartments with the
endoplasmic reticulum (8). The result is a retention of secretory
proteins in the endoplasmic reticulum, as well as a redistribution of
enzymes normally resident in the cis-, medial-,
and trans-Golgi back to the endoplasmic reticulum (9-11).
In contrast, enzymes located in the trans-Golgi network are
not redistributed back to the endoplasmic reticulum (12). Hence, BFA
may be used to distinguish between enzymatic reactions taking place in
the endoplasmic reticulum/proximal parts of the Golgi apparatus and
those taking place in the trans-Golgi network.
In the present study we have studied the effect of BFA on the
biosynthesis of proteoglycans in mouse mastocytoma cells. These cells synthesize a hybrid form of the proteoglycan serglycin, in which
both chondroitin sulfate and heparin chains are linked to the same core
protein (13). Our results suggest that, in these cells, both enzymes
involved in the biosynthesis of chondroitin sulfate and enzymes
involved in the biosynthesis of heparin are located proximally to the
trans-Golgi network. Further, we show that BFA treatment
results in undersulfation of both chondroitin sulfate and heparin.
[35S]Sulfate (carrier-free)
and D-[6-3H]glucosamine were purchased from
DuPont NEN. UDP-[14C]GlcA was prepared enzymatically from
D-[14C]glucose (uniformly labeled, 320 µCi/mmol as described previously (14)). Q Sepharose,
DEAE-Sephacel, Sepharose CL-6B, Superose 6 (HR 10/30), Sephadex G-15,
Sephadex G-50 (fine), and Sephadex G-25 (superfine) were from Pharmacia
Biotech Inc. and chondroitinase ABC from Seikagaku Kogyo Co, Japan;
brefeldin A was from Boehringer Mannheim. A transplantable mouse
mastocytoma, originally described by Furth et al.
(15), was maintained in the laboratory by routine intramuscular passage
every 10-12 days in the hind legs of (A/Sn × Leaden)F1 mice. A microsomal fraction from the tumor was
prepared according to Jacobson et al. (14).
Mouse mastocytoma cells were established in
culture after passage through an ascites stage. Solid tumor tissue was
dispersed and injected intraperitoneally into another mouse. After 12 days, cultures were established from ascites fluid and maintained in Dulbecco's modified Eagle's medium (Flow Laboratories) containing 10% inactivated fetal calf serum, 100 units of penicillin, 100 µg/ml
streptomycin, 2.5 µg/ml Fungizone, and 2 mM of glutamine (all from Life Technologies, Inc.). Cultures in 50-ml flasks (Nunc, Roskilde, Danmark) reached near confluency in ~14 days and
were then used in labeling experiments.
Mastocytoma cells were labeled for
5 h with 50 µCi/ml [35S]sulfuric acid or
[3H]glucosamine. BFA was dissolved in ethanol and diluted
to a final concentration of 1 µg/ml in standard medium. BFA was added
to the cultures 15 min prior to the radioactive precursors and was present during the entire radiolabeling period.
The proteoglycans in the cell
fraction were extracted by the addition of 4 M guanidine
HCl, containing 2% Triton X-100. Alternatively, 0.05 M
Tris-HCl, pH 8.0, containing 1% Triton X-100 and protease inhibitors
(0.002 M EDTA, 0.001 M phenylmethylsulfonyl
fluoride, 0.002 M N-ethylmaleimide, and 10 µg/ml pepstatin) was added to the cell fraction, followed by
centrifugation at 600 × g for 10 min to remove nuclei.
NaCl to a final concentration of 0.15 M was then added to
the lysate. Unincorporated radioactive precursors and guanidine HCl
were removed from the samples by gel chromatography on Sephadex G-50
(fine) columns (bed volume, 4 ml) equilibrated and eluted in
phosphate-buffered saline containing 0.5% Triton X-100 and protease
inhibitors (in concentrations as described above).
To
release the polysaccharide chains from the peptide core of the
proteoglycan, the sample was treated with 0.5 M NaOH at 4 °C for 20 h. After neutralization with 4 M HCl,
the polysaccharide chains were dialyzed against water.
Galactosaminoglycans present in the proteoglycans were degraded by
digestion with 0.2 unit of chondroitinase ABC/ml of 0.05 M
Tris-HCl, pH 8.0, containing 0.03 M sodium acetate and 0.1 mg of bovine serum albumin (16). Prior to digestion, 100 µg of chondroitin sulfate was added as a carrier. After incubation for 15 h at 37 °C, the digest was passed through a column (1 × 200 cm) of Sephadex G-25 superfine, equilibrated with 0.2 M NH4HCO3 to separate disaccharides
from undigested material. The disaccharides were freeze-dried and
analyzed further by HPLC.
Depolymerization of heparin by nitrous acid deamination at pH 1.5, cleaving the polysaccharide at N-sulfated glucosamine units (17), was performed as described elsewhere (18) on material resistant
to digestion with chondroitinase ABC. The 3H-labeled
deamination products were reduced with NaBH4 and
fractionated by gel chromatography on Sephadex G-25. Estimation of the
percentage of GlcN residues carrying N-sulfate groups was
made using weighted integration of the elution profiles on Sephadex
G-25 according to the following formula,
Department of Biochemistry, Institute of
Medical Biology, University of Tromsø, 9037 Tromsø, Norway and
¶ Department of Medical and Physiological Chemistry,
Department of Veterinary Medical Chemistry,
Materials
where dis, tetras, hexas, and octas stands for di-, tetra-,
hexa-, and octasaccharides, respectively.
(Eq. 1)
To achieve a complete depolymerization of the heparin chains into disaccharides, 3H-labeled heparin from control cells and cells incubated with BFA was N-deacetylated by hydrazinolysis. Briefly, ~50,000 cpm of each sample was N-deacetylated (19, 20) by treatment with 0.25 ml of hydrazine containing 30% (v/v) water and 1% (w/v) hydrazine sulfate, at 100 °C for 6 h. The N-deacetylated product was reisolated by gel chromatography on Sephadex G-15 in 10% ethanol, lyophilized, and cleaved with nitrous acid at pH 1.5 and 3.9 (21), followed by reduction with NaBH4. Disaccharides were recovered after gel chromatography on Sephadex G-15 (1 × 200 cm), eluted with 0.2 M NH4HCO3.
ChromatographyGel chromatography on Superose 6 (HR 10/30)
was performed in 0.1 M sodium acetate, pH 6.0, containing 4 M guanidine HCl and 0.5% Triton X-100. The column was run
at a flow rate of 0.4 ml/min, and fractions of 1 min were collected.
Gel chromatography on Sephadex G-25 (superfine grade) was done in 0.2 M NH4HCO4 at a flow rate of 6 ml/h,
and fractions of 2 ml were collected and analyzed for radioactivity. Q
Sepharose was used for analytical ion-exchange chromatography. Bound
material was eluted with a gradient of 0.15-1.2 M NaCl in
8 M urea, 0.05 M sodium acetate, pH 6.0, containing 0.5% Triton X-100. Fractions of 2 ml were collected and
analyzed for radioactivity. Conductivity was measured in every fifth
fraction. HPLC of 3H-labeled chondroitin sulfate
disaccharides was carried out using a YMC-Pack Polyamine II column,
eluted with Na2HPO4 as described in the legend
to Fig. 5, whereas HPLC of 3H-labeled heparin disaccharides
was carried out using a Whatman Partisil-10 SAX column, eluted with
KH2PO4 (18, 22) as described in the legend to
Fig. 7.
Biosynthesis of Proteoglycans in a Microsomal Fraction
5 mg
of mastocytoma microsomal protein in 0.5 ml of 50 mM Hepes,
pH 7.4, containing 10 mM MnCl2, 10 mM MgCl2, 5 mM CaCl2, 1 mM adenosine 3-phosphate 5-phosphosulfate (PAPS), 0.5 mM UDP-GlcNAc, and 0.5 mM UDP-GalNAc, were
incubated with 10 mCi UDP-[14C]GlcA for 30 min at
37 °C. BFA was added 15 min prior to the radioactive precursors. The
reaction was terminated by the addition of 0.5 ml of 8 M
guanidine HCl, containing 4% Triton X-100, followed by gel
chromatography on Sephadex G-50 (fine) columns to remove unincorporated
14C-labeled precursor. Superose 6 gel chromatography of the
[14C]macromolecules was performed after treatment with
chondroitinase ABC/alkali and nitrous acid/alkali, respectively.
Mouse mastocytoma cell cultures were metabolically labeled with
[35S]sulfate or [3H]glucosamine for 5 h in the absence or presence of BFA. Radiolabeled macromolecules were
then isolated from the culture medium and from the cells and subjected
to gel and ion-exchange chromatography. In the control cells
(radiolabeled in the absence of BFA) about 10% of the proteoglycan
molecules were secreted to the culture medium.2 In contrast, practically no
radiolabeled proteoglycans were found in the medium fraction in the BFA
treated cultures, demonstrating that BFA inhibits the secretion of
macromolecules in mouse mastocytoma cells. The total incorporation of
[3H]glucosamine into proteoglycans was reduced to about
25% in the BFA-treated cultures compared to the control (Fig.
1, right panel), whereas the reduction in
[35S]sulfate incorporation was larger, amounting to 5%
of the control (Fig. 1, right panel), indicating that the
proteoglycans synthesized in the presence of BFA were undersulfated.
Both the control cells and the cells labeled in the presence of BFA
synthesized a mixture of heparin and chondroitin sulfate. The ratio
between [3H]heparin and [3H]chondroitin
sulfate isolated from the control cells was about 1:1. The
corresponding figure was 4:1 in the BFA-treated cultures, suggesting
that BFA had a more dramatic inhibitory effect on the biosynthesis of
chondroitin sulfate than on the biosynthesis of heparin (Fig. 1,
center and left panel, respectively). This was not due to different sensitivity of the two different GAG synthesizing enzymatic systems to BFA, since dose-response experiments revealed that
maximum inhibitory effect on the biosynthesis of both chondroitin sulfate and heparin was obtained with 1 µg/ml BFA (Fig.
2). To exclude that the observed effect of BFA was due
to direct interference of BFA with enzymes involved in the GAG
synthesis, the effect of BFA on the biosynthesis of proteoglycans in a
microsomal fraction from mouse mastocytoma was studied. Microsomal
proteins were incubated in the presence of UDP-GlcNAc, UDP-GalNAc, and
UDP-[14C]GlcA in the presence of PAPS for 30 min at
37 °C. Analysis of the 14C-labeled macromolecules (see
"Experimental Procedures") demonstrated that similar amounts of the
two polysaccharides were synthesized in the presence and absence of BFA
(data not shown).
Polyanionic Properties of Proteoglycans and GAG Chains
[14C]Proteoglycans synthesized by microsomal proteins (see above) were also subjected to anion-exchange chromatography on Q Sepharose. Identical elution patterns were observed for proteoglycans isolated from control and BFA-treated microsomal fractions; the 14C-labeled proteoglycans were eluted in a single peak at a NaCl concentration of 0.8 M (data not shown).
When the control material from cell cultures was similarly analyzed,
the 35S macromolecules were also eluted at a NaCl
concentration of 0.8 M (Fig. 3, panel
A). In contrast, the proteoglycans from BFA-treated cultures were eluted as a broad peak, ranging from about 0.3-0.7 M NaCl (Fig. 3, panel D). Further, both the
[35S]chondroitin sulfate (obtained after HNO2
treatment) and the [35S]heparin chains (obtained after
chondroitinase ABC treatment) from BFA-treated cultures (Fig. 3,
panels E and F, respectively) were eluted at a
lower salt concentration than the corresponding GAG chains from control
cultures (panels B and C).
Effect of BFA on Proteoglycan Size and GAG Chain Length
To determine if BFA treatment altered the size of the proteoglycans and the chondroitin sulfate and/or heparin chains, 35S-labeled proteoglycans isolated from the cell fraction by ion exchange chromatography were analyzed by Superose 6 gel chromatography. The calculated Kav values of the proteoglycans and GAG chains are presented in Table I. After treatment with HNO2 at pH 1.5, which degrades the 35S-labeled heparin, the intact 35S-labeled chondroitin sulfate proteoglycans from BFA-treated cells were eluted later from the column than the chondroitin sulfate proteoglycans from control cells (Fig. 4, G and B; Kav = 0.59 and 0.33, respectively). The smaller size of the chondroitin sulfate proteoglycans from BFA-treated cells was at least partly due to a decrease in the chondroitin sulfate polysaccharide chain length; after alkali treatment of the chondroitin sulfate proteoglycans, the released chondroitin sulfate chains from BFA-treated cells were eluted at Kav = 0.73 (Fig. 4H), corresponding to a molecular mass of about 8 kDa, whereas control chondroitin sulfate chains were estimated to have a molecular mass of 15 kDa (Fig. 4C; Kav = 0.60). Also the size of the heparin proteoglycans was reduced by BFA treatment; after chondroitinase ABC treatment, the intact 35S-labeled heparin proteoglycans from control and BFA-treated cells were eluted at Kav = 0.26 and 0.32, respectively (Fig. 4, D and I). However, 35S-labeled heparin polysaccharide chains from BFA-treated cells, released after alkali treatment, were of larger size (Fig. 4J; Kav = 0.45; estimated molecular mass 30 kDa) than those from control cells (Fig. 4E; Kav = 0.53; estimated molecular mass 20 kDa). Hence, the proteoglycans synthesized in the presence of BFA contain a reduced number of heparin chains of larger molecular size.
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The size of [14C]polysaccharide chains synthesized by microsomal proteins was also tested by gel chromatography on Superose 6; the heparin chains from both control and BFA-treated microsomal fractions were eluted at Kav = 0.3, whereas the chondroitin sulfate chains, synthesized both in the absence and presence of BFA, were eluted at Kav = 0.7. Hence, in the microsomal fraction, neither the amount of heparin and chondroitin sulfate synthesized or the polyanionic properties and size of the GAG chains are affected by BFA. It can therefore be concluded that BFA does not directly interfere with the GAG-synthesizing enzymes.
Polysaccharide Structure3H-Labeled
alkali-released polysaccharide chains isolated from control and
BFA-treated cells were digested with chondroitinase ABC, and the
resulting unsaturated disaccharides were separated from resistant
material by gel chromatography on Sephadex G-25. The chondroitin
sulfate disaccharides were then analyzed by HPLC as described in the
legend to Fig. 5. Whereas 73% of the disaccharides from control cells
(Fig. 5, upper panel) were monosulfated,
containing 4-O-sulfate groups, 10% were disulfated and
co-eluted with the Di-diSE standard. The remaining 17%
of the disaccharides from control cells was nonsulfated. In contrast,
the BFA-treated cells (lower panel) produced low sulfated
chondroitin sulfate chains, composed of about 52% nonsulfated
disaccharides and about 46% monosulfated disaccharides which co-eluted
with the
Di-4S standard. Disulfated disaccharides were virtually
absent (less than 3%) in chondroitin sulfate chains from BFA-treated
cells. These results demonstrate that there is a reduction in both 4- and 6-sulfation of the galactosamine units in chondroitin sulfate
synthesized by mast cells cultured in the presence of BFA, compared to
cells cultured without BFA.
To gain information regarding the amounts and distribution of
N-sulfated glucosamine units in heparin synthesized by the
mast cells grown with or without the addition of BFA,
3H-labeled material resistant to chondroitinase ABC was
lyophilized and cleaved with nitrous acid at pH 1.5 (deamination of
N-sulfated regions) followed by gel chromatography on
Sephadex G-25 (Fig. 6). Heparin produced by control
cells was extensively depolymerized yielding di- and tetrasaccharides
as the major labeled products. In contrast deamination of the
polysaccharide from BFA-treated cells resulted in larger
oligosaccharides with only a small fraction of di- and
tetrasaccharides. Based on these results, the degree of
N-sulfation of glucosamine units was estimated to 62 and
29% in heparin from control and BFA-treated cultures, respectively (see "Experimental Procedures"). The 3H-labeled
disaccharides obtained after gel chromatography on Sephadex G-25 were
further analyzed by HPLC-anion exchange chromatography (Fig. 7,
A and B). As for chondroitin
sulfate, the amount of nonsulfated disaccharides increased, whereas the
amount of di-O-sulfated disaccharides decreased in
BFA-treated cells.
In a separate analysis, the total disaccharide composition was
determined. 3H-Labeled polysaccharides from control and
BFA-treated cells were N-deacetylated by hydrazinolysis,
deaminated first at pH 3.9 and then at pH 1.5 (resulting in cleavage of
all glucosaminidic linkages), and reduced with NaBH4. The
resulting disaccharides were recovered after gel chromatography and
identified by HPLC (Fig. 7, C and D; Table
II). While most of the control material appeared as
O-sulfated disaccharides with the highest yield of
di-O-sulfated
IdceA(2-OSO3)-ManR(6-OSO3),3
more than 90% of the disaccharides obtained from BFA-treated cultures
were non-O-sulfated. In addition, in the presence of BFA,
the degree of O-sulfation decreased more than the
N-sulfation, resulting in a lowered O-sulfate to
N-sulfate ratio (Table II). As also reflected in the
2-OSO3/6-OSO6 ratio, the
2-O-sulfation reaction appeared to be more sensitive to BFA
than the 6-O-sulfation reaction.
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While most proteoglycans contain either heparan sulfate or chondroitin sulfate, hybrids exist in which both heparin/heparan sulfate and chondroitin sulfate are linked to the same core protein (1). Serglycin, a proteoglycan found in hemopoietic cells, is unusual in that it occurs in various cells as "pure" chondroitin sulfate proteoglycan and in other cells the same core protein is substituted with heparin (23). In addition, serglycin may occur as a hybrid (3, 13). The aim of the present investigation was to study the localization and organization of the GAG-synthesizing enzymes in a cell type capable of adding both heparin and chondroitin sulfate to the serglycin core protein. Recently, mouse mastocytoma cells were shown to have this capacity (13), and these cells were therefore chosen for this study.
Incorporation of [3H]glucosamine and [35S]sulfate into GAGs (Fig. 1) demonstrated that both heparin and chondroitin sulfate were synthesized by mastocytoma cells in the presence of BFA. This indicates that both chondroitin sulfate- and heparin-synthesizing enzymes are located proximal to the trans-Golgi network in these cells. Since the amount of radiolabeled GAGs synthesized in the presence of BFA was reduced, it cannot be excluded that also the trans-Golgi network in these cells contain chondroitin sulfate- and/or heparin-synthesizing enzymes. Judging from the amount of [3H]chondroitin sulfate compared to the [3H]heparin recovered after [3H]glucosamine labeling of the cells (10 and 40%, respectively, compared to the control; Fig. 1), the chondroitin sulfate synthesis was more affected by the drug. However, the heparin chains synthesized in the presence of BFA were longer (30 kDa compared to 20 kDa in the control; Fig. 4 and Table I), whereas the chondroitin sulfate chains were shorter than those produced in the absence of BFA (8 kDa compared to 15 kDa in the control; Fig. 4 and Table I). Therefore, the reduction in number of GAG chains synthesized in the presence of BFA was roughly the same for heparin and chondroitin sulfate, amounting to 25 and 17%, respectively, compared to the control. The opposite effect of BFA on chondroitin sulfate and heparan sulfate/heparin chain length has also previously been observed (7, 26). Whereas the polymerases responsible for chondroitin sulfate chain elongation so far have not been characterized, the two glycosyltransferase reactions catalyzing the polymerization of heparin have been shown to reside in one protein (27, 28). Tentatively, if two separate proteins carry out chondroitin sulfate polymerization, a disorganization of the Golgi membranes may be more deleterious to chondroitin sulfate than to heparin elongation.
Previous investigations, using BFA as a tool to locate the GAG-synthesizing machineries, indicate that, whereas chondroitin sulfate biosynthesis appears to take place in the trans-Golgi network, the heparan sulfate-synthesizing enzymes are located in a more proximal part of the Golgi complex (6, 7). The presence of chondroitin sulfate-synthesizing enzymes proximal to the trans-Golgi network, as shown in the present investigation, is thus in contrast to previous results. The different location of the chondroitin sulfate-synthesizing enzymes in mastocytoma cells may tentatively suggest the existence of more than one machinery for the biosynthesis of chondroitin sulfate. This has previously been suggested for heparin/heparan sulfate biosynthesis, based on the identification of two genetically distinct enzymes catalyzing N-sulfation (29, 30). The difference in the N-terminal regions of these proteins may suggest that they are present in different Golgi subcompartments, since this region of the proteins contain Golgi retention signals. It is therefore possible that enzymes capable of synthesizing a certain glycosaminoglycan are located in different Golgi compartments in different cells. If so, it would be expected that chondroitin sulfate-synthesizing enzymes also would be present in more than one variant.
The sulfation of both chondroitin sulfate (Fig. 5) and heparin (Figs. 6 and 7) was decreased in the presence of BFA. However, all the various modification reactions occurred as shown by the presence of the same disaccharide units, although in different amounts, in polysaccharides from control and BFA-treated cells. If the structural changes in heparin induced by BFA is compared with, e.g. the lowered sulfation of heparan sulfate synthesize by CHO cell mutants with a reduced N-sulfotransferase activity (31), it is apparent that the effect of BFA is more general and/or random. In the CHO cell mutant, the O-sulfate/N-sulfate ratio is the same as in the wild type cell. This result is expected, since a N-sulfated glucosamine residue is part of the substrate recognized by the O-sulfotransferases. In the BFA-treated cells the O-sulfate/N-sulfate ratio is decreased pointing to a loss of regulation of the biosynthesis machinery, further illustrated by the larger relative decrease in 2-O-sulfation than in 6-O-sulfation.
Current views on GAG biosynthesis envisage the enzymes as part of an enzyme complex acting on the polysaccharide (5). Our results may argue against a tight association between the modification enzymes, since BFA seems to induce a less ordered and less efficient modification process. Another possibility is that the concentration of PAPS, the activated sulfate donor, may be different in the Golgi complex of control cells and the fused endoplasmic reticulum/Golgi compartment of BFA-treated cells. If less PAPS is available in BFA-treated cells, a lowered sulfation of the heparin and chondroitin sulfate should be expected. In addition, the lowered O-sulfate/N-sulfate ratio found for heparin from BFA-treated cells, and the larger relative decrease in 2-O-sulfation than in 6-O-sulfation may be explained by differences in Km values for the different enzymes.