(Received for publication, October 18, 1994; and in revised form, November 15, 1994)
From the
4-Methylumbelliferyl--xyloside (Xyl
MU) primes
glycosaminoglycan synthesis by first serving as an acceptor for the
addition of 2 galactoses and 1 glucuronic acid residue to make the
typical core structure, GlcUA
1, 3Gal
1,3Gal
1,4Xyl
MU.
To investigate the relative localization of these biosynthetic enzymes,
intact and properly oriented rat liver Golgi preparations were
incubated with Xyl
MU and 1 µM UDP-[
H]Gal and then chased with 5 µM of unlabeled UDP-Gal, UDP-GlcUA, UDP-GlcNAc, UDP-GalNAc, and
CMP-Neu5Ac. Under these conditions, no intervesicular transport occurs
and acceptor labeling depends entirely upon transporter-mediated
delivery of the labeled sugar nucleotides into the lumen of a vesicle
and co-localization of the appropriate glycosyltransferases. The
labeled products were isolated from the incubation medium and from
within the Golgi and their structures analyzed by C18, anion-exchange,
and amine adsorption high performance liquid chromatography in
combination with glycosidase digestions. Surprisingly, the major
products within the Golgi were two sialylated xylosides
(Sia
2,3Gal
1,4Xyl
MU and
Sia
2,8Sia
2,3Gal
1,4Xyl
MU) rather than the expected
group of partially completed GAG core structures. Less than 10% of the
products within the Golgi are the expected core structures containing a
second Gal residue or, in addition, GlcUA. The amount of the sialylated
products is only partially decreased if the chase is omitted or if the
chase is done in the absence of added CMP-Sia, suggesting a pool of
previously transported CMP-Sia drives synthesis of the major products.
Conversely, when detergent permeabilized vesicles are provided with
high concentration of the same sugar nucleotides, the ratio of
sialylated products is reduced and replaced by an increase in GAG-like
products. These results argue that GAG core-specific Gal transferase I
and II are not extensively co-localized within the same Golgi
compartment. By contrast, glycosaminoglycan core Gal transferase I is
substantially co-localized with an
-2,3-sialyltransferase and an
-2,8-sialyltransferase. Incubating intact Golgi vesicles with
exogenous diffusible acceptors offers a novel method to assess the
functional co-localization of glycosyltransferases of multiple pathways
within the Golgi compartments.
Most of the glycosylation reactions involved in the biosynthesis
of glycoconjugates (glycoproteins, proteoglycans, and glycolipids)
occur in the Golgi. The sugar nucleotide donors are made in the
cytoplasm, transported, and concentrated into the Golgi lumen by
specific transporters(1) . Glycosyltransferases located on the
lumenal face of the Golgi utilize the transported nucleotide sugars to
glycosylate the various glycoconjugate acceptors transiting through
different Golgi subcompartments during their biosynthetic maturation. A
popular view is that the required glycosyltransferases are sequentially
ordered in the Golgi cis, medial, trans compartments roughly
corresponding to the known order of
glycosylations(2, 3, 4, 5, 6) .
This generally accepted view primarily addresses the early steps in N-linked glycosylation(7) , but the later steps in
this pathway, O-linked glycosylation, and most of the GAG ()synthesis, may occur in the trans-Golgi (8) and
trans-Golgi network(9) . An alternative perspective of the
Golgi is that the glycosyltransferase organization is more flexible and
cell-type
dependent(10, 11, 12, 13, 14) .
Regardless of the specific distribution of glycosyltransferases, any
glycosylation step requires that a functionally active Golgi
compartment must have 1) sugar nucleotide transporters to deliver 2)
the donor sugar nucleotides to the lumen of the compartment where 3)
the acceptors and 4) glycosyltransferases are spatially and temporally
co-localized(15) . In the dynamic setting of the living cell,
vesicular trafficking (transport) continuously delivers maturing
glycoconjugates from one compartment to the next. In an in
vitro, non-transporting, static system, a functional compartment
defines itself by having all the essential components spatially
co-localized.
This concept was recently exploited by Hayes et al.(16, 17, 18) in a series of papers showing that endogenous acceptors in sealed Golgi vesicles could be radiolabeled by a sugar nucleotide transporter-mediated process. A chase with non-labeled sugar nucleotides led to further glycosylation of the labeled endogenous acceptors. Since this occurred in the absence of intervesicular transport, all of the contributing transporters and transferases involved in the labeling and subsequent glycosylation of the products must have resided in a single functional compartment as defined above.
We have extended this approach by using a small
freely diffusable glycoside in place of the normal endogenous
acceptors. We chose XylMU as a specific acceptor for the GAG
core-specific galactosyltransferase I, and then asked whether other
glycosyltransferases and sugar nucleotide transporters needed for GAG
core synthesis were also functionally co-localized with this
transferase. Xyl
MU primes glycosaminoglycan synthesis by first
serving as an acceptor for the addition of galactose and glucuronic
acid residues to make the core structure,
GlcUA
1,3Gal
1,3Gal
1,4Xyl
MU(19, 20) .
The results obtained in the present studies using the rat liver
``freeze-frame'' Golgi incubations suggest that the first GAG
core-specific galactosyltransferase is substantially co-localized with
one or more sialyltransferases and not with the second
galactosyltransferase required for GAG core synthesis. This unexpected
co-localization may explain why, in a previous study(20) ,
sialylated xylosides were the major products made by melanoma and CHO
cells incubated with Xyl
MU. Using purified Golgi vesicles and
diffusible acceptors offers a novel method to assess the functional
co-localization of glycosyltransferases within the Golgi compartments.
Purification of
Golgi membranes was monitored by assaying for galactosyltransferase
enrichment. UDP-Gal:GlcNAc Gal1,4-galactosyltransferase was
assayed exactly as described previously(16) . Two separate
preparations were used in the experiments described in this report, and
both had comparable specific activities (
2.5 milliunits/mg of
protein). Contamination of the Golgi membranes with soluble cytoplasmic
enzymes was followed by measuring lactate dehydrogenase activity.
Appropriate dilutions of the subcellular fractions were incubated in a
1-ml reaction at 23 °C containing 0.2 M Tris-HCl, pH 7.3,
1 mM sodium pyruvate, and 0.22 mM NADH. Enzyme
activity was measured by following the oxidation of NADH
spectrophotometrically at 340 nm. One unit equals 1 µmol of NADH
oxidized per min at 23 °C.
To verify that the incubations with the
Golgi-enriched fraction were dependent on the transport of nucleotide
sugars, reactions were carried out as above in the presence of 0.2%
Triton X-100. To demonstrate the absence of intercompartmental
transport, reactions were carried out as above after pretreatment of
the Golgi-enriched fraction with 2 mMN-ethylmaleimide (24) or 30 µM ilimaquinone(25) . To control for the possibility that
galactose labeled xyloside product could diffuse from one compartment
to another, reactions were carried out as above in the presence of 4
units/ml -galactosidase (jack bean).
Additional reactions were carried out after preincubating the Golgi fraction with 1 mMN-acetyllactosamine to attempt to deplete the endogenous CMP-Sia pools and thus favor the formation of GAG core products. These reactions were stopped after the primary 15-min incubation and not chased with the cold nucleotide sugars used in the standard incubation conditions described above. The controls for these incubations used the standard incubation conditions without the nucleotide sugar chase. In another reaction, the Golgi fraction was incubated using the standard conditions except that the chase contained only 5 µM UDP-Gal and UDP-GlcUA.
Transport-independent, permeabilized
Golgi reactions were carried out with the following modifications to
the standard incubation conditions. The Golgi membranes were
permeabilized by the addition of 0.2% Triton X-100, the
UDP-[H]Gal concentration was increased to 50
µM (25 µCi; 2 Ci/mmol), and the concentration of the
nucleotide sugars present during the chase was increased to 100
µM.
To verify that the purified Golgi vesicle fraction was properly oriented and sealed, protease protection of radiolabeled endogenous acceptors was assayed using the methods described previously(16) . The capacity of the Golgi vesicles to transport UDP-Gal and transfer radiolabeled Gal to endogenous acceptors was quantified as described by Perez and Hirschberg(26) .
The frozen pellets from the Golgi incubations
were resuspended by sonication in 0.5 ml 70% ethanol. The samples were
centrifuged at maximum speed in a microcentrifuge for 5 min. The pellet
was again resuspended by sonication in 0.5 ml of 70% ethanol, and the
centrifugation was repeated. The supernatants (XTs) were combined and
dried in a Savant Speed-Vac. The extracted pellets (XPs) were
solubilized by sonication in 250 µl of 2% SDS in 10 mM Tris base for 10-20 s and stored frozen at -20 °C.
The XylMU products were purified from the dried supernatants (XTs)
by reverse phase chromatography on C18 spin columns which had been
pre-washed with four column volumes each of methanol, distilled water,
and 1 M NaCl. The XTs were redissolved in 100 µl of
distilled water and applied to the top of the C18 resin (0.2-ml bed
volume) in microcentrifuge spin filter baskets. The spin columns were
microcentrifuged at 3000 rpm for 1-2 min. The columns were washed
with three 200-µl portions of 1 M NaCl followed by two
200-µl washes with distilled water. The bound Xyl
MU products
were eluted with three 200-µl portions of 40% methanol (XT40s). The
remainder of bound products were eluted with two 200-µl portions of
absolute methanol.
The presence of detergent in the supernatants of
the permeabilized Golgi incubations resulted in the elution of some of
the anionic xyloside products in the water washes during the
purification on the C18 cartridges. Initially, these were recovered by
repurification of the water washes. Subsequently, we found that these
anionic xylosides were retained on the C18 cartridges when washing was
done with 0.1 M NHCOOH, pH 6, instead of distilled
water.
Analysis of xyloside products by
amine adsorption HPLC was done using a 4.6 mm 25-cm
Microsorb-MV amino-bonded silica column. The gradient was 80-40%
acetonitrile in water over 60 min at a flow rate of 1 ml/min.
Ion
exchange HPLC analysis was carried out using the above amino column
using the following gradient. After injection, the column was washed
with water for 2 min; then a gradient of 0-50 mM sodium
phosphate pH 4.3 over 25 min; 50-150 mM over 15 min;
and, 150-250 mM over 10 min. The flow rate was 1 ml/min,
and 1-ml fractions were collected. A small amount of
Na[
S]SO
(50-200
cpm) was used as an internal marker in each run.
To further characterize the purification of the
``functional'' Golgi fraction, we monitored labeling of
endogenous acceptors. This assay requires not only the
galactosyltransferase activity, but also co-localized UDP-Gal
transporter and endogenous acceptors. As shown in Table 1, this
activity is enriched almost 1000-fold in the Golgi fraction, but not in
other fractions from the sucrose gradient. We likewise assayed the
various fractions for their ability to galactosylate XylMU and
found that only the classical Golgi fraction was active (Table 1).
To demonstrate that the labeling depends on the transport and concentration of UDP-Gal in the lumen, a portion of the Golgi fraction was incubated (see ``Experimental Procedures'') with 0.1% Triton X-100 to permeabilize the Golgi membranes. This reduced incorporation into endogenous acceptors 15-20-fold (data not shown)(16) . We also quantified the ability of the Golgi fraction to transport UDP-Gal as described by Perez and Hirschberg(26) : 187 pmol/mg of protein was transported in our standard incubation conditions (1 µM UDP-Gal, 15 min, pH 6.5 buffer). This compares well with a value of 372 pmol/mg of protein reported by Perez and Hirschberg (26) using different incubation conditions (2 µM UDP-Gal, 10 min, pH 7.5 buffer).
Fig. 1shows that the
incorporation into endogenous acceptors occurs inside the Golgi lumen.
Portions of one sample were incubated in the presence of Triton X-100
alone, Pronase alone, or Triton X-100 plus Pronase for 1 and 16 h. The
results showed that the incorporated [H]Gal was
susceptible to Pronase digestion only if the Golgi was permeabilized
with Triton X-100. The above results show that the Golgi preparations
are intact, properly oriented, and that synthesis requires
transportermediated delivery of sugar nucleotides into the lumen.
Figure 1:
Protection of the
UDP-[H]Gal labeled endogenous acceptors from
protease digestion. The Golgi vesicles from one standard incubation
with 1 µM UDP-[
H]Gal were split into
six aliquot portions. Two portions were treated with 0.1% Triton X-100;
two with 1 mg/ml Pronase; and two with 0.1% Triton X-100 and 1 mg/ml
Pronase. The samples were incubated at 37 °C and a sample from each
set analyzed after 1 and 16 h for perchloric acid-precipitable
radioactivity(16) . The dark bars represent the 1-h
incubations; the light, the 16-h incubations. The 100% of
control value is defined as the 1-h incubation in the presence of
Triton X-100 only.
Figure 2:
Fractionation scheme and flow diagram for
the isolation of [H]Gal-labeled xyloside products
from the Golgi incubations. The SE40 fraction contains the xyloside
products which diffuse from the Golgi lumen after being labeled with
[
H]Gal; the XT40 fraction, those which remain in
the Golgi lumen. The insoluble XP products are mainly
[
H]Gal-labeled N-linked oligosaccharides
attached to endogenous acceptors.
Figure 4:
C18 reverse-phase HPLC fractionation of
[H]Gallabeled xyloside products from Golgi
supernatant and pellet fractions. [
H]Gal-labeled
xyloside products from Golgi supernatant and pellet fractions (SE40 and
XT40, respectively, see Fig. 2) were fractionated by preparative
C18 HPLC. Panel A shows the products in the supernatant; Panel B, those in the Golgi pellet. The data shown are for the
100 µM Xyl
MU incubation (open symbols); the
data for the control incubation without Xyl
MU (solid
symbols) are shown for comparison. The arrows mark the
elution positions of authentic Gal
Xyl
MU and
Gal
Gal
Xyl
MU.
Fig. 3shows the incorporation of
[H]galactose into xyloside products with
different concentrations of Xyl
MU. Maximum incorporation is
reached by about 50 µM and half maximal by 18
µM. Approximately half of the labeled xyloside products
are found outside the vesicles. Since we have shown that the vesicles
are sealed to macromolecules and the GAG-core specific Gal transferase
is located within the Golgi, the products found outside must have
diffused from the lumen. This is not unexpected; since the substrate,
Xyl
MU, is freely diffusable, the product
[
H]Gal
Xyl
MU may also be able to diffuse
across the membranes, albeit more slowly. Based on our estimates and
those of others (30) of the volume of the Golgi lumen
(
0.5-1 µl/mg of Golgi protein), the concentration of the
xyloside products inside the lumen is at least 20-40 µM (averaged over the entire lumenal volume of the Golgi vesicles),
while that on the outside is only 0.04-0.08 µM. As
shown below, the xyloside products found outside the lumen consist
almost entirely of Gal
Xyl
MU, whereas those inside contain one
or more additional sugar residues. This probably reflects a higher
diffusion rate for the smaller, radiolabeled Xyl
MU product.
Figure 3:
Incorporation of
[H]Gal into xyloside products versus Xyl
MU concentration. Golgi vesicles were incubated with
varying concentrations of Xyl
MU and 1 µM UDP-[
H]Gal using the standard incubation and
chase conditions (see ``Experimental Procedures''). The
labeled xyloside products were purified from the supernatant and Golgi
pellet (see ``Experimental Procedures'' and Fig. 2).
Portions of each were assayed for radioactivity and the values
expressed as picomoles of [
H]Gal incorporated per
mg of Golgi protein.
The
labeled xyloside products from each XylMU concentration and
corresponding controls were fractionated by C18 reverse phase HPLC. Fig. 4shows a representative fractionation of products from the
incubation supernatant (Panel A) and Golgi pellet (Panel
B) samples (SE40 and XT40, respectively) using 100 µM Xyl
MU (open circles). An equal portion of a control
incubation without Xyl
MU is superimposed for comparison (filled symbols). The major product (>90%) found in the
supernatant coelutes with authentic Gal
Xyl
MU and/or
Gal
Gal
Xyl
MU (which are not resolved on this column).
Amine adsorption HPLC (Fig. 5, Panel A) shows it is
exclusively Gal
Xyl
MU. By contrast, only a minor portion
(5-10%) of the products from the Golgi pellet elutes at the
position corresponding to Gal
Xyl
MU and/or
Gal
Gal
Xyl
MU (Fig. 4, Panel B). This
product is mostly Gal
Xyl
MU (80%) and some
Gal
Gal
Xyl
MU (20%) (Fig. 5, Panel B).
Figure 5:
Amine adsorption HPLC analysis of the
neutral [H]Gal-labeled xyloside products from the
Golgi supernatant and pellet fractions. The neutral
[
H]Gal-labeled xyloside products eluting with
retention times corresponding to authentic Gal
Xyl
MU and
Gal
Gal
Xyl
MU (see Fig. 4) were analyzed by amine
adsorption HPLC. Panel A shows the analysis of the neutral
products in the supernatant; Panel B, the neutral products in
the Golgi pellet. The arrows mark the elution positions of
authentic Gal
Xyl
MU and
Gal
Gal
Xyl
MU.
The major products (50-70%) associated with the Golgi pellet
eluted between fractions 15 and 25 (Fig. 4B, peaks
II and III). These products are anionic based on their
binding to QAE-Sephadex, and 80-90% of the label in peak II was
neutralized by digestion with either the broad spectrum A.
ureafaciens sialidase or the -2,3-specific NDV sialidase. The
neutral product obtained coelutes with Gal
Xyl
MU on amine
adsorption HPLC (Fig. 6B). Taken together, these
results show that peak II is mostly (80-90%)
Sia
2,3Gal
Xyl
MU. However, approximately 3-7% of
peak II was neutralized by
-glucuronidase digestion and gives the
expected GAG core digestion product, Gal
Gal
Xyl
MU
(
60%), and Gal
Xyl
MU (
40%). These results indicate
that peak II contains 2-5% GlcUA
Gal
Gal
Xyl
MU.
Similar analyses showed that peak III was also mostly sialylated and
only a few percent could be neutralized by
-glucuronidase
digestion.
Figure 6:
HPLC analysis of neutral products obtained
after sialidase treatment of the major anionic product from the Golgi
pellet. A portion of the major anionic product from the Golgi
incubation pellet (see Fig. 4B, peak II) was
digested with AUS and the neutralized products separated by
QAE-Sephadex chromatography. Panel A shows the C18 HPLC
analysis of this neutralized product; Panel B, the amine
adsorption HPLC analysis of the same product. The arrows in Panel B mark the elution positions of authentic
GalXyl
MU and
Gal
Gal
Xyl
MU.
Approximately 20-30% of the products in the Golgi
pellet (XT40s) were not retained by the C18 HPLC column (Fig. 4B, peak I, fractions
3-6). Most of the products from the control incubated
without XylMU also did not bind. QAE-Sephadex analysis showed that
peak I was anionic. It was retained on the C18 HPLC column in the
presence of 0.1 M NH
COOH, pH 6, acting as a charge
suppression/ion-pairing agent. Fig. 7A clearly shows that
the xyloside products in peak I were efficiently separated from the
endogenous acceptor products eluting in fractions 12-22. Three
xyloside products, Ia, Ib, and Ic, were analyzed by ion exchange HPLC
and found to have a -2 charge (Fig. 7B). There was
insufficient material for further analysis of peaks Ib and Ic which are
only about 2-3% of the total xyloside products. Peak Ia was
analyzed in detail. Digestion with either the broad spectrum A.
ureafaciens sialidase or the
-2,8- and
-2,3-specific NDV
neuraminidase converted 60-70% to neutral products. Treatment
with 0.05 N HCl at 80 °C for 30 min to selectively
hydrolyze sialic acid moieties also neutralized 85-90%. Analysis
of these neutral products by amine adsorption HPLC showed that the
major desialylated species obtained was Gal
Xyl
MU (Fig. 7C). These results suggested that peak Ia was
structurally related to the Sia
2,3Gal
Xyl
MU product
described above but contained an additional sialic acid residue.
Considering the similarity of the latter product with ganglioside GM3
(Sia
2,3Gal
Glc
Cer), we reasoned that it might be similar
to ganglioside GD3 (Sia
2,8Sia
2,3Gal
Glc
Cer). To
confirm this we digested it with S. typhimurium sialidase
(which does not cleave Sia
2, 8Sia
2-X linkages but will cleave
-2,3- and
-2,6-linked Sia residues). Peak Ia was resistant to
this digestion and retained both negative charges. This means that an
-2,8-linked Sia must have been attached to and blocked digestion
of the S. typhimurium-sensitive
-2,3-linked Sia. Taken
together, these results indicate that the structure of peak Ia is most
likely Sia
2,8Sia
2,3Gal
Xyl
MU. This material
represented 10-15% of the sialylated xyloside products.
Figure 7:
HPLC analysis of the highly anionic
products from the Golgi pellet. The anionic products which were not
retained by the C18 HPLC column using the standard water/methanol
gradient were further analyzed by C18 HPLC using an ammonium
formate/methanol gradient (Panel A, open symbols). An
equal portion of the same peak from Golgi vesicles incubated without
XylMU and analyzed in parallel is shown for comparison (solid
symbols); this shows the label in fractions 12-22 is not due to
xyloside products. Peak Ia was further analyzed by anion
exchange HPLC (Panel B); the arrow labeled
-2 shows the elution position of
Na
[
S]SO
. The relative
elution position for Sia
2,3Gal
Xyl
MU is indicated for
comparison. Peak Ia was digested with AUS, the neutralized products
were isolated by QAE-Sephadex, and these neutral products were analyzed
by amine adsorption HPLC (Panel C). The arrows in Panel C mark the elution positions of authentic
Gal
Xyl
MU and
Gal
Gal
Xyl
MU.
Table 2shows the products made by the Golgi at various
concentrations of XylMU. The concentration of Xyl
MU has
little effect on the relative proportion of the products. Most products
in the Golgi lumen are sialylated (75-80%). Sialidase digestions
(see above) established the linkages shown. Since the broad spectrum
AUS and the
-2,3-specific NDV both neutralized 85-95% of the
Sia
Gal
Xyl
MU, only a small percentage (5-10%) of
the Sia could be in
-2,6 linkage. The linkage of the Gal in
Gal
Xyl
MU is
-1,4 since this product is hydrolyzed by the
-1,4-specific galactosidase from S. pneumoniae, and we
have previously shown that Xyl
MU is galactosylated only by GAG
core galactosyltransferase I(28) . The second Gal residue in
the Gal
Gal
XylMU is presumed to be a
-1,3 linkage by
analogy with the GAG core structure. This Gal residue was resistant to
hydrolysis by the S. pneumoniae
-galactosidase and the
Gal
1,3GlcNAc-specific
-galactosidase from Xanthomonas
manihotis, but was hydrolyzed by Aspergillus niger, jack
bean, and bovine testicular
-galactosidases.
Although the Golgi purification removes >99.9% of the soluble cytoplasmic proteins and presumably other soluble factors required for vesicular transport between Golgi compartments, we incubated the Golgi vesicles in the presence of either 30 µM ilimaquinone (25) or pretreated the Golgi with 2 mMN-ethylmaleimide(24) . Neither inhibitor of vesicular transport had any effect on the xyloside products made (Table 4; N-ethylmaleimide data not shown). Altogether, the above results demonstrate that the synthesis of the xyloside products in these in vitro Golgi incubations does not involve intercompartmental transport or diffusion.
Fig. 8A shows the C18 HPLC profile of the products made
by the detergent-permeabilized, transport-independent Golgi vesicles.
All of the products were in the incubation supernatant; less than 1% remained in the Golgi lumen after centrifugation. The major neutral
product was GalXyl
MU (
68%) and very little (<2%)
Gal
Gal
Xyl
MU. The peak corresponding to
Sia
2,3Gal
Xyl
MU and GlcUA
Gal
Gal
Xyl
MU
was much smaller than seen in previous incubations and represented only
about 10% of the total products as compared to about 30% in the
standard incubation, indicating its synthesis is favored by continued
presence in a single vesicle compartment. Approximately 20% of the
products did not bind to the C18 HPLC column. The control incubation
without Xyl
MU showed that endogenous acceptors accounted for less
than 1% of these products. The xyloside products were shown to be
anionic by QAE-Sephadex chromatography and, in contrast to the products
from the standard incubations, were resistant to sialidase digestion. Fig. 8B shows that these products are heterogeneous by
analysis on C18 HPLC using the ammonium formate/methanol gradient.
Figure 8:
HPLC fractionation of the
[H]Gal-labeled xyloside products made by
detergent-permeabilized, transport-independent Golgi vesicles.
Detergent-permeabilized Golgi vesicles were incubated with 50
µM UDP-[
H]Gal and 100 µM Xyl
MU then chased with unlabeled sugar nucleotides as
described under ``Experimental Procedures.'' The labeled
xyloside products were purified and analyzed by HPLC. Panel A shows the fractionation of these products by C18 HPLC using a
water/methanol gradient. The anionic xylosides which were not retained
by the C18 column (see bar in Panel A) using this
gradient system were rechromatographed by C18 HPLC using an ammonium
formate/methanol gradient (Panel
B).
Analysis of these anionic products by ion-exchange HPLC showed that
the majority contained at least two negative charges (Fig. 9A). Treatment with -glucuronidase converted
about 36% of the labeled products to neutral structures (Fig. 9B). Analysis of these neutral products by amine
adsorption HPLC showed that they co-eluted with authentic Gal
1,
3Gal
1,4Xyl
MU. This was puzzling since the
-glucuronidase
digestion appeared to have removed two negative charged moieties. One
possibility, although we know of no precedent, is that these products
contained 2 terminal
-GlcUA residues. Alternatively, they may have
contained one GlcUA and one sulfate ester, and the
-glucuronidase
preparation may have contained a contaminating sulfatase activity.
Addition of sulfate was not anticipated, since we intentionally did not
add 3`-phosphoadenylylphosphosulfate to the incubations to avoid the
difficult task of analyzing sulfated products. To examine the latter
possibility, we subjected these products to mild anhydrous methanolysis
using conditions known to cleave 4-O-sulfate esters but not
the glycosidic linkages(29) . These conditions will also
convert carboxyl groups to their methyl esters. Fig. 9C shows the products. Approximately 30% were converted to neutral,
35% were converted to structures with a single negative charge, and 35%
still contained at least two negative charges. The neutral products
obtained by this treatment were analyzed by amine adsorption HPLC and
they co-eluted with authentic Gal
1,3Gal
1,4Xyl
MU. A
parallel methanolyzed sample was treated with mild base to hydrolyze
the carboxylic methyl esters and analyzed as above. Fig. 9D shows that the product having a single negative charge (see Fig. 9C) was absent after hydrolysis of the methyl
esters; suggesting that this product may have contained both a GlcUA
residue and a sulfate ester resistant to mild methanolysis. In separate
series of analyses, treatment with
-glucuronidase neutralized
35-40% of these products; methanolysis of the
glucuronidase-resistant products neutralized an additional 25%.
Analysis of these neutralized products by amine adsorption HPLC again
showed that these co-eluted with Gal
1,3Gal
1,4Xyl
MU. The
highly specific glycosidases and sulfatases needed to structurally
define these radiolabeled products are not available. Nevertheless, we
know that at least 65% of these products contain the
Gal
Gal
Xyl-core structure and most contain GlcUA and,
probably, sulfate. On this basis, we refer to these products as
``GAG-like'' xyloside products.
Figure 9:
Anion exchange HPLC analysis of
[H]Gal-labeled xyloside products after
-glucuronidase digestion and mild acid methanolysis. The anionic
xylosides which were not retained by the C18 column (see bar in Fig. 8A) were analyzed by anion exchange HPLC (Panel A). The arrow marks the elution position of
Na
[
S]SO
(-2 charge). Panel B shows a similar analysis after digestion
with
-glucuronidase. Panel C shows the analysis after
treatment with 0.5 N methanolic HCl for 5 h at 23 °C. Panel D shows the analysis after treatment with methanolic HCl
as above followed by treating with 0.05 N NaOH at 23 °C
for 30 min to cleave glucuronate methyl
esters.
Table 5shows a
summary of the products made by the detergent-permeabilized,
transport-independent Golgi vesicles in comparison to those made under
standard conditions. The most striking difference is the relative
decrease in sialylated structures in the permeabilized Golgi and the
corresponding increase in GAG-like structures. The structure of the GAG
core region is considerably more complex than once
thought(19, 37, 38, 39) . The
galactose residues may be sulfated in the 4- or 6-position, the xylose
residue may contain a phosphate at the 2-position, and the first GalNAc
of the chondroitin sulfate core may be sulfated at the 4- or
6-position. In addition, we recently found a novel -GalNAc residue
replacing the typical
-GalNAc in the chondroitin sulfate core made
on xylosides by several different cell types. (
)Essentially
nothing is known about the biosynthesis of these modifications, their
order of addition, site of synthesis, or their permanence. The results
presented above are consistent with
[
H]Gal
Xyl
MU being converted into a
complex mixture of these anionic structures when incubated with a
detergent-permeabilized Golgi preparation.
The pioneering studies of Hirschberg showed that sugar nucleotide transporters are essential for the synthesis of glycoconjugates within the Golgi(1) . Mutant cell lines deficient in these transporters make incomplete sugar chains(33, 34) . Hayes et al.(16, 17, 18) recently showed that intact and properly oriented Golgi preparations glycosylate endogenous acceptors when supplied with low concentration of labeled sugar nucleotides. When followed by a chase with non-labeled sugar nucleotides, the labeled chains can be further modified by adding more sugar residues to the radiolabeled chains. Since intervesicular transport does not occur under these conditions, all of the transporters, transferases, and acceptors must reside in the same compartment. Most of the products analyzed in those studies were N-linked oligosaccharides whose biosynthetic pathways have been well characterized. The approach showed an unexpected co-localization of some transferase activities with respect to other studies using different methods of localization. However, it was difficult to quantify the amount of this co-localization.
We wanted
to adapt this approach to investigate the synthesis of the core regions
of GAG chains. Xylosides have been used for more than 20 years to prime
the synthesis of GAG chains and compete with endogenous acceptors. The
core region, GlcUA1,3Gal
1,3Gal
1,4Xyl
-, is formed
and then elongated by the addition of GlcUA and GalNAc or GlcNAc to
initiate heparan or chondroitin sulfate chains. We reasoned that the
freely diffusable acceptor could enter the Golgi vesicles, but would
only be glycosylated in those which contained both the UDP-Gal
transporter and the first galactosyltransferase. A subsequent chase
with other sugar nucleotides would then result in the extension of the
[
H]Gal-labeled xylosides. The residues added next
would depend upon which enzymes were substantially co-localized with
the first galactosyltransferase. Considering the known structure of the
core region, it would be reasonable to expect a series of partially
completed core structures. However, in previous studies with intact
human melanoma or Chinese hamster ovary cells(20) , we found
that all of these cell lines preferentially made and secreted a
sialylated product, Sia
2,3Gal
Xyl
MU, when incubated with
Xyl
MU and [
H]Gal. These studies showed that
the addition of Gal was catalyzed only by GAG core specific
galactosyltransferase I; a mutant cell line lacking this specific
transferase was unable to galactosylate Xyl
MU.
The results of
the studies presented here support the hypothesis that the preferential
synthesis of a sialylated, ganglioside-like structure is due to the
co-localization of the first GAG core specific galactosyltransferase
and at least one -2,3-sialyltransferase. The synthesis appears to
utilize previously transported CMP-Sia as well as donors supplied and
transported during the chase. Even when CMP-Sia is not provided in the
chase, the amount of sialylated molecules is not reduced. Most of the
molecules labeled in the absence of a chase or chased in the absence of
CMP-Sia still have only 1 Gal residue and do not have the second Gal or
the GlcUA residues. Since we have shown that no intervesicular
transport and no diffusion between vesicles occur, this strongly argues
that Gal transferase I and II are not substantially co-localized.
Previous studies (35) using subcellular fractionation and
enzyme assays support this conclusion since the two activities were
overlapping but not coincident. On the other hand there is substantial
overlap with one or more sialyltransferases. This is even seen at the
lowest concentration of xyloside (10-20 µM), and
therefore is not an artifact due to high concentrations of Xyl
MU.
We do not know which sialyltransferases carry out this reaction, but
addition of sialic acid is restricted to glycoproteins and glycolipids
and does not occur on GAG chains. As discussed previously, the major
sialylated product resembles ganglioside GM3, though we do not know
whether GM3 synthase carries out this sialylation. Approximately
10-15% of the sialylated Xyl
MU products resembles
ganglioside GD3 and the
-2,8Sia
-2,3Sia- moiety occurs only in
gangliosides and in those glycoproteins containing polysialic acid
structures (e.g. N-CAM). These results show that both
glycoprotein and/or glycolipid biosynthetic steps can occur in the same
Golgi compartment as the first galactosylation step of the GAG core
region. Previous studies by Vertel et al.(31) have
shown that xylose can be added to endogenous acceptors in the
transitional region between the ER and cis-Golgi. Our results suggest
that the next step occurs in a functional compartment that contains
both the galactosyltransferase I and sialyltransferase(s), but this
method cannot distinguish whether these reactions occur in the cis,
medial, trans, or other compartments. Results using other approaches
and other cell lines often place sialyltransferases in the later Golgi
compartments(2) . Other studies with Brefeldin A and
immunolocalization suggest that some sialyltransferases may occur in
early compartments of the Golgi(3, 20) . The studies
of Sugumaran et al.(35) place GAG core
galactosyltransferases in early (cis) Golgi compartments.
The liver and cell lines derived from it synthesize proteoglycans with typical GAG core structures(40, 41, 42) . Although these cells can use xylosides as alternate acceptors for GAG chain synthesis(43, 44) , these studies focus on the synthesis of the elongated GAG chains rather than the smaller, non-sulfated xyloside products. It is not known if they also make shorter, sialic acid terminated xyloside structures. We have previously shown that CHO and melanoma cells do make sialylated xyloside products (20) in addition to normal GAG chains, but it is not known whether any cell type can sialylate a GAG core precursor during proteoglycan biosynthesis. If sialylation does not normally occur in native proteoglycans, the biosynthetic mechanism must be able to circumvent the action of other co-localized glycosyltransferases. In this case, co-localization of glycosyltransferases does not necessarily control structure in the dynamic Golgi environment. Residence time or additional restrictions to free diffusion within a given compartment may determine whether co-localized glycosyltransferases are able to glycosylate the product of another transferase.
The maximum amount
of product appears to be made at about 50 µM XylMU.
Further increases in the amount of acceptor do not produce a
proportional increase in the amount of product. Since it is unlikely
that diffusion of the acceptor is limiting, it is more likely that the
availability of sugar nucleotide donor and/or the glycosyltransferase
becomes limiting. Since this occurs only within the vesicles that have
core Gal transferase I, this should locally restrict the amount of
available sugar nucleotide within those compartments. In effect,
Xyl
MU may act as a sink for the transported sugar nucleotides and
thus inhibit the synthesis of any endogenous acceptor that is
co-localized in the same compartment and requires available UDP-Gal.
Our preliminary analyses of the galactosylation of endogenous acceptors
in the presence of Xyl
MU indicate that there is a 25-35%
inhibition of incorporation into endogenous glycoprotein products.
Using this rationale, we are now using Xyl
MU to co-localize other
galactosyltransferases to the same compartment with GAG Gal transferase
I. The results presented here also demonstrate that Gal
Xyl
MU
also diffuses across the Golgi vesicle membranes. Using this structure
as the acceptor in similar incubations should allow us to determine if
the GAG
-1,3-glucuronyltransferase is co-localized with GAG Gal
transferase II.