From the
Heparan sulfate biosynthesis initiates by the transfer of
Chondroitin sulfate and heparan sulfate are attached to specific
serine residues in a proteoglycan through a common linkage
tetrasaccharide
(-GlcA
The CarboPac PA-1 column was equilibrated in
0.1 M NaOH and calibrated with 10
Attempts to identify oligosaccharide intermediates on native core
proteins in the mutant did not meet with success because their
concentration was too low, and O-linked and N-linked
oligosaccharides interfered with the analyses (data not shown). To
circumvent this problem, a fusion protein was made consisting of a
45-amino acid segment of betaglycan fused by its N terminus to the
286-amino acid IgG-binding domain of Staphylococcal protein A
and the signal peptide from the secreted metalloprotease, transin. This
segment of betaglycan (residues 518-562) has two GAG attachment
sites at Ser
Analysis of
The GAG chains of proteoglycans assemble by the stepwise
addition of monosaccharides to the nonreducing terminus of nascent
chains (Rodén, 1980). The sequential addition of sugars suggests
that discrete oligosaccharide intermediates should exist in
vivo. Vertel et al.(1994) found xylose on a truncated
aggrecan precursor that fails to be secreted. However, more complex
intermediates have not been detected on core proteins, possibly because
of their rapid and efficient conversion to full-length chains.
Intermediates accumulate when GAG synthesis is altered by drugs.
Spiro et al.(1991) characterized accumulated
Gal
Mutants provide another
way to find intermediates, since they should accumulate metabolites
upstream from the block imposed by the mutation. Quentin et
al.(1990) showed that xylose residues occur on a natural
proteoglycan core protein in human fibroblasts deficient in
galactosyltransferase I. The accumulation of the pentasaccharide,
GlcNAc
The
pentasaccharide that accumulates in the mutant does not contain
phosphate at C-2 of xylose (Oegema et al., 1984; Fransson
et al., 1985; Shibata et al., 1992), sulfate at C-4
or C-6 of the Gal residues (Sugahara et al., 1988; de Waard
et al., 1992), or sulfate on the
The chimera used in this study makes 40%
heparan sulfate and 60% chondroitin sulfate in wild-type cells, and
about one-half of the material contains two chains (Fig. 1).
Therefore, the amount of pentasaccharide (plus tetrasaccharide and
trisaccharide) should have represented
Transfected cells were labeled with
[6-
-D-GlcNAc from UDP-GlcNAc to the D-GlcA moiety
of the linkage tetrasaccharide,
GlcA
1-3Gal
1-3Gal
1-4Xyl
1-core
protein. The enzyme catalyzing this reaction differs from the
-GlcNAc transferase involved in chain polymerization based on
genetic and enzymatic studies of an animal cell mutant defective in
chain polymerization (Fritz, T. A., Gabb, M. M., Wei, G., and Esko, J.
D.(1994) J. Biol. Chem. 269, 28809-28814). In this
report we show that this mutant also accumulates a pentasaccharide
intermediate containing
-GlcNAc. A fusion protein was made from
the IgG-binding domain of protein A and a segment of the proteoglycan,
betaglycan. This segment contained one glycosaminoglycan attachment
site that primes only chondroitin sulfate and another that primes both
heparan sulfate and chondroitin sulfate (Zhang, L., and Esko, J.
D.(1994) J. Biol. Chem. 264, 19295-19299). Expression of
the chimera in the mutant resulted in the accumulation of an
oligosaccharide that labeled with [6-
H]GlcN. The
oligosaccharide comigrated with a pentasaccharide standard derived from
chondroitin sulfate, but acid hydrolysis gave 98%
[
H]GlcN. Heparin lyase III digestion yielded
[
H]GlcNAc, suggesting that the GlcNAc residue was
-linked to the nonreducing terminus. Enzymatic treatment of
[6-
H]Gal-labeled material yielded the
tetrasaccharide,
GlcA-[
H]Gal-[
H]Gal-xylitol.
These findings suggest that pentasaccharide had the structure,
GlcNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl.
Its accumulation in a Chinese hamster ovary cell mutant defective in
the polymerizing
-GlcNAc transferase provides in vivo evidence that two
-GlcNAc transferases catalyze the formation
of heparan sulfate.
1-3Gal
1-3Gal
1-4Xyl).
Studies of Chinese hamster ovary mutants altered in glycosaminoglycan
(GAG)
(
)
synthesis have shown that the pathways of
heparan sulfate and chondroitin sulfate synthesis share at least the
first two enzymes that initiate the chains, xylosyltransferase and
galactosyltransferase I (Esko et al., 1985, 1987; Esko, 1991).
The pathways diverge thereafter by the addition of the first
N-acetylated hexosamine residue,
-GlcNAc (heparan
sulfate) or
-GalNAc (chondroitin sulfate). Previous studies showed
that the transferase that adds the first
-GalNAc residue in
chondroitin sulfate differs from the transferase involved in chain
polymerization (Rohrmann et al., 1985). Recently, Fritz et
al.(1994) reported that the enzyme transferring
-GlcNAc to
the linkage tetrasaccharide differs from the one involved in forming
the repeating disaccharide units of heparan sulfate
(GlcA
1-4GlcNAc
1-4). Based on this finding, we
predicted that a Chinese hamster ovary mutant defective in heparan
sulfate polymerization (Lidholt et al., 1992) might accumulate
a pentasaccharide intermediate consisting of
-GlcNAc attached to
the linkage tetrasaccharide. Its accumulation in the mutant provides
strong evidence that different enzymes catalyze the initiation and
polymerization of heparan sulfate chains.
Cell Culture
Chinese hamster ovary cells (CHO-K1) were obtained from the
American Type Culture Collection (CCL-61, Rockville, MD) and
pgsD-677 was described previously (Lidholt et al.,
1992). Cells were grown in Ham's F-12 medium supplemented with
7.5% fetal bovine serum (Hyclone, Salt Lake City, UT), penicillin G
(100 units/ml), and streptomycin sulfate (100 µg/ml) under an
atmosphere of 5% CO, 95% air and 100% relative humidity.
Cells were passaged with trypsin every 3-4 days and revived
periodically from frozen stocks.
Linkage Region Oligosaccharides
Decorin from bovine skin (a generous gift from M.
Höök, Texas) was used to prepare oligosaccharide standards.
The GAG chain was released by -elimination, and the terminal
xylose residue was reduced to [
H]xylitol with
NaB
H
. Briefly, 0.5 µg of decorin was
dissolved in 50 µl of water and mixed with 50 µl of a solution
containing 0.5 M NaOH, 20 mM NaBH
, and 5
mCi of NaB
H
(12.6 Ci/mmol, DuPont NEN). After
24 h at 4 °C, 10 µl of 10 N acetic acid was added in a
fume hood to stop the reaction. Authentic chondroitin 4-sulfate (1 mg)
was added as carrier. The sample was diluted with 9 volumes of water
and applied to a 0.5-ml column of DEAE-Sephacel. The column was washed
twice with 12.5 ml of a 20 mM sodium acetate buffer (pH 6.0)
containing 0.25 M NaCl. The GAG chains were eluted with 1
M NaCl buffer (2.5 ml) and precipitated twice with 4 volumes
of ethanol at 4 °C. The hexasaccha-ride,
GlcA
1-3GalNAc
1-4GlcA
1-3Gal
1-3Gal
1-4[
H]xylitol,
was generated by chondroitinase ABC digestion (Hascall et al.
1972; Oike et al., 1980). The tetrasaccharide,
GlcA
1-3Gal
1-3Gal
1-4[
H]xylitol,
was generated by chondroitinase ACII digestion (Hascall et al.
1972; Oike et al., 1980). The pentasaccharide,
GalNAc
1-4GlcA
1-3Gal
1-3Gal
1-4[
H]xylitol,
and the trisaccharide,
Gal
1-3Gal
1-4[
H]xylitol,
were generated by treating the hexasaccharide and the tetrasaccharide
with mercuric acetate (Ludwigs et al., 1987), respectively.
The completion of each reaction and the purity of each compound was
confirmed by gel filtration and anion-exchange chromatography, as
described below.
Transfection and Radiolabeling
A chimera containing a 45-amino acid segment of betaglycan
fused to the IgG binding domain of staphylococcal protein A was
expressed transiently in pgsD-677 mutant and wild-type cells as
described previously (Zhang and Esko, 1994). In transfection
experiments, a culture dish (150 mm diameter) was seeded with 5
10
cells in Ham's F-12 medium containing 10% (v/v)
NuSerum (Collaborative Research, Inc., Bedford, MA). After 1 day, the
medium was removed, and 10 ml of Ham's F-12 medium containing
0.25 mg/ml of DEAE-dextran (Sigma D-9885), 50 mM Tris-HCl (pH
7.4), 50 µg/ml of chloroquine, and 10 µg/ml of plasmid DNA was
added. The cells were incubated at 37 °C for 2 h. The solution was
aspirated, and 10 ml of 10% (v/v) dimethyl sulfoxide (Sigma) in
phosphate-buffered saline was added. After 2 min at 23 °C, the
solution was aspirated. The cells were washed with Ham's F-12
medium without serum and grown in 30 ml of complete Ham's F-12
medium for 12 h. The transfected cells were then labeled at 37 °C
for 48 h with 50 µCi/ml
[
S]H
SO
(25-40
Ci/mg, Amersham Corp.) prepared in sulfate-free growth medium. In some
experiments, the cells were labeled with 100 µCi/ml of
D-[6-
H]glucosamine (40 Ci/mmol) or 100
µCi/ml of D-[6-
H]galactose (25.5
Ci/mmol) in medium containing 1 mM glucose (12 ml).
Fusion Protein Purification
Labeled culture medium was mixed with 120 µl of 1
M Tris-HCl (pH 7.5), 5% (v/v) Triton X-100, and 2% (w/v)
sodium azide. The samples were centrifuged at 1,100 g for 10 min (IEC HN-SII centrifuge), and the supernatant was
decanted into a fresh tube. IgG-Agarose beads (Sigma) were added (0.2
ml), and the sample was mixed end-over-end overnight at 4 °C.
Samples were centrifuged 1,100
g for 10 min, and the
supernatants were aspirated. The pellets were washed 3 times with
buffer containing 50 mM Tris-HCl (pH 8), 0.15 M NaCl,
and 0.02% (w/v) sodium azide (14 ml).
S-Labeled samples
were dissolved in 20 mM Tris buffer (pH 7.0), and an aliquot
was treated at 37 °C for 4 h with 10 milliunits of chondroitinase
ABC (Seikagaku), 2 milliunits of heparin lyase III (EC 4.2.2.8,
Seikagaku), or both enzymes in a total volume of 50 µl. The
reactions were stopped by adding SDS-PAGE sample buffer (Laemmli, 1970)
and boiling the samples in a water bath for 7 min. The samples were
loaded on a 5-16% linear gradient SDS-PAGE gel (200
160
1.5 mm) and electrophoresed at constant voltage (80 V)
overnight (Laemmli, 1970). The gel was dried on a piece of filter
paper, and the radioactive proteoglycans were visualized by
autoradiography.
Oligosaccharide Purification
All purification steps were performed at room temperature
unless otherwise indicated.
Step 1: Trypsin Treatment
[6-H]Gal
and [6-
H]GlcN-labeled betaglycan chimeras bound
to IgG-agarose beads were washed with 0.1 M ammonium
bicarbonate and suspended in bicarbonate buffer (0.5 ml) containing 0.1
mM CaCl
and 50 µg/ml
L-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin
(Sigma). After the samples were incubated at 37 °C, they were
centrifuged. The proportion of total counts in the supernatant
increased from less than 1% at the beginning of the reaction to
65-100% by 40 min. The reaction was continued for another 40 min
and then stopped by boiling the samples in a water bath for 10 min. The
beads were removed by centrifugation, and the supernatants were
collected. Chondroitin 4-sulfate (1 mg, Calbiochem, La Jolla, CA) was
added to each sample.
Step 2: DEAE-Sephacel Chromatography
DEAE-Sephacel
columns (0.5 ml) were prepared in a disposable pipette tip and
equilibrated with 0.25 M NaCl in 50 mM sodium acetate
buffer (pH 6). The samples were loaded, and the columns were washed
with 12.5 ml of buffer containing 0.25 M NaCl to remove
protein A fragments and any betaglycan segments without GAG chains.
Peptides containing chains were eluted with 2.5 ml of buffer containing
1 M NaCl and precipitated by adding 4 volumes of ethanol.
Step 3:
The precipitated
glycopeptides were resuspended in 0.5 M NaOH containing 1
M sodium borohydride (0.1 ml) and incubated at 4 °C for 24
h to liberate any O-linked GAG or oligosaccharide chains.
After -Elimination
-elimination, the samples were neutralized with acetic acid
and diluted 500-fold with water.
Step 4: DEAE-Sephacel Chromatography
The
-eliminated material was subjected to another round of
chromatography on DEAE-Sephacel to separate small oligosaccharides from
large GAG chains. The flow-through (50 ml) and 0.25 M NaCl
wash fraction (10 ml) were collected. The GAG chains were eluted with
2.5 ml of 1 M NaCl buffer and precipitated with ethanol.
Step 5: AG1-X2 Anion-exchange Chromatography
The
flow-through sample was loaded on a 0.5-ml column of AG1-X2 resin
(200-400 mesh, Bio-Rad, Hercules, CA) equilibrated with water.
The column was eluted stepwise with 5 ml each of water, 0.015, 0.05,
0.1, 0.15, 0.2, and 1 M ammonium acetate (pH 7.0). Each
fraction was collected and lyophilized.
Step 6: TSK-G2500 Gel Filtration
Chromatography
Samples collected from AG1-X2 were analyzed by
gel filtration high performance liquid chromatography (Progel
TSK-G2500, 300 7.8 mm, inner diameter, Toso Haas,
Montgomeryville, PA). The column was equilibrated in 0.5 M
pyridinium acetate buffer (pH 5.0), and radioactivity in the effluent
was detected by in-line liquid scintillation counting (Packard
Instrument Company, Downers Grove, IL). The column was calibrated with
tri-, tetra-, penta- and hexasaccharide standards prepared from
chondroitin sulfate as described above. Material that migrated in the
position of the pentasaccharide standard was collected for further
analysis.
Compositional Analysis
[6-H]GlcN-labeled material from the
mutant was acid hydrolyzed for 4 h at 100 °C as described by Hardy
et al.(1988). A sample was mixed with standard GlcN and GalN
(10 nmol) and applied to a CarboPac PA-1 column (Dionex Corp)
equilibrated with 16 mM NaOH. The column was run at 1 ml/min,
and 0.5-ml fractions were collected. The elution position of sugar
standards was monitored by pulsed amperometric detection. The
radiolabeled sugars were quantitated by liquid scintillation
spectrometry, and their elution positions were compared with that of
the sugar standards.
Heparin Lyase III Digestion
An aliquot of the intermediate labeled either with
[6-H]GlcN or [6-
H]Gal was
dissolved in 50 µl of 20 mM Tris-HCl buffer (pH 7.0)
containing 1 mM CaCl
and 10 milliunits of heparin
lyase III (Seikagaku, heparitinase I) and incubated at 37 °C
overnight. The enzyme was inactivated by boiling the sample for 10 min.
Samples were filtered through a microcon
10 microconcentrator
(Amicon, Inc., Beverly, MA), and the filtrates were applied to the
CarboPac PA-1 column.
cpm of each
linkage region standards. The samples and standards were eluted with a
gradient of sodium acetate as described by Shibata et
al.(1992). Fractions (1 ml) were collected at a flow rate of 1
ml/min, and the radiolabeled sugar was quantitated by liquid
scintillation spectrometry.
Intermediate Purification
We have described a
mutant designated pgsD-677 that lacks the -GlcNAc transferase
involved in the polymerization of heparan sulfate chains (Lidholt
et al., 1992). Enzymatic studies showed that the mutant
transferred
-GlcNAc residues to a disaccharide resembling the
terminal sugars of the linkage region (GlcA
1-3Gal-).
This finding suggested that cells use two
-GlcNAc transferases to
assemble heparan sulfate, one to initiate the chain (
-GlcNAc
transferase I) and another to polymerize the chain (
-GlcNAc
transferase II) (Fritz et al., 1994). If this idea is correct,
then pgsD-677 cells should accumulate an intermediate consisting of
-GlcNAc attached to the linkage tetrasaccharide
(GlcNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl-).
and Ser
(Zhang and Esko, 1994;
López-Casillas et al., 1994). The site at Ser
primes both heparan sulfate and chondroitin sulfate, whereas the
site at Ser
primes only chondroitin sulfate (Zhang and
Esko, 1994; López-Casillas et al., 1994). Mutant and
wild-type cells were transiently transfected with the construct and
labeled with [6-
H]GlcN. Chimeras secreted into
the growth medium were purified by affinity chromatography using
IgG-agarose (see ``Experimental Procedures''). Both cell
lines glycosylated the chimeras, but the amount of
[6-
H]GlcN-labeled material was higher in
wild-type cells than in the mutant (). This result was not
unexpected since the mutant makes chondroitin sulfate, whereas the
wild-type makes both chondroitin sulfate and heparan sulfate and most
of the label was found in the repeating disaccharide units of the GAG
chains. In contrast, comparable amounts of material was made in mutant
and wild-type cells labeled with [6-
H]Gal. This
precursor labels the internal Gal residues found in the linkage region,
suggesting that the chimeras expressed by mutant cells might contain
linkage fragments.
SO
-labeled
chimeras by SDS-PAGE (Fig. 1) revealed that wild-type cells
produced sulfated material that separated into two broad bands, typical
of proteins containing variably sized GAG chains (lane3). The upper, more slowly migrating material consisted
of chimeras containing two GAG chains since none was made in chimeras
containing only one attachment site.
(
)
Digesting
the samples with chondroitinase ABC (lane4) or
heparin lyase III (lane5) collapsed the upper band
from wild-type cells, indicating that most of these chimeras contained
one chondroitin sulfate and one heparan sulfate chain. Samples treated
with both enzymes yielded only a residual core protein (
45 kDa)
that labeled with
SO
due to sulfate-containing
linkage fragments resistant to enzyme digestion (lane6). The chimeras expressed in pgsD-677 cells mostly
contained one chondroitin sulfate chain (lane1), and
all of the material collapsed into the
45-kDa range after
chondroitinase ABC treatment (lane2). The reduced
amount of chimeras containing two GAG chains in the mutant suggested
that some molecules containing a chondroitin sulfate chain might bear
an oligosaccharide intermediate at the site where heparan sulfate
assembled in wild-type cells.
Figure 1:
SDS-PAGE of
[S]betaglycan chimeras. Mutant and wild-type
cells were transfected with the betaglycan chimera (see
``Experimental Procedures'') and labeled with
SO
. Chimeras were purified by IgG-affinity
chromatography and dissolved in SDS-PAGE protein sample buffer. The
samples were analyzed on SDS-polyacryamide (5-16%) gradient gels.
The positions of chimeras containing one and two GAG chains are
indicated to the right of the gel, and the positions of
protein molecular weight standards are indicated to the left. Each lane
contained the chimera with the insert
VVYYNSIVVQAPSPGDSSGWPDGYEDLESGDNGFPGDGDEGETAP
derived from betaglycan (López-Casillas et al.,
1991). Lane1, chimera expressed in pgsD-677 cells;
lane2, chimera from pgsD-677 cells after treatment
with chondroitinase ABC; lane3, chimera expressed in
wild-type cells; lane4, chimera from wild-type after
treatment with chondroitinase ABC; lane5, chimera
from wild-type cells after treatment with heparin lyase III; lane6, chimera from wild-type cells after treatment with both
chondroitinase ABC and heparin lyase III.
A purification scheme for finding
intermediates was devised (Fig. 2). Chimeras were first affinity
purified from cells labeled with [6-H]Gal or
[6-
H]GlcN. They were then treated with trypsin to
remove the protein A domain. This region contains potential
N-linked and O-linked glycosylation sites that might
interfere with the identification of linkage region intermediates. The
peptide derived from betaglycan does not contain Arg or Lys residues,
and the closest cleavage site was 5 amino acids into the protein A
domain (Nilsson et al., 1985). Thus, trypsin should have
generated a fragment containing 45 amino acids from betaglycan and 5
amino acids from protein A.
Figure 2:
Purification scheme for
oligosaccharides.
Next, the mixture was separated by
DEAE-Sephacel chromatography to select fragments containing a GAG
chain. This step ensured that any oligosaccharides found subsequently
would have been made on fragments that also supported the assembly of a
complete GAG chain. Treating this material with alkali liberated the
O-linked chains. A second round of chromatography on
DEAE-Sephacel separated the GAG chains from neutral or weakly charged
oligosaccharides. The latter material was then separated by strong
anion-exchange chromatography on a column of AG1-X2 resin (see
``Experimental Procedures''). Prior calibration of the column
with linkage region fragments showed that oligosaccharides containing a
charge of -1 or -2 would bind to the column and elute with
50 mM ammonium acetate. Material eluting under these
conditions was used for the analytical studies described below.
Analysis of the Intermediates
Gel filtration
chromatography showed that some of the oligosaccharides labeled with
[6-H]GlcN from mutant cells migrated in the same
position as a pentasaccharide standard derived from authentic
chondroitin sulfate (Fig. 3A). Lesser amounts of
tetrasaccharide and trisaccharide were also present. In contrast,
wild-type cells contained mostly short oligosaccharides and little if
any material migrating like pentasaccharides (Fig. 3B).
Another aliquot was analyzed by chromatography on a CarboPac PA-1
column under alkaline conditions (Shibata et al., 1992). Most
of the labeled oligosaccharides from the mutant eluted like the
pentasaccharide standard, except that its position was shifted by one
fraction (Fig. 4A). Wild-type cells did not produce any
material migrating in this position (Fig. 4B).
Figure 3:
An unique oligosaccharide exists in
pgsD-677. Chimeras containing two GAG sites were labeled with
[6-H]GlcN and purified from mutant and wild-type
cells. An aliquot of material that eluted with 50 mM ammonium
acetate from the AG1-X2 column was analyzed by TSK-G2500 gel filtration
chromatography (see ``Experimental Procedures''). The
fractions indicated by shading were pooled for further
analysis. A, mutant; B, wild-type; 3,
Gal
1-3Gal
1-4xylitol; 4,
GlcA
1-3Gal
1-3Gal
1-4xylitol; and
5,
GalNAc
1-4GlcA
1-3Gal
1-3Gal
1-4xylitol.
Figure 4:
A pentasaccharide exists in pgsD-677
mutant but not in wild-type cells. An aliquot of material purified by
AG1-X2 chromatography was analyzed on a CarboPac PA-1 column (Shibata
et al., 1992). The amount of radioactivity in each fraction
was monitored. A, pgsD-677; B, wild-type; 3,
Gal1-3Gal
1-4xylitol; 4,
GlcA
1-3Gal
1-3Gal
1-4xylitol; and
5,
GalNAc
1-4GlcA
1-3Gal
1-3Gal
1-4xylitol.
The
putative pentasaccharide intermediate represented 0.1% of the
starting material from chimeras labeled with
[6-
H]GlcN and
2% of material labeled with
[6-
H]Gal. The labeling of the pentasaccharide
with [6-
H]GlcN suggested that it contained
GlcNAc, but some GalNAc may have been present since GalNAc can be made
from GlcNAc through epimerization of the corresponding nucleotide
sugars (Kingsley et al., 1986). Acid hydrolysis, however,
released 98% [
H]GlcN and less than 2%
[
H]GalN (Fig. 5).
Figure 5:
Acid hydrolysis liberates
[H]GlcN. [6-
H]GlcN-labeled
oligosaccharide from the mutant was hydrolyzed in acid and analyzed by
CarboPac PA-1 chromatography. The arrows indicate the position
of authentic GlcN and GalN standards mixed with the sample and detected
by pulsed amperometric detection.
Fractions containing
pentasaccharides were pooled from gel filtration (Fig. 3,
shadedarea). This material migrated on the Carbopac
PA-1 column like the major oligosaccharide peak found in the material
prior to gel filtration (Fig. 4). The pentasaccharide was
resistant to -hexosaminidase (data not shown), suggesting that the
GlcNAc residue was not
-linked or that it was inaccessible. When
[6-
H]GlcN-labeled material was treated with 10
milliunits of heparin lyase III, [6-
H]GlcNAc was
released (Fig. 6A). Heparin lyase III recognizes
N-acetyl or N-sulfated GlcN residues linked
-(1-4) to GlcA in heparan sulfate chains (Nader et
al., 1990). The reaction normally liberates disaccharides with a
nonreducing terminal
-D-
-unsaturated
glucopyranosyluronic acid. Apparently, the enzyme also acts on the
terminal
GlcNAc residues. Treating
[6-
H]Gal-labeled intermediate with heparin lyase
III should liberate radiolabeled tetrasaccharide with a
unsaturated glucopyranosyluronic acid at the nonreducing end
(
-GlcA
1-3[
H]Gal
1-3[
H]Gal
1-4xylitol).
Analysis of the products of the reaction showed a single radiolabeled
product that migrated exactly at the position of standard
tetrasaccharide on the CarboPac PA-1 column (Fig. 6B).
Together, these data indicated that the pentasaccharide accumulating in
pgsD-677 cells had the structure
GlcNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl.
Figure 6:
Heparin lyase III digestion demonstrates
that the pentasaccharide in the mutant is
GlcNAc1-4GlcA
1-3Gal
1-3Gal
1-4xylitol.
The pooled pentasaccharides labeled with
[6-
H]GlcN or [6-
H]Gal were
applied to a CarboPac PA-1 column before (
) or after (
)
heparin lyase III digestion. Fractions were monitored for
radioactivity. A, samples labeled with
[6-
H]GlcN; B, samples labeled with
[6-
H]Gal; 4,
GlcA
1-3Gal
1-3Gal
1-4xylitol;
5,
GalNAc
1-4GlcA
1-3Gal
1-3Gal
1-4xylitol.
1-3Gal
1-4Xyl stubs on a chondroitin sulfate
proteoglycan core protein in M21 human melanoma cells after blocking
chondroitin sulfate elongation with brefeldin A. Takagaki et
al.(1991) and Freeze and co-workers (Freeze et al., 1993;
Manzi et al., 1995; Salimath et al., 1995) have
characterized several oligosaccharides primed on
-D-xylosides, including Gal
1-4Xyl,
Gal
1-3Gal
1-4Xyl, GlcA
1-4Xyl,
NeuAc
2-3Gal
1-4Xyl, and
GalNAc
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl,
although only the first two lie along the pathway of GAG synthesis. The
accumulation of these compounds may reflect detoxification reactions
(glucuronidation) or utilization of alternate substrates by enzymes
belonging to other biosynthetic pathways.
1-4GlcA
1-3Gal
1-3Gal
1-4Xyl
1-,
in a mutant altered in heparan sulfate polymerization suggests that the
assembly of chains normally goes through this intermediate. Its absence
in wild-type cells suggests that the conversion of pentasaccharide to
heparan sulfate occurs rapidly and quantitatively under normal
conditions. Because the mutant is defective in chain polymerization
(Lidholt et al., 1992), the presence of
-GlcNAc-terminated pentasaccharide provides another piece of
evidence that a unique
-GlcNAc transferase catalyzes the addition
of the first
-GlcNAc residue (Fritz et al., 1994).
-GlcNAc residue
(Sugahara et al., 1992a). Hexasaccharides from chondroitin
sulfate containing these modifications would have eluted from the
AG1-X2 column with
1 M ammonium acetate. Thus, if
phosphorylation or sulfation had occurred, we would have detected the
modified oligosaccharides. The lack of phosphorylation and sulfation is
not surprising since it does not occur universally or
stoichiometrically in chondroitin sulfate and heparan sulfate (Sugahara
et al., 1988; de Waard et al., 1992; Fransson et
al., 1990; Shibata et al., 1992; Sugahara et
al., 1992a, 1992b).
40% of material labeled
with [6-
H]Gal in pgsD-677 cells instead of only
2% (). Several possibilities may explain this
discrepancy. Core proteins with immature chains may be degraded in the
endoplasmic reticulum/Golgi by proteolysis (Su et al., 1993),
by endolytic cleavage of the carbohydrate chains (Villers et
al., 1994), or by removal of the terminal
-GlcNAc residue by
an unknown mechanism. Addition and removal of sugars occurs normally
during the processing of N-linked oligosaccharides (Kornfeld
and Kornfeld, 1985), in the modification of cytosolic proteins (Kearse
and Hart, 1991), and in the sorting of lysosomal glycoproteins (Reitman
and Kornfeld, 1981; Varki and Kornfeld, 1980). Additional experiments
are needed to determine if linkage fragments from GAG chains also
undergo dynamic processing reactions.
Table:
Yield of [6-H]GlcN
or [6-
H]Gal-labeled oligosaccharides in wild-type
and mutant pgsD-677 cells
H]GlcN or [6-
H]Gal, and
the chimeras were purified by IgG-agarose affinity chromatography (see
``Experimental Procedures''). The recovery of radioactive
material through the various preparation steps was measured. The data
represent a typical experiment (n = 3).
GlcA, D-
-unsaturated
glucopyranosyluronic acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.