From the Division of Cellular and Molecular Medicine, Glycobiology Program, University of California, San Diego, La Jolla, California 92093-0687
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ABSTRACT |
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The proteoglycans of animal cells typically
contain one or more heparan sulfate or chondroitin sulfate chains.
These glycosaminoglycans assemble on a tetrasaccharide primer,
-GlcA The assembly of the glycosaminoglycans
(GAGs),1 heparan sulfate and
chondroitin sulfate, initiates by the transfer of xylose to specific
serine residues in core proteins, which then gives rise to the
so-called linkage tetrasaccharide,
GlcA Several of the enzymes involved in glycosaminoglycan assembly have been
purified and cloned, and some of them appear to be members of families
of related genes. For example, at least three different GlcNAc
N-deacetylase/N-sulfotransferases (20-24), three GlcNAc 6-O-sulfotransferases
(25),2 and as many as five
3-O-sulfotransferases exist (26-28). Recent reports claim
that multiple copolymerases forming the heparan sulfate backbone may
exist as well (29, 30). Multiple isozymes make possible tissue-specific
and developmentally regulated expression of the corresponding enzyme
activities. Since the enzymatic properties of the isozymes may differ
in subtle ways (27), their selective expression may be responsible for
unique saccharide sequences found in different tissues (31-37).
In contrast to the amount of information available for these late
reactions, relatively little is known about the structure and
expression of the transferases that act early in the pathway (1).
Mutational studies of GAG assembly in Chinese hamster ovary cells have
shown that mutants in the first two steps (catalyzed by
xylosyltransferase (XylT) and galactosyltransferase I (GalT-I)) fail to
make both heparan sulfate and chondroitin sulfate chains (38-40). This
finding suggests that a single set of enzymes catalyze the biosynthesis
of the linkage region, without regard to the eventual composition of
the chain. To obtain information about other enzymes involved in
linkage region formation, we have developed a powerful selection and
screening method to find mutants in these reactions. One new class of
mutants does not synthesize glycosaminoglycan chains due to a
deficiency of glucuronosyltransferase I (GlcAT-I). Like mutants
defective in XylT or GalT-I, the new mutants fail to make both heparan
sulfate and chondroitin sulfate, suggesting that each of the early
steps in linkage region formation is catalyzed by a single isozyme.
Cell Cultures--
Chinese hamster ovary cells (CHO-K1) were
obtained from the American Type Culture Collection (CCL-61, Rockville,
MD). Mutants pgsA-745 (xylosyltransferase-deficient) and pgsB-761
(galactosyltransferase I-deficient) were characterized previously (38,
39). Cells were grown under an atmosphere of 5% CO2 in air
and 100% relative humidity in Ham's F-12 growth medium (Life
Technologies, Inc.) supplemented with 7.5% (v/v) fetal bovine serum
(HyClone Laboratories), 100 µg/ml streptomycin sulfate, and 100 units/ml penicillin G. Defined F-12 medium was prepared from individual
components (41), substituting chloride salts for sulfate, and
supplemented with fetal bovine serum that had been dialyzed
106-fold against phosphate-buffered saline (PBS) (42). Low
glucose medium containing 1 mM instead of 10 mM
glucose was used for labeling cells with radioactive sugars.
Deprivation of the cells for sulfate and glucose in this way had no
effect on GAG composition but raised the level of radiolabeling by
10-fold or more.
Mutant Screening--
Wild-type CHO cells were mutagenized with
ethylmethanesulfonate and frozen under liquid nitrogen (43). A portion
of cells was thawed, propagated for 3 days, and used to screen for
mutants. Approximately 1 × 105 mutagen-treated cells
were added to multiple 100-mm diameter tissue culture dishes filled
with complete growth medium containing 2 µg/ml FGF2-SAP, a
recombinant chimera consisting of FGF2 fused to the plant cytotoxin
saporin-6 (Selective Genetics, Inc., La Jolla, (44). After 1 day, the
cells were overlaid with a stack of polyester disks and glass beads in
order to generate colony replicas (43). The plates were incubated at
37 °C, and the FGF2-SAP-containing medium was changed every 4 days.
Under these conditions, 50-100 colonies arose on each plate after
~10 days. The disks were then removed from the plates and transferred
to a fresh plate containing sulfate-deficient medium supplemented with
10 µCi/ml 35SO4. After 4 h incubation at
37 °C, radiolabeled proteoglycans in each colony were precipitated
by incubating the disks in a few milliliters of 10% (w/v)
trichloroacetic acid. The disks were washed by replacing the solution
three times with 2% trichloroacetic acid and then water. After
staining the colonies with Coomassie Blue (43), the disks were dried
and exposed to x-ray film (Fuji). Tentative mutants were identified as
blue-stained colonies that failed to incorporate
35SO4. Candidates were picked from the original
master plates using cloning cylinders and trypsin (43). While the disks
were being screening, the master plates were stored at 33 °C filled
with complete F-12 growth medium supplemented with 2.5 µg/ml
Fungizone and 10 units/ml Nystatin (Life Technologies, Inc.).
Complementation Assay--
Complementation tests were carried
out by cell hybridization (43). Approximately 2 × 105
cells of each strain were added to individual wells of a 24-well plate
along with an equal number of pgsA-745 or pgsB-761 cells. After
overnight incubation, the mixed cell monolayers were treated for 1 min
with 50% (w/w) polyethylene glycol (Mr = 3350)
in F-12 medium. The cells were incubated for 1 day, replated in 100 mm-diameter tissue culture dishes to obtain ~300 colonies per dish,
and then overlaid with polyester cloth. The replica-plated colonies
were incubated with 35SO4 as described above.
Complementation was assessed by the appearance of colonies that
regained the capacity to incorporate 35SO4 into
acid-precipitable material, as judged by autoradiography.
Purification of Glycosaminoglycan Chains--
Cells were labeled
for 24 h with 10 µCi/ml Na35SO4 (25-40
Ci/mg, NEN Life Science Products) in growth medium containing serum or
with 10 µCi/ml D-[6-3H]glucosamine HCl (40 Ci/nmol, NEN Life Science Products) in low glucose medium. Radiolabeled
GAG chains were isolated as described (45) and analyzed by
anion-exchange high pressure liquid chromatography using a 7.5-mm
diameter × 7.5-cm column of DEAE-3SW (Tosohaas, Montgomeryville,
PA). The column was equilibrated in 10 mM
KH2P04 buffer (pH 6.0) containing 0.2% (w/v)
Zwittergent 3-12 and 0.2 M NaCl. GAGs were eluted with a
linear gradient of NaCl (0.2-1 M) in the same buffer using
a flow rate of 1 ml/min and by increasing the NaCl concentration by 10 mM/min. The effluent from the column was monitored for
radioactivity with an in-line radioactivity detector (Radiomatic Flo
one/beta, Packard Instruments) with sampling rates every 6 s and
data averaged over 1 min.
Isolation of Metabolically Labeled Xyloside-primed
Oligosaccharides--
CHO cells were grown to near confluence and then
incubated with low glucose F-12 medium supplemented with different
concentrations of naphthol- Separation and Characterization of Neutral and Charged
Products--
The dried samples were resuspended in 2 mM
Tris base and separated on a 0.5-ml column of QAE-Sephadex (Amersham
Pharmacia Biotech) equilibrated with 2 mM Tris base and
packed into a disposable 1-ml pipette tip. The flow-through and wash
fraction (10 ml of 2 mM Tris base) were collected as
neutral products (47), and the bound material (charged products) was
eluted with 0.4 M NaCl in 2 mM Tris base. The
fractions were desalted and concentrated on Sep-Pak C18 cartridges.
The oligosaccharides were characterized by treating samples with
various enzymes at 37 °C in a final volume of 25-100 µl. Neutral
species were incubated for 4 and 24 h with 5 milliunits of
Biotinylation of FGF2--
FGF2 (0.5 mg, Escherichia
coli recombinant material, Selective Genetics, Inc.) was protected
with heparin (0.4 mg) in 0.2 M HEPES buffer (pH 8.4) and
mixed with biotin hydrazide (Pierce Chemical, Long arm, water-soluble,
40 µg) in a final volume of 0.2 ml. After 2 h at room
temperature, 40 µl of 10 mg/ml glycine solution was added to stop the
reaction. The sample was diluted with 30 ml of 20 mM HEPES
buffer (pH 7.4) containing 0.5 M NaCl and 0.2% bovine
serum albumin and loaded onto a 1-ml column of heparin-Sepharose CL-6B
(Amersham Pharmacia Biotech). The column was washed with 30 ml of
buffer and eluted with 2.5 ml of solution adjusted to 3 M
NaCl. The sample was desalted on a PD-10 column (Amersham Pharmacia
Biotech) equilibrated with 20 mM HEPES buffer (pH 7.4)
containing 0.2% bovine serum albumin. Inclusion of
125I-FGF2 (~4 × 105 cpm, Ref. 48)
showed that the overall recovery of material was 30-40%.
Stable Transfection of Glucuronosyltransferase I--
A cDNA
for glucuronosyltransferase I (GlcAT-I) was cloned from CHO cells and
inserted into pCDNA3 (49). Mutant cells were transfected using
Lipofectin (Life Technologies, Inc.) according to the manufacturer's
instructions, and stable transfectants were selected with geneticin
(G418). Drug-resistant colonies were isolated and further characterized
by measuring the binding of biotinylated FGF2 in the following way.
Cells were detached with 5 mM EDTA in PBS, washed twice
with 10 ml of PBS, and resuspended in 0.2 ml of F-12 medium containing
~0.5 µg/ml biotin-FGF2 and 1% bovine serum albumin. The cells were
incubated for 1 h at 4 °C with constant shaking, washed twice
with 0.5 ml of cold 20 mM NaH2PO4
(pH 7.4) buffer containing 150 mM NaCl, and resuspended in
0.2 ml of buffer containing 5 µg/ml fluorescein/avidin/DCS (Vector
Laboratories). After shaking the cells in the dark at 4 °C for 20 min, they were washed and resuspended in cold phosphate buffer. Cell
sorting was done on a FACS Star Sorter (Becton Dickinson) and strongly positive clones were picked for further analysis.
Northern Analysis--
mRNA was isolated from mutant and
wild-type cells using QuickPrep Micro mRNA Purification Kit
(Amersham Pharmacia Biotech). The mRNA was denatured at 65 °C in
a solution of 50% (v/v) formamide, 6% (v/v) formaldehyde, and 20 mM MOPS (pH 7.0) and separated on a 1.2% (w/v) agarose gel
containing 6% (v/v) formaldehyde. The gels were blotted for 18 h
onto a Nytran plus nylon membrane (Schleicher & Schuell). The blotted
RNA was fixed by UV-catalyzed cross-linking and prehybridized for
2 h at 42 °C in a solution containing 50% formamide, 20 mM sodium phosphate (pH 6.8), 5× SSC, 1× Denhardt's solution, 1% SDS, 5% dextran sulfate, and 100 µg/ml denatured salmon sperm DNA. A double-stranded DNA probe was labeled with [32P]dCTP with random oligonucleotide primers (Prime-IT
II labeling kit, Stratagene) using the GlcAT-I cDNA (1.5 kilobase
pairs) as template (49), and the probe was purified on an Elute-tip
(Schleicher & Schuell). Hybridization was carried out overnight at
42 °C in the same buffer as the prehybridization but containing
~1 × 106 cpm/ml 32P-labeled probe. The
membrane was washed twice for 30 min at 42 °C with 2× SSC
containing 0.1% SDS and then twice for 30 min at 65 °C with 0.2×
SSC and 0.1% SDS. Bound probe was visualized on a PhosphorImager
(Storm 860, Molecular Dynamics) after overnight exposure.
Glucuronosyltransferase I Assay--
The activities of GlcAT-I
was measured in crude cell-free homogenates. Cells were grown to
confluence, rinsed three times with cold PBS, and detached with a
rubber policeman in 50 µl of solution containing 0.25 M
sucrose, 50 mM Tris-HCl (pH 7.4), 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride. Aliquots of the cell extracts were stored at Selection of Glycosaminoglycan-deficient Mutants--
We have
identified previously a number of CHO cell mutants altered in GAG
biosynthesis using a "brute force" replica plating technique (43).
In this procedure, cell colonies were grown on plastic tissue culture
dishes in the presence of an overlaying disk of polyester cloth.
Colonies forming on the plate also developed on the cloth, thus
providing two copies of each colony. The cloth "replica" was then
incubated in growth medium containing 35SO4,
and incorporation of 35SO4 into proteoglycans
was measured by autoradiography after acid precipitating radioactive
proteoglycans on the disk. Binding of 125I-FGF2 or
125I-antithrombin to colonies could be measured as well,
providing a way to obtain mutants in steps that affect sulfation of the chains (48, 50). Using these techniques, mutants altered in linkage
region assembly (designated pgsA and pgsB), sulfate transport (pgsC),
heparan sulfate polymerization (pgsD), and GlcN N-sulfation (pgsE), uronic acid 2-O-sulfation (pgsF), and GlcNAc
3-O-sulfation of heparan sulfate chains (38-40, 45, 48,
50-53). pgsA and pgsB mutants, defective in xylosyltransferase (XylT)
and galactosyltransferase I (GalT-I), respectively, fail to make both
chondroitin sulfate and heparan sulfate. Mutants in the second
galactosylation step (GalT-II) and glucuronosyltransferase I (GlcAT-I),
however, have not yet been identified, possibly due to their low
incidence in mutagen-treated populations of cells.
In order to identify these rarer mutants, a higher capacity screening
method was needed. Previous studies have shown that cells lacking fully
sulfated heparan sulfate did not bind FGF2 (54, 55). Thus, heparan
sulfate-deficient mutants should also be resistant to a recombinant
mitotoxin composed of FGF2 and saporin-6, a type 1 ribosome-inactivating protein from Saponaria officinalis seeds (44, 56). To test if this idea was correct, wild-type CHO cells
and GAG-deficient pgsA-745 cells were treated with different concentrations of the mitotoxin (FGF2-SAP). As shown in Fig.
1, wild-type CHO cells succumbed with an
ED50 of ~1 µg/ml for the mitotoxin, whereas the
GAG-deficient mutant pgsA-745 survived under these conditions.
After chemical mutagenesis ("Experimental Procedures"), the
incidence of FGF2-SAP-resistant mutants was ~10
To sort the various strains into different groups, we tested the
ability of the cells to prime oligosaccharides on
naphthol- The Oligosaccharides Assembled on
Analysis of the material by anion-exchange chromatography revealed
neutral and anionic oligosaccharides bearing one or two charges (see
"Experimental Procedures"). The neutral species from both wild-type
and mutant cells resolved into two peaks when analyzed by reverse
phase-high pressure liquid chromatography (Fig.
2, top two panels). Both peaks
were sensitive to overnight digestion with
Approximately 50% of the xyloside-primed oligosaccharides bore a
charge in pgsG mutants, somewhat less than found in wild-type cells
(~70%).3 The charged
oligosaccharides obtained from the mutant separated into two peaks of
material after reverse phase chromatography (Fig.
3B). This material was
completely sensitive to mild acid or sialidase (Fig. 3C),
but it was resistant to
Wild-type cells also generated charged oligosaccharides (Fig.
4B), but these were >95%
resistant to sialidase treatment (and mild acid hydrolysis) (Fig.
4C). The material was also resistant to Deficient GlcAT-I in pgsG Mutants--
An optimized assay for
GlcAT-I in CHO cell-free extracts was developed using synthetic
Gal
Recently, GlcAT-I was cloned from human placenta and CHO cells (49,
70). One of the mutants was stably transfected with the CHO cDNA
clone, and individual colonies were screened (see "Experimental
Procedures"). Assay for enzyme activity in the transfectants showed
that they contained normal levels of GlcAT-I (Table IV). As shown in
Fig. 5, the synthesis of heparan sulfate
and chondroitin sulfate was also restored and in this example to levels
that were somewhat higher than wild type. Northern blot analysis of
poly(A+) mRNA from mutant and wild-type cells showed
that the level of transcript was comparable, suggesting that both
transcription and message stability were comparable in mutant and
wild-type cells (Fig. 6). These findings
support the idea that the defect in GAG synthesis in pgsG cells was due
to the loss of GlcAT-I and that the mutant most likely has a defect in
the structural gene encoding the enzyme.
FGF2-SAP Selection Yields New GAG-deficient Mutants--
In
previous studies, we identified GAG-deficient mutants of CHO cells
using a replica plating technique in which individual colonies were
screened for failure to incorporate sulfate into GAG chains or by
altered binding of 125I-FGF2 (38-40, 45, 48, 51-53).
Although this technique has yielded interesting mutants, replica
plating has a relatively low capacity (~105 colonies) due
to the physical requirements of colony transfer, assay, and
autoradiography. In this report, we have described a direct selection
protocol using a chimeric mitotoxin prepared from FGF2 and saporin-6, a
toxic plant lectin. Direct selection has the advantage that very large
populations of cells can be manipulated (~108), which
should make it possible to detect even rare mutations in GAG
biosynthesis. Other selection techniques have been described that
depend on resistance to viruses that bind to heparan sulfate during
infection (71-74). The advantage of the present method is that it
targets GAG synthesis selectively.
In theory, conjugates composed of any GAG-binding protein and a
suitable toxin can be used to identify interesting mutants in GAG
synthesis. By careful selection of the binding component, it should be
possible to obtain mutants in the fine structure of the chains in
addition to the more dramatic mutants described here. For example,
chimeras prepared with antithrombin might yield mutants altered in
3-O-sulfation of GlcNAc, uronic acid epimerization, and
other reactions required for the generation of the high affinity binding site (50, 75). Although we have not yet characterized all of
the mutants resistant to FGF2-SAP, preliminary studies indicate the
most prevalent class of mutations affects the processing of the heparan
sulfate chains (the so-called "partial" mutants). Some of the these
strains may bear defects in other previously unidentified genes
required for binding site assembly.
The dramatic deficiency in GAG synthesis in the 13 mutants reported
here indicates that they have defects in early steps common to heparan
sulfate and chondroitin sulfate assembly. Three of 13 mutants
identified lacked GlcAT-I. Presumably, the other strains have defects
in other early steps, such as XylT (76, 77), the decarboxylase that
converts UDP-GlcA to UDP-xylose (78, 79), or the UDP-xylose transporter
(80-83). Other studies have suggested that the linkage region of both
heparan sulfate, chondroitin sulfate, and dermatan sulfate may undergo
sulfation of the Gal residues (5, 7-13) or phosphorylation at C-2 of
the xylose (2-6). If these reactions are essential for GAG synthesis,
then in theory some mutants altered in GAG assembly could have defects in one of these steps as well.
GAG Synthesis Depends on the Linkage Region
Tetrasaccharide--
The analysis of pgsG strains supports our earlier
conclusion that only single isozymes of each transferase are involved
in linkage region formation. Defects in XylT, GalT-I, and GlcAT-I fail
to produce the two major GAG chain types in CHO cells, chondroitin sulfate and heparan sulfate. These findings indicate that the initial
steps in the synthesis of these GAGs are catalyzed by a shared set of
enzymes, at least in CHO cells. Since heparin and dermatan sulfate are
more highly modified forms of heparan sulfate and chondroitin sulfate,
respectively, we conclude that all of the major GAGs linked by way of
xylose (and containing uronic acids) probably use a common set of
enzymes to form the linkage region. Mutants in the second
galactosyltransferase (GalT-II) are needed to confirm this conclusion,
and efforts are underway to determine if such strains are present in
the new collection of mutants. Divergence of the pathways presumably
occurs at the addition of the first unique sugar,
Recent cloning experiments have yielded cDNA clones for human and
Chinese hamster GlcAT-I (49, 70). This gene has high homology with
GlcAT-P, an enzyme selectively expressed in brain and neurons that is
responsible for the formation of HNK-1 determinants (3OSO3GlcA Unusual Xyloside-primed Oligosaccharides--
The accumulation of
unusual oligosaccharides on
In addition to these intermediates, xylosides will stimulate the
formation of unusual oligosaccharides, such as
Sia PgsG Mutations May Help Define the Catalytic Domain--
Northern
blot analysis of pgsG cell mRNA indicates that all of the mutants
make normal amounts of message for GlcAT-I. Although transcriptional
rates were not measured, the data suggest that the mutants most likely
have a missense or nonsense mutation that does not alter transcription
or message stability. Sequencing cDNA clones prepared from pgsG
mRNA will help confirm this idea. Point mutations altering GlcAT-I
are quite desirable since they may help define the catalytic domain of
the protein. The three mutants reported here represent a good starting
point for structure/function studies of this interesting and essential
enzyme for GAG biosynthesis.
1,3Gal
1,3Gal
1,4Xyl
-O-, attached to
specific serine residues in the core protein. Studies of Chinese
hamster ovary cell mutants defective in the first or second enzymes of
the pathway (xylosyltransferase and galactosyltransferase I) show that
the assembly of the primer occurs by sequential transfer of single
monosaccharide residues from the corresponding high energy nucleotide
sugar donor to the non-reducing end of the growing chain. In order to
study the other reactions involved in linkage tetrasaccharide assembly,
we have devised a powerful selection method based on induced resistance
to a mitotoxin composed of basic fibroblast growth factor-saporin. One
class of mutants does not incorporate 35SO4 and
[6-3H]GlcN into glycosaminoglycan chains. Incubation of
these cells with naphthol-
-D-xyloside
(Xyl
-O-Np) resulted in accumulation of linkage region
intermediates containing 1 or 2 mol of galactose (Gal
1,
4Xyl
-O-Np and Gal
1, 3Gal
1,
4Xyl
-O-Np) and sialic acid (Sia
2,3Gal
1, 3Gal
1,
4Xyl
-O-Np) but not any GlcA-containing oligosaccharides.
Extracts of the mutants completely lacked UDP-glucuronic acid:Gal
1,3Gal-R glucuronosyltransferase (GlcAT-I) activity, as
measured by the transfer of GlcA from UDP-GlcA to
Gal
1,3Gal
-O-naphthalenemethanol (<0.2
versus 3.6 pmol/min/mg). The mutation most likely lies in the structural gene encoding GlcAT-I since transfection of the mutant
with a cDNA for GlcAT-I completely restored enzyme activity and
glycosaminoglycan synthesis. These findings suggest that a single GlcAT
effects the biosynthesis of common linkage region of both heparan
sulfate and chondroitin sulfate in Chinese hamster ovary cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,3Gal
1,3Gal
1,4Xyl
-O-Ser (1). In some
proteoglycans, the linkage tetrasaccharide undergoes phosphorylation at
C-2 of xylose (2-6) or sulfation at C-4 of the galactose residues (5,
7-13). The linkage tetrasaccharide serves as a primer for the
formation of heparan sulfate and chondroitin sulfate chains, which
initiate by the attachment of either GlcNAc or GalNAc, respectively
(14, 15). The chains then assemble by the alternating addition of GlcA
and GlcNAc (heparan sulfate) or GlcA and GalNAc (chondroitin sulfate).
During polymerization, the chains undergo a series of modification
reactions that include N-deacetylation and
N-sulfation of GlcNAc residues, epimerization of some GlcA
residues to iduronic acid, and addition of sulfate groups at various
positions (1). The arrangement of sulfated and non-sulfated residues
and the uronic acid epimers gives rise to specific binding sites for
various protein ligands (reviewed in Refs. 16-19).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-xyloside (46) and 20 µCi/ml D-[6-3H]galactose (34.6 Ci/mmol)
(NEN Life Science Products). After 4 h incubation, the medium was
collected, centrifuged to remove floating cells, adjusted to 0.5 M NaCl, and applied to Sep-Pak Vac
C18-cartridges (Waters, 100 mg). The cartridges were washed with 10-25 ml of deionized water, and bound material was eluted with
50% methanol and dried. The cell layers were rinsed three times with
PBS and lysed in 0.1 N NaOH. An aliquot was taken for protein assay using a kit (Bio-Rad) with bovine serum albumin as standard.
-galactosidase (bovine testes, Oxford Glycosystems) in 0.1 M citrate/phosphate buffer (pH 4.0). Charged materials were
treated with (i) 10 milliunits of sialidase (Arthrobacter
ureafaciens, Oxford Glycosystems) for 16 h in 100 mM sodium acetate (pH 5.0); some samples were desialylated
by boiling in 10 mM HCl for 30 min; (ii) 100 units of
-glucuronidase (bovine liver, Sigma) overnight in 100 mM
sodium acetate (pH 5.0) buffer containing 0.3 M NaCl and 8 mM D-galactonic acid
-lactone to inhibit
contaminating
-galactosidase activity; and (iii) 1 milliunit of
-N-acetylgalactosaminidase (chicken liver, Oxford
Glycosystems) overnight in 0.1 M citrate/phosphate buffer
(pH 4.0). In some samples, the incubation was adjusted to pH 5.0 and
incubated with
-glucuronidase as described above. After enzymatic
treatment, samples were boiled for 10 min, dissolved in 200 µl of
water, and injected into a reverse phase C18 column (Tosohaas RP18, 4.6 mm × 25 cm) connected to Rainin HPXL solvent delivery system. The
column was first washed with water (flow rate 0.5 ml/min) and
then with increasing concentrations of acetonitrile in water.
Radioactivity in the eluate was monitored by Radiomatic Flo-one
Beta detector connected in line to the column. Some samples were
analyzed by QAE-Sephadex anion-exchange chromatography
20 °C. The
donor, UDP[1-3H]glucuronic acid, and the substrate,
Gal
1,3Gal
-O-naphthalenemethanol, were synthesized as
described (49).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
FGF2-saporin inhibition of cell growth.
Mutant pgsA-745 and wild-type CHO cells were plated in a 96-well dish
at 1 × 103 cells/well. Different concentrations of
FGF2-saporin were added. After 10 days incubation, the cells were fixed
with 10% trichloroacetic acid and stained with Coomassie Blue.
4, which
therefore makes possible direct selection of large numbers of mutants.
Approximately 105 mutagen-treated cells were added to
individual tissue culture plates, treated with 2 µg/ml FGF2-SAP, and
overlaid with polyester cloth. The resulting colonies that arose on the
replica disks were screened by 35SO4
incorporation into proteoglycans using the colony autoradiography procedure described above (see "Experimental Procedures") (43). Thirteen colonies showed dramatic loss of 35SO4
incorporation (data not shown) and had stable phenotypes after repurification by limiting dilution. Many more colonies exhibited partial reduction in 35SO4 incorporation
presumably from defects in specific sulfotransferases or mutations that
did not completely inactivate a particular enzyme. Only the mutants
that showed dramatic reduction in 35SO4
incorporation were characterized further.
-D-xyloside added to the culture medium.
Xylosides are known to stimulate GAG synthesis by substituting for the
natural xylosylated core protein intermediates (57). Thus, providing
xyloside to XylT-deficient pgsA-745 CHO cells stimulates GAG synthesis
by bypassing the GAG deficiency in this strain (38). In contrast,
providing xyloside to pgsB-761 cells does not stimulate GAG synthesis
since these cells lack GalT-I that adds the next sugar in the linkage
region (39). Thus, xylosides can be used to map if a defect in a mutant lies upstream or downstream of XylT. Of the 13 mutants identified above, 10 were responsive to xyloside treatment and three were not
(Table I). The three unresponsive strains
were then fused pair-wise to mutants pgsA-745 and pgsB-761 to test for
genetic complementation. All three strains complemented both pgsA-745 and pgsB-761, indicating that the defects were downstream from GalT-I.
When fused to each other, the mutants failed to complement, suggesting
that they belonged to the same complementation group, which was
formally designated pgsG. When metabolically labeled with both
35SO4 and [6-3H]glucosamine, all
three strains failed to incorporate the precursors into GAG chains to
the same extent as the pgsA mutant (Table I). Thus, the new mutants did
not make either chondroitin sulfate or heparan sulfate, consistent with
the idea that they have defects in a shared step early in the
pathway.
GAG content of mutant and wild-type cells
-D-xyloside
(Xyl
-O-Np), and the GAG was isolated from the cells and
the growth medium (see "Experimental Procedures"). The amount of
radioactive material was normalized to the amount of cell protein
analyzed.
-D-Xylosides
Suggest a Defect in GlcA Addition--
In addition to priming mature
GAG chains, many cell types accumulate biosynthetic intermediates on
xylosides that resemble those made during chain assembly (58-64).
Thus, knowing the composition of these intermediates could help
pinpoint the enzymatic defect in the mutants. When incubated with 3 µM naphthol-
-D-xyloside and
[6-3H]galactose, mutant and wild-type cells secreted
labeled oligosaccharides covalently linked to the primer. The amount of
secreted material in all three pgsG mutants was approximately the same
as in wild-type cells (Table II). As
expected, the formation of the oligosaccharides was not dependent on
XylT, since comparable amounts of material were recovered in pgsA-745
cells. However, it was entirely dependent on GalT-I, since pgsB cells,
which lack the enzyme, did not accumulate any primed
oligosaccharides.
Xyloside-stimulated oligosaccharide formation in mutant and
wild-type cells
-D-xyloside
and labeled with [6-3H]galactose for 4 h. The
conditioned medium was passed through Sep-Pak C18 cartridges, and the
oligosaccharides generated on the primer were eluted and separated into
neutral and charged species by anion-exchange chromatography (see
"Experimental Procedures").
-galactosidase, with the
counts shifting to the position of unbound [3H]galactose
(Fig. 2, lower panel). However, the individual peaks differed significantly in their sensitivity to
-galactosidase; peak
II was completely digested after only 4 h, whereas peak I was only
partially hydrolyzed in this time period. Some of the counts shifted to
the same position as peak II, suggesting that peak I consisted of a
galactosylated form of material in peak II. Since peak II comigrated
exactly in the same position as authentic Gal
1,4Xyl
-O-naphthol and was sensitive to
-galactosidase, the findings suggested that peak I was most likely
Gal
1,3Gal
1,4Xyl
-O-naphthol. The actual linkages
were not directly determined but inferred from the established
structure of the linkage region tetrasaccharide (1). The proposed
structure is also consistent with the relative resistance of Gal-linked
1,3 versus
1,4 (65). The presence of galactosylated
oligosaccharides in the mutant indicated that the second
galactosyltransferase (GalT II) was functioning normally in pgsG cells
(Table III).
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Fig. 2.
Analysis of neutral oligosaccharides primed
on naphthol- -D-xyloside.
Cells were fed 3 µM naphthol-
-D-xyloside
and [6-3H]galactose (see "Experimental Procedures").
Radioactive oligosaccharides generated on the primer were concentrated
from the growth medium and analyzed by reverse phase-high pressure
liquid chromatography. Peak II comigrated with authentic
Gal
1,4Xyl
-O-naphthol. The individual peaks were
collected and digested with
-galactosidase for 4 h and
rechromatographed. In the lower panel, the material from
both peaks was combined and treated with
-galactosidase
overnight.
Oligosaccharides primed on naphthol--D-xyloside
-glucuronidase,
-N-acetylgalactosaminidase, or a combination of both
enzymes (Fig. 3, D, E and F). Sialidase shifted
the counts to material eluting in the position of the neutral
trisaccharide (compare Fig. 3, A and C),
suggesting the structure,
Sia
2,3Gal
1,3Gal
1,4Xyl
-O-naphthol. The faster
eluting, relatively minor peak in Fig. 3B also was sensitive
to sialidase treatment, suggesting that this material may represent
desialylated oligosaccharide.
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Fig. 3.
Analysis of charged oligosaccharides primed
on naphthol- -D-xyloside by mutant
cells. A, the distribution of neutral oligosaccharides
for comparison; B, the elution position of charged
oligosaccharides; C, after treatment with A. ureafasciens sialidase; D, after treatment with
-glucuronidase; E, after treatment with
-N-acetylgalactosaminidase; and F, after
treatment with both
-N-acetylgalactosaminidase and
-glucuronidase. All of the charged material was sensitive to
sialidase and resistant to the other enzymes.
-glucuronidase
(Fig. 4D) and
-N-acetylgalactosaminidase (Fig. 4E). However, it was sensitive to the combination of these
two enzymes (Fig. 4F), resulting in the counts shifting to
the position of the neutral trisaccharide (compare with Fig.
4A). These findings suggested that the majority of charged
intermediates in the wild-type most likely had the structure,
GalNAc
1,3GlcA
1,3Gal
1,3Gal
1,4Xyl
-O-naphthol, which was described previously (62, 66-69). In contrast the mutant lacked this oligosaccharide. The proportion of neutral and charged oligosaccharides in mutant and wild-type cells is summarized in Table
III. Taken together, the data suggested that the mutant probably lacked
GlcAT-I.
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Fig. 4.
Analysis of charged oligosaccharides primed
on naphthol- -D-xyloside by
wild-type cells. A, the distribution of neutral
oligosaccharides for comparison; B, the elution position of
charged oligosaccharides; C, after treatment with A. ureafasciens sialidase; D, after treatment with
-glucuronidase; E, after treatment with
-N-acetylgalactosaminidase; and F, after
treatment with both
-N-acetylgalactosaminidase and
-glucuronidase. Only a small amount of material was sensitive to
sialidase. The majority of material was cleaved by a combination of
-N-acetylgalactosaminidase and
-glucuronidase.
1,3Gal
-O-naphthalenemethanol as an acceptor (see
"Experimental Procedures"). Enzyme activity was undetectable in the
mutants compared with the wild-type (<0.2 pmol/min/mg in the mutant
versus 3.7 pmol/min/mg in wild-type cells) (Table
IV). When equal amounts of wild-type and
mutant cell extract were assayed together, the enzyme-specific activity was 1.7 pmol/min/mg, exactly the amount predicted by simple mixing. This finding indicated that the mutant did not produce a soluble inhibitor that acted on wild-type GlcAT-I in the extracts. Likewise, the wild type did not seem to produce a soluble activator of the enzyme
that might have been missing in the mutant.
GlcAT-I activity in mutant and wild-type CHO cells
1,
3Gal-naphthalenemethanol as acceptor (see "Experimental
Procedures"). Each value represents the average at least two
independent determinations plus and minus the standard error from the
mean.
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Fig. 5.
Anion-exchange chromatography of GAG from
cells. Mutant pgsG-110, wild-type, and mutant cells stably
transfected with GlcAT-I cDNA were labeled with
35SO4. The 35S-GAG chains were
isolated from proteoglycans in the medium and the cells and analyzed by
anion-exchange high pressure liquid chromatography (see "Experimental
Procedures"). , mutant pgsG-110;
, GlcAT-I transfected
pgsG-110;
, wild-type cells.
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Fig. 6.
Northern blot analysis of the GlcAT-I in pgsG
mutants and wild-type CHO cells. ~5 µg of poly(A+)
RNA from each strain was electrophoresed and blotted to nitrocellulose
("Experimental Procedures"). A, hybridization results
using 32P-labeled full-length GlcAT-I cDNA as probe;
B, hybridization with the -glyceraldehyde-3-phosphate
dehydrogenase (
-GADPH) probe. Kb, kilobase
pair.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-GlcNAc in the
case of heparan sulfate and heparin and
-GalNAc in the case of
chondroitin and dermatan sulfate (14, 15, 84).
1,3Gal-) (85). Substrate specificity studies
showed that GlcAT-I is highly selective for substrates that resemble the linkage region tetrasaccharide, namely Gal
1,3Gal-terminated oligosaccharides (49). In contrast, GlcAT-P tends to be more promiscuous in vitro, using these substrates just as well as
(or better than) glycoproteins bearing N-linked or
O-linked (mucin-type) chains with terminal
-linked Gal
residues (49). Transfection experiments showed that GlcAT-P will in
fact substitute for GlcAT-I by enabling pgsG-110 cells to form GAG
chains (49). Thus, in brain and other tissues that express GlcAT-P, two
isozymes may participate in forming the linkage region of
proteoglycans. Determining the relative contribution of each of these
enzymes in neural tissues will require additional experiments,
including mutation of the genes in neuronal cell lines or in mice by
tissue-specific gene ablation.
-D-xylosides deserves
comment. Previous studies from a number of laboratories have shown that
xylosides will induce the formation of GAG chains and various
intermediates that lie along the pathway (58-64). PgsG mutants
accumulate normal intermediates, such as Gal
1,4Xyl
-O-R and Gal
1,3Gal
1,4Xyl
-O-R (Fig. 3). Interesting,
linkage tetrasaccharides (GlcA
1,3Gal
1,3Gal
1,
4Xyl
-O-R) were not observed in this study (in wild-type
cells) but have been found at low concentrations in other cell types
fed other types of xylosides (58, 62, 86). These findings suggest that
once GlcA is added to the nascent chain, the intermediate is
efficiently consumed by downstream enzymes in the pathway and that in
CHO cells GlcAT-I may be rate-limiting. As shown in Fig. 5,
transfection of the mutant with GlcAT-I cDNA augmented GAG
synthesis to levels about 2-fold higher than seen in wild-type cells,
consistent with this idea.
2,3Gal
1,4Xyl
-O-R, Xyl
1,3Xyl
-O-R, GlcA
1,3Xyl
-O-R,
3OSO3GlcA
1,3Xyl
-O-R, and GalNAc
1,3GlcA
1,3Gal
1,3Gal
1,4Xyl
-O-R
(60-64, 66). In this report, we found that CHO cells also make
Sia
2,3Gal
1,3Gal
1,4Xyl
-O-R and possibly a
disialylated species of unknown structure (86). To date, none of these
oligosaccharides have been found on endogenous proteoglycan,
glycoprotein, or glycolipids. Presumably, their accumulation reflects
the alternate use of substrates by resident transferases under
conditions where the substrate concentration is driven to very high
levels with exogenously added xyloside. The formation of these
compounds depends to a certain extent on the concentration of xyloside
provided to the cells, aglycone, and the type of cell (62, 64, 86).
However, at the lowest concentration tested (3 µM), the
unusual
-GalNAc-capped linkage tetrasaccharide actually predominates
over GAG chains and other precursors. This finding suggests that the
-GalNAc transferase (68, 69) must be quite abundant in CHO cells,
but somehow it is normally sequestered from endogenous proteoglycan
intermediates since this unusual modification has not yet been found on
a native proteoglycan.
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ACKNOWLEDGEMENTS |
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We thank Mark D'Andrea and Barbara Sosnowski of Selective Genetics, Inc., for their preparation and generous provision of FGF2-saporin and Hud Freeze for many helpful discussions.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
On leave from the Dept. of Biochemistry and Molecular Genetics,
University of Alabama, Birmingham, AL 35294.
§ To whom correspondence should be addressed: Division of Cellular and Molecular Medicine, Dept. of Medicine, The Glycobiology Program, University of California, San Diego, 9500 Gilman Dr., CMM East 1055, La Jolla, CA 92093-0687. Tel.: 619-822-1100; Fax: 619-534-5611; E-mail: jesko{at}ucsd.edu.
2 K. Kimata, personal communication.
3
The relative amount of charged material was
dependent on the concentration of xyloside in the growth medium;
~50% of the oligosaccharides in wild-type cells and ~20% in the
mutant were charged when the xyloside concentration was increased to
10 µM.
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ABBREVIATIONS |
---|
The abbreviations used are:
GAG, glycosaminoglycan;
CHO, Chinese hamster ovary;
PBS, phosphate-buffered
saline;
FGF2-SAP, basic fibroblast growth factor-saporin-6 chimera;
GlcAT-I, UDP-glucuronic acid:Gal1,3Gal-R glucuronosyltransferase;
GlcAT-P, UDP-glucuronic acid:glycoprotein glucuronosyltransferase;
MOPS, 3-[N-morpholino]propanesulfonic acid;
XylT, xylosyltransferase;
GalT-I, galactosyltransferase I.
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REFERENCES |
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