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INTRODUCTION |
Chondroitin sulfate proteoglycan is found in various tissues and
thought to play important roles in various cellular interactions involving cell adhesion (1, 2), regulation of neurite outgrowth (3-6),
migration of neural crest cells (7), binding of phospholipase A2 (8), and an adherence receptor for Plasmodium
falciparum-infected erythrocytes (9). In most of mammalian and
avian chondroitin sulfate proteoglycans, the glycosaminoglycan chains
bear sulfate at position 4 or 6 of N-acetylgalactosamine
residues. The ratio of chondroitin 4-sulfate/chondroitin 6-sulfate has
been reported to vary with the development of animals (10-12),
malignant change (13), and leukocyte differentiation (14). Sulfation of
positions 4 and 6 of GalNAc residue was shown to be catalyzed by
different sulfotransferases (15); chondroitin 4-sulfotransferase
(C4ST)1 and chondroitin
6-sulfotransferase (C6ST). Characterization of the purified
sulfotransferases and molecular cloning of these cDNAs are
basically important to reveal the functional roles of these chondroitin
sulfate isomers. We have purified C6ST from the culture medium of chick
chondrocytes (16) and cloned the cDNA (17). Unexpectedly, the
purified C6ST was found to catalyze not only chondroitin but also
keratan sulfate (18) and sialyl N-acetyllactosamine
oligosaccharides (19). We previously observed that C4ST was also
secreted to the culture medium of chick chondrocytes (20), but the
purification of C4ST from the culture medium of chick chondrocytes has
been hampered by the presence of an excess amount of C6ST. Unlike chick
chondrocytes, rat chondrosarcoma cells were reported to synthesize
chondroitin 4-sulfate as a major component (21-23). The Swarm rat
chondrosarcoma cell line RCS-LTC was shown to display a stable,
differentiated, chondrocyte-like phenotype (24). We investigated
whether C4ST was secreted from the chondrosarcoma cells, and found that
C4ST, but not C6ST, was actively secreted from these cells. We report
here the purification and characterization of C4ST from the serum-free
culture medium of the chondrosarcoma cells.
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EXPERIMENTAL PROCEDURES |
Materials
The following commercial materials were used:
H235SO4 was from DuPont NEN;
chondroitinase ACII, chondroitinase ABC, chondroitin sulfate A (whale
cartilage), chondroitin sulfate C (shark cartilage), dermatan sulfate
(pig skin), heparan sulfate (bovine kidney), completely desulfated
N-resulfated heparin (CDSNS-heparin),
Di-0S,
Di-6S,
Di-4S,
Di-diSD,
Di-diSB, and
Di-diSE were from Seikagaku Corp. (Tokyo, Japan);
Partisil SAX-10 was from Whatman; Dulbecco's modified Eagle's medium
and fetal bovine serum were from Life Technologies, Inc. (Life Tech
Oriental, Tokyo, Japan); Cosmedium-001 (a commercial serum-free culture
medium developed for the culture of hybridoma, which contains human
transferrin and bovine insulin as growth factors) was from Cosmo-Bio
(Tokyo, Japan); Hanks' solution was from Nissui Pharmaceutical Co.
Ltd. (Tokyo, Japan); trypsin (from bovine pancreas, type III),
penicillin G, streptomycin sulfate, 3',5'-ADP, unlabeled PAPS,
3',5'-ADP-agarose, and molecular weight standards for SDS-PAGE and gel
chromatography were from Sigma-Aldrich Japan (Tokyo, Japan);
recombinant N-glycanase was from Genzyme (Cambridge, MA);
Hiload Superdex 30 HR 16/60, fast desalting column HR 10/10, and
heparin-Sepharose CL-6B were from Amersham Pharmacia Biotech; Toyopearl
HW-55 Superfine was from Tosoh (Tokyo, Japan); Matrex gel red A
(cross-linked 6% agarose with covalently coupled dye) was from Amicon
Inc. (Beverly, MA).
[35S]PAPS was prepared as described (25). Chondroitin
(squid skin) was prepared previously described (26). Keratan sulfate (bovine cornea) was generously gifted from Seikagaku. Partially desulfated dermatan sulfate was prepared from pig skin dermatan sulfate
according to the method of Nagasawa et al. (27). Solvolysis with dimethyl sulfoxide was carried out at 100 °C for 60 min. The
degree of the desulfation was calculated as 83% from the proportion of
Di-0S to the total unsaturated disaccharides formed after chondroitinase ABC digestion. When the desulfated dermatan sulfate was
digested with chondroitinase ACII, the yield of
Di-0S was only 5%
of the total unsaturated disaccharides formed after chondroitinase ABC
digestion (see Fig. 10, B and C). Chondroitin
sulfate E (squid cartilage), which was eluted with 1.5 M
NaCl from DEAE-Sephadex A-50, was prepared as described (28).
Culture of Chondrosarcoma Cells and Preparation of the Medium
Fraction
Rat chondrosarcoma cells (frozen cell line LTC) (23) were plated
in 10-cm culture dishes (Falcon) at a density of 2 × 106 cells/dish. Volume of the medium was 10 ml. The medium
in which the cells were plated consisted of Dulbecco's modified
Eagle's medium adjusted to pH 7.4 containing penicillin (100 units/ml), streptomycin (50 µg/ml), 5% fetal bovine serum, and 5 µg/ml insulin, and cells were grown at 37 °C in 5%
CO2, 95% air. The medium was changed every other day. When
the cells grew to confluence, they were treated with Hanks' solution
containing 0.1% trypsin and 0.1% collagenase, and replated to the 200 10-cm dishes at a density of 2.0 × 105 cells/dish in
the same medium. On day 6, the cells were grown to 5.2 × 106 cells/dish, and thereafter, the culture medium was
replaced with Cosmedium-001. The culture of chondrosarcoma cells in
Cosmedium-001 was continued for 4 days, and the spent culture medium
was recovered on days 2 and 4. For determining the sulfotransferase
activity contained in the cell layer, the cell layer was scraped in
0.01 M Tris-HCl, pH 7.2, containing 0.25 M
sucrose and 0.5% Triton X-100, homogenized with a Dounce homogenizer,
and centrifuged for 20 min at 10,000 × g. The
resultant supernatant fraction was used as the cell layer extract. The
culture of chondrosarcoma cells using 200 10-cm dishes described above
was carried out three times. The spent medium was pooled and
centrifuged at 10,000 × g for 10 min. To the
supernatant solution the following materials were added to final
concentrations indicated: 10 mM Tris-HCl, pH 7.2, 0.1%
Triton X-100, 20 mM MgCl2, 10 mM
2-mercaptoethanol, 20% glycerol, and a mixture of protease inhibitors
(5 µM
N
-p-tosyl-L-lysine
chloromethyl ketone, 3 µM
N-tosyl-L-phenylalanine chloromethyl ketone, 30 µM phenylmethylsulfonyl fluoride, and 3 µM
pepstatin A). The pooled medium containing these additives was
designated the buffered medium fraction and stored at 4 °C until
purification was started.
Purification of Chondroitin 4-Sulfotransferase
All operations were performed at 4 °C.
Step 1: Chromatography on Heparin-Sepharose CL-6B--
The
buffered medium fraction prepared as above (4.7 liters obtained from
200 10-cm dishes) was applied to a column of heparin-Sepharose CL-6B
(2.2 × 28 cm) equilibrated with 0.15 M NaCl in buffer
A (10 mM Tris-HCl, pH 7.2, 20 mM
MgCl2, 2 mM CaCl2, 10 mM 2-mercaptoethanol, 0.1% Triton X-100, and 20%
glycerol). The column was washed with 1000 ml of 0.15 M
NaCl in buffer A. The absorbed materials were eluted with 1000 ml of
buffer A containing 0.4 M NaCl. 10-ml fractions were
collected. The heparin-Sepharose CL-6B chromatography was carried out
three times, and the 0.4 M NaCl fractions were combined.
Step 2: Chromatography on Matrex Gel Red A--
Half of the
combined fraction from Step 1 was applied to a Matrex gel red A column
(2.2 × 9.5 cm) equilibrated with 0.4 M NaCl in buffer
A. The column was eluted stepwise with 200 ml of buffer A containing
0.4 M NaCl, 200 ml of buffer A containing 0.75 M NaCl, and 200 ml of buffer A containing 1 M
guanidine hydrochloride. All C4ST activity was eluted in the 1 M guanidine fractions. The fractions containing C4ST
activity were pooled and dialyzed against 0.05 M NaCl in
buffer A. Chromatography on Matrex gel red A was repeated once more,
and dialyzed solution was combined. To the combined dialyzed solution
10% Triton X-100 was added to a final concentration of 2%.
Step 3: Chromatography on 3',5'-ADP-Agarose--
The dialyzed
fraction from Step 2 containing 2% Triton X-100 was applied to a
3',5'-ADP-agarose column (1.2 × 11 cm) equilibrated with modified
buffer A containing 0.05 M NaCl. The composition of
modified buffer A was the same as that of buffer A, except that the
concentration of Triton X-100 was increased to 2%. The column was
washed with 150 ml of the modified buffer A containing 0.05 M NaCl. The sulfotransferase activity was eluted with 150 ml of modified buffer A containing 0.1 mM 3',5'-ADP. The
fractions containing sulfotransferase activity were pooled and applied
to the second heparin-Sepharose CL-6B column.
Step 4: Second Heparin-Sepharose CL-6B Chromatography--
The
fraction containing the sulfotransferase activity from Step 3 was
applied to a column of heparin-Sepharose CL-6B (0.9 × 6.7 cm)
equilibrated with 0.05 M NaCl in buffer A. The column was
washed with 50 ml of 0.05 M NaCl in buffer A. The absorbed sulfotransferase was eluted with 25 ml of buffer A containing 0.4 M NaCl. The collected fraction was dialyzed against buffer A containing 0.05 M NaCl. The purified enzyme was stored at
20 °C.
Assay of Sulfotransferase Activity
C4ST and C6ST activity were assayed by the method described
previously (16). The standard reaction mixture contained 2.5 µmol of
imidazole-HCl, pH 6.8, 1.25 µg of protamine chloride, 0.1 µmol of
dithiothreitol, 25 nmol (as glucuronic acid) of chondroitin, 50 pmol of
[35S]PAPS (~5.0 × 105 cpm), and
enzyme in a final volume of 50 µl. For determining the activity for
various glycosaminoglycans, chondroitin was replaced with 25 nmol (as
galactosamine for chondroitin sulfate, dermatan sulfate, and desulfated
dermatan sulfate or as glucosamine for heparan sulfate, CDSNS-heparin,
and keratan sulfate) of glycosaminoglycans. The reaction mixtures were
incubated at 37 °C for 20 min, and the reaction was stopped by
immersing the reaction tubes in a boiling water bath for 1 min. After
the reaction was stopped, 35S-labeled glycosaminoglycans
were isolated by the precipitation with ethanol followed by gel
chromatography with a fast desalting column as described previously,
and radioactivity was determined. For determining C6ST and C4ST
activity, 35S-labeled chondroitin was digested with
chondroitinase ACII, and the radioactivity of unsaturated disaccharides
(
Di-4S and
Di-6S) separated with paper chromatography was
counted. The chondroitinase ACII-digested materials were also analyzed
with HPLC using a Whatman Partisil 10-SAX column as described below. 1 unit of enzyme activity is defined as the amount required to catalyze
the transfer of 1 pmol of sulfate/min.
SDS-Polyacrylamide Gel Electrophoresis
Polyacrylamide gel electrophoresis of proteins in SDS was
carried out on 10% polyacrylamide gels under reducing or nonreducing conditions as described (29). Protein bands were detected by silver
stain or Coomasie Brilliant Blue.
Gel Chromatography of the Sulfotransferase on Toyopearl HW-55
A Toyopearl HW-55 column (1.4 × 99 cm) was equilibrated
with a buffer containing 2 M NaCl, 10 mM
Tris-HCl, pH 7.2, 20 mM MgCl2, 2 mM
CaCl2, 0.1% Triton X-100, and 20% glycerol (buffer B).
0.8 ml of sample was applied to the column and eluted with buffer B. 1.2-ml fractions were collected.
Superdex 30 Chromatography, Paper Chromatography, and HPLC
A Superdex 30 16/60 column was equilibrated with 0.2 M NH4HCO3. The flow rate was 1 ml/min. 1-ml fractions were collected. On the analysis of the
desulfated dermatan sulfate and chondroitinase ACII-digested desulfated
dermatan sulfate, the eluates were monitored with absorption at 232 nm
and with orcinol color reaction using glucuronic acid as a standard
(30). Paper chromatography was performed on Toyo 51A paper using a
solvent system, 1-butanol/acetic acid/1 M NH3
(2:3:1, by volume). Regions containing unsaturated disaccharides on the
paper chromatogram were visualized under a UV lamp and excised, and the
radioactivity was determined. Separation of the degradation products
formed from 35S-labeled chondroitin and
35S-labeled desulfated dermatan sulfate after digestion
with chondroitinase ACII or chondroitinase ABC was carried out by HPLC
using a Whatman Partisil 10-SAX column (4.5 mm × 25 cm)
equilibrated with 35 mM KH2PO4. The
column was developed with gradient elution as shown in Fig. 7. The flow
rate was 1 ml/min, and the column temperature was 40 °C. 0.5-ml
fractions were collected.
Assay of Protein
Protein was determined by the method of Bradford using bovine
serum albumin as a standard (31). Protein assay reagent was obtained
from Bio-Rad. The protein concentration of the second heparin-Sepharose
CL-6B fraction was too low to be determined directly; therefore,
samples were concentrated as described previously (16). The protein
concentration of 3',5'-ADP-agarose fraction was not shown in Table I,
because the high concentration of Triton X-100 in the 3',5'-ADP-agarose
fraction hampered the accurate determination of protein.
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RESULTS |
Secretion of Chondroitin 4-Sulfotransferase into the Serum-free
Culture Medium--
Fig. 1 shows
sulfotransferase activity contained in the spent culture medium and
cell layer when chondrosarcoma cells were cultured in Cosmedium-001 for
10 days. The level of C4ST activity secreted to the serum-free medium
was decreased rapidly. On day 4 in the serum-free medium, the activity
of C4ST in the medium dropped to 48% of the initial activity. In
contrast, C4ST activity retained in the cell layer was nearly constant
during the whole culture period. The decrease in C4ST activity in the
medium during the culture in the serum-free medium seems not to be
caused by cell death or cell detachment from the dish, because the
cellular protein increased gradually during 10-day culture in the
serum-free medium. Unlike C6ST from the culture medium of chick
chondrocytes (20), secretion of C4ST was not stimulated by the addition
of ascorbate to the serum-free medium (data not shown). When
35S-labeled chondroitin formed after incubation with the
spent medium or the extracts from the cell layer was separated with
paper chromatography, small radioactivity was observed at the position
of
Di-6S, but this radioactivity was not attributable to
Di-6S,
because no radioactivity was detected at the retention time of
Di-6S
when the chondroitinase ACII digests were separated with HPLC (Fig. 2). The small peak observed at ~23 min
in HPLC has not been identified yet. C6ST activity thus could not be
detected both in the culture medium and in the cell layer.

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Fig. 1.
Secretion of chondroitin 4-sulfotransferase
from the cultured chondrosarcoma cells to the serum-free medium.
The culture of chondrosarcoma cells was carried out as described under
"Experimental Procedures," except that the cells were plated in
5-cm dishes at 1 × 105 cells/dish. On day 6 and
thereafter, culture medium was replaced every other day with fresh
Cosmedium-001. In this figure, day 6 from the start of the culture was
set as day 0. The spent Cosmedium-001 recovered every other day was
stored separately. The cell layer was scraped, homogenized with a
buffer containing 0.5% Triton X-100, and centrifuged as described
under "Experimental Procedures." C4ST activity in the spent medium
(closed circle) and in the cell layer (open
circle) and protein content of the cell layer (open
square) were determined.
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Fig. 2.
HPLC separation of chondroitinase ACII
digests of 35S-labeled chondroitin formed after the
incubation with the spent medium (A) or the extracts from
cell layer (B). The culture of chondrosarcoma cells
was carried out as in Fig. 1. On day 2 in the serum-free medium, the
spent medium was recovered, and the cell extracts were prepared as
described under "Experimental Procedures." 35S-Labeled
chondroitin formed after sulfotransferase reaction under the standard
conditions was digested with chondroitinase ACII and separated with
HPLC. Conditions of HPLC were as described under "Experimental
Procedures." The broken line depicts concentration of
KH2PO4. The arrows indicate the
elution position of: 1, Di-0S; 2, Di-6S;
3, Di-4S; 4, Di-diSD;
5, Di-diSB; and 6,
Di-diSE.
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Purification of Chondroitin 4-Sulfotransferase--
C4ST was
purified to apparent homogeneity ~1900-fold over the specific
activity of the pooled spent culture medium. Table I summarizes the purification of the
sulfotransferases from 14.2 liters of the buffered medium fraction.
As observed in most of glycosaminoglycan sulfotransferase, C4ST was
also absorbed to heparin-Sepharose CL-6B. A large volume of the
buffered medium fraction was efficiently concentrated with the
heparin-Sepharose CL-6B step. C4ST bound to Matrex gel red A was not
eluted with 0.75 M NaCl and eluted with 1 M
guanidine hydrochloride. At this step, C4ST was still stable in 1 M guanidine hydrochloride. Stability of C4ST in 1 M guanidine hydrochloride, however, decreased as the purity
of the enzyme increased. Fig. 3 shows the
elution pattern of sulfotransferase from 3',5'-ADP-agarose. Unlike C6ST
from the culture medium of chick chondrocytes, efficient absorption of
C4ST to 3',5'-ADP-agarose was achieved only in the presence of 2%
Triton X-100. Because the high concentration of Triton X-100 disturbed
further analysis of the purified C4ST, the concentration of the
detergent was decreased to 0.1% during the second heparin-Sepharose
CL-6B chromatography. 3',5'-ADP, which is a strong inhibitor of the
sulfotransferase, was also removed from the purified sample after the
second heparin-Sepharose CL-6B chromatography. The final fraction was
used for the later experiments as the purified C4ST fraction.

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Fig. 3.
Affinity chromatography on 3',5'-ADP-agarose
of the fraction eluted from Matrex gel red A. The fractions eluted
from the Matrex gel red A with 1 M guanidine hydrochloride
were pooled and dialyzed. After the concentration of Triton X-100 was
adjusted to 2%, the pooled fraction was applied to a 3',5'-ADP-agarose
column as described under "Experimental Procedures." After the
column was washed with modified buffer A containing 0.05 M
NaCl, C4ST was eluted stepwise with modified buffer A containing 0.1 mM 3',5'-ADP (arrow). C4ST activity
(closed circle), and protein concentration (open
circle) of each fraction were assayed.
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Purity of the Chondroitin 4-Sulfotransferase--
The different
purified fractions of C4ST were analyzed by SDS-PAGE under nonreducing
conditions and stained with silver nitrate stain (Fig.
4, lanes 1-4). For the
routine analysis of the purified C4ST with SDS-PAGE, we removed
2-mercaptoethanol from the sample buffer to avoid an artifact of silver
staining. A broad protein band of 50 kDa was predominantly stained in
the second heparin Sepharose CL-6B fraction, and a weaker protein band
of 54 kDa was also detected (Fig. 4, lane 4). When the
purified C4ST was treated with sample buffer containing
2-mercaptoethanol and stained with Coomasie Brilliant Blue, two protein
bands of 60 and 64 kDa were detected (Fig. 4, lane 5). The
protein bands of 50 and 54 kDa disappeared after N-glycanase
digestion, and protein bands of 33 kDa as a major band and 35 kDa as a
minor band appeared (Fig. 4, lane 7), indicating that the
purified protein contained N-linked oligosaccharides. A
protein band of 70 kDa also appeared after N-glycanase
digestion (Fig. 4, lane 7). This protein band seems to be a
dimer formed from the deglycosylated 35 kDa protein. To confirm that
the protein bands observed in SDS-PAGE corresponded to C4ST, the
purified C4ST was applied to a Toyopearl HW-55 column, and elution
profiles of the C4ST activity and protein were determined (Fig.
5). The protein bands of 50 and 54 kDa
appeared almost exclusively when the peak fractions (tubes 43 and 44 in
Fig. 5A) were subjected to SDS-PAGE (Fig. 5B).
The molecular mass of the peak fraction determined from the elution
position in the Toyopearl HW-55 chromatography was 50 kDa, which agreed
well with molecular mass determined by SDS-PAGE. These results suggest
that C4ST may behave as a monomer. A small part of C4ST activity was
also observed around tubes 31-38 in the Toyopearl HW-55 chromatography
(Fig. 5A), but only the protein bands of 50 and 54 kDa were
faintly detected in SDS-PAGE (data not shown). These observations
suggest that C4ST may tend to aggregate as observed previously in chick
C6ST (16).

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Fig. 4.
SDS-polyacrylamide gel electrophoresis of
C4ST. Lanes 1-4, different purified fractions of C4ST were
analyzed under nonreducing conditions: lane 1, buffered
medium fraction; lane 2, first heparin-Sepharose CL-6B
fraction; lane 3, Matrex gel red A fraction; and lane
4, second heparin-Sepharose CL-6B fraction. Lane 5, the
second heparin-Sepharose CL-6B fraction was analyzed under reducing
conditions. Lanes 6-8, the second heparin-Sepharose CL-6B
fraction was analyzed under nonreducing conditions before (lane
6) or after (lane 7) N-glycanase digestion.
Lane 8, control with N-glycanase alone. Proteins
were visualized with silver nitrate stain (lanes 1-4,
6, and 7) or Coomasie Brilliant Blue
(lane 5). Molecular size standards were the following:
myosin (205 kDa), -galactosidase (116 kDa), phosphorylase
b (97.4 kDa), bovine serum albumin (66 kDa), egg albumin (45 kDa), and carbonic anhydrase (29 kDa).
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Fig. 5.
Toyopearl HW-55 gel chromatography of
chondroitin 4-sulfotransferase. A, C4ST eluted from the
second heparin-Sepharose CL-6B column (1 µg as protein) was applied
to a Toyopearl HW-55 column and eluted with buffer B as described under
"Experimental Procedures." C4ST activity was assayed after each
fraction was dialyzed against buffer A containing 0.05 M
NaCl. The arrows indicate the elution position of
-amylase (200 kDa), bovine serum albumin (66 kDa), and carbonic
anhydrase (29 kDa). B, protein contained in each fraction
was precipitated with 10% trichloroacetic acid. The precipitates were
washed with acetone and analyzed by SDS-PAGE under nonreducing
conditions. Proteins were visualized with silver nitrate stain.
Molecular size standards are the same as used in Fig. 3.
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Properties of the Purified Chondroitin
4-Sulfotransferase--
Dithiothreitol was found to stimulate C4ST
activity (Fig. 6A). Other
sulfhydryl compounds such as 2-mercaptoethanol and reduced glutathione
also showed a stimulatory effect on C4ST activity (data not shown).
Optimum pH was ~7.2 (Fig. 6B). The Km for PAPS was 2.7 × 10
7 M (Fig.
6C). C4ST was activated not only with protamine but also with various metal ions such as Ca2+, Fe2+,
Mn2+, Ba2+, and Sr2+. When added at
5 mM, Ca2+ was most effective among these ions.
Mg2+ was less effective. Co2+, which was
activated C6ST (18), was inhibitory. The purified C4ST was less stable
than the purified C6ST from the culture medium of chick chondrocytes;
after 4 months at
20 °C, the activity of the purified C4ST was
decreased to ~20% of the original activity.

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Fig. 6.
Effects of dithiothreitol (A) and
pH (B) on C4ST activity and the determination of
Km for PAPS (C). A, the
sulfotransferase activity was determined as described under
"Experimental Procedures," except that the concentration of
dithiothreitol was varied. B, 0.05 M
imidazole-HCl contained in the standard reaction mixture was replaced
with 0.05 M buffers with various pH values: sodium acetate
(filled square), 4-morpholineethanesulfonic acid (open
square), imidazole-HCl (filled circle), and Tris-HCl
(open circle). C, the sulfotransferase activity
was determined as described under "Experimental Procedures," except
that the concentration of [35S]PAPS was varied. Values of
the ordinate of the double reciprocal plot represent
1/(pmol/min/µg of protein).
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Acceptor Substrate Specificity of Chondroitin
4-Sulfotransferase--
For determining acceptor specificity, the
purified C4ST was incubated with different glycosaminoglycans in the
presence of varying amounts of protamine chloride. Fig.
7 shows that the purified C4ST was able
to transfer sulfate to chondroitin and desulfated dermatan sulfate.
Slight activity was observed when chondroitin sulfate A and chondroitin
sulfate C were used as acceptors. Dermatan sulfate, keratan
sulfate, chondroitin sulfate E, heparan sulfate, and CDSNS-heparin did
not serve as acceptors. Optimum protamine concentration was varied with
the kind of glycosaminoglycans used as acceptors. The lowest optimum
concentration was observed for chondroitin (Fig. 7).

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Fig. 7.
Incorporation of [35S]sulfate
from [35S]PAPS into exogenous glycosaminoglycan acceptors
by the purified C4ST. Incorporation into the polysaccharide
fraction was determined as described under "Experimental
Procedures," except that chondroitin was replaced with various kinds
of glycosaminoglycans (25 nmol as galactosamine or 25 nmol as
glucosamine), and the concentration of protamine chloride was varied.
Chondroitin (filled circle), desulfated dermatan sulfate
(open circle), chondroitin sulfate A (filled
triangle), chondroitin sulfate C (open triangle), and
dermatan sulfate (filled square) were used as acceptors.
Incorporation into keratan sulfate, heparan sulfate, CDSNS-heparin, and
chondroitin sulfate E was below the incorporation into dermatan
sulfate.
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To determine the position of the sulfate group transferred to
chondroitin and desulfated dermatan sulfate, we digested
35S-labeled glycosaminoglycans with chondroitinase ACII or
chondroitinase ABC and analyzed the digestion products with SAX HPLC
(Fig. 8). When the
35S-labeled chondroitin and the 35S-labeled
desulfated dermatan sulfate were digested with chondroitinase ACII and
chondroitinase ABC, respectively, radioactivity was detected only at
the position of
Di-4S (Fig. 8, A and B). When
35S-labeled desulfated dermatan sulfate was digested with
chondroitinase ACII, ~50% of the total radioactivity was recovered
in
Di-4S, and the remainder appeared in minor peaks with higher
retention time (Fig. 8C). The digestion products derived
from the 35S-labeled desulfated dermatan sulfate were also
separated with Superdex 30 gel chromatography (Fig.
9). After digestion with chondroitinase
ABC, radioactivity was only detected in
Di-4S (Fig. 9B).
Approximately 50% of the total radioactivity was recovered in
Di-4S
after chondroitinase ACII as observed in the SAX HPLC. Oligosaccharides
observed in Fig. 9C appeared to correspond to the minor
peaks observed in Fig. 8C. One oligosaccharide with retention time of 76 min (Fig. 9C, peak 6) was
eluted between chondroitin sulfate hexasaccharide and chondroitin
hexasaccharide, and another oligosaccharide with retention time of 82 min (Fig. 9C, peak 7) was eluted between
chondroitin sulfate tetrasaccharide and chondroitin tetrasaccharide.
Comparison of the retention times of these oligosaccharides with those
of standard oligosaccharides allowed us to deduce that peaks 6 and 7 may be a monosulfated hexasaccharide and a monosulfated
tetrasaccharide, respectively. To clear the sensitivity to
chondroitinase ACII of the partially desulfated dermatan sulfate used
as acceptor, we digested it with chondroitinase ACII and separated with
Superdex 30 (Fig. 10). Desulfated dermatan sulfate was slightly depolymerized with chondroitinase ACII
(Fig. 10E), but the UV-absorbing materials were detected
only at the position of
Di-0S. The proportion of
Di-0S to the
total unsaturated disaccharide formed after chondroitinase ABC
digestion was only 5%. After digestion with chondroitinase ABC, no
UV-absorbing materials other than
Di-4S and
Di-0S were formed
from the partially desulfated dermatan sulfate (Fig. 10B),
suggesting that structural changes in the polymer, which would make the
polymer resistant to chondroitinase ABC, were not likely to be
introduced after chemical desulfation.

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Fig. 8.
HPLC separation of chondroitinase ACII or
chondroitinase ABC digests of 35S-labeled
glycosaminoglycans derived from chondroitin (A) and
desulfated dermatan sulfate (B and C) by
incubation with [35S]PAPS and the purified C4ST.
35S-Labeled glycosaminoglycans were digested with
chondroitinase ACII (A and C) or chondroitinase
ABC (B). The sulfotransferase reaction was carried out as
described under "Experimental Procedures," except that the amount
of protamine chloride was increased to 10 µg when desulfated dermatan
sulfate was used as acceptor. Conditions of HPLC were as described
under "Experimental Procedures." The broken line depicts
concentration of KH2PO4. The arrows
indicate the elution position of: 1, Di-0S; 2,
Di-6S; 3, Di-4S; 4, Di-diSD;
5, Di-diSB; and 6,
Di-diSE.
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Fig. 9.
Superdex 30 chromatography of chondroitinase
ACII or chondroitinase ABC digests of 35S-labeled products
derived from the desulfated dermatan sulfate by incubation with
[35S]PAPS and the purified C4ST. The
sulfotransferase reaction was carried out as described under
"Experimental Procedures," except that 25 nmol (as galactosamine)
of desulfated dermatan sulfate instead of chondroitin was added and
that the amount of protamine chloride was increased to 10 µg. The
35S-labeled, desulfated dermatan sulfate was applied to the
Superdex 30 column before (A) or after digestion with
chondroitinase ABC plus chondroitinase ACII (B) or
chondroitinase ACII alone (C). Arrows indicate
the elution position of: Vo, blue dextran; 1,
chondroitin sulfate A hexasaccharide; 2, chondroitin
hexasaccharide; 3, chondroitin sulfate A tetrasaccharide;
4, chondroitin tetrasaccharide; and 5, Di-4S.
Peaks 6 and 7 shown in C were deduced
to be monosulfated hexasaccharide and monosulfated tetrasaccharide,
respectively.
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Fig. 10.
Superdex 30 chromatography of chondroitinase
ACII or chondroitinase ABC digests of the partially desulfated dermatan
sulfate. The partially desulfated dermatan sulfate (1 µmol as
GalNAc) was digested in the reaction mixture containing 0.2 unit of
chondroitinase ABC or 0.2 unit of chondroitinase ACII in a final volume
of 100 µl for 2 h and applied to a column of Superdex 30. The
eluate was monitored with absorption at 232 nm (A-C) and
with orcinol color reaction for uronic acid (D and
E). A and D, undigested control;
B, after digestion with chondroitinase ABC; C and
E, after digestion with chondroitinase ACII.
Arrows indicate the elution position of: 1, blue
dextran; 2, Di-4S; and 3, Di-0S.
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DISCUSSION |
In the present study, we have purified chondroitin
4-sulfotransferase from the culture medium of rat chondrosarcoma cells. The purified preparation showed broad protein bands of 50 kDa as a
major component and 54 kDa as a minor component in SDS-PAGE under
nonreducing conditions. Under reducing conditions, these protein bands
shifted to the slower moving bands of 60 and 64 kDa, suggesting that
these proteins may contain intramolecular disulfide bonds. Both protein
bands disappear after N-glycanase digestion, and protein
bands of 33 and 35 kDa appeared, indicating that these proteins contain
N-linked oligosaccharides. From the decrease in the
molecular mass observed after N-glycanase digestion, the
contents of N-linked oligosaccharides could be estimated as ~35%. Although the relation of the two protein bands is not clear at
present, it may be possible that both protein bands correspond to C4ST,
because both protein bands coeluted with C4ST activity from Toyopearl
HW-55. We have previously found that the purified preparations of C6ST
(17), heparan sulfate 6-sulfotransferase (32), and heparan sulfate
2-sulfotransferase (33) were composed of two protein bands, and that
amino-terminal amino acid sequences of the two protein bands were
identical in the case of C6ST (17) and heparan sulfate
6-sulfotransferase (34). The GalNAc-4-sulfotransferase responsible for
the sulfation of GalNAc
1-4GlcNAc-bearing oligosaccharides was
purified from submaxillary gland, but this enzyme seems to be quite
distinct from C4ST, because the molecular mass of the GalNAc-4-sulfotransferase was 128 kDa (35).
Before starting the purification of C4ST from the culture medium of
chondrosarcoma cells, we tried to purify the enzyme from the culture
medium of chick embryo chondrocytes. After the 3',5'-ADP agarose
chromatography, however, the C4ST fraction from the culture medium of
chick chondrocytes was still contaminated by a large amount of C6ST,
and it was difficult to remove C6ST activity from the C4ST preparation.
In contrast, the culture medium of rat chondrosarcoma cells contained
C4ST exclusively and, therefore, was an excellent source for the
purification of C4ST. A major difference in the purification procedures
between C4ST and C6ST was in the composition of the buffer used for the
3',5'-ADP agarose chromatography; the concentration of Triton
X-100 was 2% for C4ST and 0.1% for C6ST. In the absence of the high
concentration of Triton X-100, efficient absorption of C4ST could not
be achieved.
Desulfated dermatan sulfate was efficiently sulfated by the purified
C4ST. After digestion with chondroitinase ABC, only
Di-4S was
detected as the degradation product, indicating that desulfated dermatan sulfate was also sulfated at position 4 of GalNAc residues. The 35S-labeled, desulfated dermatan sulfate was also
sensitive to the digestion with chondroitinase ACII. After digestion
with chondroitinase ACII,
Di-4S and higher oligosaccharides were
formed; ~50% of the total radioactivity was recovered in
Di-4S.
On the other hand, the yield of
Di-0S after chondroitinase ACII
digestion of the desulfated dermatan sulfate used as acceptor was only
5% of the total unsaturated disaccharides formed after chondroitinase ABC digestion (see "Materials"). Because formation of
Di-4S from dermatan sulfate by chondroitinase ACII digestion is possible only when
the GalNAc-4-sulfate residue is flanked with GlcA residues on both
reducing and nonreducing sides, the relatively high yield of
35S-labeled
Di-4S after chondroitinase ACII digestion
suggests that C4ST may preferentially transfer sulfate to position 4 of GalNAc residues flanked with GlcA residues on both reducing and nonreducing sides. However, the presence of GlcA residues on both sides
of the targeting GalNAc residue is not necessarily requisite for the
activity, because, in addition to
Di-4S, higher oligosaccharides, which were deduced as monosulfated hexasaccharide and monosulfated tetrasaccharide, were formed after chondroitinase ACII digestion. However, the possibility of the presence of subpopulation with regard
to size, which serves as substrate for C4ST rather efficiently, could
not be excluded, because the elution profile of
35S-labeled, desulfated dermatan sulfate (Fig.
9A) was slightly shifted to more retarded fractions compared
with the profile of the substrate (Fig. 10D).
The purified C4ST was stimulated by dithiothreitol. We have previously
found that C4ST preparations obtained from chick embryo cartilage (15)
and the culture medium of chick embryo chondrocytes (20) were also
activated by sulfhydryl compounds. Various glycosaminoglycan sulfotransferases have been reported to be affected by sulfhydryl compounds differently. C6ST (16) and heparan sulfate 2-sulfotransferase were not affected (33); whereas heparan sulfate 6-sulfotransferase was
strongly inhibited (32). Heparan sulfate glucosaminyl
3-O-sulfotransferase was reported to be inactivated by
incubation with dithiothreitol (36). GalNAc 4-sulfotransferase, which
catalyzes the formation of SO4-4GalNA
1-4GlcNAc
1-2Man
chain found in glycoprotein hormones, was reported to be stimulated by
2-mercaptoethanol (37). The intracellular level of sulfhydryl compound,
especially reduced form of glutathione, might contribute to the
regulation of sulfation of glycosaminoglycans.
C4ST has been obtained from chick embryo cartilage (15), microsomal
fraction of chick chondrocyte (42), and the culture medium of chick
chondrocytes (20). C4ST purified from the rat chondrosarcoma cells in
the present study shares several common features with chick cartilage
C4ST (15); both preparations were stimulated with protamine and
sulfhydryl compounds and had similar optimum pH and
Km values for PAPS. These observations suggest that
the protein nature of C4ST obtained from the rat chondrosarcoma cells
may be similar to that of C4ST from chick cartilage. A marked
difference between the purified C4ST and chick cartilage C4ST was
solubility in buffers; the chick cartilage C4ST was almost insoluble in
buffers unless guanidine hydrochloride was included in the buffer. We
have previously found that C6ST secreted from chick chondrocytes
appeared to be truncated at the transmembrane domain (17). C4ST from
the culture medium of the rat chondrosarcoma cells may also be cleaved
during secretion and may have lost its putative hydrophobic
transmembrane domain and, as a result, may become a soluble form. The
microsomal C4ST required the presence of detergents in the reaction
mixture for the maximum activity (42), but C4ST obtained from the
culture medium did not (20). C4ST secreted to the culture medium as a
soluble form might be derived from C4ST that was bound to the Golgi
membrane. We have achieved purification of C4ST to apparent homogeneity
for the first time. By using homogeneous preparation, we found that
C4ST could also sulfate desulfated dermatan sulfate. If molecular
cloning of C4ST is attained from the amino acid sequence of the
purified C4ST, not only strict substrate specificity of C4ST but also a
relationship of C4ST between the secreted and Golgi forms and
differences and similarities among different species will be revealed.
Several reports have suggested that the functional roles of chondroitin
4-sulfate may be different from those of chondroitin 6-sulfate.
Chondroitin 4-sulfate inhibited the cytoadherence of P. falciparum-infected erythrocytes to melanoma cells, whereas chondroitin 6-sulfate had little or no effect (38). Chondroitin 4-sulfate, but not chondroitin 6-sulfate, exhibited an inhibitory effect during Cu2+-mediated low density lipoprotein
oxidation (39). Chondroitin 6-sulfate inhibited neurite growth from rat
cerebellar and dorsal root ganglion neurons, whereas chondroitin
4-sulfate stimulated elongation of dorsal root ganglion neurites (40).
Chondroitin 6-sulfate was shown to inhibit moderately the binding of
6B4 proteoglycan, an extracellular variant of a receptor-like
protein-tyrosine phosphatase (PTP
/RPTP
), to pleiotrophin, but
chondroitin 4-sulfate scarcely influenced the binding (41). Molecular
cloning of C4ST would offer new approaches for investigating the
function of chondroitin 4-sulfate.