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(Received for publication, March 20, 1995; and in revised form, May 15, 1995) From the
Chondroitin 6-sulfotransferase (C6ST) catalyzes the transfer of
sulfate from 3`-phosphoadenosine 5`-phosphosulfate to position 6 of the N-acetylgalactosamine residue of chondroitin. The enzyme has
been purified previously to apparent homogeneity from the serum-free
culture medium of chick chondrocytes. The purified enzyme also
catalyzed the sulfation of keratan sulfate. We have now cloned the cDNA
of the enzyme. This cDNA contains a single open reading frame that
predicts a protein composed of 458 amino acid residues. The protein
predicts a Type II transmembrane topology similar to other
glycosyltransferases and heparin/heparan sulfate N-sulfotransferase/N-deacetylases. Evidence that the
predicted protein corresponds to the previously purified C6ST was the
following: (a) the predicted sequence of the protein contains
all of the known amino acid sequence, (b) when the cDNA was
introduced in a eukaryotic expression vector and transfected in COS-7
cells, both the C6ST activity and the keratan sulfate sulfotransferase
activity were overexpressed, (c) a polyclonal antibody raised
against a fusion peptide, which was expressed from a cDNA containing
the sequence coding for 150 amino acid residues of the predicted
protein, cross-reacted to the purified C6ST, and (d) the
predicted protein contained six potential sites for N-glycosylation, which corresponds to the observation that the
purified C6ST is an N-linked glycoprotein. The amino-terminal
amino acid sequence of the purified protein was found in the
transmembrane domain, suggesting that the purified protein might be
released from the chondrocytes after proteolytic cleavage in the
transmembrane domain.
Chondroitin sulfate proteoglycan is abundantly found in
cartilage and is thought to contribute to the expression and the
maintenance of the phenotype of chondrocytes(1) . Chondroitin
sulfate proteoglycan is also present in the various tissue other than
cartilage and thought to play an important role in cellular
interaction(2) . Major chondroitin sulfate found in the
mammalian or avian tissues bears sulfate groups at position 6 or
position 4 of acetylgalactosamine residue. The ratio of
6-sulfation/4-sulfation varies with development of
animals(3, 4) , malignant change(5) , or
susceptibility to atherosclerosis(6) . The presence of two
sulfate groups per one repeating disaccharide unit was also reported.
The GalNAc(4,6-bis-SO We have
previously purified chondroitin 6-sulfotransferase (C6ST), (
Figure 2:
A, oligonucleotide primer sequences,
derived from peptide 1 and 2, used for the PCR experiment shown in Fig. 2B. At the 5`-end of oligonucleotide 1s and 3a,
recognition site of HindIII and EcoRI, respectively,
were introduced. B, agarose gel electrophoresis of the PCR
products. Lane 1, the template was poly(A)
Reaction products were
subjected to agarose gel electrophoresis (Fig. 2B). The
amplified DNA band (indicated by an arrowhead in Fig. 2B) was cut out and the DNA fragment was recovered
from the gel, digested with HindIII and EcoRI, and
subcloned into these sites of Bluescript (Stratagene). Subclones were
characterized by sequencing. The radioactive probe for screening the
cDNA library was prepared from the PCR product by the random
oligonucleotide-primed labeling method (24) using
[
Figure 3:
Nucleotide sequence of the C6ST cDNA, and
the predicted amino acid sequence and hydropathy plot of the protein. A, the predicted amino acid sequence is shown below the
nucleotide sequence. Peptides from which amino acid sequence data were
obtained are underlined. Six potential N-linked
glycosylation sites are underlined with double solid
lines. The putative transmembrane hydrophobic domain is boxed. The site at which C6ST might be cleaved during the
secretion is indicated with a triangle. B, the hydropathy plot
was calculated by the method of Kyte and Doolittle (34) with a
window of 11 amino acids.
Figure 1:
N-Glycanase
digestion of the purified chondroitin 6-sulfotransferase. Chondroitin
6-sulfotransferase (0.6 µg as protein) was digested with N-glycanase as described under ``Experimental
Procedures,'' and 0.2 µg of protein was analyzed by SDS-PAGE.
Proteins were visualized with silver nitrate stain. Lane 1,
undigested control; lane 2, protein digested with N-glycanase for 1 h; lane 3, protein digested with N-glycanase for 2 h; lane 4, protein digested with N-glycanase for 12 h. Molecular size standards were the
following: myosin (205 kDa),
Figure 4:
Immunoblots of the purified C6ST and the
22-kDa fusion peptide. Lanes 1 and 4, 12 µg (as
protein) each of the serum-free culture medium of chondrocytes; lanes 2 and 5, 0.4 and 0.8 µg, respectively, of
the purified C6ST; and lanes 3 and 6, 0.1 and 0.5
µg, respectively, of the 22-kDa fusion peptide. Lanes
1-3, immunoblot stained with anti-22-kDa fusion peptide
polyclonal antibody; lanes 4-6, Amido Black staining.
Molecular size standards were the following: bovine serum albumin (66
kDa), egg albumin (45 kDa), carbonic anhydrase (29 kDa), trypsinogen
(24 kDa), soybean trypsin inhibitor (20.1 kDa), and
Figure 5:
Northern blot analysis. Polyadenylated RNA
(5 µg) prepared from the cultured chick embryo chondrocytes was
subjected to Northern blot analysis, using hybridization and wash
conditions described under ``Experimental Procedures.'' The
blot was probed with the 465-bp fragment that was obtained by PCR. The arrowheads indicate the position of different mRNAs: 5.8, 4.5,
3.2, and 2.5 kilobases. The positions of ribosomal RNAs are indicated
at the left.
We have cloned a cDNA that encodes the C6ST. Different lines
of evidence indicated that the cloned cDNA corresponds to the C6ST
previously purified from the culture medium of chondrocytes: (a) the predicted sequence of the protein contains all of the
known amino acid sequence, (b) both the C6ST activity and the
KSST activity were overexpressed when the cDNA was introduced into a
eukaryotic expression vector and transfected in COS-7 cells, (c) a polyclonal antibody raised against a fusion peptide,
which was expressed from a cDNA containing the sequence coding for 150
amino acid residues of the predicted protein, cross-reacted to the
purified C6ST, and (d) the predicted protein contained six
potential N-linked glycosylation sites, which fits with the
observation that the purified C6ST is an N-linked
glycoprotein. The predicted protein showed no sequence homology to
other sulfotransferases, including N-sulfotransferase/N-deacetylases. The sequence
observed in most of sulfotransferases, GXXGXXK(R) (19) , was not found in C6ST. LEKCGR, which was reported as a
peptide sequence at the biding site for PAPS in aryl sulfotransferase
IV(35) , was not present in C6ST. The amino-terminal
sequence contains two in-frame ATG codons. When the sequence
surrounding the first ATG codon is compared to the eukaryotic consensus
translation sequence(36) , the purine at -3, is not
conserved, but G in position +4 is conserved. Kozak showed that,
in the absence of a purine in position -3, G in position +4
was essential for efficient translation(37) . The sequence
surrounding the second ATG codon (Met The hydrophobic transmembrane domain of C6ST
contains 14 amino acid residues; the length of the transmembrane domain
of C6ST seems to be shorter than those of most of cloned
glycosyltransferases(38) . Eckhardt et
al.(39) , however, recently reported that a stretch of 13
hydrophobic amino acids was found within the NH When COS-7 cells were transfected with the expression vector
containing the cDNA of C6ST, C6ST activity in the transfected cells was
20-fold above that of the control transfections, but C4ST activity in
the cells was not increased at all, indicating that C4ST is quite a
different enzyme from C6ST. We found previously that the purified C6ST
catalyzed the transfer of sulfate to keratan sulfate (16) and
that position 6 of the Gal residue of keratan sulfate was sulfated by
the purified C6ST. (
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s)
D49915[GenBank].
Volume 270,
Number 31,
Issue of August 04, pp. 18575-18580, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) residue was found in chondroitin
sulfate from mast cell(7) , rat glomeruli(8) , and
subcultured chick chondrocytes(9) .
GalNAc(4,6-bis-SO) located at the nonreducing end was found
in chondroitin sulfate from rat cartilage(10) , cultured chick
chondrocytes(11) , and thrombomodulin(12) .
GlcA(2SO)-GalNAc(6SO) unit was contained in
chondroitin sulfate from cultured mast cells (13) . Such
observed diversity in sulfation patterns of chondroitin sulfate may
reflect the molecular basis of its function. As reported in heparan
sulfate N-sulfotransferase(14, 15) ,
chondroitin 6-sulfotransferase(16) , and heparan sulfate
6-sulfotransferase(17) , a specific sulfotransferase seems to
be involved in the sulfation of the particular position of sugar
residues of glycosaminoglycans. On the other hand, Wlad et al.(18) presented evidence suggesting that the same enzyme
may catalyze the sulfation of both 2-O of L-iduronic
acid residue and 6-O of D-glucosamine residue of
heparin. Cloning and expression of glycosaminoglycan sulfotransferase
seems to offer the direct evidence about the acceptor substrate
specificity. Of various glycosaminoglycan sulfotransferases, N-sulfotransferase/N-deacetylase has been cloned from
rat liver(19) , heparin-producing cell line(20) , and
mouse mastocytoma (21) and expressed in COS cells.
)which catalyzes the transfer of sulfate from
3`-phosphoadenosine 5`-phosphosulfate to position 6 of the N-acetylgalactosamine residue of chondroitin, to apparent
homogeneity(16) . This enzyme was also found to catalyze the
sulfation of keratan sulfate. In this paper we report the cloning of
the cDNA encoding chick chondrocyte C6ST and the expression of it in
COS-7 cells.
Assay and Purification of Chondroitin
6-Sulfotransferase
C6ST was purified from the serum-free culture
medium of chick embryo chondrocytes as described
previously(16) . Activity of C6ST, C4ST, and KSST was
determined as described previously (16) .Digestion of the Purified Sulfotransferase with
N-Glycanase
The purified sulfotransferase was precipitated with
10% trichloroacetic acid. The precipitates were washed with acetone and
digested with recombinant N-glycanase (Genzyme) by the methods
recommended by the manufacturer. After digestion, the reaction mixture
was analyzed by SDS-PAGE as described by Laemmli(22) .Determination of Amino Acid Sequence of the Purified
Protein
The purified C6ST (10 µg as protein) was digested
with N-glycanase as described above for 12 h. The resulting
deglycosylated C6ST and the intact C6ST (20 µg as protein) were
subjected to 10% SDS-PAGE and transferred to a PVDF membrane. After the
membrane was stained with Coomassie Blue, the bands of the 49- and
47-kDa protein formed after N-glycanase digestion and the band
of the 75-kDa intact C6ST were cut out and used for amino acid
sequencing. The limited digestion of the purified C6ST with protease V8
was carried out according to Cleveland et al.(23) .
Briefly, the purified protein (30 µg) was separated with SDS-PAGE
using 10% gel. After staining the gel with Coomassie Blue, the 75-kDa
protein band was cut out and inserted in the wells of another 16% gel.
After a buffer containing protease V8 (sequencing grade, Boehringer
Mannheim) in the ratio of 0.05 µg/µg of the purified protein
was layered on the inserted gel, SDS-PAGE was started. When the dye
front reached the edge of the separation gel, the power was turned off.
After 30 min, electrophoresis was resumed. The peptides formed by the
limited protease digestion were transferred to a PVDF membrane. After
staining the membrane with Coomassie Blue, the 19-kDa peptide band was
excised. The PVDF membrane containing the protein and peptide samples
were sent to Takara Shuzo Co. Ltd., Kyoto, Japan for determining the
amino-terminal amino acid sequence.Oligonucleotides and Polymerase Chain
Reaction
Degenerated oligonucleotide primers were designed as
indicated in Fig. 2A. Two sense primers (1s and 2s) and
one antisense primer (3a) were prepared from the amino acid sequence of
the 75-kDa protein and the 19-kDa peptide, respectively. At 5`-end of
oligonucleotide 1s and 3a, restriction enzyme recognition sites were
introduced; HindIII site for 1s and EcoRI site for
3a. The first strand of cDNA was synthesized by the reverse
transcriptase reaction using poly(A) RNA from
chondrocyte as a template and degenerated oligonucleotide 3a as a
primer. The PCR reaction was carried out in a final volume of 100
µl containing 50 pmol each of oligonucleotide 1s and 3a, 10 µl
of the reverse transcriptase reaction mixture in which the first strand
cDNA was synthesized, 100 µM each of four deoxynucleoside
triphosphates, 2.5 units of AmpliTaq polymerase (Perkin-Elmer).
Amplification was carried out by 30 cycles of 94 °C for 1 min, 45
°C for 1 min, and 55 °C for 3 min.
RNA from chondrocytes; lane 2 and 3, the
template was the 400-bp PCR product of lane 1 which was
indicated by an arrowhead.
-P]dCTP (Amersham Corp.) and a DNA random
labeling kit (Takara Shuzo).
Construction of
Total RNA was
prepared from the chick embryo chondrocytes cultured for 11 days in
DMEM containing 10% fetal bovine serum as described previously (25, 26, 27) by the guanidine
thiocyanate/CsCl methods(28) . Poly(A)gt11 Library
RNA was
purified by oligo(dT)-cellulose column chromatography. The synthesis of
cDNA and ligation of the cDNA to EcoRI-digested
gt11
(Pharmacia) was carried out using TimeSaver cDNA synthesis kit
(Pharmacia). Random oligonucleotide primers were used for the reverse
transcriptase reaction. The ligated DNA was packaged in vitro using a Stratagene Gigapack II packaging extract and plated on Escherichia coli Y1088. The library was used for cDNA
screening without further amplification.
Screening of
Approximately 5
gt11 Library
10
plaques were screened. Hybond N nylon membrane (Amersham) replicas of the plaques from the
gt11 cDNA library were fixed by the alkali fixation method
recommended by the manufacturer, prehybridized in a solution containing
50% formamide, 5
SSPE (SSPE, sodium chloride/sodium
phosphate/EDTA buffer), 5
Denhardt's solution, 0.5% SDS,
0.04 mg/ml of denatured salmon sperm DNA, and 0.004 mg/ml E. coli DNA for 3.5 h at 42 °C. Hybridization was carried out in the
same buffer containing
P-labeled probe for 16 h at 42
°C. The filters were washed at 55 °C in 1
SSPE, 0.1%
SDS, and subsequently in 0.1
SSPE, 0.1% SDS, and positive
clones were detected by autoradiography.
DNA Sequence Analysis
DNA from gt11 positive
clones were isolated and cut with EcoRI, which excised the
cDNA insert in a single fragment. The fragments were inserted into
Bluescript plasmid, and deletion clones were prepared as described (29, 30) using a DNA deletion kit (Takara Shuzo). The
complete nucleotide sequence was determined independently on both
strands using the dideoxy chain termination method (31) with
[
-P]dCTP and Sequenase (U. S. Biochemical
Corp.). DNA sequences were compiled and analyzed using the Gene Works
computer programs (IntelliGenetics).
Construction of pCXNC6ST
To construct the plasmid
containing the C6ST cDNA named pCXNC6ST, the EcoRI fragment
containing the 2354-bp cDNA indicated in Fig. 3A was
excised from the Bluescript plasmid and ligated into the EcoRI
site of pCXN2 expression vector (pCXN2 vector was constructed by Dr.
Jun-ichi Miyazaki, Department of Disease-related Gene Regulation,
Faculty of Medicine, University of Tokyo (32) and was a gift
from Dr. Yasuhiro Hashimoto, Tokyo Metropolitan Institute of Medical
Sciences). JM109 cells were transformed with the ligation mixture and
plated on LB ampicillin plates. Recombinant plasmids were analyzed by
restriction mapping using BamHI to confirm the correct
orientation of pCXNC6ST. The recombinant plasmids used for the
transfection were purified with CsCl/ethidium bromide equilibrium
centrifugation three times. The plasmid that contained the cDNA
fragment in the reversed orientation was named as pCXNC6ST2 and used
for control experiments.
Transient Expression of Chondroitin 6-Sulfotransferase
cDNA in COS-7 Cells
COS-7 cells (obtained from Riken Cell Bank,
Tsukuba, Japan) were plated in 100-mm culture dishes at a density of 8
10
cells/dish. The volume of the medium was 10 ml.
The medium used was DMEM containing penicillin (100 units/ml),
streptomycin (50 µg/ml), and 10% fetal bovine serum (Life
Technologies, Inc.), and cells were grown at 37 °C in 5%
CO
, 95% air. When the cell density reached 3
10
cells/dish (48 h after plating), COS-7 cells were
transfected with pCXNC6ST or pCXNC6ST2. The transfection was performed
using the DEAE-dextran method(33) . 5 ml of the prewarmed DMEM
containing 10% Nu serum (Collaborative Biomedical Products) was mixed
with 0.2 ml of PBS containing 10 mg/ml DEAE-dextran plus 2.5 mM chloroquine solution. 15 µg of the recombinant plasmid was
mixed with the solution, and the mixture was added to the cells. The
cells were incubated for 4 h in a CO incubator. The medium
was then replaced with 5 ml of 10% dimethyl sulfoxide in PBS. After the
cells were left at room temperature for 2 min, the dimethyl sulfoxide
solution was aspirated, and 25 ml of DMEM containing penicillin (100
units/ml), streptomycin (50 µg/ml), and 10% fetal bovine serum was
added. The cells were incubated for 67 h, washed with DMEM alone,
scraped, and homogenized with a Dounce homogenizer in 1.5 ml/dish of
0.25 M sucrose, 10 mM Tris-HCl, pH 7.2, and 0.5%
Triton X-100. The homogenates were centrifuged at 10,000 g for 20 min, and the activities of C6ST, C4ST, and KSST in the
supernatant fractions were measured.
Preparation of Polyclonal Antibody against a Fusion
Peptide
A DNA fragment which codes for 150 amino acid residues
(Glu to Ile
shown in Fig. 3A) was amplified by PCR using the 465-bp DNA
fragment as a template which was amplified from poly(A)
RNA as described above. At 5`-end of the oligonucleotide primers,
restriction enzyme recognition sites were introduced: BamHI
site for the sense primer beginning at Glu
and EcoRI site for the antisense primer beginning at
Ile
. Amplification was carried out by 30 cycles of 94
°C for 1 min, 45 °C for 2 min, and 72 °C for 2 min. The PCR
product was digested with EcoRI and BamHI and
subcloned into these sites of pRSET A plasmid (Invitrogen). The
resulting plasmid was transfected in E. coli DE3, and the
fusion peptide produced was purified by a ProBond Resin (Invitrogen) by
the method described by the manufacturer. The fusion peptide purified
with the affinity column was still contaminated by other proteins. The
final purification of the fusion peptide was achieved with 15%
SDS-PAGE. The 22-kDa fusion peptide eluted from the polyacrylamide gel
with 50 mM Tris-HCl, pH 8.0, containing 150 mM NaCl,
100 mM EDTA, 0.1% SDS, and 5 mM dithiothreitol was
precipitated with 5 volumes of acetone. The precipitate was dissolved
in 50 mM Tris-HCl, pH 8.0, containing 6 M guanidine
HCl, 150 mM NaCl, 0.1 mM EDTA, 0.1% Nonidet P-40, and
1 mM dithiothreitol and dialyzed against PBS. The dialyzed
sample was injected into mice intraperitoneally. Polyclonal antibody
was obtained after twice boost injections.
Immunoblotting
The purified C6ST and the 22-kDa
fusion peptide were subjected to 12% SDS-PAGE and transferred to a
nitrocellulose filter. Protein bands were visualized by Amido Black.
For immunostaining, the filter was blocked and incubated with the
diluted antiserum (1:1000) raised against the 22-kDa fusion peptide as
described above. Bound antibodies were visualized with an enzyme
reaction using peroxidase-conjugated anti-mouse immunoglobulin goat IgG
(Cappel) as a secondary antibody.Immunoprecipitation of C6ST
The mouse antiserum (3
µl) against the 22-kDa fusion peptide was added to 31 µl of
Buffer A (10 mM Tris-HCl, pH 7.2, 130 mM NaCl, 10
mM MgCl, 2 mM CaCl, 0.1%
Triton X-100, 20% glycerol) containing 23 ng of the purified C6ST. The
mixtures were incubated at 4 °C overnight. 6 µl of anti-mouse
IgG rabbit IgG (Cappel) was added to the mixtures. After further
incubation for 1 h at 0 °C, the reaction mixtures were mixed with
20 µl of a 50% (v/v) suspension of protein A-Sepharose (Pharmacia),
which was equilibrated with Buffer A, and were rocked for 30 min at 4
°C. The immune complexes on the protein A beads were removed by
centrifugation and C6ST activity remaining in the supernatant solution
was assayed.Northern Blot Hybridization
Poly(A) RNA prepared from chick chondrocytes as described above was
denatured in 50% formamide (v/v), 5% formaldehyde (v/v), 20 mM MOPS, pH 7.0, at 65 °C for 10 min, electrophoresed in 1.2%
agarose gel containing 5% formaldehyde (v/v), and transferred to a
Hybond N
nylon membrane overnight. The RNA was fixed
by baking at 80 °C for 2 h and then prehybridized in a solution
containing 50% formamide, 5
SSPE, 5
Denhardt's
solution, 0.5% SDS, and 0.1 mg/ml of denatured salmon sperm DNA for 3 h
at 42 °C. Hybridization was carried out in the same buffer
containing
P-labeled probe for 14 h at 42 °C. The
radioactive probe was the same as the probe used for the screening of
the cDNA library described above. The filters were washed at 65 °C
in 2
SSPE, 0.1% SDS, and subsequently in 1
SSPE, 0.1%
SDS. The membrane was exposed to x-ray film for 26 h with a
intensifying screen at -80 °C.
Amino Acid Sequence of Chondroitin
6-Sulfotransferase
The purified C6ST gave a broad protein band
on SDS-PAGE (Fig. 1, lane 1). Microheterogeneity of N-linked oligosaccharides attached to the enzyme probably
cause the width of the band. After the sulfotransferase was digested
with N-glycanase, the protein band of 75 kDa disappeared, and
two sharp bands (49 and 47 kDa) appeared as shown in Fig. 1, lane 2-4. A broad protein band of 56 kDa appeared to be
a partially N-deglycosylated product, since the band became
weaker as the period of the digestion with N-glycanase became
longer. The amino-terminal amino acid sequence of the 49-kDa protein
was identical with that of the 47-kDa protein as far as the
identification was possible; therefore we determined the amino-terminal
amino acid sequence of the 75-kDa intact C6ST without further
separation (Table 1). We also prepared the 19-kDa peptide from
the intact protein by the limited digestion with protease V8, and the
amino acid sequence of the 19-kDa peptide was determined (Table 1).
-galactosidase (116 kDa),
phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), egg
albumin (45 kDa), and carbonic anhydrase (29 kDa). 2-Mercaptoethanol
was removed from sample buffer for SDS-PAGE to avoid artifact of silver
staining. The arrowhead indicates the position of N-glycanase.
Generation of PCR Probe to Screen cDNA
Library
When primer 1s and 3a (for the nucleotide sequences of
the primers, see Fig. 2A) were used in a PCR with
poly(A) RNA from chick chondrocytes as template, DNA
fragments of 400 and 600 bp were obtained (Fig. 2B, lane 1). The 400-bp product, which is indicated by an arrowhead in Fig. 2B, lane 1,
appeared to be specific because, when primer 2s and 3a were used in a
PCR with the 400-bp fragment as template, a fragment slightly shorter
than the template was amplified (Fig. 2B, lane
2). Further proof that the 400-bp fragment was amplified from the
mRNA of the purified C6ST protein was obtained by sequencing the 400-bp
fragment; the fragment contained 465 nucleotides in which a nucleotide
sequence corresponding to the amino acid sequence encoded by primer 2s
was present adjacent to the sequence corresponding to primer 1s.
Screening of
The above described
465-bp PCR product was labeled with
[gt11 Library
-P]dCTP by the random
oligonucleotide-primed labeling method and used as a probe to screen a
gt11 library obtained as described under ``Experimental
Procedures.'' About 90 positive clones were observed from 5
10
plaques. Sixteen independent clones were
isolated and subcloned into Bluescript. The nucleotide sequence of the
largest cDNA insert (2.3 kilobases) was determined.cDNA and Predicted Protein Sequence of the Chondroitin
6-Sulfotransferase
The nucleotide sequence of the C6ST cDNA and
the predicted amino acid sequence are shown in Fig. 3A.
The amino-terminal sequence contains two in-frame ATG codons. A single
open reading frame beginning at the first ATG codon predicts a protein
of 458 amino acid residues with a molecular mass of 52,193 Da with six
potential N-linked glycosylation sites. To determine the
location of any transmembrane domain, a hydropathy plot was generated
from the translated sequence. Analysis of the plot revealed one
prominent hydrophobic segment in the amino-terminal region, 14 residues
in length, that extends from amino acid residues 24-37 (Fig. 3B). The amino-terminal-amino acid sequence of
the purified C6ST was found in the transmembrane domain. The molecular
mass of the truncated protein at the site in the transmembrane domain
was calculated as 47,885 Da, and it agreed well with the molecular mass
of the protein formed after N-glycanase digestion. All the
amino acid sequences that have been obtained from the purified protein
were found in the predicted protein sequences, confirming that the cDNA
clone encodes the purified C6ST protein.Expression of Chondroitin 6-Sulfotransferase cDNA in
COS-7 Cells
Direct evidence demonstrating that the isolated cDNA
encodes the chondroitin 6-sulfotransferase protein was obtained by
expressing it in COS-7 cells. COS-7 cells were transfected with the
pCXNC6ST, a recombinant plasmid containing the isolated cDNA in the
mammalian expression vector pCXN2. The transfected cells were scraped
at 67 h after transfection, homogenized, and centrifuged. Activities of
C6ST, C4ST, and KSST contained in the supernatant fractions were
determined in the presence or absence of sulfate acceptors. Control
experiments without vector, and with vector containing the cDNA in the
reversed orientation (pCXNC6ST2), were also done. As shown in Table 2, when the vector containing the isolated cDNA was used,
C6ST activity and KSST activity in the transfected cells was 20- and
9-fold, respectively, above that of the control transfections. In
contrast, C4ST activity in the transfected cells was not increased at
all. These results demonstrate that the isolated cDNA encodes a protein
with both the C6ST activity and the KSST activity.
Immunoblotting and Immunoprecipitation
A
polyclonal antibody raised against the 22-kDa fusion peptide, which was
expressed from the sequence coding for 150 amino acid residues of the
predicted protein, cross-reacted to the purified C6ST with molecular
mass of 75 kDa (Fig. 4, lane 2). This antibody did not
stain any proteins contained in the culture medium of chondrocytes (lane 1). C6ST, which was contained in the culture medium, was
not detected on the immunoblot; this result may be due to the trace
amount of C6ST in the culture medium. When the antibody was added to
the purified C6ST solution and the immunocomplexes on protein A were
removed by centrifugation, a large part of the C6ST activity was
depleted from the soluble fractions (Table 3). These results
clearly indicate that C6ST contains a polypeptide sequence whose
antigenicity is indistinguishable from the 22-kDa fusion peptide and
confirm that the isolated cDNA encodes C6ST protein.
-lactalbumin
(14.2 kDa).
Northern Analysis
A Northern blot of
poly(A) RNA from the cultured chick chondrocytes was
made and hybridized with a radioactive probe prepared from the
PCR-amplified DNA fragment with 465 bp described above by the random
oligonucleotide-primed labeling method. As can be seen in Fig. 5, four bands with a relative size of 5.8, 4.5, 3.2, and
2.5 kilobases were obtained.
in Fig. 3A) also partially fits with the consensus
sequence; position -3 of the second ATG codon is A, whereas
position +4 is not G but A. It remains to be studied whether both
of the two in-frame ATG codon contained in C6ST cDNA could function as
the initiation codon.
-terminal
domain of eukaryotic polysialyltransferase-1, and they supposed that
the hydrophobic domain represents a Golgi retention signal, because the
cationic borders characteristic for type II transmembrane proteins were
also found. Cationic amino acid residues (Lys and
Lys
in Fig. 3A) were also found at both
sides of the hydrophobic domain of C6ST. Since the microsomal C6ST from
the cultured chondrocytes was markedly activated by detergents such as
Triton X-100 or Brij 58(40, 41, 42) , C6ST
appeared to be a membrane protein, although the transmembrane domain
seemed to be rather unusual. The amino-terminal amino acid sequence of
the purified C6ST was found in the predicted transmembrane domain,
suggesting that the purified protein might be released from the
chondrocytes after proteolytic cleavage within the transmembrane
domain.
)The sulfotransferase activity toward
keratan sulfate was also expressed when pCXNC6ST was transfected to
COS-7 cells, suggesting that position 6 of GalNAc residue in
chondroitin and position 6 of Gal residue in keratan sulfate may be
sulfated by the same enzyme.
We thank Dr. Jun-ichi Miyazaki, Department of
Disease-related Gene Regulation, Faculty of Medicine, University of
Tokyo and Dr. Yasuhiro Hashimoto, Tokyo Metropolitan Institute of
Medical Sciences, for donating pCXN2 expression vector; Dr. Keiichi
Yoshida, Tokyo Research Institute of Seikagaku Corp., for generous gift
of bovine corneal keratan sulfate; and Dr. Naomi Yamakawa, Institute
for Molecular Medical Science, Aichi Medical University, for useful
suggestions about the pRSET vector.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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A. H. K. Plaas, L. A. West, S. Wong-Palms, and F. R. T. Nelson Glycosaminoglycan Sulfation in Human Osteoarthritis. DISEASE-RELATED ALTERATIONS AT THE NON-REDUCING TERMINI OF CHONDROITIN AND DERMATAN SULFATE J. Biol. Chem., May 15, 1998; 273(20): 12642 - 12649. [Abstract] [Full Text] [PDF]
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H. Habuchi, M. Kobayashi, and K. Kimata Molecular Characterization and Expression of Heparan-sulfate 6-Sulfotransferase. COMPLETE cDNA CLONING IN HUMAN AND PARTIAL CLONING IN CHINESE HAMSTER OVARY CELLS J. Biol. Chem., April 10, 1998; 273(15): 9208 - 9213. [Abstract] [Full Text] [PDF]
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E. Ong, J.-C. Yeh, Y. Ding, O. Hindsgaul, and M. Fukuda Expression Cloning of a Human Sulfotransferase That Directs the Synthesis of the HNK-1 Glycan on the Neural Cell Adhesion Molecule and Glycolipids J. Biol. Chem., February 27, 1998; 273(9): 5190 - 5195. [Abstract] [Full Text] [PDF]
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H. Bakker, I. Friedmann, S. Oka, T. Kawasaki, N. Nifant'ev, M. Schachner, and N. Mantei Expression Cloning of a cDNA Encoding a Sulfotransferase Involved in the Biosynthesis of the HNK-1 Carbohydrate Epitope J. Biol. Chem., November 21, 1997; 272(47): 29942 - 29946. [Abstract] [Full Text] [PDF]
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M. Kobayashi, H. Habuchi, M. Yoneda, O. Habuchi, and K. Kimata Molecular Cloning and Expression of Chinese Hamster Ovary Cell Heparan-sulfate 2-Sulfotransferase J. Biol. Chem., May 23, 1997; 272(21): 13980 - 13985. [Abstract] [Full Text] [PDF]
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K. Honke, M. Tsuda, Y. Hirahara, A. Ishii, A. Makita, and Y. Wada Molecular Cloning and Expression of cDNA Encoding Human 3'-Phosphoadenylylsulfate:galactosylceramide 3'-Sulfotransferase J. Biol. Chem., February 21, 1997; 272(8): 4864 - 4868. [Abstract] [Full Text] [PDF]
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J. Liu, N. W. Shworak, L. M.S. Fritze, J. M. Edelberg, and R. D. Rosenberg Purification of Heparan Sulfate D-Glucosaminyl 3-O-Sulfotransferase J. Biol. Chem., October 25, 1996; 271(43): 27072 - 27082. [Abstract] [Full Text] [PDF]
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M. Kobayashi, H. Habuchi, O. Habuchi, M. Saito, and K. Kimata Purification and Characterization of Heparan Sulfate 2-Sulfotransferase from Cultured Chinese Hamster Ovary Cells J. Biol. Chem., March 29, 1996; 271(13): 7645 - 7653. [Abstract] [Full Text] [PDF]
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H. Kitagawa, M. Fujita, N. Ito, and K. Sugahara Molecular Cloning and Expression of a Novel Chondroitin 6-O-Sulfotransferase J. Biol. Chem., July 7, 2000; 275(28): 21075 - 21080. [Abstract] [Full Text] [PDF]
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N. Hiraoka, H. Nakagawa, E. Ong, T. O. Akama, M. N. Fukuda, and M. Fukuda Molecular Cloning and Expression of Two Distinct Human Chondroitin 4-O-Sulfotransferases That Belong to the HNK-1 Sulfotransferase Gene Family J. Biol. Chem., June 23, 2000; 275(26): 20188 - 20196. [Abstract] [Full Text] [PDF]
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K. Honke, M. Tsuda, S. Koyota, Y. Wada, N. Iida-Tanaka, I. Ishizuka, J. Nakayama, and N. Taniguchi Molecular Cloning and Characterization of a Human beta -Gal-3'-sulfotransferase That Acts on Both Type 1 and Type 2 (Galbeta 1-3/1-4GlcNAc-R) Oligosaccharides J. Biol. Chem., January 5, 2001; 276(1): 267 - 274. [Abstract] [Full Text] [PDF]
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S. Bhakta, A. Bartes, K. G. Bowman, W.-M. Kao, I. Polsky, J. K. Lee, B. N. Cook, R. E. Bruehl, S. D. Rosen, C. R. Bertozzi, et al. Sulfation of N-Acetylglucosamine by Chondroitin 6-Sulfotransferase 2 (GST-5) J. Biol. Chem., December 15, 2000; 275(51): 40226 - 40234. [Abstract] [Full Text] [PDF]
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T. Okuda, S. Mita, S. Yamauchi, M. Fukuta, H. Nakano, T. Sawada, and O. Habuchi Molecular Cloning and Characterization of GalNAc 4-Sulfotransferase Expressed in Human Pituitary Gland J. Biol. Chem., December 15, 2000; 275(51): 40605 - 40613. [Abstract] [Full Text] [PDF]
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T. O. Akama, J. Nakayama, K. Nishida, N. Hiraoka, M. Suzuki, J. McAuliffe, O. Hindsgaul, M. Fukuda, and M. N. Fukuda Human Corneal GlcNAc 6-O-Sulfotransferase and Mouse Intestinal GlcNAc 6-O-Sulfotransferase Both Produce Keratan Sulfate J. Biol. Chem., May 4, 2001; 276(19): 16271 - 16278. [Abstract] [Full Text] [PDF]
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F. M. El-Fasakhany, K. Uchimura, R. Kannagi, and T. Muramatsu A Novel Human Gal-3-O-Sulfotransferase. MOLECULAR CLONING, CHARACTERIZATION, AND ITS IMPLICATIONS IN BIOSYNTHESIS OF (SO4-3)Galbeta 1-4(Fucalpha 1-3)GlcNAc J. Biol. Chem., July 13, 2001; 276(29): 26988 - 26994. [Abstract] [Full Text] [PDF]
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K. Uchimura, K. Kadomatsu, H. Nishimura, H. Muramatsu, E. Nakamura, N. Kurosawa, O. Habuchi, F. M. El-Fasakhany, Y. Yoshikai, and T. Muramatsu Functional Analysis of the Chondroitin 6-Sulfotransferase Gene in Relation to Lymphocyte Subpopulations, Brain Development, and Oversulfated Chondroitin Sulfates J. Biol. Chem., January 4, 2002; 277(2): 1443 - 1450. [Abstract] [Full Text] [PDF]
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Y. Ito and O. Habuchi Purification and Characterization of N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase from the Squid Cartilage J. Biol. Chem., October 27, 2000; 275(44): 34728 - 34736. [Abstract] [Full Text] [PDF]
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