Molecular Cloning and Characterization of a Human Uronyl
2-Sulfotransferase That Sulfates Iduronyl and Glucuronyl Residues
in Dermatan/Chondroitin Sulfate*
Masashi
Kobayashi
§,
Geetha
Sugumaran§¶
,
Jian
Liu
,
Nicholas W.
Shworak¶**,
Jeremiah E.
Silbert§¶
, and
Robert D.
Rosenberg
¶**
From the
Department of Biology, Massachusetts
Institute of Technology, Cambridge, Massachusetts 02139, the
§ Edith Nourse Rogers Memorial Veterans Hospital,
Bedford, Massachusetts 01730, the ¶ Department of Medicine,
Harvard Medical School,
Brigham and Women's
Hospital, Division of Rheumatology, Immunology and Allergy, and
** Beth Israel Hospital, Boston, Massachusetts 02215
 |
ABSTRACT |
A partial-length human cDNA with a predicted
amino acid sequence homologous to a previously described heparan
sulfate iduronyl 2-sulfotransferase (Kobayashi, M., Habuchi, H.,
Yoneda, M., Habuchi, O., and Kimata, K. (1997) J. Biol.
Chem. 272, 13980-13985) was obtained by searching the expressed
sequence-tagged data bank. Northern blot analysis was performed using
this homologous cDNA as a probe, which demonstrated ubiquitous
expression of messages of 5.1 and 2.0 kilobases in a number of human
tissues and in several human cancer cell lines. Since the human
lymphoma Raji cell line had the highest level of expression, it was
used to isolate a full-length cDNA clone. The full-length cDNA
was found to contain an open reading frame that predicted a type II
transmembrane protein composed of 406 amino acid residues.
The cDNA in a baculovirus expression vector was expressed in
Sf9 insect cells, and cell extracts were then incubated together
with 3'-phosphoadenosine 5'-phospho[35S]sulfate and
potential glycosaminoglycan acceptors. This demonstrated substantial
sulfotransferase activity with dermatan sulfate, a small degree of
activity with chondroitin sulfate, but no sulfotransferase activity
with desulfated N-resulfated heparin. Analysis of
[35S]sulfate-labeled disaccharide products of chondroitin
ABC, chondroitin AC, and chondroitin B lyase treatment demonstrated
that the enzyme only transferred sulfate to the 2-position of uronyl
residues, which were preponderantly iduronyl residues in dermatan
sulfate, but some lesser transfer to glucuronyl residues of chondroitin sulfate.
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INTRODUCTION |
Dermatan sulfate is a glycosaminoglycan polysaccharide consisting
of N-acetylgalactosamine (GalNAc) residues alternating
with varying proportions of glucuronyl
(GlcA)1 and iduronyl
(IdceA) residues that are formed from the GlcA by epimerization during
polymerization and GalNAc 4-sulfation (1-4). Thus dermatan sulfate can
be considered as a variant of chondroitin 4-sulfate, containing some
IdceA as well as GlcA, with the IdceA only found next to 4-sulfated
GalNAc residues (5). In addition the IdceA of dermatan sulfate is
frequently 2-sulfated (6). Some 2-sulfation of GlcA on chondroitin
sulfate has also been found but only next to GalNAc 6-sulfate rather
than GalNAc 4-sulfate. Proteoglycans containing dermatan sulfate are
ubiquitously present in most tissues, where the dermatan sulfate
portion may be involved in various biological activities presumably
relating in great part to its fine structure. Activities include
interaction with heparin cofactor II (7, 8) requiring repeating
2-sulfated iduronyl-containing disaccharide units (7), hepatocyte
growth factor/scatter factor (9), and promotion of fibroblast growth factor-2 during wound repair (10). Although there is little information
concerning detailed biological activities based on the structural
diversity of the dermatan sulfate, the 2-sulfation of IdceA would
appear to be of special interest.
The only galactosaminoglycan sulfotransferase that has been cloned to
date is a chondroitin 6-sulfotransferase (11). However, several
glucosaminoglycan sulfotransferases have been cloned (12-19), including an IdceA 2-sulfotransferase for heparan sulfate (15, 20). It
seemed likely that this enzyme would have similarities to IdceA
2-sulfotransferase for dermatan sulfate. Therefore, in an attempt to
find such an IdceA 2-sulfotransferase, we employed the heparan sulfate
IdceA 2-sulfotransferase sequence to obtain a related expressed
sequence-tagged (EST) clone. This provided for the molecular cloning of
a human cDNA which we found to encode a uronyl 2-sulfotransferase.
We have found this enzyme to have no 2-sulfotransferase activity with
heparan sulfate but to be involved in the sulfation of the IdceA
residues of dermatan sulfate with some lesser activity in 2-sulfation
of GlcA residues in chondroitin sulfate.
 |
EXPERIMENTAL PROCEDURES |
Heparan Sulfate 2-Sulfotransferase Homologous cDNA and
Generation of PCR Probe to Isolate Full-length Clone--
The National
Center for Biotechnology Information (NCBI) data bank of I.M.A.G.E.
Consortium (Lawrence Livermore National Laboratory) EST cDNA clones
(21) was probed with a deduced CHO cell heparan sulfate 2- sulfotransferase sequence (15). Clone ID HE9MJ06 was obtained from the
TIGR/ATCC Special Collection (ATCC). This was a partial-length EST
clone from a 9-week-old human embryo, in which only 248 bp of sequence
(positions 473-720, Fig. 1) was present
in the data base (22). The cDNA from this clone was inserted into
the EcoRI and XhoI sites of the Bluescript SK
plasmid and carried in an Escherichia coli host strain. The
pBluescript plasmid DNA was purified from the host bacteria using
QIAfilter plasmid kits (Qiagen) and used to prepare a PCR probe
consisting of a 614-bp fragment at positions 473-1086 as shown in Fig.
1 using 20-bp oligonucleotides of both ends as primers. The PCR was
carried out in a 50-µl volume containing 0.5 µM each
primer, 40 ng of the template, 0.2 mM each dNTP, and 1.25 units of Taq2000TM DNA polymerase (Stratagene)
under the conditions of 30 cycles of denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, and extension at 72 °C for 1 min. The products were subjected to agarose gel electrophoresis, and an
amplified DNA band of ~600 bp was excised, recovered from the gel
using QIAEX II (Qiagen), and radiolabeled for the probe using
[
-32P]dATP and a Prime-It II random primer labeling
kit (Stratagene).

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Fig. 1.
Nucleotide sequence and predicted amino acid
sequence of the presumptive sulfotransferase cDNA. The
predicted amino acid sequence is shown below the nucleotide
sequence. The putative transmembrane hydrophobic domain (see Fig. 2) is
boxed, and the polyadenylation signal is
underlined with a dashed line. The five possible
sites for N-glycosylation are shown with black
dots. A point mutation found in the isolated clone is
underlined with a solid line.
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Northern Blot Hybridization--
To obtain full-length clones,
we first scanned tissue-specific and cell type-specific expression of
the cDNA by Northern blot hybridization using the
32P-labeled PCR probe. Tissue type-specific expression of
presumptive 2-sulfotransferase cDNA was analyzed with human RNA
Master BlotTM and human Multiple Tissue Northern
(MTNTM) Blot (CLONTECH) membranes and
cell type-specific expression with a human cancer cell line Multiple
Tissue Northern (MTNTM) Blot (CLONTECH)
membrane. Each Northern analysis was carried out according to the
manufacturer's protocol with some modifications. The Master Blot
membrane was prehybridized in an ExpressHyb solution containing 100 µg/ml denatured sheared salmon testes DNA (Sigma) at 65 °C for 30 min, hybridized in the same solution containing the denatured
32P-labeled probe, 6 µg/ml human Cot-1 DNA (Boehringer
Mannheim), and 0.2× SSC at 65 °C for 16 h, and washed four
times with 2× SSC, 1% SDS at 65 °C for 20 min, twice with 0.1×
SSC, 0.5% SDS at 55 °C for 20 min. The human and cancer cell line
MTN Blot membranes were prehybridized in an ExpressHyb at 68 °C for
30 min, hybridized in the same solution containing the
32P-labeled probe at 68 °C for 16 h, and washed
twice with 2× SSC, 0.05% SDS at 22 °C for 20 min, twice with 0.1×
SSC, 0.1% SDS at 50 °C for 20 min. The membranes were then exposed
to x-ray film with an intensifying screen at
80 °C.
Screening of cDNA Library--
The human lymphoma 5'-STRETCH
PLUS cDNA library (CLONTECH) was constructed
from mRNA from Burkitt's lymphoma Raji cell line at
EcoRI-cloning sites of the
gt11 vector by the priming
method with oligo(dT) and random primers. The host strain Y1090r
cells were infected with phage from the library, plated at 4 × 105 plaque-forming units/dish, and approximately 1.2 × 106 plaques were screened. Colony/Plaque
screenTM (NEN Life Science Products) membrane replicas of
the plaques were fixed by the rapid autoclave method recommended by the
manufacturer, prehybridized in an ExpressHybTM
hybridization solution (CLONTECH) for 30 min at
68 °C, and hybridized in the same solution containing the denatured
32P-labeled probe at 68 °C according to the
manufacturer's protocol with a 16-h modification. The filters were
washed twice with 2× sodium citrate-sodium chloride (SCC), 0.05%
sodium dodecyl sulfate (SDS) for 20 min at 22 °C, and then twice
with 0.1× SCC, 0.1% SDS for 20 min at 50 °C. The positive clones
were detected by autoradiography.
Characterization of cDNA Clones--
Plaque solutions from
the positive clones were initially characterized with LD-insert
screening amplimer sets (CLONTECH) according to the
manufacturer's PCR protocol. The resultant PCR products were subjected
to agarose gel electrophoresis to determine the sizes of the inserts,
recovered from the gel, and sequenced.
gt11 DNA of the clones was
isolated from its plate lysate using a Qiagen lambda kit (Qiagen),
subcloned into pcDNA3 vector (Invitrogen) at the EcoRI
sites, and sequenced again to confirm sequence data. For DNA
sequencing, the 5' and 3' insert regions were enzymatically sequenced
from flanking primer sites of the respective PCR fragments or vectors.
The remaining sequences of both strands were obtained with internally
priming oligonucleotides. Primers were spaced no more than 400 bp apart
with a 200-bp offset between sense and antisense strands. Automated
fluorescence sequencing was performed with Perkin-Elmer Applied
Biosystems models 373A and 477 DNA Sequencers. The DNA sequence files
obtained were aligned and compiled with Sequencher (Gene Codes Corp.)
and GENETYX-MAC (Software Development Corp.) computer programs.
Sequence comparison searches were performed on the data bases of
GenBankTM, EMBL, PDB, SwissProt, SPupdate, PIR, and dbEST.
Construction of Baculovirus Expression Vector--
The
PvuII-EcoRI fragment containing the coding region
from positions 193 to 1,382 shown in Fig. 1 was excised from the
gt11 cloning vector, blunted with T4 DNA polymerase, and ligated
into the StuI site of the pFASTBACTM HTa plasmid
(Life Technologies, Inc.). The recombinant bacmid-sulfotransferase (presumptive) molecules were then produced by Tn7-mediated
site-specific transposition when MAX EFFICIENCY DH10BACTM
competent cells (Life Technologies, Inc.) were transformed with the
recombinant pFASTBAC HTa donor plasmid according to the manufacturer's instructions. The recombinant molecules were isolated and analyzed by
agarose gel electrophoresis and PCR with vector and gene-specific primers to confirm the presence of bacmid high molecular weight DNA and
the correct orientation of the inserted cDNA. Recombinant bacmid-heparan sulfate 2-sulfotransferase DNA that contained the entire
coding region of its cDNA (SacII-AflII
fragment) (15) was also constructed and used for control experiments.
Expression of cDNA--
cDNA was expressed using a
BAC-TO-BACTM HT baculovirus expression system (Life
Technologies, Inc.) according to the manufacturer's instructions with
slight modifications. Sf9 insect cells (Invitrogen) seeded onto
35-mm culture dishes containing 2 ml of SF-900 II SFM (Life
Technologies, Inc.) were transfected with bacmid-sulfotransferase (presumptive) or bacmid-heparan sulfate 2-sulfotransferase using CELLFECTINTM reagent (Life Technologies, Inc.). The medium
was replaced with Grace's insect medium (Invitrogen), 10% fetal
bovine serum (JRH Biosciences), and the culture was continued for
another 3 days at 27 °C. The spent medium was centrifuged for 5 min
at 500 × g to obtain the virus-containing supernatant
as viral stock. 150-mm Petri dishes of Sf9 cells were then
infected by each recombinant viral stock and incubated at 27 °C for
3 days. After collecting the spent medium, the 150-mm Petri dishes of
infected Sf9 cells were washed with phosphate-buffered saline,
scraped, and homogenized in a 3-ml solution of 10 mM
Tris-HCl, pH 7.4, 0.5% (w/v) Triton X-100, 0.15 M NaCl, 10 mM MgCl2, 2 mM CaCl2,
20% (v/v) 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) as described previously (15, 20). After 1 h of gentle
stirring at 4 °C, the homogenate was centrifuged at 4 °C for 30 min at 10,000 × g. Sulfotransferase activities in the
supernatant fractions (cell extracts) were measured as described below.
Protein contents of the cell extracts were estimated by a micro-BCA
protein assay reagent kit (Pierce) using bovine serum albumin as a standard.
Assay for Sulfotransferase Activity--
Completely desulfated
and N-resulfated heparin (CDSNS-heparin) and shark cartilage
chondroitin sulfate C (4-sulfate:6-sulfate, 10:90) were obtained from
Seikagaku; porcine skin dermatan sulfate was obtained from Sigma.
Chemical desulfation (23) was used to obtain dermatan and chondroitin,
which resulted in apparent complete desulfation of the chondroitin but
left small amounts of 4-sulfate on the dermatan. A standard reaction
mixture (25 µl) contained 1.25 µmol of imidazole HCl, pH 6.8, 1.88 µg of protamine chloride, 12.5 µg of glycosaminoglycan, 0.5 nmol
(2.5 µCi/nmol) of 3'-phosphoadenosine
5'-phospho[35S]sulfate (NEN Life Science Products, Inc.),
and 5 µl of the cell extract. After incubation at 37 °C for 30 min, the reaction mixtures were directly spotted on Whatman No. 1 paper
and chromatographed in ethanol, 1 M ammonium acetate (5:2
(v/v)) overnight. The origins, which contained the sulfated products,
were assayed for radioactivity as described previously (24).
Structural Analysis of 35S-Labeled Products--
In
order to obtain sufficient labeled products for detailed analyses,
higher specific activity 5'-phospho[35S]sulfate (~150
µCi/nmol) prepared as described previously (25) was used. After
phenol:chloroform:isoamyl alcohol (25:24:1) treatment and subsequent
ethanol precipitation, the 35S-labeled
glycosaminoglycans were digested with protease-free chondroitin ABC
lyase (20 milliunits/µg substrate), chondroitin AC I lyase (10 milliunits/µg substrate), or chondroitin B lyase (100 milliunits/µg
substrate) (Seikagaku Corp.) for 16 h at 37 °C (30 °C for B
lyase) and boiled at 100 °C for 1 min to terminate the reaction.
Products (~9,000 cpm) were applied on Bio-Gel P-2 (Bio-Rad) columns
(0.75 × 200 cm) that were equilibrated and eluted with 0.1 M ammonium bicarbonate at a flow rate of 4 ml/h and assayed for radioactivity. Aliquots of the boiled ABC lyase degradation mixtures were then incubated for an additional 16 h at 37 °C
with (a)
4,5hexuronate-2-sulfatase (10 milliunits/µg substrate (provided by K. Yoshida, Seikagaku Corp.)
(26), (b) chondro-4-sulfatase (30 milliunits/µg
substrate), or (c) chondro-6-sulfatase (30 milliunits/µg substrate (Seikagaku Corp.). 35S-Labeled digests (~3,000
cpm) before and after sulfatase treatment were then analyzed by high
performance liquid chromatography (HPLC) on a column of YMC-Pack
Polyamine II (0.46 × 25 cm) (YMC), injected together with
unsaturated disaccharide standards (Seikagaku Corp.) and eluted with a
linear gradient from 16 to 530 mM
NaH2PO4 over a 60-min period at a flow rate of
1.0 ml/min (27). Fractions of 0.5 ml were collected and mixed with 6 ml
of EcoLumeTM (ICN Biomedicals), and the radioactivity
was determined.
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RESULTS |
cDNA and Predicted Protein Sequence of the
Presumptive Sulfotransferase--
In order to screen for IdceA
2-sulfotransferases the NCBI Data Bank of EST cDNA clones was
probed with the deduced amino acid sequence of CHO cell heparan sulfate
IdceA 2-sulfotransferase cDNA (15). As described under
"Experimental Procedures," a human partial-length cDNA clone,
Clone ID HE9MJ06, was found, encoding a novel related species. The
cDNA from this clone was 3,743-bp (positions 473-4215) in length
as shown in Fig. 1. The 614-bp (positions 473-1086) PCR probe for
library screening and Northern hybridization was generated as described
under "Experimental Procedures." Approximately 1.2 × 106 plaques of a
gt11 human lymphoma Raji cell cDNA
library were screened using this PCR fragment as a probe, resulting in
25 positive clones. Sixteen insert cDNAs of these clones were
selected, amplified, and sequenced as described under "Experimental
Procedures," but only one (1.4 kb) appeared to have the complete
coding sequence of the presumptive sulfotransferase.
The amino-terminal sequence of this clone was found to contain three
in-frame ATG codons and a TGA stop codon in frame at position 56 upstream from the first ATG codon. A single open reading frame
beginning at the first ATG codon predicted a protein of 406 amino acid
residues with a molecular mass of 47,672 Da with five potential
N-linked glycosylation sites. Hydropathic analysis (28) of
the predicted amino acid sequence of the presumptive sulfotransferase
revealed that it had a prominent type II membrane protein hydrophobic
segment in the amino-terminal region, 18 residues in length at
positions 48-65 (Figs. 1 and 2).

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Fig. 2.
Hydropathic analysis of the predicted
protein. Hydrophobicity values were obtained according to the
algorithm of Kyte and Doolittle (28). Positive values represent
increased hydrophobicity.
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Comparison of the sequence of this human presumptive sulfotransferase
with CHO cell heparan sulfate IdceA 2-sulfotransferase (GenBankTM accession number D88811) (15) revealed ~30%
identity and ~50% similarity at the amino acid level. In particular,
extensive homology existed across 210-amino acid residues from 114 to
320 for a consensus sequence in the middle region of these enzymes, which included the 5'-phosphosulfate binding motif, its catalytic Lys,
and 3'-phosphate binding motif corresponding to the reports of new
algorithms using PAPS on substrates (29-31) (Fig.
3). There was no significant homology at
the nucleotide level. In addition there was considerable homology and
identity with a protein from Caenorhabditis elegans
(GenBankTM accession number Z81479) (32) and
Drosophila melanogaster segregation distorter protein
(GenBankTM accession number P25722) (33) (Fig. 3). It
shared little overall sequence similarity and no common sequence
elements with any glycosaminoglycan sulfotransferase previously
reported (11-14, 16-19) other than the heparan sulfate IdceA
2-sulfotransferase (15), indicating that it was also most likely an
IdceA 2-sulfotransferase.

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Fig. 3.
Sequence comparison of the presumptive
sulfotransferase with heparan sulfate IdceA 2-sulfotransferase and
sequences from C. elegans and D. melanogaster. The program Pileup was used to align
amino acid sequence of the presumptive sulfotransferase
(DS2ST) with sequences from heparan sulfate IdceA
2-sulfotransferase (HS2ST), C. elegans
(Ce), and D. melanogaster (Dm).
Consensus residues (shaded) are indicated for each position
where at least three candidates exhibit identical or similar amino
acids. Numeration is given for each and for a consensus sequence. The
5'-phosphosulfate binding motif (5'PSB) and 3'-phosphate
binding motif (3'PB) for PAPS (29) are labeled, and the
catalytic lysines are shown with a black dot (30).
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Expression of the Presumptive 2-Sulfotransferase in a Baculovirus
System--
Extracts from cells infected with recombinant viral stocks
containing the bacmid-presumptive 2-sulfotransferase showed (Table I) incorporation of
[35S]sulfate from 5'-phospho[35S]sulfate
into dermatan sulfate, with lesser incorporation into dermatan and
chondroitin sulfate, and no incorporation into chondroitin. There was
also no net incorporation into CDSNS-heparin by this cell extract over
that incorporated by control cell extract which contained significant
endogenous heparan sulfate sulfotransferase activity. Cells infected
with recombinant viral stocks containing the bacmid-heparan sulfate
IdceA 2-sulfotransferase showed net incorporation of sulfate only into
CDSNS-heparin, with none into dermatan sulfate, dermatan, or
chondroitin sulfate. These results indicated that the isolated cDNA
encoded a protein that presumably had dermatan IdceA 2-sulfotransferase
activity with lesser amounts of chondroitin GlcA 2-sulfotransferase
activity.
Northern Blot Analysis--
Human RNA Master Blot analysis
demonstrated ubiquitous expression of the gene across a wide range of
human tissues (data not shown). Human MTN Blot membrane analysis (Fig.
4A) demonstrated a major band
of 5.1 kb and a minor band of 2.0 kb for human tissues. Analysis with
the human cancer cell line MTN Blot (Fig. 4B) showed the
same two bands except with promyelocytic leukemia HL-60 and chronic
myelogenous leukemia K-562. Burkitt's lymphoma Raji cell line showed
the greatest expression. For this reason the cDNA library of this
lymphoma cell line was chosen as the cDNA source to isolate the
present gene.

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Fig. 4.
Northern blot analysis of the presumptive
2-sulfotransferase messages. A, tissue type-specific
analysis with a human MTN Blot membrane was performed as described
under "Experimental Procedures" on 2 µg of poly(A)+
RNA from human heart (lane 1), brain (lane 2),
placenta (lane 3), lung (lane 4), liver
(lane 5), skeletal muscle (lane 6), kidney
(lane 7), and pancreas (lane 8). B,
cell type-specific analysis with a human cancer cell line MTN Blot
membrane was performed on 2 µg of poly(A)+ RNA from
promyelocytic leukemia HL-60 (lane 1), HeLa cell S3
(lane 2), chronic myelogenous leukemia K-652 (lane
3), lymphoblastic leukemia MOLT-4 (lane 4), Burkitt's
lymphoma Raji (lane 5), colorectal adenocarcinoma SW480
(lane 6), lung carcinoma A549 (lane 7), and
melanoma (lane 8). Membranes were hybridized with a
32P-labeled presumptive 2-sulfotransferase-specific probe
for autoradiography as described under "Experimental Procedures."
The positions of the molecular size standards are indicated at the
left.
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Characterization of Dermatan [35S]Sulfate and
Chondroitin [35S]Sulfate--
35S-Labeled
dermatan/chondroitin sulfate glycosaminoglycans were digested with
chondroitin ABC lyase alone and with chondroitin ABC lyase immediately
followed by disaccharide 2-sulfatase, 4-sulfatase, or 6-sulfatase.
The digests were then analyzed by HPLC on a YMC-Pack Polyamine II
column as described under "Experimental Procedures." The major
35S-labeled disaccharide from dermatan sulfate was found to
chromatograph with standard
Di-2,4S (
HexA-2S-GalNAc-4S) (Fig.
5A). Following 2-sulfatase
digestion, the radioactivity was shifted to the position of free
sulfate (Fig. 5B); following 4-sulfatase it was shifted to
the position of
Di-2S (
HexA-2S-GalNAc)(Fig. 5C); but
following 6-sulfatase it did not shift (Fig. 5D). These
results established that the enzyme was a uronyl 2-sulfotransferase.
Chondroitin B lyase, which degrades between GalNAc-4S and IdceA or
IdceA-2S but not if there is GlcA or GlcA-2S, provided disaccharides as the only 35S-labeled product (not shown). This confirmed
that the enzyme was an IdceA 2-sulfotransferase.

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Fig. 5.
HPLC of disaccharide products formed by
digestion of dermatan [35S]sulfate with chondroitin ABC
lyase and sulfatases. 35S-Labeled products were
chromatographed with standards as described under "Experimental
Procedure" after digestion of dermatan [35S]sulfate
with chondroitin ABC lyase (A), followed by 2-sulfatase
(B), 4-sulfatase (C), or 6-sulfatase
(D). The broken line indicates the concentration
of NaH2PO4. The arrows indicate the
elution positions of standards.
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Desulfated dermatan sulfate and chondroitin sulfate C used similarly as
potential [35S]sulfate acceptors were degraded, and
disaccharide products were characterized in the same fashion.
Comparison with the disaccharide products from the dermatan sulfate
2-sulfation are shown (Table II). The
predominant disaccharides from 35S-labeled desulfated
dermatan sulfate were shown to be
Di-2S and
Di-2,4S, indicating
that 2-sulfation could take place on IdceA residues adjacent to
non-sulfated GalNAc as well as next to the small amount of GalNAc-4S
that apparently had remained following chemical desulfation to
prepare the dermatan. B lyase had no action on the chondroitin sulfate
C, but there were 35S-sulfated disaccharides equally
produced by ABC lyase or AC lyase that were found to consist almost
entirely of
Di-2,6S (
HexA-2S-GalNAc-6S). This indicated that some
sulfation of GlcA residues had taken place but essentially only if
there were an adjacent GalNAc-6S. This was confirmed by showing that
there was no incorporation of sulfate into GlcA residues of the
desulfated chondroitin. It was of interest to note that the need for
GalNAc-6S adjacent to a GlcA was in contrast to the sulfation of IdceA
of dermatan sulfate that occurred mainly where there was an adjacent
GalNAc-4S. We also used chondroitin sulfate A (Seikagaku Corp.) as an
acceptor. However, chondroitin AC lyase did not degrade it completely,
showing that it contained considerable dermatan residues as well as
chondroitin 4-sulfate and chondroitin 6-sulfate. Analysis of the small
amount of 2-sulfated AC lyase products indicated that there was mainly
Di-2,6S, a minor amount of
Di-2,4S, and no
Di-2S (data not shown).
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DISCUSSION |
Comparison of the amino acid sequences of the dermatan/chondroitin
sulfate uronyl 2-sulfotransferase with the CHO cell heparan sulfate
IdceA 2-sulfotransferase (15) as well as the C. elegans (32)
and D. melanogaster proteins (33) showed ~50% similarity, which was concentrated on the middle region across 210 amino acid residues. This was where the 5'-phosphosulfate binding motif, its
catalytic Lys, and 3'-phosphate binding motif were found, corresponding
to the previous reports concerning the heparan sulfate IdceA
2-sulfotransferase (29, 30) (Fig. 3). The homologous regions of these
two uronyl 2-sulfotransferases can be partially characterized as
containing four invariant cysteines (Fig. 3, consensus sequence 247, 255, 268, 274), whereas the sulfotransferase domain of heparan sulfate
GlcNS 3-sulfotransferase isoforms (16, 18), 6-sulfotransferase (17),
and N-sulfotransferase isoforms (12-14) contained no more
than two invariant cysteines. In addition, neither the sequence of the
so-called "P-loop," GXXGXXK(R), often observed in sulfotransferases and thought to be a possible ATP- or
GTP-binding site (34), nor LEKCGR, the putative PAPS-binding site found
in arylsulfotransferase IV (35), was found in either of the two IdceA
2-sulfotransferases.
The amino-terminal sequence of the dermatan/chondroitin sulfate
2-sulfotransferase cDNA contains three in-frame ATG codons (Fig.
1). When the sequence surrounding the first ATG codon is compared with
the eukaryotic consensus translation sequence (36, 37), the purine G at
position
3 is conserved, whereas G at position +4 is not. The
sequence surrounding the second and third ATG codons (Met-21 and Met-52
in Fig. 1) also partially fit the consensus sequence; the nucleotide at
position
3 of these ATG codons is not a purine, whereas the
nucleotide at position +4 is G. The third ATG codon (Met-52), however,
is unlikely to be an initiation site because of its location in the
amino-terminal transmembrane domain (Fig. 1). It remains to be
determined which ATG codons could function as the initiation codon.
Northern analysis showed two transcripts of 5.1 and 2.0 kb (Fig. 4),
similar to heparan sulfate IdceA 2-sulfotransferase (5.0 and 3.0 kb)
(15). Such multiple transcripts of different sizes are also observed in
other glycosaminoglycan sulfotransferases (11-13, 18, 38) and are
likely to be due to the difference in size and sequence of the
untranslated regions. The possible existence of largely different
untranslated regions may be important in the function and distribution
of the transcripts (38).
The expressed dermatan/chondroitin sulfate uronyl 2-sulfotransferase
catalyzed some 2-sulfation of the IdceA residues of IdceA-GalNAc and
better sulfation of IdceA-GalNAc-4S. No 2-sulfation of dermatan-6S or
4,6S residues was found. The enzyme also had some activity in
2-sulfation of GlcA residues of GlcA-GalNAc-6S of chondroitin sulfate,
but essentially no 2-sulfation of unsulfated disaccharide residues and
little or no sulfation of 4S disaccharide residues (Table II). The
activity on chondroitin sulfate raises the possibility that this enzyme
functions in vivo for 2-sulfation of chondroitin 6-sulfate,
but alternatively it is possible that this is due to a certain degree
of nonspecificity. The results conform with the 2-sulfation of
IdceA and GlcA found in connective tissue of many species, where
IdceA-2S has not been described in the absence of GalNAc-4S, and
GlcA-2S has not been described in the absence of GalNAc-6S. The
differences in GalNAc sulfation specificities are apparently due to
differences in the conformation of IdceA and GlcA with their
positioning relative to the GalNAc-4S and GalNAc-6S, respectively. The
results show that 2-sulfation of IdceA preferentially occurs next to
GalNAc-4S rather than non-sulfated GalNAc, and 2-sulfation of GlcA
requires GalNAc-6S with no 2-sulfation next to non-sulfated GalNAc.
Thus the biosynthetic order apparently proceeds by prior GalNAc
4-sulfation for 2-sulfation of IdceA and prior GalNAc 6-sulfation for
2-sulfation of GlcA.
Tissue-specific patterns of epimerization and 4- and 6-sulfation in
dermatan sulfate have not been reported in detail, and no preferred
chain location or distribution of 2-sulfated IdceA has been reported.
Comparison between decorin dermatan sulfate and biglycan dermatan
sulfate was reported to show a greater correlation to tissue source
than to the core protein (39). It is likely, however, that contiguous
disaccharide sequences containing IdceA or IdceA-2S residues might
account for the weak interaction reported with many heparin/heparan
sulfate-binding proteins such as basic fibroblast growth factor (40),
histidine-rich glycoprotein, platelet factor 4 (41), fibronectin (42),
interleukin-7 (43), and protein C inhibitor (44). In contrast to these
weak interactions, a comparable high affinity interaction of dermatan
sulfate has been demonstrated with heparin cofactor II (7, 8) and with hepatocyte growth factor/scatter factor (9). Furthermore, the affinity
for heparin cofactor II was shown to be dependent upon 2-sulfation of
the IdceA residues (45). Sulfation profiles of chondroitin sulfate have
been shown to change with concomitant specific spatiotemporal patterns
in various tissues, suggesting that differences in sulfation position
and degree might have distinct functions in development (46).
The GlcA 2-sulfated chondroitin has been shown to be expressed by
immature glial cells of the central nervous system to promote neurite
outgrowth (47) and to be expressed in the cerebellum and the
telencephalon of adult mouse (48). This would suggest that our finding
of IdceA/GlcA 2-sulfotransferase expression in brain and especially
cerebellum (data not shown) could account for the 2-sulfation of
chondroitin sulfate in brain. Disaccharide residues of
GlcA-2S-GalNAc-6S have also been identified in mouse mast cells derived
from immune lymph nodes and function as an important phenotypic marker
distinguishing different mast cell subsets (49) and mouse tooth germ
basement membrane (46).
Characteristic oligosaccharide sequences including 2-sulfated IdceA or
GlcA residues in DS and CS may serve as functional domain structures
recognized by some protein ligands as in heparin/heparan sulfate.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Keiichi Yoshida
(Seikagaku Corp.) for providing
4,5hexuronate-2-sulfatase and to members of the HHMI/MIT
Biopolymers Laboratory for sequencing DNA samples. We also thank Drs.
Koji Kimata, Hiroko Habuchi (Aichi Medical University), and Rosenberg Laboratory members for insightful suggestions.
 |
FOOTNOTES |
*
This research was supported by National Institutes of Health
Grant HL-66385 (to R. D. R.), by the Veterans Affairs Medical Research Service, and by National Institutes of Health Grant AR-41649 (to G. S.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB020316.

To whom correspondence and reprint requests should be
addressed: Massachusetts Institute of Technology, 68-480, 77 Massachusetts Ave., Cambridge, MA 02139. Tel.: 617-253-8803; Fax:
617-258-6553.
 |
ABBREVIATIONS |
The abbreviations used are:
GlcA, D-glucuronic acid;
PAPS, 3'-phosphoadenosine
5'-phosphosulfate;
CHO, Chinese hamster ovary;
EST, expressed
sequence-tagged;
PCR, polymerase chain reaction;
SSC, sodium
citrate-sodium chloride;
HPLC, high performance liquid chromatography;
CDSNS-heparin, completely desulfated and
N-resulfated-heparin;
HexA, 4-deoxy-
-threo-hex-4-enepyranosyluronic acid;
Di-2S, 2-acetamido-2-deoxy-3-O-(
-D-Glc-4-enepyranosyluronic
acid-2-O-sulfo)-D-galactose;
Di-2, 4S,
2-acetamido-2-deoxy-3-O-(
-D-Glc-4-enepyranosyluronic
acid-2-O-sulfo)-4-O-sulfo-D-galactose;
Di-2, 6S,
2-acetamido-2-deoxy-3-O-(
-D-Glc-4-enepyranosyluronic
acid-2-O-sulfo)-6-O-sulfo-D-galactose;
Di-2, 4,6S,
2-acetamido-2-deoxy-3-O-(
-D-Glc-4-enepyranosyluronic
acid-2-O-sulfo)-4-O-sulfo-6-O-sulfo-D-galactose;
bp, base pair(s);
kb, kilobase pair(s).
 |
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