(Received for publication, August 7, 1995; and in revised form, January 11, 1996)
From the
Heparan sulfate 2-sulfotransferase, which catalyzes the transfer
of sulfate from adenosine 3`-phosphate 5`-phosphosulfate to position 2
of L-iduronic acid residue in heparan sulfate, was purified
51,700-fold to apparent homogeneity with a 6% yield from cultured
Chinese hamster ovary cells. The isolation procedure included a
combination of affinity chromatography on heparin-Sepharose CL-6B and
3`,5`-ADP-agarose, which was repeated twice for each, and finally gel
chromatography on Superose 12. Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis of the purified enzyme showed two protein bands
with molecular masses of 47 and 44 kDa. Both proteins appeared to be
glycoproteins, because their molecular masses decreased after N-glycanase digestion. When completely desulfated and N-resulfated heparin and mouse Engelbreth-Holm-Swarm tumor
heparan sulfate were used as acceptors, the purified enzyme transferred
sulfate to position 2 of L-iduronic acid residue but did not
transfer sulfate to the amino group of glucosamine residue or to
position 6 of N-sulfoglucosamine residue. Heparan sulfates
from pig aorta and bovine liver, however, were poor acceptors. The
enzyme showed no activities toward chondroitin, chondroitin sulfate,
dermatan sulfate, and keratan sulfate. The optimal pH for the enzyme
activity was around 5.5. The enzyme activity was minimally affected by
dithiothreitol and was stimulated strongly by protamine. The K value for adenosine 3`-phosphate
5`-phosphosulfate was 0.20 µM.
Heparan sulfate and heparin bind to a variety of proteins, such
as growth factors and protease inhibitors, suggesting their involvement
not only in a fundamental cellular behaviors such as cell growth,
differentiation, and cell adhesion, but also in the anticoagulation
process and some pathological processes such as viral infections (1, 2, 3) . Bindings of those ligands to
heparan sulfate or heparin seem to be mediated via specific structures
in heparan sulfate or heparin. For example, basic fibroblast growth
factor (FGF-2) ()interacts with a cluster of
GlcNSO
-IdoA(2SO
) in heparan
sulfate(4, 5, 6, 7, 8) ,
and, in addition, its high affinity receptor appears to interact with
some specific sites containing
GlcNSO
(6SO
)-IdoA(2SO
) in heparan
sulfate(8) . It has recently been suggested that heparan
sulfate proteoglycans on cell surfaces as well as in extracellular
matrix may have such specific structures and regulate the biological
activity of basic fibroblast growth factor(9) . In fact, the
response of neural cells to either acidic or basic fibroblast growth
factor (FGF-1 or FGF-2, respectively) seems to be regulated by
developmentally modulated forms of heparan sulfate
proteoglycans(10) . Thus, microheterogeneity in the heparan
sulfate structures, particularly in the sulfation positions may be an
important factor to control the biological activity of basic fibroblast
growth factor. In this regard, it is important to study how the
microheterogeneity is caused and regulated.
Various types of sulfotransferases have been shown to be responsible for the sulfation of heparin and heparan sulfate: sulfation of 2-N(11, 12, 13, 14) , 6-O(13, 15, 16) , and 3-O(17) of glucosamine residue, sulfation of 2-O(16) of L-iduronic acid residue, and sulfation of 2-O(18) of D-glucuronic acid residue. The sulfate donor in these reactions is adenosine 3`-phosphate 5`-phosphosulfate (PAPS) that is synthesized in the cytosol and transported into the lumen of the Golgi to serve as substrate(19) . N-Sulfotransferases have been purified to homogeneity from rat liver and mouse mastocytoma(11, 12) . Recently, molecular cloning studies have suggested that these N-sulfotransferases were closely related to but were clearly distinct from each other(20, 21, 22, 23) , suggesting that the biosynthesis of heparan sulfate and heparin may be catalyzed by different enzymes and independently regulated.
It has been
suggested that O-sulfation is the final step in the
modification of the structure during the biosynthesis of heparin and
heparan sulfate in the lumen of the Golgi
apparatus(24, 25) . We recently purified heparan
sulfate 6-sulfotransferase (HS6ST) that catalyzes the transfer of
sulfate to position 6 of N-sulfoglucosamine residue in heparan
sulfate, with a high yield from the serum-free culture medium of
Chinese hamster ovary (CHO) cells(15) . However, the enzyme
activity to catalyze the 2-O-sulfation of L-iduronic
acid residue of heparin and heparan sulfate was hardly detected in the
culture medium of CHO cells, although heparan sulfate prepared from CHO
cells contained HexA(2SO) and
GlcNSO
(6SO
) residues in a proportion of 4:3.
These observations have interested us in the intracellular O-sulfotransferases of CHO cells. We found in the present
study that in CHO cell culture more than 90% of activities of heparan
sulfate O-sulfotransferases in the cell layer catalyzed the
transfer of sulfate to position 2 of L-iduronic acid residue.
In this report, we describe the purification to apparent homogeneity
and some properties of this sulfotransferase from the cultured CHO
cells (this enzyme was designated as heparan sulfate 2-sulfotransferase
(HS2ST)).
For
purification of HS2ST, CHO cells were plated onto 10-cm culture dishes
at a density of 2.0 10
cells/dish in 10 ml of
CHO-S-SFM-II containing antibiotics. After 4 days the CHO cells were
transferred to 500-ml spinner flasks (Techne, Cambridge, United
Kingdom) at a density of 3.0
10
cells/ml and
continued culturing with constant agitation of 90 rpm for 4 days in 500
ml of the same medium. The CHO cells were harvested and washed with
phosphate-buffered saline. The cell pellet was suspended in the
solution (55 ml/1
10
cells) of 10 mM Tris-HCl, pH 7.2, 0.5% (w/v) Triton X-100, 10 mM MgCl
, 2 mM CaCl
, 0.15 M NaCl, 20% (v/v) glycerol, and a mixture of protease inhibitors (5
µMN
-p-tosyl-L-lysine
chloromethyl ketone, 3 µMN-tosyl-L-phenylalanine chloromethyl ketone, 30
µM phenylmethylsulfonyl fluoride, and 3 µM pepstatin A). Extraction of sulfotransferases from the cells was
carried out by homogenizing the cell suspension in the above buffer
with a glass homogenizer on ice. After the gentle stirring at 4 °C
for 1 h, the homogenate was centrifuged at 4 °C for 30 min at
10,000
g. The supernatant was pooled (1.8 liters),
designated as the crude extract, and stored at -20 °C until
use.
Figure 1:
First heparin-Sepharose
CL-6B chromatography of the crude extract from cultured CHO cells. The
crude extract from cultured CHO cells was applied to a
heparin-Sepharose column as described under ``Experimental
Procedures.'' After wash with buffer A containing 0.15 M NaCl, the column was eluted with a linear gradient of NaCl.
Fractions of 13 ml were collected. Sulfotransferase activity in the
absence () or presence (
) of 10 mM DTT, and protein
concentration(- - - -) of each fraction were assayed. The broken
line(- - -) indicates the concentration of
NaCl.
Figure 2:
Second
heparin-Sepharose CL-6B chromatography of the first 3`,5`-ADP-agarose
fractions. The fractions eluted from the first 3`,5`-ADP-agarose column
with 0.2 mM 3`,5`-ADP in buffer A containing 0.05 M NaCl were applied to a heparin-Sepharose column as described under
``Experimental Procedures.'' After wash with buffer A
containing 0.15 M NaCl, the column was eluted with a linear
gradient of NaCl. Fractions of 4 ml were collected. Sulfotransferase
activity in the absence () or presence (
) of 10 mM DTT, and protein concentration (- - -) of each fraction were
assayed. The broken line (- - -) indicates
the concentration of NaCl.
Figure 3:
Superose 12 gel chromatography of the
second 3`,5`-ADP-agarose fraction. A, the fraction eluted from
the second 3`,5`-ADP-agarose column with 0.2 mM 3`,5`-ADP in
buffer A containing 0.05 M NaCl was applied to a small
heparin-Sepharose CL-6B column equilibrated with 0.1 M NaCl in
buffer A. After the column was eluted with 2.5 ml of 1 M NaCl
in buffer B, the eluate was concentrated and dialyzed against 1 M NaCl in buffer A. The dialysate was injected into a Superose 12
column and eluted with buffer A containing 2 M NaCl as
described under ``Experimental Procedures.'' Fractions of 250
µl were collected. Sulfotransferase activity in the absence
() or presence (
) of 10 mM DTT of each fraction was
assayed. The arrows indicate the elution positions of bovine
serum albumin (67 kDa)(1) , ovalbumin (43 kDa)(2) , and
chymotrypsinogen A (25 kDa) (3) under the same chromatographic
conditions as described above. B, aliquots of every other
fraction that showed the activity were analyzed by SDS-PAGE. Proteins
were visualized with silver nitrate stain. Molecular size standards
were phosphorylase b (94 kDa), bovine serum albumin (67 kDa),
ovalbumin (43 kDa), carbonic anhydrase (30 kDa), and soybean trypsin
inhibitor (20.1 kDa).
The details for each purification step are as follows.
Figure 4:
Heparin-Sepharose CL-6B chromatography of
the second 3`,5`-ADP-agarose fraction. A, an aliquot of the
second 3`,5`-ADP-agarose fraction in buffer A containing 0.1 M NaCl was applied to a heparin-Sepharose column (bed volume, 2.5
ml) equilibrated with 0.1 M NaCl in buffer A at a flow rate of
6 ml/h. After wash with 7.5 ml of buffer A containing 0.1 M NaCl, the column was eluted with a linear gradient from 0.1 to 1 M NaCl in buffer A (total volume, 75 ml). Fractions (1 ml)
were collected. Sulfotransferase activity () in the presence of 10
mM DTT of each fraction was assayed. The broken line indicates the concentration of NaCl. B, aliquots of every
fourth fraction that showed the activity were analyzed by SDS-PAGE.
Proteins were visualized with silver nitrate stain. Molecular size
standards were the same as described in Fig. 3B.
Samples at each purification step (0.47
µg of protein for each) were also analyzed by SDS-PAGE (Fig. 5A). Both protein bands of M 47,000 and 44,000 were stained more intensely with silver nitrate
in the samples at the higher fold purification, and were the most
predominantly stained in the Superose 12 fraction (``purified
HS2ST'') (Fig. 5A, lane 7). In addition,
the fraction that passed through the second 3`,5`-ADP-agarose column
and had the little activity exhibited little if any of the two protein
bands on SDS-PAGE (Fig. 5A, lane 6). Such a
concomitancy between the two protein bands and the HS2ST activity again
suggested that the two protein bands correspond to HS2ST.
Figure 5: SDS-PAGE of heparan sulfate 2-sulfotransferase fractions at various purification steps (A) and of the purified heparan sulfate 2-sulfotransferase before and after treatment with N-glycanase (B). A, 0.47 µg of protein was loaded onto each lane. Lane 1, the crude extract; lane 2, protein eluted with a NaCl gradient from the first heparin-Sepharose CL-6B column; lane 3, protein eluted with 0.2 mM 3`,5`-ADP from the first 3`,5`-ADP-agarose column; lane 4, protein eluted with a NaCl gradient from the second heparin-Sepharose CL-6B column; lane 5, protein eluted with 0.2 mM 3`,5`-ADP from the second 3`,5`-ADP-agarose column; lane 6, protein that passed through the second 3`,5`-ADP-agarose column (no sulfotransferase activity); lane 7, protein eluted with 2 M NaCl in buffer A from Superose 12 column (purified HS2ST). B, lane 1, 6.6 ng of protein eluted with 2 M NaCl in buffer A from Superose 12 column (purified enzyme); lane 2, digests of 6.6 ng of the purified enzyme treated with N-glycanase; lane 3, the same amount of N-glycanase alone as in lane 2. Proteins were visualized with silver nitrate stain. Molecular size standards were the same as described in Fig. 3B.
When the N-glycanase digest of the purified HS2ST was subjected to SDS-PAGE, the two protein bands of 47 and 44 kDa disappeared, but two sharp protein bands of 38 and 34 kDa appeared newly (Fig. 5B), indicating that both proteins are glycoproteins containing almost equal contents of carbohydrates (approximately 20%). The result further suggested that both proteins are closely related to each other.
However, it was worthy of note
that the molecular weights of the two protein bands were half or
one-third as much as the molecular weight of the HS2ST activity
determined by gel chromatography on Superose 12 using standard M proteins (Fig. 3A). When the
purified HS2ST was again applied to the Superose 12 gel column, which
was this time equilibrated with buffer A containing 4 M guanidine HCl (known to be a ``dissociative'' solvent),
the HS2ST activity mostly disappeared in the fractions around M
130,000 but, instead of it, a little but
significant activity (<0.3% of the starting activity) was recovered
in the fractions where ovalbumin (43 kDa) was eluted. Subsequent
SDS-PAGE of the fractions revealed that the two proteins of M
47,000 and 44,000 newly appeared in the
fractions around 43 kDa (data not shown). The observed discrepancy
between their elution position on gel chromatography and their
mobilities on SDS-PAGE could be explained by the possibility that HS2ST
may exist as a dimer or trimer, although other possibilities could also
be considered.
Figure 6:
HPLC on PAMN column of
heparitinase-digests of S-labeled products formed from
CDSNS-heparin (A) and mouse EHS tumor heparan sulfate (B) by incubation with [
S]PAPS and the
purified heparan sulfate 2-sulfotransferase. The products of the
sulfotransferase reactions were digested with a mixture of
heparitinases and subjected to HPLC on a PAMN column as described under
``Experimental Procedures.'' The broken line indicates the concentration of KH
PO
. The arrows indicate the elution positions of standards;
Di-6S (1),
Di-NS (2),
Di-(6)diS (3),
Di-(U)diS (4), and
Di-(6,U)triS (5).
Figure 7:
HPLC on Partisil-10 SAX column of
disaccharides produced by nitrous acid degradation at pH 1.5 of S-labeled products formed from CDSNS-heparin (A)
and mouse EHS tumor heparan sulfate (B) by incubation with
[
S]PAPS and the purified heparan sulfate
2-sulfotransferase. The products of sulfotransferase reaction were
degraded by nitrous acid at pH 1.5 and treated with mild acid, and
subjected to gel filtration on a Superdex 30. Then the disaccharide
fraction was applied to a Partisil-10 SAX column as described under
``Experimental Procedures.'' The broken line indicates the concentration of KH
PO
. The arrows indicate the elution positions of HexA-AMan
(1), GlcA(2SO
)-AMan
(2), GlcA-AMan
(6SO
) (3), IdoA-AMan
(6SO
) (4), and
IdoA(2SO
)-AMan
(5).
Figure 8:
Sulfation site in heparan sulfate by
heparan sulfate 2-sulfotransferase. An arrow indicates the
sulfation site by purified HS2ST. Sulfations of position 2 of GlcA (a) and of position 2 of IdoA adjacent to
GlcNSO(6SO
) (b) were not
observed.
Figure 9:
Properties of the purified heparan sulfate
2-sulfotransferase. Sulfotransferase activities were determined using
0.21 ng of the purified enzyme as described under ``Experimental
Procedures.'' A, pH dependence of the sulfotransferase
activity. The pH of the reaction mixtures was varied using 2.5 µmol
of Tris-HCl (), 2.5 µmol of imidazole HCl (
), 2.5
µmol of Mes (
), or 2.5 µmol of potassium acetate
(
) buffer. Relative activity was expressed as a ratio to the
sulfotransferase activity obtained from the standard reaction mixture. B, effect of DTT on the sulfotransferase activity. The
reaction mixtures contained various amounts of DTT. Relative activity
was expressed as a ratio to the sulfotransferase activity obtained from
the standard reaction mixture without DTT. C, effect of NaCl
on the sulfotransferase activity. The reaction mixtures contained
various concentrations of NaCl. Relative activity was expressed as a
ratio to the sulfotransferase activity obtained from the standard
reaction mixture without NaCl. D, effect of protamine on the
sulfotransferase activity. The reaction mixtures contained various
amounts of protamine. Relative activity was expressed as a ratio to the
sulfotransferase activity obtained from the standard reaction mixture
without protamine. E, dependence of the sulfotransferase
activity on PAPS concentration. Sulfotransferase activities were
determined using 0.15 ng of the purified enzyme as described under
``Experimental Procedures'' except that the reaction mixtures
contained various amounts of PAPS.
We have purified HS2ST to an apparent homogeneous level from the extract of the cultured CHO cells by affinity chromatography with heparin-Sepharose and 3`,5`-ADP-agarose. As was also the case with heparan sulfate/heparin N-sulfotransferase, HS6ST and chondroitin 6-sulfotransferase(11, 12, 15, 32) , affinity chromatography on those columns yielded successful purification of this enzyme. Furthermore, gel chromatography on Superose 12 resulted in the effective separation of the HS2ST activity from HS6ST.
The purified HS2ST fraction was found to transfer
sulfate exclusively to position 2 of the L-iduronic acid
residue of IdoA-GlcNSO unit in CDSNS-heparin or EHS tumor
heparan sulfate (Fig. 8), and none of the activity was observed
to transfer sulfate to position 2 of D-glucuronic acid
residue, position 6 of N-sulfoglucosamine residue or amino
group of glucosamine residue. Wlad et al.(33) have
recently purified
60-kDa enzyme capable of catalyzing both the
2-O- and 6-O-sulfotransferase reactions from mouse
mastocytoma tissue. A proteolytic fragment (
20 kDa) of this
original enzyme remains capable of promoting 2-O-sulfation but
has lost the 6-O-sulfotransferase activity, indicating that
the two reactions are catalyzed by separate active sites derived from a
single protein. We previously showed that HS6ST from the culture medium
of CHO cells transfers sulfate exclusively to position 6 of N-sulfoglucosamine residue(15) . The difference
between the reports by Wlad et. al. and us suggests that our
HS6ST and HS2ST from CHO cells are distinct from their enzyme from
mouse mastocytoma tissue capable of catalyzing both the 2-O-
and 6-O-sulfotransferase reactions. Supposing that HS2ST and
HS6ST be derived from a composite enzyme containing distinct domains,
each committed to a specific O-sulfotransferase reaction, the
composite enzyme would be expected to have a molecular mass of
90-100 kDa. This again suggests that our sulfotransferases may be
genetically different from the sulfotransferase purified by Wlad et
al. It is very likely that sulfotransferases prepared from CHO
cells are engaged in the biosynthesis of heparan sulfate and the enzyme
from mouse mastocytoma tissue is mainly involved in the biosynthesis of
heparin. This is also the case with N-sulfotransferases(20, 21, 22, 23) .
Considering these results, the molecular organization of the O-sulfation process may differ between heparin and heparan
sulfate.
Modification reactions in the biosynthesis of
heparin/heparan sulfate are thought to occur in the following
sequences: N-deacetylation/N-sulfation of
glucosamine, epimerization of D-glucuronic to L-iduronic acid, 2-O-sulfation of L-iduronic
acid, and finally, 6-O- and 3-O-sulfation of
glucosamine residue(1) . In these sequential reactions, each
product in the respective reaction becomes the substrate for the
subsequent reaction and may be controlled by the substrate
specificities of the enzymes involved. HS6ST has a capacity to catalyze
6-O-sulfation of N-sulfoglucosamine residue
irrespective of the 2-O-sulfation of the neighboring L-iduronic acid residue(15) , while HS2ST is unable to
catalyze 2-O-sulfation of L-iduronic acid residue
adjacent to GlcNSO(6SO
) residue. This
difference strongly supports the above modification reaction sequence.
In relation to this control mechanism, it should be of note that HS2ST
was much less active toward heparan sulfates from pig aorta and bovine
liver (Table 3). There seem to be some differences in the
acceptor efficiency between EHS tumor heparan sulfate and the above two
heparan sulfates as the good and poor acceptor substrates,
respectively. The content of HexA-GlcNSO
unit in heparan
sulfate may be one of factors affecting the substrate efficiency,
because the content of this unit in EHS tumor heparan sulfate is the
highest (Table 4). However, heparan sulfates from pig aorta and
bovine liver contain significant amount of HexA-GlcNSO
unit, although they are very poor acceptors, suggesting that
other factors such as the higher content of IdoA-GlcNSO
unit or a longer oligosaccharide sequence containing
IdoA-GlcNSO
unit may be required for the recognition by
HS2ST. Furthermore, HS2ST may be negatively controlled by its reaction
products, IdoA(2SO
)-GlcNSO
and
IdoA(2SO
)-GlcNSO
(6SO
) units,
because heparin, a form of heparan sulfate containing the highest
contents of these units, appeared to act as an inhibitor and, in
addition, heparan sulfates from pig aorta and bovine liver, poorer
acceptors had the relatively higher contents of
IdoA(2SO
)-GlcNSO
and
IdoA(2SO
)-GlcNSO
(6SO
) units (8.2
and 37.2%, respectively) than EHS tumor heparan sulfate (3.0%) (Table 4). Since the purified HS2ST showed no activity toward
dermatan sulfate, the enzyme does not appear to transfer sulfate to
position 2 of L-iduronic acid residue adjacent to N-acetylgalactosamine residue.
It is interesting to compare
the properties of HS2ST with those of other purified glycosaminoglycan
sulfotransferase. DTT had little effect on HS2ST, while it
substantially inhibited HS6ST(15) . This finding was useful to
monitor activities of both sulfotransferases separately in the present
study. In contrast, DTT stimulated chondroitin
4-sulfotransferase(34) . HS2ST appeared to exist as a dimer or
trimer, because the enzyme behaved on gel chromatography 2-3
times larger than on SDS-PAGE (Fig. 3). However, considering the
effect of Triton X-100 micelles on molecular mass determinations of
membrane proteins, there is another possibility that HS2ST may exist as
a monomer. Mass determinations for proteins embedded in the micelles
might have caused the overestimation by the mass of the detergent
micelle. The guanidine treatment would have dissociated the micelles,
and, therefore, the enzyme might have run as a monomer independently of
the detergent. Similarly to HS2ST, chick chondrocyte chondroitin
6-sulfotransferase apparently showed a single broad protein band of M 75,000 on SDS-PAGE although it was eluted at the
position with an apparent molecular mass of 160,000 on gel
chromatography(32, 35) . A majority of proteins
intrinsic to the Golgi apparatus membrane appear to be dimers in
situ(36) . However, this is not always the case. HS6ST
from CHO cells may be a monomer(15) . Rat liver heparan sulfate N-deacetylase/N-sulfotransferase is also a
monomer(11, 37) . The apparent K
value for PAPS of the purified HS2ST was 2.0
10
M, which was in the same order of that
of HS6ST (4.4
10
M)(15) .
In contrast, that of heparan sulfate N-sulfotransferase of rat
liver was 1.08
10
M(38) .
HS2ST as well as HS6ST appeared to have a higher affinity for PAPS than
heparan sulfate N-sulfotransferase. The large difference in
the K
value for PAPS between our sulfotransferases
and heparan sulfate N-sulfotransferase, however, might be due
to the differences in the assay conditions used. For example, we added
cationic activator, protamine, to the assay mixture in this study,
while the assay mixture reported for heparan sulfate N-sulfotransferase (38) included Mg
and Mn
, instead of protamine. We previously
showed that cationic proteins such as protamine and histone stimulated
chondroitin 6-sulfotransferase by decreasing the K
value for PAPS(27) . The low K
values for PAPS of HS2ST as well as HS6ST may have been caused by
the presence of protamine.
SDS-PAGE of the purified HS2ST gave only two protein bands of 47 and 44 kDa, which were both sensitive to N-glycanase digestion to the same extent (Fig. 5), as was also observed with HS6ST (15) and chondroitin 6-sulfotransferase(32, 35) . Both proteins were always comigrated in the SDS gel whenever the fractions containing the HS2ST activity were subjected to SDS-PAGE (see Fig. 3and Fig. 4for the Superose 12 column chromatography and the heparin-Sepharose CL-6B column chromatography, respectively). These findings strongly support that both the 47- and 44-kDa proteins bear HS2ST activity and the size difference may be due to some protein modification such as limited proteolytic cleavage as we discussed previously in the case of HS6ST (15) . Several glycosyltransferases that exhibit catalytically active multiple forms with different molecular weights have been shown to be derived from the single genes(39, 40, 41, 42) . However, since our several trials to detect the HS2ST activity in the gel segments after SDS-PAGE of the purified HS2ST were unsuccessful, probably owing to a difficulty of renaturation of the enzyme activity (data not shown), one could still argue that either or both of the 47- and 44-kDa proteins represent contaminants. Relating to this possibility, one could also argue that the extent of purification was rather low (51,700-fold), compared with those of other Golgi enzymes. Because HS6ST was purified 10,700-fold to apparent homogeneity from the culture medium of CHO cells, the obtained extent of purification for the HS2ST from the Triton X-100 extract of cultured CHO cells is not far from the expected range.
It should be noted that in cultured CHO cells more than 90% of the HS6ST activity was secreted into the medium, while 97% of the HS2ST activity was retained in the cells. Several reports have shown that the stem regions are cleaved off proteolytically when proteins originally present in the Golgi apparatus such as glycosyltransferases are secreted(42, 43) . The molecular cloning of chondroitin 6-sulfotransferase suggests that the enzyme may be released from the Golgi apparatus by proteolytic cleavage at the specific sequence of the transmembrane domain(35) . HS6ST may have such a sequence, while HS2ST may lack it. Molecular cloning of both sulfotransferases should provide us with a clue to answer these possibilities.