(Received for publication, September 9, 1994; and in revised form, November 28, 1994)
From the
Heparan sulfate 6-sulfotransferase, which catalyzes the transfer
of sulfate from 3`-phosphoadenylyl sulfate to position 6 of N-sulfoglucosamine in heparan sulfate, was purified
10,700-fold to apparent homogeneity with a 40% yield from the
serum-free culture medium of Chinese hamster ovary cells. The isolation
procedure included affinity chromatography of the first
heparin-Sepharose CL-6B column (stepwise elution), 3`,5`-ADP-agarose,
and the second heparin-Sepharose CL-6B column (gradient elution).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the
purified enzyme showed two protein bands with molecular masses of 52
and 45 kDa. Both proteins appeared to be glycoproteins, because their
molecular masses decreased after N-glycanase digestion. When
completely desulfated and N-resulfated heparin was used as
acceptor, the purified enzyme transferred sulfate to position 6 of N-sulfoglucosamine residue but did not transfer sulfate to the
amino group of glucosamine residue or to position 2 of the iduronic
acid residue. Heparan sulfate was also sulfated by the purified enzyme
at position 6 of N-sulfoglucosamine residue. Chondroitin and
chondroitin sulfate did not serve as acceptors. The optimal pH for
enzyme activity was around 6.3. The enzyme activity was inhibited by
dithiothreitol and was stimulated strongly by protamine. The K value for adenosine 3`-phosphate
5`-phosphosulfate was 0.44 µM.
Heparan sulfate and heparin interact with a variety of proteins,
such as growth factors, extracellular matrix components, and protease
inhibitors, suggesting their involvement not only in a variety of
cellular aspects, 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) . Interactions of heparan
sulfate/heparin with those ligands seem to be mediated by the binding
of ligands to specific structures in heparan sulfate/heparin. For
example, the basic fibroblast growth factor interacts with a cluster of
GlcNSO-IdoA(2SO
) in heparan sulfate (4, 5, 6, 7, 8) , (
)and 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 chains of cell surface and extracellular matrix heparan sulfate
proteoglycan, which could be distinguished from any known proteoglycan
such as syndecan, fibroglycan, or glypican, regulate basic fibroblast
growth factor receptor binding and thereby regulate the biological
activity of basic fibroblast growth factor(9) . The response of
neural cells to either acidic or basic fibroblast growth factor appears
to be regulated by developmentally modulated forms of heparan sulfate
proteoglycans (10) . Heparan sulfate from highly metastatic
tumor cells exhibited a higher degree of sulfation than that from low
metastatic tumor cells, which was due to increased contents of
6-O-sulfated glucosamine residue(11) .
Microheterogeneity in the heparan sulfate structure, particularly in
sulfation patterns at various positions, may play an important role in
these cellular aspects. Thus, it is important to study how the
microheterogeneity is yielded and regulated. It is suggested that O-sulfation is the final step in the modification of the
structure during the biosynthesis of heparin and probably heparan
sulfate as well(12, 13) . Therefore, O-sulfation at various positions of heparan sulfate is an
important step in determining the structure of each functional domain
in heparan sulfate. Various types of sulfotransferases have been shown
to be responsible for the sulfation of heparin and heparan sulfate:
sulfation of
2-N(14, 15, 16, 17) ,
6-O(16, 19) , and 3-O(20) of glucosamine residue, sulfation of 2-O(19) of L-iduronic acid residue, and sulfation of
2-O(21) of D-glucuronic acid residue (see Fig. 1). However, only N-sulfotransferases have been
purified to homogeneity from rat liver and mouse
mastocytoma(14, 15) . More recently, molecular cloning
studies have suggested that these N-sulfotransferases were
closely related but were clearly distinct from each
other(22, 23, 24, 25) , suggesting
that the biosyntheses of heparan sulfate and heparin may be catalyzed
by different enzymes and independently regulated.
Figure 1:
Partial structures of heparan sulfate
with possible sulfation positions and the sulfation sites by heparan
sulfate 6-sulfotransferase. Arrows indicate the sulfation
sites by purified heparan sulfate 6-sulfotransferase. It remains to be
determined whether or not purified heparan sulfate 6-sulfotransferase
catalyzes the transfer of sulfate to position 6 of GlcNSO adjacent to GlcA (broken
arrow).
We recently purified chondroitin 6-sulfotransferase with a high yield from the serum-free culture medium of chick chondrocytes(26) . We have found in the present study that heparan sulfate 6-sulfotransferase (Fig. 1) was likewise secreted into the serum-free culture medium of CHO cells, although the amount of secreted heparan sulfate 6-sulfotransferase was only one-fiftieth of the amount of chondroitin 6-sulfotransferase secreted from the chondrocytes. In this paper, we describe the purification to apparent homogeneity and some properties of heparan sulfate 6-sulfotransferase from the serum-free culture medium of CHO cells.
Figure 3:
3`,5`-ADP-agarose affinity chromatography
of the first heparin-Sepharose fractions. The fractions eluted from the
heparin-Sepharose column with buffer A containing 1 M NaCl
were concentrated, dialyzed exhaustively against 0.05 M NaCl
in buffer A, and applied to a 3`,5`-ADP-agarose column as described
under ``Experimental Procedures.'' Fractions of 2 ml were
collected. Heparan sulfate O-sulfotransferase activity
() and protein concentration (
) of each fraction were
assayed. The broken line indicates the concentration of
3`,5`-ADP. The arrow indicates the elution with buffer A
containing 1 M NaCl. The horizontal bar indicates the
fractions that were pooled for further
purification.
Figure 4:
Second heparin-Sepharose CL-6B
chromatography of the 3`,5`-ADP-agarose fraction. A, the
sulfotransferase fraction from 3`,5`-ADP-agarose (indicated by horizontal bar in Fig. 3) was applied to a
heparin-Sepharose column as described under ``Experimental
Procedures.'' Fractions of 2 ml were collected. After washing with
buffer A containing 0.25 M NaCl, the column was eluted with a
linear gradient of NaCl. Chondroitin sulfotransferase activity ()
and heparan sulfate O-sulfotransferase activity (
) of
each fraction were assayed. The broken line indicates the
concentration of NaCl. Protein concentration was not determined because
of very low content. B, aliquots of every three fractions that
showed the activity were analyzed by SDS-PAGE (10% gel). 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 sharp band at 55 kDa in the fractions is
likely due to the artifact of silver staining because this sharp band
did not appear reproducibly.
Figure 2:
Analysis of S-labeled
products derived from the incubation of CDSNS-heparin,
[
S]PAPS, and the enzymes. The enzymes were
prepared from the spend medium of CHO cells (A and C), and FM3A cells (B and D), respectively,
as described in Table 1. The sulfotransferase reaction, digestion
of the product with a mixture of heparitinase, and subsequent HPLC of
the digested products on a polyamine-bound silica PAMN column were
carried out as described under ``Experimental Procedures'' (A and B). Broken lines indicate a gradient
concentration of KH
PO
. Arrows indicate
the elution positions of:
Di-6S (1),
Di-NS (2),
Di-(N,6)diS (3),
Di-(N,U) diS (4),
Di-(N,6,U)triS (5). Products of the
degradation with nitrous acid at pH 1.5 were subjected to paper
electrophoresis as described under ``Experimental
Procedures'' (C and D). The dotted lines indicate the migration of
S-free sulfate as
standard.
Figure 5: SDS-PAGE of heparan sulfate 6-sulfotransferase fractions at various purification steps. Lane 1, 0.6 µg of protein of the buffered medium fraction; lane 2, 0.6 µg of protein eluted with 1 M NaCl in buffer A from the first heparin-Sepharose CL-6B column; lane 3, 0.6 µg of protein eluted with a 3`,5`-ADP gradient from 3`,5`-ADP-agarose column; lane 4, 0.15 µg of protein eluted with a NaCl gradient from the second heparin-Sepharose CL-6B column; lane 5, 14 µg of protein from the same fraction as in lane 4, except the sample in lane 5 was reduced only with 5% mercaptoethanol before electrophoresis. Lanes 1-4 were stained with silver nitrate. Lane 5 was stained with Coomassie Brilliant Blue. Molecular size standards were the same as in Fig. 4B.
Figure 6:
Superose 12 gel chromatography of heparan
sulfate 6-sulfotransferase. Heparan sulfate 6-sulfotransferase eluted
from the second heparin-Sepharose CL-6B column was concentrated and
dialyzed against buffer A containing 2 M NaCl. 200 µl of
concentrated solution was injected into a Superose 12 column and eluted
with buffer A containing 2 M NaCl as described under
``Experimental Procedures.'' Heparan sulfate
6-sulfotransferase activity () of each fraction was assayed. The arrows indicate the elution positions of bovine serum albumin
(68 kDa) (1), ovalbumin (45 kDa) (2), and
chymotrypsinogen A (25 kDa) (3).
Figure 7: SDS-PAGE of heparan sulfate 6-sulfotransferase treated with N-glycanase. Lane 1, 0.15 µg of the purified enzyme protein; lane 2, digests of 0.15 µg of the purified enzyme protein with N-glycanase; lane 3, the same amount of N-glycanase as in lane 2. Proteins were stained with silver nitrate. Molecular size standards were the same as in Fig. 4B.
Figure 8:
HPLC on PAMN column of S-labeled CDSNS-heparin produced by incubation with
[
S]PAPS and the purified heparan sulfate
6-sulfotransferase (A) and HPLC on Partisil-10 SAX column of
the products of degradation with nitrous acid at pH 1.5 (B). A, the products of sulfotransferase reaction were digested
with a mixture of heparitinases and subjected to PAMN column as
described under ``Experimental Procedures.'' The broken
line indicates the concentration of KH
PO
.
The arrows indicate the same as in Fig. 2. B,
the products of nitrous acid degradation at pH 1.5 were subjected to
gel filtration, and the disaccharide fraction was applied to a
Partisil-10 SAX column. The conditions of HPLC were as described under
``Experimental Procedures.'' The broken line indicates the concentration of KH
PO
. The arrows indicate the elution position of HexA-AMan
(1), GlcA(2SO
)-AMan
(2), GlcA-AMan
(6SO
) (3), IdoA-AMan
(6SO
) (4),
IdoA(2SO
)-AMan
(5), and
IdoA(2SO
)-AMan
(6SO
) (6).
Figure 9:
Analysis of S-labeled heparan
sulfate produced by incubation with [
S]PAPS and
the purified heparan sulfate 6-sulfotransferase. After the
sulfotransferase reaction, the samples were digested with a mixture of
heparitinases and applied to HPLC as described under
``Experimental Procedures.'' The conditions of HPLC on a
polyamine-bound silica PAMN column were as described under
``Experimental Procedures.'' The broken line indicates the concentration of KH
PO
. The arrows are the same as indicated in Fig. 2.
Figure 10:
Properties of the purified heparan
sulfate 6-sulfotransferase. A, pH dependence of heparan
sulfate 6-sulfotransferase activity. The sulfotransferase activities
were determined as described under ``Experimental
Procedures,'' except that the pH 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. B, effect of DTT on the sulfotransferase activity. The
reaction mixtures contained various amounts of DTT. C, effect
of NaCl on the sulfotransferase activity. The reaction mixture
contained various concentrations of NaCl with (
) or without
(
) 2 mM DTT. D, effect of protamine on the
sulfotransferase activity. The reaction mixture contained various
amounts of protamine.
Figure 11:
K value of
heparan sulfate 6-sulfotransferase for PAPS. The sulfotransferase
activity was determined using 2.12 ng of the purified enzyme as
described under ``Experimental Procedures'' except that
various amounts of PAPS were added. The inset shows the
reciprocal plot.
In our present experiments, heparan sulfate 6-sulfotransferase was purified to an apparent homogeneous level from the culture medium of CHO cells. Chondroitin 6-sulfotransferase was also purified from the culture medium of chondrocytes. Although the mechanisms of the secretion of these sulfotransferases are not clear, the results may suggest that conditioned media may be useful sources for the purification of glycosaminoglycan sulfotransferases. As was also the case with heparan sulfate/heparin N-sulfotransferase and chondroitin 6-sulfotransferase(14, 15, 26) , affinity chromatography with heparin-Sepharose and 3`,5`-ADP-agarose yielded successful purification of heparan sulfate 6-sulfotransferase.
When
CDSNS-heparin was used as an acceptor, the purified sulfotransferase
was found to transfer sulfate exclusively to position 6 of N-sulfoglucosamine residue and not to transfer sulfate to
position 2 of hexuronic acid residue or position 2 of glucosamine
residue. The evidences for this conclusion are that: 1) digestion of S-labeled product derived from CDSNS-heparin with a
heparitinase mixture yielded
Di-(N,6)diS as a major
S-labeled disaccharide product, 2) degradation with
nitrous acid at pH 1.5 yielded IdoA-AMan(6SO
) as a major
S-labeled disaccharide product, and 3) no free
SO
was released when the
S-labeled product derived from CDSNS-heparin was treated
with nitrous acid at pH 1.5. Wlad et al. (35) have
recently reported that both the 6-sulfotransferase and the
2-sulfotransferase from mouse mastocytoma may be part of the same
protein. The observed difference of the enzyme specificity between
their and our sulfotransferases might be due to the following reasons.
Because our enzyme was obtained from culture medium, the
6-sulfotransferase might have been produced as a secretary protein by
protease processing from the single protein that contained both O-sulfotransferase activities. Because these enzymes were
produced by different types of cells, they might be different gene
products. We prepared the 6-sulfotransferase from CHO cells, which are
engaged in the biosynthesis of heparan sulfate. On the other hand, Wald et al.(35) prepared the enzyme from mouse mastocytoma
tissue, which is mainly involved in the biosynthesis of heparin. As
with N-sulfotransferases(22, 23, 24, 25) ,
it is possible that O-sulfation of heparin and heparan sulfate
may be catalyzed by different enzymes.
When heparan sulfate was used
as acceptor for the purified enzyme, digestion of the S-labeled product with the heparitinase mixture yielded
nearly equal amounts of
Di-(N,6)diS and
Di-(N,6,U)triS.
Because the purified sulfotransferase preparation was unable to
transfer sulfate to position 2 of the hexuronic acid residue of the
HexA-GlcNSO
unit in CDSNS-heparin, it is possible that the
S-labeled
HexA(2SO
)-GlcNSO
(6SO
) unit in the
S-heparan sulfate product was formed by the sulfation of
position 6 of N-sulfoglucosamine residue in the
HexA(2SO
)-GlcNSO
unit. The content of the
HexA(2SO
)-GlcNSO
unit was 4.4 and 0.2% in
heparan sulfate (pig aorta) and CDSNS-heparin, respectively. The
relatively higher content of the HexA(2SO
)-GlcNSO
unit in the heparan sulfate may have caused the higher production
of the
S-labeled
HexA(2SO
)-GlcNSO
(6SO
) unit.
However, the possibility cannot be completely ruled out that the
HexA(2SO
)-GlcNSO
(6SO
) unit was
formed by the transfer of sulfate to position 2 of hexuronic acid
residue of the HexA-GlcNSO
(6SO
) unit. If
heparan sulfate 6-sulfotransferase is able to catalyze the transfer of
sulfate not only to position 6 of N-sulfoglucosamine residue
in the HexA-GlcNSO
unit but also to position 6 of N-sulfoglucosamine residue in the
HexA(2SO
)-GlcNSO
unit, this specificity of the
enzyme could be consistent with the previous observations that the
IdoA(2SO
)-GlcNSO
(6SO
) unit was
formed by the sulfation of position 6 of GlcNSO
residue
after or simultaneously with the sulfation of position 2 of the IdoA
residue(18) . Kusche et al., using various
pentasaccharides
GlcNSO
-GlcA/IdoA-GlcNSO
-GlcA/IdoA-GlcNSO
as acceptors and mouse mastocytoma microsome as an enzyme, also
reported that sulfation at the position 6 of the internal glucosamine
unit took place, irrespective of the structure of the adjacent
hexuronic acid residue(19) . The purified heparan sulfate
6-sulfotransferase can catalyze the transfer of sulfate at least to
position 6 of GlcNSO
adjacent to the iduronic acid unit.
However, it remains to be determined whether or not the purified
heparan sulfate 6-sulfotransferase catalyzes the transfer of sulfate to
position 6 of GlcNSO
adjacent to GlcA. It has also been
noticed that, when heparan sulfate was used as an acceptor, the
S-labeled GlcA-GlcNAc(6SO
) unit was not
detected in the products, although the acceptor contained 64% of the
GlcA-GlcNAc unit (Fig. 9). The result suggests that the C-6
sulfation of GlcNAc in heparan sulfate may be performed by a different
sulfotransferase.
It is interesting to compare the properties of heparan sulfate 6-sulfotransferase with those of other purified glycosaminoglycan sulfotransferase. DTT inhibited heparan sulfate 6-sulfotransferase but not chondroitin 6-sulfotransferase(36) . On the contrary, DTT stimulated chondroitin 4-sulfotransferase(36) .
Heparan sulfate 6-sulfotransferase
appears to be a monomer protein, because the molecular weight of the
purified sulfotransferase determined by SDS-PAGE was consistent with
that determined by Superose 12 gel chromatography (Fig. 6). Rat
liver heparan sulfate N-deacetylase/N-sulfotransferase is a monomer (14, 37) and chick chondrocyte chondroitin
6-sulfotransferase may be a dimer. A majority of proteins intrinsic to
the Golgi apparatus membrane appear to be dimers in
situ(38) . The apparent K value for
PAPS of the purified heparan sulfate 6-sulfotransferase was 4.4
10
M, whereas that of heparan sulfate N-sulfotransferase of rat liver was 1.08
10
M(39) . Heparan sulfate
6-sulfotransferase, therefore, appears to have higher affinity to PAPS
than heparan sulfate N-sulfotransferase. The difference in the
value of K
for PAPS between heparan sulfate
6-sulfotransferase and heparan sulfate N-sulfotransferase,
however, might be due to the difference in the assay conditions used.
For example, as a cationic activator, protamine was added to the
present assay mixture for heparan sulfate 6-sulfotransferase, whereas
Mg
and Mn
were added to the
reported assay mixture for heparan sulfate N-sulfotransferase.
We previously observed that cationic proteins such as protamine and
histone stimulated chondroitin 6-sulfotransferase by decreasing the K
value for PAPS(28) . The low K
value for PAPS of heparan sulfate
6-sulfotransferase may have been caused by the presence of protamine.
SDS-PAGE of the purified enzyme fraction gave two protein bands of
52 and 45 kDa. Both protein bands were always comigrated in the gel
when the peak fractions containing the activity of heparan sulfate
6-sulfotransferase from heparin-Sepharose or Superose 12 chromatography
were subjected to SDS-PAGE. Both protein bands decreased their
molecular masses by treatment with N-glycanase, suggesting
that the difference in the molecular masses of the two proteins may not
be attributed solely to the difference in the content of N-glycosidic carbohydrate chains. When proteins were extracted
from the gels following SDS-PAGE and assayed for 6-sulfotransferase
after renaturation, the significant activity of 6-sulfotransferease was
recovered from the gels containing these two protein bands. The results
suggested that both the 52- and 45-kDa proteins bear 6-sulfotransferase
activity. Our preliminary studies on the amino-terminal amino acid
sequences of the two proteins showed that at least 11 amino acid
residues were completely identical, ()which suggested a
close relationship between these two proteins. Several
glycosyltransferases that are derived from the single genes exhibit
catalytically active multiple forms with different molecular
weights(40, 41, 42, 43) . This size
difference has been supposed to be due to limited proteolytic cleavage,
binding of detergents or lipids, or aggregation. In addition, there are
some reports suggesting that the stem regions are cleaved off
proteolytically when proteins originally present in the Golgi
apparatus, such as glycosyltransferases, were
secreted(43, 44) . For now, it remains to be studied
whether these two proteins of heparan sulfate 6-sulfotransferase are
closely related proteins or distinct ones.
Heparan sulfate prepared
from CHO cells contains HexA(2SO) residue and
GlcNSO
(6SO
) residue in a proportion of 4:3.
However, IdoA 2-O-sulfotransferase activity was only 4% of
total heparan sulfate O-sulfotransferase activity in the
culture medium of CHO cells. It is not certain why IdoA
2-O-sulfotransferase activity was hardly detected in the
cultured medium of CHO cells. The following are possible reasons to
explain why scarce activity of IdoA 2-O-sulfotransferase was
detected in the culture medium: 1) the processing of IdoA
2-O-sulfotransferase to soluble forms may not be as efficient
as the processing of heparan sulfate 6-sulfotransferase; 2) IdoA
2-O-sulfotransferase may need cofactors for its enzyme
activity that were not secreted into the culture medium; or 3) there
may be no relationship between the enzyme activity and the amount of
the product. These possibilities remain to be studied.