(Received for publication, March 3, 1995; and in revised form, July 5, 1995)
From the
A soluble sulfotransferase that could 6-sulfate both chondroitin
sulfate and corneal keratan sulfate was purified 27,500-fold using a
sequence of affinity chromatographic steps with heparin-Sepharose,
wheat germ agglutinin-agarose, and 3`,5`-ADP-agarose. The essentially
pure enzyme had a specific activity 40 times greater than the most
purified chondroitin 6-sulfotransferase previously reported and
exhibited a single sharp Coomassie Blue-stained and a heavy
silver-stained protein band of 75 kDa on SDS-polyacrylamide gel
electrophoresis. Chromatography of the purified enzyme on Sephacryl
demonstrated a size of 150 kDa, which indicated that the native enzyme
exists as a dimer. In addition to 6-sulfation of nonsulfated GalNAc,
the purified serum enzyme had the ability to sulfate GalNAc 4-sulfate
residues to give GalNAc 4,6-disulfate residues. The purified enzyme
exhibited a K of 40 µM for
adenosine 3`-phosphate 5`-phosphosulfate when either chondroitin
sulfate or corneal keratan sulfate were used as the acceptors. Use of
both chondroitin sulfate and keratan sulfate in the same experiment
demonstrated mutual competition, establishing that the sulfation of
these substrates is by the same enzyme. Photoaffinity labeling of the
purified enzyme with 2-azidoadenosine
3`,5`-di[5`-
P]phosphate occurred only with the
75-kDa protein, confirming that this is the chondroitin
6-sulfotransferase/keratan sulfotransferase.
Proteoglycans contain sulfated glycosaminoglycans that are covalently attached to a wide range of core protein families. In addition to the considerable variation and heterogeneity in the size and number of glycosaminoglycan chains that are linked to a core protein, there is heterogeneity in the position and degree of sulfation. The multiple functions of these molecules appear to be contributed, at least in part, by defined sulfate substitution of the glycosaminoglycan chains, so that the process of sulfation may have a direct effect on function.
During biosynthesis of the polysaccharide portions of proteoglycans, sulfation of nascent polysaccharide chains is considered to be accomplished by specific sulfotransferases that are located in juxtaposition to the polymer-forming enzymes in the Golgi(1, 2) . Thus, sulfation in chondroitin is a complicated process that can result in the presence of GalNAc 4-sulfate and GalNAc 6-sulfate in the same glycosaminoglycan chains (3) by the presence of occasional GalNAc 4,6-disulfate and GlcA 2-sulfate (4) or by the presence of iduronic 2-sulfate (5) when dermatan sulfate is formed. Different chondroitin sulfotransferases are involved for each type of sulfate substituent, and a number of these have been examined(6, 7, 8, 9, 10) . It has been generally assumed in the case of keratan sulfate that the sulfotransferase involved in the sulfation of GlcNAc residues is different from the sulfotransferase involved in the sulfation of the Gal residues(11) . However, this has not been clearly established. With heparin or heparan sulfate there are three distinct O-sulfotransferases and one N-sulfotransferase that are involved in the modification of these polymers(12) .
Although there had been a number of reports on the utilization of soluble or solubilized sulfotransferases with soluble polysaccharide substrates, it became apparent many years ago that sulfation in intact cells only takes place efficiently with microsomal membrane-bound enzymes(13) . Thus during biosynthesis, the nascent membrane-bound proteoglycan is presented to the membrane-bound sulfotransferases for sulfation to take place during the process of polymerization(14) . During cell growth and/or turnover, there is a release of soluble sulfotransferases into the circulatory system of intact animals (8, 9, 15, 16) or into the media of cell cultures(9, 17) , but this released enzyme plays no apparent role in biosynthesis. In order to understand the specificity and mechanism of these membrane-bound enzymes and their secreted forms, one needs to obtain purified enzymes.
Previous work with partially purified chondroitin
6-sulfotransferases from a variety of sources (8, 15, 18, 19) has suggested that
the sulfation of Gal residues in keratan sulfate may be catalyzed by
the same enzyme, since the activities were not separable. In addition,
it has been previously shown that the two enzyme activities show
similar properties (18, 20) and similar
developmentally associated changes (16) . However, other work
with cornea has suggested that the chondroitin 6-sulfotransferase and
keratan sulfotransferase may be separate(11) . Furthermore,
partially purified chondroitin 6-sulfotransferase from mouse liver was
reported to have no activity toward corneal keratan
sulfate(21) . The most highly purified chondroitin
6-sulfotransferase obtained to date was from the culture medium of
chick embryo chondrocytes (19) and was found to transfer small
but significant amounts of sulfate from adenosine 3`-phosphate
5`-phosphosulfate to keratan sulfate. Although this enzyme was purified
1,430-fold, the major 75 kDa band on SDS-PAGE ()was quite
broad, raising questions regarding its purity. Thus it is still not
clear whether two separate sulfotransferases are involved in the
6-sulfation of GalNAc residues in chondroitin sulfate and 6-sulfation
of Gal residues in keratan sulfate or whether both glycosaminoglycans
are substrates for the same enzyme.
In this report, we present our
results on the purification of chondroitin 6-sulfotransferase/keratan
Gal 6-sulfotransferase from chicken serum. Our 27,500-fold purified
enzyme preparation had a specific activity 40 times greater than that
previously reported(19) . The two activities exhibited
identical chromatographic properties throughout the purification steps.
In addition, we have found that the two substrates are mutually
competitive in accepting [S]sulfate, indicating
that the two activities are associated with the same enzyme molecule.
In combination with the purification steps, a
2-N
-[
P]cADP photoaffinity ligand was
used to confirm that the purified protein of 75 kDa was chondroitin
6-sulfotransferase/keratan Gal 6-sulfotransferase. This is the first
description of the use of this analog in photoaffinity labeling of
sulfotransferases.
Origins containing the sulfated
glycosaminoglycan products were eluted with water and lyophilized, and
the [S]sulfate-labeled glycosaminoglycans were
incubated with 50 µl of chondroitin AC II lyase (0.15 units),
chondroitin ABC lyase (0.15 units), or keratanase (1 unit) in buffer
containing 0.05 M Tris-HCl, pH 7.5 and 0.1 mg/ml bovine serum
albumin for 2 h at 37 °C. The products of chondroitinase
degradation were spotted on Whatman No. 1 paper together with standards
of
Di-4S,
Di-6S, and
Di-0S and chromatographed in
1-butanol, acetic acid, 1 M NH
OH (2:3:1)
overnight(25) . The positions of the standards were located
under UV light. The paper was cut into 1-cm strips and analyzed for
radioactivity. The products of keratanase degradation were analyzed by
chromatography on Sephadex G-50 columns.
Purity of the various active fractions was determined by 10% SDS-PAGE. Conventional staining of protein was performed with Coomassie Blue R-250 followed by destaining with isopropyl alcohol, acetic acid, water (1:1:8 (v/v/v)) and by silver staining.
When the eluted protein was added to a WGA-agarose column, less than 50% of the activity was bound. However, after mixing the enzyme for 3 h with WGA-agarose, all of the activity was bound and could be specifically eluted with 0.5 M GlcNAc with no loss of activity. Both chondroitin 6-sulfotransferase and keratan sulfotransferase activities had identical elution profiles from the WGA-agarose column. However, unlike a previously reported 40% loss in activity in purification of chondrocyte growth medium chondroitin 6-sulfotransferase(19) , we found no loss in activity. This could be attributed to our procedure of mixing the WGA-agarose with the enzyme solution for 3 h, which provided a 100% binding. Like other glycosaminoglycan sulfotransferases(19, 27, 28) , the bound enzyme from the WGA-agarose column could be specifically eluted with the use of GlcNAc, suggesting that the enzyme is a glycoprotein.
Because 3`,5`-ADP is one of the products of the sulfotransferase reaction, the next two chromatographic steps with 3`,5`-ADP-agarose were chosen. Only 5% of the total protein from the previous step bound to this column (Fig. 1), and could be eluted with 1.0 M NaCl with a recovery of approximately 35% activity of both chondroitin 6-sulfotransferase and keratan sulfotransferase from the previous step. The eluted enzyme was further purified by adsorbing on the same column after removal of salt and eluting specifically with 3`,5`-ADP (Fig. 2). Chondroitin 6-sulfotransferase and keratan sulfotransferase had an identical elution profile from this column (Fig. 2). The eluted fractions did not contain enough protein for detection by measurement of the absorbance at 280 nm. We found that eluting directly with 3`,5`-ADP without eluting first with NaCl resulted in only a 12,000-fold purification. However, as shown in Table 1, by using the NaCl elution step prior to the 3`,5`-ADP elution, we obtained a total 27,500-fold purification of chondroitin 6-sulfotransferase with an overall recovery of 6.8%. The final specific activity with chondroitin as acceptor was 18 µmol of sulfate incorporated per min/mg of protein (40 times higher than that previously reported(19) ) and with keratan sulfate as acceptor was 12 µmol of sulfate incorporated per min/mg of protein. Chondroitin 6-sulfotransferase and keratan sulfotransferase were inseparable throughout the purification, maintaining a constant activity ratio of approximately 1.5:1.
Figure 1:
Elution of sulfotransferase from a
3`,5`-ADP-agarose column by 1 M NaCl. Dialyzed
sulfotransferase from a WGA-agarose column was mixed with
3`,5`-ADP-agarose for 5 h. After washing the column, elution was
performed with 1.0 M NaCl as described under
``Experimental Procedures.'' Fractions of 2 ml were
collected and assayed for chondroitin 6-sulfotransferase () and
keratan sulfotransferase (
) activities. Protein was monitored by
measuring the absorbance at 280 nm (
). Elution with 1.0 M NaCl was started at fraction 31.
Figure 2:
Elution of sulfotransferase from a
3`,5`-ADP-agarose column by 3`,5`-ADP. Dialyzed sulfotransferase from
the first 3`,5`-ADP-agarose column (Fig. 1) was applied to a
3`,5`-ADP-agarose column as described under
``Experimental Procedures.'' Beginning with fraction
31, the column was eluted with 0.5 mM 3`,5`-ADP as described
under ``Experimental Procedures.'' Fractions of 2 ml
were collected and assayed for chondroitin 6-sulfotransferase ()
and keratan sulfotransferase (
).
Coomassie Blue R-250 staining of the SDS gel after eluting directly with 3`,5`-ADP resulted in a broad doublet around 75 kDa (not shown), while the use of the NaCl elution step prior to the 3`,5`-ADP elution resulted in a single, sharp protein band of 75 kDa after the final step of purification (Fig. 3A). A similar heavy band was found on silver staining (Fig. 4), with one light band and perhaps another at 50 and 40 kDa, respectively. The approximate molecular weight of the purified sulfotransferase, as determined by gel chromatography on a Sephacryl S-300 column (not shown), was found to be 150 kDa, confirming the previous report that the native enzyme exists as a dimer(19) . It is of note that rat liver heparan sulfate N-sulfotransferase (27) and mouse mast cell heparin N-sulfotransferase (28) as well as heparin O-sulfotransferases appear to be monomers(29, 30) .
Figure 3:
SDS-PAGE analysis of photoaffinity-labeled
preparations of chondroitin 6-sulfotransferase. Protein fractions from
the different steps in the purification were subjected to photolabeling
with 2-N-[
P]cADP, and the
photolabeled samples were electrophoretically separated on a 10%
SDS-PAGE as described under ``Experimental
Procedures.'' The following standards were used: rabbit muscle
phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin
(45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor
(21.5 kDa), and lysozyme (14.4 kDa). A, proteins visualized by
staining the gel with Coomassie Blue R-250. Lane 1, 65 µg
of protein eluted from a heparin-Sepharose column; lane 2, 30
µg of protein eluted from a WGA-agarose column; lane 3, 1
µg of protein eluted with 1 M NaCl from a
3`,5`-ADP-agarose column; lane 4, 0.2 µg of protein eluted
with 0.5 mM 3`,5`-ADP from a 3`,5`-ADP-agarose column. B, radioactivity of the same bands visualized by
autoradiography of the gel.
Figure 4: SDS-PAGE analysis of chondroitin/keratan 6-sulfotransferase preparations by silver staining. Proteins visualized by silver staining as follows: lane 1, 400 µg of protein eluted from a heparin-Sepharose column; lane 2, 320 µg of protein eluted from a WGA-agarose column; lane 3, 30 ng of protein eluted with 1 M NaCl from a 3`,5`-ADP-agarose column; lane 4, 18 ng of protein eluted with 0.5 mM 3`,5`-ADP from a 3`,5`-ADP-agarose column. Standards were as in Fig. 3
Figure 5:
Incorporation of
sulfate from PAPS into exogenous
glycosaminoglycan acceptors. Reaction mixtures containing varying
amounts of glycosaminoglycan acceptors (0.4-40 µg) and 0.05 M MES buffer, pH 6.5, 0.015 M MnCl
, 0.1%
Triton X-100, 1.0 mM PAP
S (1.5 Ci/mmol), 20 ng of
purified enzyme protein in a total volume of 15 µl were incubated
at 37 °C for 30 min. The reaction mixtures were then spotted on
Whatman No. 1 paper and chromatographed as described under
``Experimental Procedures.'' The origins containing the
sulfated glycosaminoglycans were eluted with water and analyzed for
radioactivity.
, chondroitin;
, chondroitin 4-sulfate;
, chondroitin 6-sulfate;
, dermatan sulfate;
, bovine
corneal keratan sulfate; and
, human costal cartilage keratan
sulfate.
We have previously
reported an apparent K of 500 µM for
PAPS when a chondroitin hexasaccharide was used as the acceptor with
soluble enzyme(34) . However, in the present experiments using
desulfated chondroitin or keratan sulfate with the purified enzyme, the K
for PAPS was found to be decreased significantly
to 40 µM. These results would indicate that interaction of
a full-length chondroitin with the enzyme had an effect upon the K
for PAPS.
All sulfation of chondroitin, as
determined by analyzing the products of chondroitin ABC lyase
degradation, was found to be at the GalNAc 6-position with no
detectable transfer to the 4-position. However, approximately
10-15% of the S-labeled disaccharides from
chondroitin ABC lyase degradation of the labeled chondroitin 4-sulfate
and dermatan sulfate chromatographed as
Di-4,6S, indicating that
the chondroitin 6-sulfotransferase can add a 6-sulfate to an already
4-sulfated GalNAc residue.
As shown in Fig. 5, human costal
cartilage did not serve as an acceptor for the sulfotransferase, while
corneal keratan sulfate served as an efficient acceptor. Furthermore,
keratanase degradation of S-labeled bovine corneal keratan
sulfate did not yield any disaccharide products, consistent with the
previous report(19) , indicating that the enzyme had no
activity on any sulfated internal residue. It has previously been
established that human costal cartilage keratan sulfate is essentially
100% Gal 6-sulfated(20) , while 50% of the Gal residues of
bovine corneal keratan sulfate are unsulfated(35) , and it has
also been established that Pseudomonas keratanase has no
activity on internal residues where the Gal is 6-sulfated(36) .
Thus our results indicate that the sulfate was only incorporated onto
the Gal of the keratan sulfate. In order to substantiate these results,
we incubated PAP
S with p-nitrophenyl galactoside, p-nitrophenyl N-acetylgalactosaminide, and p-nitrophenyl N-acetylglucosaminide. Incorporation of
[
S]sulfate into these glycosides was found to be
17,000 and 22,000 cpm for the galactoside and N-acetylgalactosaminide, respectively, but less than 500 cpm
for the N-acetylglucosaminide. These results confirm that the
sulfate was transferred to the 6-position of Gal residues in keratan
sulfate.
Competition experiments with both chondroitin sulfate and keratan sulfate in the same incubation mixture were performed in order to determine whether the purified enzyme had a single catalytic site that is involved in the sulfation of both GalNAc and Gal residues or whether the enzyme was bifunctional with separate catalytic sites for the sulfation of chondroitin and keratan. We found (Table 2) that the presence of increasing concentrations of chondroitin sulfate at a fixed concentration of 40 µg of keratan sulfate resulted in increasing inhibition of keratan sulfotransferase activity, and the presence of increasing keratan sulfate concentrations inhibited the chondroitin 6-sulfotransferase activity. These results, demonstrating that the two substrates were mutually competitive, indicate that the same catalytic site in the enzyme is involved in sulfation of both GalNAc and Gal residues.
Our purification, competition experiments,
and the photoaffinity labeling have now provided definitive information
regarding a single catalytic site for the 6-sulfation of GalNAc
residues in chondroitin sulfate and 6-sulfation of Gal residues in
keratan sulfate. Furthermore, our experiments have validated the use of
2-N-[
P]cADP as a specific
photoaffinity probe for a sulfotransferase and would indicate that this
analog might be useful as a specific photoprobe in characterizing other
sulfotransferases.