From the
The pituitary glycoprotein hormone lutropin is characterized by
its pulsatile appearance in the bloodstream which is important for the
expression of its biological activity in the ovary. We have previously
shown that lutropin bears unique Asn-linked oligosaccharides
terminating with GalNAc-4-SO
The pituitary glycoprotein hormone lutropin (LH)
We have
determined previously that LH from four different animal species bears
unique Asn-linked oligosaccharides terminating with the sequence
SO
The
presence of sulfated oligosaccharides on LH reflects the sequential
action of two highly specific enzymes expressed in the pituitary. A
glycoprotein hormone-specific GalNAc-transferase, which recognizes a
specific peptide motif in the underlying
protein(12, 13) , transfers GalNAc to the synthetic
intermediate GlcNAc
We have shown that both the GalNAc-transferase and the
GalNAc-4-sulfotransferase are present in tissues other than the
pituitary, including submaxillary gland, lacrimal gland, and kidney
(16), suggesting the existence of terminal GalNAc-4-SO
Bovine submaxillary glands were purchased from Pel-Freez
(Rogers, AR). Wheat germ agglutinin (WGA)-Sepharose (10 mg of WGA/ml of
gel) was made by coupling WGA (Sigma) to CNBr-activated Sepharose 4B
(Pharmacia Biotech Inc.). 3`,5`-ADP-agarose was obtained from Sigma.
[
All steps were carried out at 4 °C.
GalNAc-4-sulfotransferase reactions (50 µl) were carried
out as described (14) at 28 °C for 2 h, and contained 15
mM HEPES, pH 7.4, 1% Triton X-100, 40 mM
A standard GalNAc-4-sulfotransferase reaction was done as
described above, omitting unlabeled PAPS and using partially-purified
bovine pituitary sulfotransferase or homogeneous bovine submaxillary
sulfotransferase as the enzyme source. Reactions were incubated at 28
°C overnight and the sulfated GGnM-MCO product was isolated on
Sep-Paks as described previously(14) . Partial mild acid
hydrolysis and separation of sulfated monosaccharides by HPLC on a
CarboPak PA1 column (Dionex) were carried out as described
previously(14) .
Photolabeling reactions (50 µl) were carried out in
Buffer B containing the indicated amounts of protein, 4 mM
MgCl
Oligosaccharide acceptors attached to a hydrophobic aglycone
tail (-MCO) were chemically synthesized as described
previously(19, 20) , terminating with GlcNAc.
After solubilization of
GalNAc-4-sulfotransferase activity in Triton X-100, the extract was
chromatographed on DEAE-Sepharose resulting in 7.1-fold purification
over the homogenate. An additional 13-fold purification was achieved by
chromatography on WGA-Sepharose with elution in 0.5 M GlcNAc.
This was followed by passage of the partially-purified enzyme over a
sulfonamide-agarose column which removes carbonic anhydrase VI from the
preparation resulting in an additional 1.6-fold purification. Removal
of carbonic anhydrase at this stage is essential to the overall success
of this purification scheme, and is necessitated by the fact that
carbonic anhydrase VI is a major contaminant of the affinity-purified
enzyme preparation if not removed. Carbonic anhydrase VI, which we have
shown is a substrate for the GalNAc-4-sulfotransferase(17) , is
an abundant protein synthesized in the submaxillary and parotid
salivary glands (23). Sulfonamides are specific inhibitors of carbonic
anhydrases and have been used extensively as affinity ligands in the
purification of several members of this class of
enzymes(23, 24, 25) . Step 6, hydrophobic
chromatography on phenyl-Sepharose, facilitated removal of the salt
from the enzyme mixture; however, no fold enrichment is given in for this step because the high concentrations of Triton
X-100 required for elution prevented accurate protein quantitation.
Analysis by SDS-PAGE of the proteins present in purification steps
1-5 is shown in Fig. 2, lanes 1-5.
Similar results
were obtained when these acceptor oligosaccharides were tested using
partially-purified bovine pituitary sulfotransferase as the enzyme
source. The pituitary enzyme was found to have a K
The pituitary glycoprotein hormone lutropin is crucial to the
regulation of a number of physiological processes involved in
reproduction. Essential to the expression of LH bioactivity is its
pulsatile rise and fall in the bloodstream, which is in turn dependent
on its ability to be rapidly cleared from the
circulation(1, 2) . Our laboratory has previously shown
that this rapid clearance is attributable to the presence of unique
sulfated oligosaccharides on LH and a GalNAc-4-SO
In our purification scheme, two rounds of
affinity chromatography on 3`,5`-ADP-agarose resulted in the isolation
of a protein which migrates at 128 kDa on SDS-PAGE. The identification
of this protein as the GalNAc-4-sulfotransferase is supported by
experiments in which a 128-kDa protein was specifically photolabeled
with [
Several lines of evidence indicate that the
GalNAc-4-sulfotransferase purified from submaxillary gland is the same
enzyme responsible for the synthesis of sulfated oligosaccharides on LH
in the pituitary. 1) Analysis of sulfated monosaccharides derived from
sulfotransferase assay products shows that both the submaxillary and
the pituitary transferases are highly specific enzymes which transfer
sulfate exclusively to the 4-hydroxyl of terminal
Using a
panel of synthetic oligosaccharide acceptors to investigate acceptor
substrate specificity we have shown that the disaccharide
GalNAc
Sulfate has been shown to occur in a number of different linkages
and to different underlying sugars on N-linked
oligosaccharides (34-38). However, this is the first
sulfotransferase to be isolated which is responsible for the sulfation
of N-linked glycans. Obtaining the peptide sequence of this
purified sulfotransferase will enable us to isolate its cDNA clone and
compare its sequence to that of the N-heparan
sulfotransferase, the only mammalian saccharide-specific
sulfotransferase cloned to date(39) . More importantly, cloning
of the GalNAc-4-sulfotransferase will provide a crucial tool for
investigating the regulation of sulfation of
GalNAc
which allow the hormone to be
rapidly cleared from the bloodstream via a specific receptor in the
liver, thus contributing to its pulsatile appearance in the
circulation. Furthermore, we have found that carbonic anhydrase VI,
synthesized by the submaxillary gland and secreted into the saliva,
also bears Asn-linked oligosaccharides terminating with
GalNAc-4-SO
, suggesting that this unique sulfated structure
mediates other biological functions in addition to rapid clearance from
the circulation. We report here the purification of a
GalNAc-4-sulfotransferase which transfers sulfate to terminal
1,4-linked GalNAc on Asn-linked oligosaccharides. We show that the
purified submaxillary gland enzyme has kinetic parameters identical to
the pituitary enzyme, indicating that the same sulfotransferase is
responsible for the sulfation of lutropin oligosaccharides in pituitary
and carbonic anhydrase VI oligosaccharides in submaxillary gland. This
GalNAc-4-sulfotransferase has an apparent molecular mass of 128 kDa and
can be specifically photoaffinity radiolabeled with 3`,5`-ADP, a
competitive inhibitor of sulfotransferase activity. The acceptor
specificity of this GalNAc-4-sulfotransferase indicates that it is able
to transfer sulfate to terminal GalNAc
1,4GlcNAc on both N- and O-glycosidically linked oligosaccharides,
suggesting that this enzyme is also responsible for the sulfation of O-linked glycans on proopiomelanocortin.
(
)is essential for the regulation of a number of
physiological processes involved in reproduction, including follicular
maturation, ovulation, and the secretion of estradiol and progesterone.
LH exerts its effects by binding to and stimulating the
lutropin/chorionic gonadotropin (LH/CG) receptor in the ovary. One of
the distinctive features of the biology of LH is its pulsatile pattern
of appearance in the bloodstream which is thought to be necessary for
the in vivo expression of its bioactivity (1, 2). Like other G
protein-coupled receptors, the LH/CG receptor is desensitized upon
ligand binding(3) ; thus, the rise and fall of LH levels in the
circulation may be crucial for maintaining maximal stimulation of the
LH/CG receptor in the ovary. Many factors are important in producing
this pulsatile rise and fall in the bloodstream, including frequency of
stimulation of LH release from the pituitary by LH-releasing hormone,
the amount of LH released during the secretory burst, and the
circulatory half-life of LH(4, 5) .
-4-GalNAc
1,4GlcNAc
1,2Man
in contrast to
the more commonly found terminal sequence sialic
acid-Gal
1,4GlcNAc
1,2Man
(6, 7, 8, 9) .
Subsequent clearance studies showed that native bovine (b) LH, bearing
oligosaccharides which terminate with GalNAc-4-SO
, is
cleared from the circulation 4-fold faster than recombinant bLH bearing
oligosaccharides which terminate with sialic acid(10) . The
rapid clearance of native bLH is mediated by a receptor expressed in
hepatic reticuloendothelial cells which recognizes oligosaccharides
terminating with GalNAc-4-SO
and removes the hormone from
the bloodstream (11). The presence of sulfated rather than sialylated
oligosaccharides on LH results in a shorter circulatory half-life,
which contributes to the pulsatile appearance of the hormone in the
bloodstream and has a significant impact on in vivo hormone
bioactivity. The presence of sulfated oligosaccharides on LH is
therefore crucial to the biologic function of this hormone.
Man
GlcNAc
Asn.
The specificity of this transferase accounts for the addition of GalNAc
to LH and thyroid stimulating hormone (7, 8) but not to
other pituitary glycoproteins. A GalNAc-4-sulfotransferase, which
utilizes 3`-phosphoadenosine 5`-phosphosulfate (PAPS) as the sulfate
donor, accounts for the addition of sulfate to the 4-hydroxyl of
terminal GalNAc residues of LH(14) . Unlike the
GalNAc-transferase, the GalNAc-4-sulfotransferase does not appear to be
protein specific(14) . Both GalNAc-transferase and
GalNAc-4-sulfotransferase levels are up-regulated in rat pituitary in
concert with rising LH levels following ovariectomy, resulting in the
maintenance of terminal glycosylation of LH oligosaccharides with
GalNAc-4-SO
(15). Thus, synthesis of the sulfated
oligosaccharides on LH is highly regulated, further supporting the
importance of these unique oligosaccharides to the biological function
of LH. In order to better understand the regulation of the synthesis of
this sulfated structure, these transferases must be purified and
cloned.
on
glycoproteins from other tissues. Furthermore, we have recently shown
that oligosaccharides terminating with GalNAc-4-SO
are
present on a secreted form of carbonic anhydrase from submaxillary
gland where there are also high levels of both
transferases(17) . Here we report the purification of the
GalNAc-4-sulfotransferase to homogeneity from bovine submaxillary gland
and present evidence that this is the same enzyme which is responsible
for the sulfation of LH oligosaccharides in the pituitary. In addition,
we demonstrate that this sulfotransferase is not limited in specificity
to Asn-linked oligosaccharides, but also has the ability to transfer
sulfate to
1,4-linked GalNAc on O-glycosidically linked
oligosaccharide acceptors. These results suggest that this
sulfotransferase is therefore responsible for the sulfation of N-linked oligosaccharides on LH and carbonic anhydrase VI and O-linked oligosaccharides on proopiomelanocortin(18) .
S]PAPS was enzymatically synthesized as
described previously (14) using
[
S]SO
from ICN (Costa Mesa, CA).
[
P]3`,5`-ADP (3000 Ci/mmol) was purchased from
ICN. The synthesis of GGnM-MCO has been previously
published(19, 20) .
Purification of GalNAc-4-sulfotransferase from Bovine
Submaxillary Gland
Step 1: Preparation of Triton X-100 Extract
600 g of
bovine submaxillary glands were thawed, passed through a meat grinder,
homogenized, and extracted in 1% (v/v) Triton X-100 as described
previously by Schwyzer and Hill (21) for purification of a
porcine submaxillary gland GalNAc-transferase.
Step 2: DEAE-Sepharose
The Triton X-100 extract
(1010 ml) was diluted to 6 liters in Buffer A (25 mM Tris, pH
7.4, 13% glycerol, 0.1% Triton X-100) and was applied to a 1-liter
DEAE-Sepharose (Pharmacia) column equilibrated in Buffer A. After
washing in 4 liters of Buffer A the column was eluted in 4 liters of
0.5 M NaCl in Buffer A.
Step 3: WGA-Sepharose
The DEAE-Sepharose eluate
was applied to a 100-ml WGA-Sepharose column which was washed with 1
liter of 0.25 M KCl in Buffer A and then with 500 ml of Buffer
B (15 mM HEPES, pH 7.4, 13% glycerol, 0.1% Triton X-100). The
column was batch-eluted in 500 ml of 0.5 M GlcNAc, 0.25 M NaCl in Buffer B.
Step 4: Sulfonamide-agarose
The WGA-Sepharose
eluate was passed over a 20-ml p-aminomethylbenzene
sulfonamide-agarose column (Sigma) and the flow-through was collected.
The column was washed with 100 ml of 0.25 M NaCl in Buffer B,
and the wash fraction was combined with the flow-through.
Step 5: Phenyl-Sepharose
The combined flow-through
and wash fractions from the previous step were diluted to 3 liters in
Buffer C (15 mM HEPES, pH 7.4, 13% glycerol) and loaded at a
flow rate of 5 ml/min onto an 80-ml phenyl-Sepharose column (Pharmacia)
equilibrated in Buffer C. The column was washed with 500 ml of Buffer C
and batch-eluted in 200 ml of 2% (v/v) Triton X-100 in Buffer C.
Step 6: 3`,5`-ADP-agarose: NaCl Gradient Elution
1
mM GDP was added to the phenyl-Sepharose eluate, which was
incubated overnight with 10 ml of 3`,5`-ADP-agarose (1.9 µmol/ml
gel). The resin was collected in a 1.5-cm diameter column and washed
first with 50 ml of 1 mM GDP in Buffer D (15 mM HEPES, pH 7.4, 4 mM magnesium acetate, 0.1% Triton X-100,
13% glycerol) and then with 100 ml of Buffer D. The column was eluted
in a 0-1 M NaCl gradient in Buffer D at a flow rate of
0.5 ml/min in a total volume of 80 ml. 1-ml fractions were collected
and assayed for protein and for GalNAc-4-sulfotransferase activity.
Step 7: Phenyl-Sepharose Chromatography
The active
fractions from step 6 were pooled and diluted with 2 volumes of Buffer
C and adsorbed overnight to 6 ml of phenyl-Sepharose equilibrated in
Buffer C. The resin was collected in a 1.5-cm diameter column, washed
with 30 ml of Buffer C, and eluted in 30 ml of 2% Triton X-100 in
Buffer C at a flow rate of 0.5 ml/min. 1-ml fractions were collected
and assayed for GalNAc-4-sulfotransferase activity.
Step 8: 3`,5`-ADP-agarose Chromatography: Elution with
5`-ADP
The pooled active fractions from the previous step were
brought to 1 mM GDP and were adsorbed to 2 ml of
3`,5`-ADP-agarose overnight. The resin was collected in a 1.0-cm
diameter column and was washed with 20 ml of 1 mM GDP in
Buffer D, followed by 50 ml of Buffer D. The column was eluted in 20 ml
of 20 mM 5`-ADP in Buffer D at a flow rate of 0.5 ml/min.
0.5-ml fractions were collected and assayed for the presence of protein
and GalNAc-4-sulfotransferase activity.
Step 9: DEAE-Sepharose Chromatography
The pooled
active fractions from step 8 were applied to a 0.3-ml DEAE-Sepharose
column. The column was washed with 1 ml of 20 mM 5`-ADP in
Buffer D and the flow-through and wash fractions were combined.
Step 10: Hydroxylapatite Chromatography
The
combined flow-through and wash fractions from the previous step were
applied to a 0.3-ml hydroxylapatite column (Bio-Rad) which was washed
with 2 ml of Buffer D and eluted with 2 ml of 0.2 M sodium
phosphate, pH 7.4, 0.1% Triton X-100, 13% glycerol.
Assay of GalNAc-4-sulfotransferase
-mercaptoethanol, 10 mM NaF, 1 mM ATP, 4
mM magnesium acetate, 13% glycerol, protease inhibitors, 2
µM unlabeled PAPS, 1
10
cpm of
[
S]PAPS, 20 µM GGnM-MCO, and
sulfotransferase source.
[
S]SO
-GGnM-MCO was separated from
[
S]PAPS and from labeled endogenous acceptors by
passage over a Sep-Pak C
cartridge (Waters) as described
previously(14) . Control reactions were done in the absence of
GGnM-MCO.
GalNAc-4-sulfotransferase Product Proof
Photoaffinity Labeling with
[
P]3`,5`-ADP
, and 1 µCi of [
P]3`,5`-ADP,
in the presence or absence of 1 mM 3`,5`-ADP or 1 mM ATP. Reactions were incubated on ice in a 96-well microtiter plate
for 5 min and then exposed to ultraviolet light for 30 min at 1-cm
distance using a hand-held UV lamp set at 254 nm. Duplicate reactions
were not exposed to UV. Reactions containing DEAE-Sepharose and
WGA-Sepharose elutions were precipitated with 10% trichloroacetic acid
and the pellets were boiled in SDS-PAGE sample buffer (10% glycerol, 5%
2-mercaptoethanol, 2% SDS, 0.003% bromphenol blue, and 62.5 mM Tris, pH 6.8) and loaded onto a 7.5% SDS gel. The reaction
containing 3`,5`-ADP-agarose NaCl gradient elution was not
trichloroacetic acid precipitated, but was boiled in 1
sample
buffer following the reaction and was loaded directly onto the gel. Due
to differences in background, autoradiogram exposure times were
optimized individually for each of the purification steps and varied
between 12 and 48 h.
Acceptor Substrate Specificity
1,4-Linked GalNAc was added using
1,4-galactosyltransferase
(Sigma) and UDP-GalNAc (Sigma) as described previously(22) .
GalNAc-4-sulfotransferase reactions were carried out using pure
GalNAc-4-sulfotransferase as described above, except that the acceptor
substrate was varied. K
and V
values for each oligosaccharide were
calculated from double-reciprocal plots of 1/Vversus 1/[S], where [S] varied from 3.3 to 40
µM. For oligosaccharides on which there are two terminal
1,4-linked GalNAc residues, the K
values were calculated based on whole oligosaccharide
concentration. Correlation coefficients for double-reciprocal plots
ranged from 0.96 to 0.99.
Purification of GalNAc-4-sulfotransferase
summarizes the purification of
GalNAc-4-sulfotransferase from 600 g of bovine submaxillary glands. The
transferase was purified 1576-fold in 10 steps, with a yield of 0.24%
over the total homogenate. Bovine submaxillary gland was chosen as the
source for this purification because of its high expression of
GalNAc-4-sulfotransferase relative to other tissues, including
pituitary(16) .
Figure 2:
SDS-PAGE of GalNAc-4-sulfotransferase
purification fractions. Lane 1, 10 µg of Triton extract; Lane 2, 10 µg of DEAE-Sepharose eluate; Lane 3, 5
µg of WGA-Sepharose eluate; Lane 4, 5 µg of
sulfonamide-agarose flow-through fraction; Lane 5,
phenyl-Sepharose 1 eluate; Lane 6, 2 µg of pooled
fractions from 3`,5`-ADP-agarose NaCl gradient elution; Lane
7, phenyl-Sepharose 2 eluate; Lane 8, 0.5 µg of
pooled fractions from 3`,5`-ADP-agarose 5`-ADP elution. Lane
9, 2 µg of hydroxylapatite column elution. Proteins were
visualized by silver nitrate staining.
The
partially-purified GalNAc-4-sulfotransferase eluted from
phenyl-Sepharose was affinity-purified on 3`,5`-ADP-agarose with
elution in a 0-1 M NaCl gradient (Fig. 1A), resulting in a total purification of 695-fold
over the homogenate and enrichment of a predominant protein which
migrates at 128 kDa on SDS-PAGE (Fig. 2, lane 6). A
significant proportion (>50%) of the GalNAc-4-sulfotransferase
activity does not bind to the 3`,5`-ADP-agarose during this first round
of affinity chromatography resulting in a significant loss of enzyme
activity at this step (only 12% of the activity loaded is recovered in
the gradient elution). We have identified and raised monoclonal
antibodies to a specific GalNAc-4-sulfotransferase binding protein
which is present in the partially-purified enzyme mixture and which
passes through the 3`,5`-ADP-agarose column. Preliminary results
indicate that this binding protein, when complexed with the
sulfotransferase, prevents it from interacting efficiently with the
affinity ligand thus explaining the low yields obtained in this
purification step.(
)Following hydrophobic
chromatography to remove salt, the GalNAc-4-sulfotransferase was
subjected to a second round of affinity chromatography on
3`,5`-ADP-agarose with elution in 20 mM 5`-ADP (Fig. 1B). We utilized 5`-ADP rather than 3`,5`-ADP as
the affinity eluent because GalNAc-4-sulfotransferase activity is not
detectable in the presence 3`,5`-ADP, a competitive inhibitor, even at
concentrations below 1 mM.
The pooled active
fractions from this step were next chromatographed on a small (0.3 ml)
DEAE-Sepharose column. Although the transferase is bound to and eluted
from DEAE-Sepharose earlier in the purification (Step 2), in the
presence of 20 mM 5`-ADP, 50% of the GalNAc-4-sulfotransferase
activity loaded passes through DEAE-Sepharose at this later stage. This
step results in the removal of relatively minor contaminants at 150 and
200 kDa which remain bound to the resin. In order to concentrate the
enzyme, the combined flow-through and wash fractions from this step
were next applied to a hydroxylapatite column. The eluate from this
step yielded GalNAc-4-sulfotransferase which was purified 1576-fold
over the total homogenate, and gave a single, broad band centered
around an apparent molecular mass of 128 kDa (Fig. 2, lane
9; this lane was overloaded in order to enhance detection of any
minor contaminating proteins). The intensity of staining of this
128-kDa band in the individual hydroxylapatite column elution fractions
also correlated with the amount of GalNAc-4-sulfotransferase activity
in these fractions (data not shown). The diffuse nature of the purified
sulfotransferase may be due to a large number of O-linked
chains since quantitative amino acid analysis of the purified
transferase shows serine and threonine combined to comprise 11% of the
total amino acid content.
Figure 1:
Affinity chromatography of
GalNAc-4-sulfotransferase on 3`,5`-ADP-agarose. A,
partially-purified sulfotransferase from step 5 was bound to a
3`,5`-ADP-agarose column which was eluted in a gradient of 0-1 M NaCl. B, enzyme from step 7 was applied to a second
3`,5`-ADP-agarose column which was eluted with 20 mM 5`-ADP as
indicated by the arrow. Pooled fractions are indicated with a bar.
To confirm that the purified
sulfotransferase transfers sulfate exclusively to the 4-OH of terminal
GalNAc, we characterized the linkage of the sulfate transferred to
GGnM-MCO during the assay reaction.
[S]SO
-GGnM-MCO was subjected to mild
acid hydrolysis under conditions which cleave glycosidic bonds more
rapidly than sulfate esters, and the sulfated monosaccharides were
isolated and analyzed by HPLC on a CarboPak PA1 column as described
previously(14) . A single sulfated monosaccharide peak which
comigrated with authentic GalNAc-4-SO
standard was obtained
from the assay product generated by purified submaxillary
sulfotransferase (Fig. 3A), thus confirming the identity
of this enzyme as a GalNAc-4-sulfotransferase. Sulfated monosaccharide
generated using a partially-purified pituitary extract as the
sulfotransferase source gave an identical pattern (Fig. 3B).
Figure 3:
GalNAc-4-sulfotransferase product proof.
Purified GalNAc-4-sulfotransferase (A) or partially-purified
pituitary extract (B) was used to generate
[S]SO
-GGnM-MCO which was subjected
to mild acid hydrolysis. Sulfated monosaccharides were analyzed by HPLC
on a CarboPak PA1 column (Dionex). The elution positions of authentic
standards are: 1, GlcNAc-3-SO
; 2,
SO
; 3, GalNAc-3-SO
; 4, GalNAc-4-SO
; 5,
GlcNAc-6-SO
; 6, SO
-GGNM-MCO; 7, GalNAc-6-SO
.
Photoaffinity Labeling with
[
[P]3`,5`-ADP
P]3`,5`-ADP
was used as a specific photoaffinity probe to confirm that the purified
GalNAc-4-sulfotransferase activity corresponds to a protein of 128 kDa.
Radiolabeled PAPS and PAPS analogs have been used as specific
photolabeling probes in studies of several other sulfotransferases (26) as well as in the identification and purification of the
PAPS transporter(27) . 3`,5`-ADP is a PAPS analog which, in
addition to being a by-product of the sulfotransferase reaction, is a
potent competitive inhibitor of GalNAc-4-sulfotransferase activity (K
= 2.4 µM). A band
migrating at 128 kDa which is photolabeled with
[
P]3`,5`-ADP copurifies with
GalNAc-4-sulfotransferase activity during DEAE-Sepharose,
WGA-Sepharose, and 3`,5`-ADP-agarose chromatography (Fig. 4A,
lanes 2, 4, and 6). This labeling is UV-dependent, as
control reactions done in the absence of UV do not show any
radiolabeling (Fig. 4A, lanes 1, 3, and 5). In
addition, the 128-kDa radiolabeled protein becomes enriched relative to
other proteins which are also radiolabeled during this procedure (Fig. 4A, compare lanes 2 and 6).
UV-dependent radiolabeling of the 128-kDa band can be competed by 1
mM PAPS but not by 1 mM ATP, demonstrating the
specificity of the labeling reaction (Fig. 4B). These
results imply that the 128-kDa labeled protein has a relatively high
affinity for 3`,5`-ADP consistent with the identification of this
protein as the GalNAc-4-sulfotransferase.
Figure 4:
Photoaffinity labeling of a 128-kDa
protein with [P]3`,5`-ADP. A, composite
autoradiogram of protein from several purification steps subjected to
photolabeling with [
P]3`,5`-ADP. Lanes 1 and 2, 20 µg of DEAE-Sepharose eluate; Lanes 3 and 4, 20 µg of WGA-Sepharose eluate; Lanes 5 and 6, 2 µg of pooled fractions from
3`,5`-ADP-agarose NaCl gradient elution. Control reactions (Lanes
1, 3, and 5) were done in the absence of UV light. B, 20 µg of protein eluted from WGA-Sepharose was
subjected to photolabeling in the absence of any nucleotide (Lanes
1 and 2), in the presence of 1 mM PAPS (Lane
3), or in the presence of 1 mM ATP (Lane 4). The
reaction in Lane 1 was not subjected to UV. The 128-kDa
labeled protein is indicated with an arrow.
Acceptor Substrate Specificity of
GalNAc-4-sulfotransferase
The substrate specificity of the
GalNAc-4-sulfotransferase was assessed using a series of
oligosaccharide acceptors chemically and enzymatically synthesized as
described previously(19, 20) . summarizes
the results obtained using purified submaxillary gland transferase.
Each of these synthetic oligosaccharides terminates with GalNAc in a
1,4-linkage to an underlying GlcNAc. The sequences underlying
terminal GalNAc
1,4GlcNAc are representative of the sequences
observed in dibranched and tribranched Asn-linked oligosaccharides with
the exception of the sequence
GalNAc
1,4GlcNAc
1,6(Gal
1,3)GalNAc, which has been found O-glycosidically linked to the 16-kDa amino-terminal fragment
of bovine proopiomelanocortin(28) . Previous results using crude
bovine pituitary membrane extracts indicated that the trisaccharide
GalNAc
1,4GlcNAc
1,2Man
(GGnM) contains sufficient
information to allow recognition and transfer by the
GalNAc-4-sulfotransferase(14) . Purified submaxillary
GalNAc-4-sulfotransferase has a K
of 15.0
µM for GGnM (), in good agreement with our
previous results. The submaxillary sulfotransferase has a K
of 31.1 µM for the
terminal disaccharide GalNAc
1,4GlcNAc
(GGn), indicating that
the underlying mannose is not essential for recognition or transfer of
sulfate. The purified transferase has a K
of between 10 and 51 µM for each of the
remaining acceptor oligosaccharides tested, all of which terminate with
GGn. Up to 20 mM monomeric GalNAc did not inhibit
GalNAc-4-sulfotransferase activity (not shown), consistent with the
disaccharide GGn as the fundamental unit necessary for
GalNAc-4-sulfotransferase recognition. Interestingly,
GalNAc-4-sulfotransferase has a K
of 17.9
µM for GalNAc
1,4GlcNAc
1,6(Gal
1,3)GalNAc, an O-glycosidically linked structure(28) , as compared to
a K
of 15.0 µM for GGnM. The
GalNAc-4-sulfotransferase is, therefore, capable of recognizing and
transferring sulfate to terminal
1,4-linked GalNAc on O-glycans as well as N-glycans.
of 19.2 and 42.5 µM for GGnM and GGn,
respectively, and a K
of 18 µM for the O-linked structure
GalNAc
1,4GlcNAc
1,6(Gal
1,3)GalNAc. The purified
submaxillary sulfotransferase and partially-purified pituitary
sulfotransferase have similar K
values
for PAPS (1.1 µM for purified submaxillary transferase and
4 µM for pituitary). These and other data strongly suggest
that the submaxillary and the pituitary GalNAc-4-sulfotransferase are
the identical enzyme.
-specific
receptor in hepatic endothelial cells(10, 11) . We have
also shown that carbonic anhydrase VI, an abundant protein synthesized
by the submaxillary gland and secreted into the saliva(23) ,
bears N-linked oligosaccharides which terminate with
GalNAc-4-SO
(17) . We have now purified the
sulfotransferase responsible for synthesis of terminal
GalNAc-4-SO
on the N-linked oligosaccharides of LH
and carbonic anhydrase VI to apparent homogeneity from bovine
submaxillary gland.
P]3`,5`-ADP. The fact that a 1576-fold
purification is sufficient to achieve homogeneity indicates that the
submaxillary gland GalNAc-4-sulfotransferase is an abundant enzyme
relative to other purified glycosyltransferases and
sulfotransferases(29) , and corresponds to our finding that
submaxillary gland homogenates have a 15-fold higher specific activity
than pituitary homogenates(16) . This is not surprising given
the relative abundance of carbonic anhydrase VI, which we have shown to
be an endogenous substrate for the GalNAc-4-sulfotransferase in
submaxillary gland(17) . The salivary gland-specific form of
carbonic anhydrase, which is stored in granules and secreted into the
saliva(24, 25, 30, 31, 32) , is
expressed at very high levels(23) , and furthermore, greater
than 50% of its Asn-linked oligosaccharides terminate with
GalNAc-4-SO
(17) . Thus, the relative abundance of
GalNAc-4-sulfotransferase in submaxillary gland correlates with the
efficient sulfation of carbonic anhydrase VI oligosaccharides.
1,4-linked
GalNAc. 2) Submaxillary and pituitary sulfotransferase have identical
acceptor substrate specificities and virtually identical K
values for these acceptors.
Furthermore, the K
values for PAPS are
similar. 3) Carbonic anhydrase VI, an endogenous glycoprotein of the
submaxillary gland, has been shown to bear oligosaccharides terminating
with GalNAc-4-SO
(17) , thus demonstrating the in
vivo presence of sulfated oligosaccharides in the submaxillary
gland. 4) Carbonic anhydrase VI synthesized in bovine parotid glands,
which express the GalNAc-transferase but not the
GalNAc-4-sulfotransferase, bears oligosaccharides terminating with
GalNAc rather than GalNAc-4-SO
(17) .
1,4GlcNAc contains sufficient information for recognition
and transfer by the GalNAc-4-sulfotransferase, and thus can account for
recognition of native oligosaccharide acceptors. We and others have
previously shown that in addition to the pituitary glycoprotein
hormones LH and thyroid-stimulating hormone, proopiomelanocortin
synthesized in the pituitary also bears Asn-linked oligosaccharides
terminating with GalNAc-4-SO
(18, 33) . Other
studies have also shown that the major O-glycan attached to
Thr
of the 16-kDa amino-terminal fragment of bovine
proopiomelanocortin has the structure
SO
-4-GalNAc
1,4GlcNAc
1,6(Gal
1,3)GalNAc(28) .
Our results here show that both submaxillary and pituitary
GalNAc-4-sulfotransferase can transfer sulfate to the structure
GalNAc
1,4GlcNAc
1,6(Gal
1,3)GalNAc, demonstrating that
this GalNAc-4-sulfotransferase can account for sulfation of both N-linked and O-linked glycans on proopiomelanocortin.
1,4GlcNAc-bearing oligosaccharides on LH and other
glycoproteins.
Table: Purification of GalNAc-4-sulfotransferase
Table: GalNAc-4-sulfotransferase acceptor substrate
specificity
1,4GlcNAc
1,2Man
-O(CH
)
COOCH
;
CG, chorionic gonadotropin; WGA, wheat germ agglutinin; HPLC, high
performance liquid chromatography; PAGE, polyacrylamide gel
electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.