From Glycomed, Inc., Alameda, California 94501 and the Biochemistry and Molecular Biology Graduate Program, University of California, Davis, California 95616
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ABSTRACT |
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N-Deacetylase-N-sulfotransferases
(NDANST) catalyze the two initial modifications of the polysaccharide
precursor in the biosynthesis of heparin and heparan sulfate. These
modifications are the gating steps in establishing growth factor
protein-binding domains of these glycosaminoglycans. We have undertaken
a structure-activity analysis of the 841-amino acid Golgi-luminal
portion of the rat liver NDANST to localize the two enzymatic
functions. Each activity can be assayed in vitro
independently of the other when provided with the appropriate
substrate, and N-ethylmaleimide treatment selectively
inactivates the deacetylase activity. In this study, dithiothreitol
treatment of the rat liver NDANST was shown to inactivate the
sulfotransferase function, while stimulating deacetylase activity
2-3-fold over the native protein. Site-directed mutagenesis of the
eight cysteine (Cys) residues in the rat liver NDANST that are
conserved in the mouse mastocytoma protein produced three important
findings regarding the localization of each enzymatic function: 1)
derivatization of Cys486 with N-ethylmaleimide
resulted in total inactivation of the deacetylase activity based on
steric hindrance of the active site (this residue was shown not to be
involved in enzymatic catalysis), 2) substitution of either
Cys159 or Cys486 with alanine resulted in
enhanced activity of the deacetylase to the level obtained by
dithiothreitol treatment, and 3) alanine substitution of
Cys818 or Cys828 completely inactivated the
sulfotransferase activity, while substitution of Cys586 or
Cys601 resulted in a 90% loss in activity. These findings
suggest that the two enzymatic domains within the NDANST localize to
different portions of the protein, with two disulfide pairs toward the
COOH-terminal half of the protein necessary for the sulfotransferase
activity, and Cys residues within the NH2-terminal half
influencing or located near the active site of the deacetylase functionality.
Heparan sulfate is a ubiquitous component residing on the surface
of mammalian cells. It binds and modulates the activity of many
proteins, most notably growth factors and cytokines (1-7). The binding
of heparan sulfate to these proteins requires the presence of specific
structural domains contained within the polymer. The specificity for
protein binding is influenced by both the length of the polysaccharide
fragments and the presence of specific sulfate groups. For example, a
pentasaccharide containing both 2-O- and
6-O-sulfates is required for acid fibroblast growth factor (FGF-1)1 binding, while for
basic FGF (FGF-2), only 2-O-sulfate is required (8-11). The
presence of certain protein-binding domains may be regulated so that
cells can respond to different protein factors at different
developmental stages or physiological states (12). However, little is
known about how the biosynthesis of these protein-binding domains is regulated.
The backbone modification of heparan sulfate consists of a series of
enzymatic reactions, which occur in a sequential manner (13). The first
step is N-deacetylation and N-sulfation of
selective N-acetylglucosamine residues. Sulfation of these
residues is the prerequisite for the subsequent reactions, which
include C-5 epimerization of glucuronic acid, sulfation at the
2-hydroxyl group of the resulting iduronic acid, and the 6-hydroxyl
group of N-sulfated glucosamine residues. The process of
N-sulfation is not random; N-sulfated residues
tend to be clustered, and subsequent modifications are primarily
centered in these regions (14). N-Deacetylation and N-sulfation of N-acetylglucosamine of the heparan
sulfate polysaccharide precursor are catalyzed by a single protein
called heparan sulfate N-deacetylase-N-sulfotransferase (NDANST) (15,
16). Two highly homologous NDANSTs, one derived from rat liver and
another derived from a mouse mast cell line, have been cloned and their
enzymatic properties extensively characterized (17-19). These two
enzymes are 70% identical at the amino acid level, but they display
dramatically distinct kinetic properties that may account for the
different degrees of sulfation found on heparan sulfate and heparin.
Although the two activities of an NDANST must be tightly coupled
in vivo, each activity can be studied separately in
vitro using different substrates and reaction conditions. The two
activities are easily differentiated. The optimal pH for the
deacetylase activity is 6.5, while that for the sulfotransferase is
7.5; the deacetylase activity is inhibited by
N-ethylmaleimide, while the sulfotransferase is not affected
(15, 16). These enzymatic properties strongly suggest that there are
two separate enzymatic domains on the polypeptide. In this study,
experiments to localize these enzymatic domains in a chimeric form of
the Golgi-luminal domain were undertaken utilizing a site-directed
mutagenesis approach.
Site-directed Mutagenesis of Rat Liver
N-Deacetylase-N-sulfotransferase--
A mammalian expression vector
pCDM8 (Invitrogen) containing a chimeric gene encoding protein A fused
to the amino terminus of the Golgi-luminal domain of
N-deacetylase-N-sulfotransferase (15) was used as
a template for site-directed mutagenesis experiments. Site-directed
mutagenesis was performed with the Transformer kit (CLONTECH) using the manufacturer's recommended
procedure with slight modifications; the restriction site
XbaI was used as the selection restriction site. To reduce
the number of plasmids required for sequencing after the mutagenesis
protocol, a XbaI/SalI double restriction enzyme
digest was used to select clones for sequencing. Each mutant was
confirmed by DNA sequencing.
Expression of Recombinant Proteins--
Plasmids containing the
coding sequence of the protein A-NDANST fusion genes modified to
replace single cysteine residues with alanine were transiently
transfected into COS-7 cells by the DEAE-dextran transfection method
(20, 21). Briefly, 5 µg of DNA was transfected into cells seeded
24 h previously in a 100-mm tissue culture plate in Nu-serum
(Collaborative Biomedical Products). The DNA was mixed by vortexing
with 5 ml of DMEM containing 10 mg/ml DEAE-dextran and 2.5 mM chloroquine. The mixture was then added to the cells,
and, 4 h later, the transfection mixture was aspirated and cells
were incubated with 5 ml of Me2SO in phosphate-buffered saline for 2 min. Ten milliliters of DMEM with 10% serum were added
into each plate. Forty-eight hours after transfection, the conditioned
medium was discarded, 10 ml of fresh DMEM with 10% serum were added,
and the conditioned medium harvested after 24 h. This last step
was repeated once.
Recombinant Protein Purification--
Conditioned medium
combined from each plate was centrifuged to remove cellular debris.
Stabilizing buffer was added to a final concentration of 50 mM Tris-HCl (pH 7.4), 0.03% sodium azide, 0.1% Triton
X-100. The medium was passed through a column of IgG-agarose pre-equilibrated with the stabilizing buffer. The column was then washed with washing buffer containing 50 mM Tris-HCl (pH
7.4), 100 mM NaCl, and 0.1% Triton X-100. To elute the
protein from the IgG beads, agarose in the column was transferred to a
2-ml filtration unit with a 0.2-mm filter (Costar), and washed twice with 10 volumes of 1 mM Tris-HCl (pH 7.0) by brief
centrifugation. Sodium citrate (pH 4.0, 100 mM) was added
to the beads in the upper chamber to elute the protein, mixed by
tituration 20 times, and the unit was immediately spun at 8000 rpm to
elute the protein. An appropriate volume of 1 M Tris-HCl
buffer (pH 8.4) containing 20% glycerol was placed at the lower
chamber of the filtration unit. The protein preparations were stored at
Substrate Preparation, Enzyme Activity Assays, and Kinetic
Analysis--
Polysaccharide consisting of repeating
glucuronyl-N-acetylglucosamine disaccharides was isolated
from Escherichia coli strain K5 using a protocol provided by
Dr. Jeff Esko (University of California, San Diego, CA). Chemical
deacetylation of K5 polysaccharide and reacetylation with
[3H]acetic anhydride were performed as described
previously (22). The specificity of the labeled substrate was 400 cpm/ng (dry weight). N-Deacetylase activity was measured by
determining the release of radioactive acetic acid from the labeled K5
polysaccharide as described previously (22). Briefly, the deacetylation
reaction was carried out at 37 °C for 30 min with 40 ng of purified
recombinant enzyme as determined by the BCA assay. The released
[3H]acetic acid was extracted with ethyl acetate, and the
radioactivity was determined. N-Sulfotransferase activity
was measured by incorporation of [35S]PAPS into the
chemically deacetylated polysaccharide (23). Briefly, enzyme reactions
were carried out at 37 °C for 15 min with 40 ng of purified
recombinant enzyme as determined by the BCA assay. The unincorporated
PAPS was separated from the [35S]sulfated K5
polysaccharide by paper chromatography. The radioactivity incorporated
as sulfated polysaccharide was determined using a scintillation
counter. For NEM inhibition studies, enzyme was incubated in the
presence of an increasing concentration of inhibitor for 30 min on ice.
The deacetylase substrate was then added to start the reaction. To
determine the effect of dithiothreitol on enzymatic activities,
purified recombinant enzyme was incubated with 0-10 mM DTT
for 30 min on ice prior to reaction. Enzyme kinetic analyses were
performed using the Enzyme Kinetics Program (Trinity Software, Fort
Pierce, FL). For the analyses, the incubation times used gave initial
rates at all substrate concentrations.
DTT and NEM Treatment Allow for Functional Separation of the
N-Sulfotransferase and N-Deacetylase Activities--
NEM treatment of
both tissue-derived or recombinant NDANST results in selective loss of
the N-deacetylase function (15, 16, 19). Using a protein A
chimera containing the liver-derived NDANST, the role of disulfide
bonds in maintaining either enzyme function was investigated by
treating the protein with DTT (Fig. 1).
Remarkably, the N-sulfotransferase activity was selectively lost after treatment, and the N-deacetylase activity was
enhanced 2-3-fold. These complementary observations based on NEM and
DTT treatment suggested that probing the placement and secondary
structure imparted by individual Cys residues might provide insight
into the physical relationship of the enzymatic functions of the
protein.
Alanine-for-cysteine Substitutions in the COOH-terminal Half of
NDANST Selectively Abolish Sulfotransferase Activity--
There are
eight conserved cysteines in the NDANSTs derived from rat liver and
mastocytoma cells (17-19). In the rat liver enzyme, these cysteines
are Cys56, Cys159, Cys486,
Cys586, Cys601, Cys751,
Cys818, and Cys828 (Fig.
2). Using site-directed mutagenesis, a
codon for alanine was substituted separately for each of the cysteine
codons in the cDNA encoding this protein A-enzyme chimera, and each
was transfected into COS cells. All alanine-substituted chimeric
proteins were found to be transiently expressed and secreted into the
culture medium after transfection. An aliquot of each affinity-purified protein was analyzed by SDS-polyacrylamide gel electrophoresis under
reducing conditions (Fig. 3). Six of the
altered proteins exhibited a single band coincident with the native
chimeric protein at approximately 120 kDa. C586A primarily consisted of
a 70-kDa band in addition to a smaller amount of the 120-kDa species,
while the mass of C601A was split approximately equally between the two
sizes of protein, suggesting significant proteolytic degradation of
both chimeric proteins had occurred. When the electrophoresis was
performed under non-reducing conditions, approximately 70% of the wild
type protein migrated to the position of the 120-kDa monomer (data not
shown). This was also observed for C818A and C828A, indicating that the
loss of individual Cys residues did not promote dimerization of the
protein. Approximately 40% of C601A and C586A migrated as monomer,
again reflecting increased proteolysis of these proteins.
The effect of substituting an alanine for individual cysteine residues
in the protein on the deacetylase activity is shown in Table
I. In general, the loss of individual
cysteines did not cause a significant loss in enzyme activity, except
for C586A, where the total loss in activity can be attributed to
aberrant formation of disulfide bonds. The deacetylase enhancing effect of DTT was mapped to two residues in the NH2-terminal half
of the protein, C159A or C486A. In contrast, a 90% or greater loss of
the N-sulfotransferase activity was evident for several of the altered proteins (Table II). The
complete loss of activity in C818A and C828A, as well as the 90%
reduction in activity imparted by C586A and C601A, suggest that the
sulfotransferase functionality relies heavily on secondary structural
determinants contributed by Cys residues in the COOH-terminal portion
of the protein.
NEM Sensitivity of the N-Deacetylase Activity Is Based upon the
Location, but Not the Direct Participation, of Cys486 in
Catalysis--
The most surprising and unexpected result from Table I
was the absence of a protein with properties of the native protein treated with NEM, i.e. retaining full
N-sulfotransferase activity with a loss in
N-deacetylase activity. This observation suggested that no
free cysteine in the protein directly participates in the deacetylase
catalytic reaction. To gain further insight into the role of free
cysteine residues, each mutant (except for Cys586, which
has neither enzyme activity) was treated with NEM and assayed for the
deacetylase activity. Of all the mutants, only C486A was resistant to
treatment with NEM (Fig. 4), indicating that Cys486 is probably the target for NEM inactivation. To
eliminate the possibility that a non-conserved cysteine may also
contribute to the NEM inactivation of N-deacetylase, we
examined the effect of mutation of Cys195. No change in NEM
sensitivity was observed (data not shown). These observations suggest
that Cys486 is located near the deacetylase catalytic
center.
The Size of Amino Acid Side Chain at Position 486 Affects
Deacetylase Activity--
The above results raised the possibility
that the mechanism of inhibition by NEM is due to steric interference
with catalysis carried out by other amino acid side chains in close
proximity. To further test this hypothesis, Cys486 was
replaced with other amino acids of different size and charge. When
either arginine and tryptophan was placed at position 486, the
deacetylase activity was lost completely (Table
III). The protein with C486F displayed a
reduction in activity of 70% compared with the native enzyme, while a
significant increase was detected for proteins with C486A and C486V. In
total, the data demonstrate a correlation between increasing the size
of the amino acid side chain and a loss in deacetylase activity.
Replacement with the smallest amino acid, glycine, resulted in a slight
decrease, rather than increase, in deacetylase activity. This may be
explained by the fact that glycine is also the most flexible amino acid and disrupts The NDANSTs catalyze two concerted reactions that initiate
modification of the polysaccharide chain of the heparin and heparan sulfate precursors. The selective sulfation of clustered glucosamine residues is a requirement for the formation of protein-binding domains
in the fully processed glycosaminoglycans. In this study, we have
investigated the role played by eight cysteine residues conserved in
NDANSTs on both enzymatic functions. The impetus for this undertaking
was based largely on the previous demonstration of the selective loss
in deacetylase activity on treatment with NEM (15, 16, 19), and the
selective loss in sulfotransferase activity and enhancement in
deacetylase activity obtained after DTT treatment presented in Fig. 1.
Using a site-directed mutagenesis approach, each of the eight conserved
cysteines in the NDANST from rat liver was converted to an alanine, and
the effect of these changes on both enzymatic activities was examined.
The basis for the susceptibility of the N-deacetylase
activity to inactivation by NEM was determined by showing that
Cys486 was located in close proximity to the catalytic
center of the N-deacetylase active site, and played no
direct role in the N-deacetylase reaction. The enhancement
in deacetylase activity after treatment with DTT was mapped to two Cys
residues in the NH2-terminal half of the protein, while the
selective sensitivity of the N-sulfotransferase to DTT was
pinpointed to Cys residues toward the COOH-terminal portion of the
protein. These findings suggest that the two enzymatic functions are
differentiated both in their dependence on secondary structure imparted
by disulfide bonds and the localization of Cys residues to different
portions of the protein sequence.
Pettersson et al. (16) first demonstrated that treatment of
the mouse mastocytoma-derived NDANST with NEM resulted in 80% loss in
the N-deacetylase activity. Alanine substitution for each of
the conserved Cys residues in our chimeric construct failed to produce
an enzyme with a selective loss in the N-deacetylase function. Treatment of these altered proteins with NEM revealed that
the presence of an alanine at position 486 in the substituted protein
protected this enzymatic function from deactivation by NEM.
Cys486, therefore, did not appear to be required for enzyme
activity since it could be replaced by alanine, and it suggested that
NEM inhibits the deacetylase activity by physically blocking or
occupying space in the active site upon covalent modification of the
Cys486 side chain. This hypothesis by was tested by
replacing Cys486 with amino acids of differing sizes, and
showing that increasing the size of the side chains correlated with
loss in the deacetylase activity. The physiological significance of the
free cysteine is not yet clear. It has been proposed that strategically
placed free cysteines can participate in the regulation of cytosolic sulfotransferase activity by reacting with cellular glutathione (24).
Although there is no direct evidence that Cys486 is
involved in redox regulation of
N-deacetylase-N-sulfotransferase activity, its
location does make it an ideal candidate for controlling the rate of
the deacetylase activity through such a mechanism.
In contrast to the N-deacetylase activity, the
N-sulfotransferase activity was shown to be sensitive to DTT
treatment, and the cysteine residues in question were pinpointed to the
COOH-terminal portion of the protein. Substitution of
Cys818 or Cys828 with alanine had no effect on
the deacetylase activity, but completely abolished the sulfotransferase
activity. The sulfotransferase activity appears to require a specific
secondary structure localized to the COOH-terminal portion of the
protein. Although our data do not provide direct experimental proof, it
is tempting to speculate that Cys818 and Cys828
form a disulfide bond that brings residues in closer proximity within
the highly conserved sequences at both sides of the presumptive disulfide bond to form a sulfotransferase motif. This would be consistent with the sequence information recently reported for the
heparan sulfate 3-O-sulfotransferase (25). Although an
O-sulfotransferase, this enzyme is approximately 50%
identical to the carboxyl-terminal portion of
N-deacetylase-N-sulfotransferase and has only one
pair of cysteines, which correspond to the
Cys818-Cys828 pair in
N-deacetylase-N-sulfotransferases (Fig.
5). For completeness, the possibility
that the sulfotransferase domain might reside in part in the
GXXGXXK between residues 65 and 71 in rat liver N-deacetylase-N-sulfotransferase was evaluated.
This sequence had been proposed on the basis that it is a part of the
PAPS-binding domain shared among members of the cytosolic
sulfotransferases (26). Substituting alanine for individual glycine and
lysine residues in the GXXGXXK sequence had no
effect on sulfotransferase activity (data not shown).
INTRODUCTION
Top
Abstract
Introduction
References
MATERIALS AND METHODS
20 °C until used in enzyme assays.
RESULTS
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Fig. 1.
Effect of DTT on N-deacetylase
and N-sulfotransferase activities.
N-Deacetylase ( ) and N-sulfotransferase (
)
activities were assayed as described under "Materials and Methods."
Enzymes were incubated with 0-10 mM DTT for 30 min at
4 °C before assaying for activity.
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Fig. 2.
Position of the conserved cysteines in
heparan sulfate
N-deacetylase-N-sulfotransferases.
A, rat liver enzyme; B, mastocytoma enzyme;
C, construct of the protein A-NDANST fusion gene. The
protein A gene fused with that encoding the rat liver NDANST
Golgi-luminal domain was prepared as described previously (15).
Positions of cysteines that are conserved between the enzymes from rat
liver and mastocytoma cells are shown.
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Fig. 3.
SDS-polyacrylamide gel electrophoresis of
overexpressed NDANST with cysteine-for-alanine substitutions. Five
microliters of purified mutant enzyme was subjected to
SDS-polyacrylamide gel (4-12% gradient) electrophoresis under
reducing conditions. The gel was stained with Coomassie Blue dye. The
molecular size of protein markers is in kilodaltons.
Kinetic analysis of cysteine mutants: N-deacetylase activity
Kinetic analysis of cysteine mutants: N-sulfotransferase activity
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Fig. 4.
Effect of NEM on N-deacetylase
activity of cysteine mutants. N-Deacetylation assays
were performed as described under "Materials and Methods." Native
enzyme (--- ---) and substituted proteins: C56A (×), C159A
(---
---), C486A (
), C601A (··
··), C751A
(··
··), C818A (
), and C828A (
), were incubated with an
increasing concentration of NEM for 30 min on ice before addition of
3H-labeled K5 polysaccharide.
-helices (24). It should be noted that the adjacent residue 485 is also a glycine in the peptide sequence. As expected, none of the amino acid substitutions at position 486 had any
significant effect on the sulfotransferase activity of these proteins
(data not shown).
Effect of Cys486 substitution on N-deacetylase activity
DISCUSSION
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Fig. 5.
Proposed domain distribution in heparan
sulfate NDANST.
The identification of Cys486 as a residue in close
proximity to the deacetylase active center in the
NH2-terminal half of the protein, and the importance of Cys
residues for sulfotransferase activity in the COOH-terminal half,
suggest that the two enzyme domains are physically independent.
However, we have observed that removal of as little as 15 amino acid
residues from the COOH terminus resulted in complete loss of not only
the sulfotransferase activity, but also the deacetylase activity (data
not shown). It will be important to evaluate whether the portion
COOH-terminal to the proposed sulfotransferase motif is involved in
carbohydrate substrate coordination that allows for the concerted
reaction carried out by the two enzyme activities.
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ACKNOWLEDGEMENT |
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We are extremely grateful to Dr. Bruce Macher for helpful discussions and for making his laboratory available to complete this work.
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Note Added in Proof |
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During the review stage of our manuscript, two articles (Berninsone, P., and Hirschberg, C. B. (1998) J. Biol. Chem. 273, 25556-25559; and Seuyoshi, T., Kakuta, Y., Pedersen, L. C., Wall, F. E., Pedersen, L. G., and Negishi, M. (1998) FEBS Lett. 433, 211-214) have been published that demonstrated that the COOH-terminal portion of the rat liver NDANST contains the N-sulfotransferase activity.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Present address:
ChemoCentryx, 1539 Industrial Rd., San Carlos, CA 94070.
§ Present address: BioMarin Pharmaceutical, Inc., Novato, CA 94949.
The abbreviations used are: FGF, fibroblast growth factor; NEM, N-ethylmaleimide; DTT, dithiothreitol; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; DMEM, Dulbecco's modified Eagle's medium; NDANST, N-deacetylase-N-sulfotransferase; GlcA, glucuronic acid; GlcNAc, N-acetylglucosamine.
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REFERENCES |
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