Functional Analysis of Conserved Cysteines in Heparan Sulfate N-Deacetylase-N-sulfotransferases*

Zheng WeiDagger and Stuart J. Swiedler§

From Glycomed, Inc., Alameda, California 94501 and the Biochemistry and Molecular Biology Graduate Program, University of California, Davis, California 95616

    ABSTRACT
Top
Abstract
Introduction
References

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.

    INTRODUCTION
Top
Abstract
Introduction
References

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.

    MATERIALS AND METHODS

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 -20 °C until used in enzyme assays.

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.

    RESULTS

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.


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Fig. 1.   Effect of DTT on N-deacetylase and N-sulfotransferase activities. N-Deacetylase (black-square) 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.

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.


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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.

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.

                              
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Table I
Kinetic analysis of cysteine mutants: N-deacetylase activity
Deacetylase activity was assayed as described under "Materials and Methods." Enzyme activity is expressed as micromoles of [3H]acetyl group released from the deacetylation substrate K5 polysaccharide per minute. For calculation of the concentration of acetyl units on the K5 polysaccharide, it is assumed that there are on average 100 disaccharide units of GlcA-GlcNAc per K5 polysaccharide molecule. Results are the average of two independent assays. WT, wild type.

                              
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Table II
Kinetic analysis of cysteine mutants: N-sulfotransferase activity
N-sulfotransferase activity was assayed as described under "Materials and Methods." Enzyme activity is expressed as micromoles of [35S]sulfate incorporated into deacetylated K5 polysaccharide per minute. Results are the average of two independent assays. WT, wild type.

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.


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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 (---black-diamond ---) and substituted proteins: C56A (×), C159A (---black-triangle---), C486A (black-square), C601A (··black-triangle··), C751A (··black-diamond ··), C818A (open circle ), and C828A (bullet ), were incubated with an increasing concentration of NEM for 30 min on ice before addition of 3H-labeled K5 polysaccharide.

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 alpha -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).

                              
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Table III
Effect of Cys486 substitution on N-deacetylase activity
Deacetylase activity was assayed as described under "Materials and Methods." Enzyme activity is expressed as micromoles of [3H]acetyl group released from the deacetylation substrate K5 polysaccharide per minute. For calculation of the concentration of acetyl units on the polysaccharide, it is assumed that there are on average 100 disaccharide units of GlcA-GlcNAc per K5 molecule. Results are the average of two independent assays. WT, wild type.


    DISCUSSION

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).


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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.

    ACKNOWLEDGEMENT

We are extremely grateful to Dr. Bruce Macher for helpful discussions and for making his laboratory available to complete this work.

    Note Added in Proof

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.

    FOOTNOTES

* 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.

Dagger 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.
    REFERENCES
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Abstract
Introduction
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