J Biol Chem, Vol. 274, Issue 36, 25608-25612, September 3, 1999

Structure and Function of HNK-1 Sulfotransferase
IDENTIFICATION OF DONOR AND ACCEPTOR BINDING SITES BY SITE-DIRECTED MUTAGENESIS^*

Edgar Ong, Jiunn-Chern Yeh, Yili Ding, Ole Hindsgaul, Lars C. Pedersen, Masahiko Negishi, and Minoru Fukuda^§

From the Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, California 92037 and the  Laboratory of Reproductive and Developmental Toxicology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

HNK-1 glycan, sulfo3GlcA13Gal14GlcNAcR, is uniquely enriched in neural cells and natural killer cells and is thought to play important roles in cell-cell interaction. HNK-1 glycan synthesis is dependent on HNK-1 sulfotransferase (HNK-1ST), and cDNAs encoding human and rat HNK-1ST have been recently cloned. HNK-1ST belongs to the sulfotransferase gene family, which shares two homologous sequences in their catalytic domains. In the present study, we have individually mutated amino acid residues in these conserved sequences and determined how such mutations affect the binding to the donor substrate, adenosine 3'-phosphate 5'-phosphosulfate, and an acceptor. Mutations of Lys¹²⁸, Arg¹⁸⁹, Asp¹⁹⁰, Pro¹⁹¹, and Ser¹⁹⁷ to Ala all abolished the enzymatic activity. When Lys¹²⁸ and Asp¹⁹⁰ were conservatively mutated to Arg and Glu, respectively, however, the mutated enzymes still maintained residual activity, and both mutant enzymes still bound to adenosine 3',5'-diphosphate-agarose. K128R and D190E mutant enzymes, on the other hand, exhibited reduced affinity to the acceptor as demonstrated by kinetic studies. These findings, together with those on the crystal structure of estrogen sulfotransferase and heparan sulfate N-deacetylase/sulfotransferase, suggest that Lys¹²⁸ may be close to the 3-hydroxyl group of -glucuronic acid in a HNK-1 acceptor. In contrast, the effect by mutation at Asp¹⁹⁰ may be due to conformational change because this amino acid and Pro¹⁹¹ reside in a transition of the secondary structure of the enzyme. These results indicate that conserved amino acid residues in HNK-1ST play roles in maintaining a functional conformation and are directly involved in binding to donor and acceptor substrates.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

HNK-1 glycan is a unique carbohydrate expressed in a cell-type specific manner (1, 2). In particular, HNK-1 glycan has been found in a number of neural cell adhesion molecules including the neural cell adhesion molecule, myelin-associated glycoprotein, L1, contactin, and P0 (3-7). Carbohydrate structural analysis of P0 showed that HNK-1 glycan is present in biantennary bisected N-glycan as sulfoGlcA13Gal14GlcNAc12Man16[RMan13(GlcNAc14)]Man14GlcNAc14GlcNAcAsn (8). The capping structure of this HNK-1 epitope is the same as those found in glycolipids such as sulfo3GlcA13Gal14GlcNAc13Gal14Glcceramide (9, 10). The expression of HNK-1 glycan is spatially and temporally regulated and is found on migrating neural crest cells, cerebellum, and myelinating Schwann cells in motor neurons but not on those in the sensory neurons (11-13). Neural outgrowth of mouse motor neurons was facilitated much better by HNK-1 glycolipid substratum than sulfatide or GD1b ganglioside substratum (14). Complementary to this finding, the inhibition of neurite outgrowth by HNK-1 glycan was not observed once the sulfate group was removed (15). It has also been shown that the homophilic interaction of P0 depends on carbohydrate-protein interaction and anti-HNK-1 antibody inhibits this interaction (16, 17).

To elucidate the roles of HNK-1 glycan in molecular details, we and others have cloned a sulfotransferase HNK-1ST that forms HNK-1 glycan on neural cell adhesion molecule and glycolipids (18, 19). This was possible because 1,3-glucuronosyltransferase that forms a HNK-1 precursor structure was cloned before (20). When we compared the amino acid sequence of HNK-1ST to those of Golgi-associated sulfotransferases cloned before (21-24), we noticed that amino acid sequence motifs, including the sequence RDP, are shared by those enzymes (18). Although we did not initially detect its homology with cytosolic sulfotransferases, subsequent studies revealed that there are two regions in the catalytic domain that are shared by all sulfotransferases cloned so far (25, 26). This homology was also proposed because the crystal structure of a mouse estrogen sulfotransferase became available (27), and the amino acid residues involved in the interaction with the 5'-phosphosulfate or 3'-phosphate group of PAPS were identified (Fig. 1). The studies thus revealed that a lysine or arginine residue is shared by all sulfotransferases cloned to date in the 5'-phosphosulfate binding (5'-PSB)¹ site and an arginine residue is shared by almost all sulfotransferases at the 3'-phosphate binding (3'-PB) motif (25-27). Moreover, the entirely conserved serine residue at the 3'-PB loop apparently provides a hydrogen bond with a hydroxyl group of 3'-phosphate (27). These studies, however, did not reveal how those or other amino acids are involved in the interaction with an acceptor substrate.

View larger version (51K): [in this window] [in a new window]   Fig. 1.   Comparison of amino acid sequences of sulfotransferases. PAPS binding sites of cytosolic and Golgi-associated sulfotransferases are shown based on the crystal structure of estrogen sulfotransferase and heparan sulfate N-deacetylase/sulfotransferase (27, 28). 3'-PB sites were also suggested by comparison of Golgi-associated sulfotransferases (18). The following sequences are compared: mouse estrogen sulfotransferase, moEST (36); mouse phenol sulfotransferase, moPST (37); mouse hydroxysteroid sulfotransferase, moHST (38); rabbit amine sulfotransferase, rbAST (39); plant flavonol sulfotransferase, FST (40); Rhizobium sp. sulfotransferase, NodH (41); rat heparan sulfate N-deacetylase/sulfotransferase, raHSND/NST (42); chicken chondroitin sulfate D-N-acetylglucosamine-6-O-sulfotransferase, chCS6ST (21); human galactosylceramine 3'-sulfotransferase, huGalCerST (22); hamster heparan sulfate L-iduronic acid 2-O-sulfotransferase, haHS2OST (23); human heparan sulfate D-glucosamine 3-O-sulfotransferase, huHS3OST (24); human keratan sulfate D-galactose 6-O-sulfotransferase, huKSGal6ST (43); human heparan sulfate D-N-sulfoglucosamine 6-O-sulfotransferase, huHS6OST (44); human HNK-1 sulfotransferase, huHNK-1ST (18); mouse N-acetylglucosamine 6-O-sulfotransferase, moGlcNAc6ST (45); human tyrosylprotein sulfotransferase, huTPST (46); mouse L-selectin ligand sulfotransferase, moLSST (47); Drosophila segregation disorder protein, SD (48).

In the present study, we individually mutated five different amino acid residues in the 5'-PSB and 3'-PB sites of HNK-1ST by site-directed mutagenesis. We first assayed those mutant enzymes for transfer of [³⁵S]sulfate from [³⁵S]PAPS, and then catalytic properties were obtained. Those mutants were also assayed for their binding to a PAP analogue of PAPS. Finally, these results were interpreted in relation to a three-dimensional structure inferred from the crystal structures determined for estrogen sulfotransferase and the human heparan sulfate N-deacetylase/sulfotransferase, HSNST (27, 28). Our results strongly suggest that these two regions of the catalytic domain, in particular those amino acids in 5'-PSB site, are involved in the interaction with both acceptor and donor substrates.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Site-directed Mutagenesis-- The cDNA encoding HNK-1ST was cloned as described previously (18). A cDNA encoding a catalytic domain of HNK-1ST fused with an IgG-binding domain of protein A was constructed as described before (18). The plasmid, pcDNAI-A·HNK-1ST (18), encoding a soluble protein A-HNK-1ST fusion protein was digested with BamHI and SpeI to release a 1.6-kilobase pair cDNA fragment. pBluescript II KS⁺ vector (Stratagene, La Jolla, CA) was digested with the same restriction enzymes and ligated to the HNK-1ST fragment to give plasmid pBSII KS⁺-HNK-1ST. Site-directed mutagenesis was performed on this plasmid using a Chameleon^TM double-stranded, site-directed mutagenesis kit (Stratagene) (29, 30). Phosphorylated oligonucleotides covering conservative or nonconservative amino acid replacement were synthesized and purified by denaturing polyacrylamide gel electrophoresis before use. Following site-directed mutagenesis of the required amino acid residue on pBSII KS⁺-HNK-1ST, a 0.53-kilobase pair fragment flanked by Bsu36I and BamHI and containing the specific codon replacement was sequenced to confirm the correct sequence. This 0.53-kilobase pair DNA fragment was released by digestion with Bsu36I and BamHI and was used to replace the corresponding sequence in the wild type DNA sequence in pcDNAI-A·HNK-1ST.

Production of HNK-1ST-- The plasmid pcDNAI-A·HNK-1ST containing the appropriate mutation was transfected into COS-1 cells using LipofectAMINE^® Plus reagents (Life Technologies, Inc.) (18). Following 24 h of growth in a complete medium at 37 °C, the medium was replaced with serum-free OptiMEM solution (Life Technologies, Inc.). After a 48-h incubation at 37 °C, the culture medium containing the fusion protein was collected and filtered through a 0.22-µm membrane. The fusion protein was isolated by adsorption to IgG-Sepharose 6FF beads (Amersham Pharmacia Biotech) (31). After washing, the fusion protein-containing beads were resuspended in 100 mM Tris-HCl buffer, pH 7, at 50% suspension volume. This was used as the enzyme source. Appropriate controls (both wild type and mock-transfected reactions) were performed.

HNK-1ST and the mutant enzymes released were separated by SDS-polyacrylamide gel electrophoresis, and their amount was estimated by Western blot analysis using peroxidase-conjugated rabbit anti-goat IgG antibodies (30) purchased from ICN.

Sulfotransferase Assay-- The acceptor, GlcA13Gal14GlcNAc1octyl was synthesized as described previously (18, 32). Appropriate amounts of the acceptor and the donor, adenosine 3'-phosphate 5'-phospho[³⁵S]sulfate, were mixed and dried down under vacuum. The material was then redissolved in 40 µl of a solution containing 100 mM Tris-HCl, pH 7, 0.1% Triton X-100, 2.5 mM ATP, and 10 mM MnCl₂. To this, 10 µl of the 50% suspension of the enzyme solution, prepared as described above, was added. The mixture was incubated at 37 °C for 1 h with constant, gentle agitation. The reaction was stopped by adding 450 µl of 250 mM ammonium formate, pH 4. The labeled products were applied to C18 reverse-phase chromatography columns (Alltech) and eluted with 70% methanol. A portion of the eluted product was subjected to scintillation counting. Relative activity was measured and standardized against the values obtained for the wild type after subtraction of the background activity for the mock samples. The K_m for wild type HNK-1ST and mutant enzymes exhibiting detectable enzymatic activity was determined using a Lineweaver-Burk plot at various concentrations of the acceptor (2.5 µM to 2000 µM) and donor (1 µM to 2000 µM).

Binding of HNK-1ST to Immobilized Adenosine Diphosphate-- Following the transfection protocol as described above, the culture medium (20 ml) from the transfected cells was concentrated approximately 20-fold using Centriprep-10 spin columns (Amicon). The concentrated medium containing the enzyme (100 µl) was mixed with 900 µl of 100 mM Tris-HCl, pH 7, containing 5 mM MnCl₂, applied to 1 ml of adenosine-3', 5'-diphosphate-agarose (Sigma), washed, and equilibrated in 100 mM Tris-HCl, pH 7, as described previously (33). After washing the beads with 4 ml of 100 mM Tris-HCl, pH 7, the bound enzyme was eluted with a NaCl linear gradient (0-500 mM) with increasing NaCl at 25 mM/ml. The eluted sample (200 µl) from each fraction (1 ml) was coated onto a 96-well microtiter plate and incubated at 4 °C overnight. After blocking the wells with 1% bovine serum albumin for 2 h at room temperature, a 1:1000 dilution of rabbit anti-goat IgG-horseradish peroxidase conjugate (200 µl) was added and incubated for 1 h at room temperature. Following three washes with Tris-buffered saline containing 0.2% Tween 80, 200 µl of a chromogenic substrate (0.1 mg of o-phenylenediamine/ml of 50 mM phosphate-citrate buffer, pH 5, containing 0.003% H₂O₂) was added to each well. After incubation at room temperature to allow sufficient color to develop, the reaction was stopped with 50 µl of 2 N sulfuric acid. The absorbance at 490 nm was read using a microtiter plate reader (Bio-Rad).

Modeling of HNK-1ST Active Sites-- The active site of HNK-1ST was modeled using the estrogen sulfotransferase and the HSNST coordinates (27, 28). The program "O" was utilized to make the changes to the sequence using the program Molscript (34).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation of Mutant HNK-1ST and Its Assay-- Previously we have shown that HNK-1ST can be expressed as a soluble protein fused with protein A (18). Such a soluble chimeric protein has a significant activity and is judged to reflect approximately the catalytic activity of the enzyme present in Golgi (18). In the present study, we took advantage of this property, and a soluble chimeric HNK-1ST was used throughout the studies.

As shown in Fig. 2, the wild type enzyme exhibits an activity profile typical for a transferase, and substrate inhibition or acceptor inhibition was not observed. We then individually mutated amino acid residues Lys¹²⁸ in the 5'-PSB site and Arg¹⁸⁹, Asp¹⁹⁰, Pro¹⁹¹, and Ser¹⁹⁷ in the 3'-PB motif (see Fig. 1 also). After these mutated enzymes and the wild type enzyme were expressed, the expression of each enzyme was estimated by Western blot analysis using rabbit IgG as a probe because the protein A portion of the chimeric protein can bind to rabbit IgG (Fig. 3). After adjusting the amount of the enzyme, the catalytic activity of each enzyme preparation was assayed using standard incubation conditions, as described under "Experimental Procedures."

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Dependence of HNK-1ST activity on the concentrations of acceptor or donor substrate, PAPS. A and B, dependence of HNK-1ST activity of wild type and its mutants on the concentration of the acceptor, GlcA1 3Gal1 4GlcNAc1 octyl (A) or PAPS (B). C and D, Lineweaver-Burk plots of A and B, respectively. The activity was measured for wild type (), K128R (), and D190E () mutant enzymes.

View larger version (90K): [in this window] [in a new window]   Fig. 3.   Comparison of the amount of wild type and mutant HNK-1ST used. The chimeric enzymes were released from IgG-Sepharose by heating in the sample buffer and separated by SDS-polyacrylamide gel electrophoresis. The gel was blotted onto nitrocellulose membrane. The proteins were detected by horseradish peroxidase-conjugated rabbit anti-goat IgG antibodies. Molecular mass markers are on the right.

As shown in Fig. 4, almost no activity remained when Lys¹²⁸, Arg¹⁸⁹, Asp¹⁹⁰, Pro¹⁹¹, and Ser¹⁹⁷ were individually mutated to an alanine residue. In contrast, the conservative mutation of K128R and D190E maintained 15 and 60% of the activity compared with that of wild type. Mutation of R189K or P191G, on the other hand, was not tolerated, and the mutant enzymes completely lost the activity.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Comparison of the enzymatic activities of wild type and mutant HNK-1STs. The enzymatic activity was measured using the conditions described under "Experimental Procedures."

Amino Acid Mutations Affect Binding to Donor-- To determine how these mutations cause a change in the binding to PAPS, the wild type HNK-1ST and each mutated enzyme were individually applied to a column of PAP-agarose, and the bound enzyme was eluted by a linear gradient of NaCl. The eluted enzymes were immunochemically detected using rabbit IgG-peroxidase conjugate. As shown in Fig. 5A, the wild type enzyme bound to the column in a biphasic way, and the majority of the bound enzyme was eluted after the gradient elution of NaCl started. Similar results were reported in a flavonol sulfotransferase (33). In contrast, K128A mutant barely bound to PAP-agarose, whereas the K128R mutant enzyme bound to the same column as much as the wild type enzyme.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Binding of wild type and mutant HNK-1ST enzymes to PAP-agarose. The chimeric enzymes fused with protein A were concentrated from culture medium and applied to a column of adenosine 3',5'-diphosphate-agarose. After washing the column with 100 mM Tris-HCl, pH 7, the bound enzyme was eluted with a linear gradient of NaCl (0-500 mM) () as described under "Experimental Procedures." The eluted enzyme was detected by incubating with rabbit anti-goat IgG-horseradish peroxidase conjugate. The elution profiles are shown for A, wild type (), K128R (), K128A (); B, R189K (), R189A (); C, D190E (); D, S197T () and S197A () mutant enzymes.

In a similar assay, the R189K, R189A, S197T, and S197A mutant enzymes did not bind to the PAP-agarose column (Fig. 5, B and D), whereas D190E bound well to the same column (Fig. 5C). These results demonstrated that K128R and D190E mutations did not significantly affect the binding to PAPS, whereas the conservative mutation of R189K and S197T dramatically reduced the binding to PAPS.

Mutation on Lys¹²⁸ and Asp¹⁹⁰ Affects Binding to Acceptor Substrate-- The above results indicate that the conservative mutation at Lys¹²⁸ and Asp¹⁹⁰ did not significantly affect the binding to PAPS but reduced the catalytic activity. To determine whether such a decrease in activity was due to impaired binding to the acceptor, the enzyme activity was determined using varying concentrations of donor substrate PAPS or the acceptor, GlcA13Gal14GlcNAc1octyl.

The results shown in Fig. 2, A and B, were replotted for the Lineweaver-Burk plot as seen in Fig. 2, C and D. The K_m for the donor substrate PAPS is almost the same between the wild type (66 µM) and the D190E mutant (70 µM) and is even better for the K128R mutant (12 µM) than for the wild type enzyme. In contrast, the K_m value for the acceptor was 316 µM for the wild type enzyme and increased 5.6-fold for K128R mutant (1776 µM) and 2.4-fold for D190E mutant (744 µM), respectively. These results suggest that Lys¹²⁸ and Asp¹⁹⁰ may be involved in the binding to the acceptor or very close to the acceptor binding site (see also under "Discussion").

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Comparison of sulfotransferases cloned to date revealed that there are two amino acid sequences that are highly conserved among different sulfotransferases (Fig. 1). In the present study, we first demonstrated that mutations of Arg¹⁸⁹, Pro¹⁹¹, and Ser¹⁹⁷ inactivate the enzyme, indicating that these amino acid residues are important for either binding to the donor PAPS or maintaining the conformation of the enzyme for transfer of the sulfate group to the acceptor molecule. All mutations of these amino acids, including conservative replacement, resulted in the loss of PAPS binding in these mutant enzymes (Fig. 5). These results confirm the previous conclusions that 5'-phosphosulfate and 3'-phosphate binding sites are critical for the binding to PAPS (25-28).

More importantly, the present study also revealed that the mutations at Lys¹²⁸ and Asp¹⁹⁰ can be tolerated well for the binding to PAPS, and such a binding became even better after mutation of Lys¹²⁸ to Arg. The excellent binding of these mutant enzymes to PAPS can be seen in both affinity chromatography using PAP-agarose (Fig. 4) and in kinetic studies (Fig. 2). In contrast, the same kinetic studies showed that these mutant have lower affinity to the acceptor, GlcA13Gal14GlcNAc1octyl (Fig. 2). These results suggest that these two amino acids may be more intimately involved in the binding of an acceptor than in the binding of a donor substrate.

No studies are available on the mutation of Asn¹³¹ in the estrogen sulfotransferase, which corresponds to Asp¹⁹⁰ in HNK-1ST, although crystal structural studies showed that Arg¹³⁰, which is next to Asn¹³¹, interacts with the 3'-phosphate group of PAP (27). Our mutation experiments demonstrated that the mutation of Arg¹⁸⁹ to either Lys or Ala resulted in the complete loss of the enzymatic activity (Fig. 2). The loss of the activity was accompanied by the loss in the binding to PAP (Fig. 5).

The present study also demonstrated that mutation of Asp¹⁹⁰ to Glu did not inactivate the enzyme, whereas mutation of Arg¹⁸⁹ to Lys completely inactivated the enzyme. These results may suggest that Arg¹⁸⁹ is critical in the binding to a 3'-phosphate group of PAPS, whereas Asp¹⁹⁰ is involved in the binding to an acceptor. However, these results need to be evaluated in relation to the three-dimensional structure of HNK-1ST. The crystal structures of the estrogen sulfotransferase and HSNST demonstrated that the structure of the 5'-PSB and 3'-PB sites is well conserved in these two different enzymes (27, 28). These results, together with the results obtained in the present study, allow us to propose a possible three-dimensional structural model of HNK-1ST, as shown in Fig. 6.

View larger version (38K): [in this window] [in a new window]   Fig. 6.   Model of the HNK-1ST active site. Side chains that have been mutated in this study are displayed around the PAP molecule. A model was created by changing the estrogen sulfotransferase side chains that differ from HNK-1ST using the program O (34). The positions of the mutated side chains are based on the corresponding positions of the side chains in both the HSNST and mouse estrogen sulfotransferase structures (27, 28). The figure was created using the program Molscript (49). Lys128, Arg189, Asp190, Pro191, and Ser197 are shown. The position of PAP and the -glucuronic residue (shown by 3'-OH) is also postulated. A sulfate group attached to PAP is presumably at the position between the 5'-phosphate group and 3'-OH but not shown.

Concerning the 3'-PB site, Arg¹⁸⁹ and Ser¹⁹⁷ can interact directly with the 3'-phosphate group of PAP in the model, thus, mutations of Arg¹⁸⁹ and Ser¹⁹⁷ can profoundly alter PAPS binding, affecting HNK-1ST activity. Asp¹⁹⁰ and Pro¹⁹¹ are not in a position to interact directly with the PAP molecule. They reside in a transition of secondary structure from a -sheet to an -helix, making for a very tight turn of about 90° (Fig. 6). Through its interactions with backbone N atoms of residues 192 and 193 in the following -helix, Asp¹⁹⁰ may help to stabilize this turn. Thus, mutation of Asp¹⁹⁰ may alter the core structure of the PAP binding site, affecting indirectly HNK-1ST activity. Pro¹⁹¹ may also be involved in the stabilization because its mutation abolished HNK-1ST activity. These residues (Arg¹⁸⁹, Ser¹⁹⁷, Asp¹⁹⁰, and Pro¹⁹¹) are located more than 20 Å from the nearest atom in the glucuronic acid molecule and the PAP molecule than the glucuronic acid molecule. They are not in a position to interact directly with the acceptor molecule for HNK-1ST. It would be more of an indirect effect, if any mutations of these residues affected the binding of the acceptor. The R(D/N)P sequence may thus be conserved to maintain the structure of the PAPS binding site.

Lys¹²⁸ (or Arg in some sulfotransferases) is conserved in the 5'-PSB site of all known sulfotransferases (Fig. 1). It can form hydrogen bonds with the 5'-phosphate group of PAP and is involved in catalysis. Mutations of this residue inactivated HNK-1ST, except that K128R still maintains some of the original activity. Lys¹²⁸ is 9 Å away from the 3-hydroxyl group of the glucuronic acid molecule and is unlikely to interact directly with the acceptor of HNK-1ST. In the mouse estrogen sulfotransferase, the K48R mutation in the 5'-PSB site did not change K_m of the enzyme for either estrogen or PAP (35). In contrast, the K128R mutation of HNK-1ST increased K_m toward glucuronic acid in the acceptor molecule more than 5-fold. In the model, the end of the side chain of Arg (at the position of Lys¹²⁸) can be moved to within 3.2 Å of the 3-hydroxyl group of the glucuronic acid molecule (Fig. 6). A bulkier nonplaner substrate of HNK-1ST may actually come closer to the position of the mutated Arg. Thus the K128R mutation could have an effect on the binding of the acceptor in HNK-1ST.

In the mouse estrogen sulfotransferase, Lys⁴⁸ corresponds to Lys¹²⁸ in HNK-1ST. In contrast to the results obtained in the present study, the mutation of Lys⁴⁸ to Arg did not change the K_m toward estrogen or PAPS in a previous study (34). The importance of Lys⁴⁸ was, however, demonstrated because its mutation to methionine completely inactivated the enzyme in the same study. These results indicate that Lys¹²⁸ in the HNK-1ST is closer to an acceptor than Lys⁴⁸ in the estrogen sulfotransferase. This difference is probably due to the difference in acceptor structures (Fig. 6).

The present study demonstrated that HNK-1ST and estrogen sulfotransferase share many of the structural requirements for the enzymatic activity. The study, on the other hand, also indicates that acceptor binding sites of different enzymes are distinct from one another. Further studies on the three-dimensional structure of HNK-1ST will be of significance to determine how the acceptor is recognized by the enzyme and provide critical information on the three-dimensional structure of the acceptor oligosaccharide.

	ACKNOWLEDGEMENTS

We thank Susan Fanno and Julie Howe for organizing the manuscript.

	FOOTNOTES

^* This work was supported by Grants RO1 CA33895 and PO1 CA71932 awarded by the NCI, National Institutes of Health. This article is dedicated to Professor Pierre Sina on the occasion of his 62nd birthday.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: The Burnham Institute, 10901 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3144; Fax: 858-646-3193; E-mail: minoru@burnham-inst.org.

	ABBREVIATIONS

The abbreviations used are: 5'-PSB, 5'-phosphosulfate binding; 3'-PB, 3'-phosphate binding; HNK-1ST, HNK-1 sulfotransferase; PAPS, adenosine 3'-phosphate 5'-phosphosulfate; PAP, adenosine 3',5'-diphosphate; HSNST, human heparan sulfate N-deacetylase/sulfotransferase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES