Multiple Isozymes of Heparan Sulfate/Heparin GlcNAc N-Deacetylase/GlcN N-Sulfotransferase

STRUCTURE AND ACTIVITY OF THE FOURTH MEMBER, NDST4*

Jun-ichi AikawaDagger §, Kay GrobeDagger , Masafumi Tsujimoto§, and Jeffrey D EskoDagger ||

From the Dagger  Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California 92093-0687 and the § Cellular Biochemistry Laboratory, RIKEN, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan

Received for publication, October 20, 2000, and in revised form, November 20, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

We report the cloning and partial characterization of the fourth member of the vertebrate heparan sulfate/heparin: GlcNAc N-deacetylase/GlcN N-sulfotransferase family, which we designate NDST4. Full-length cDNA clones containing the entire coding region of 872 amino acids were obtained from human and mouse cDNA libraries. The deduced amino acid sequence of NDST4 showed high sequence identity to NDST1, NDST2, and NDST3 in both species. NDST4 maps to human chromosome 4q25-26, very close to NDST3, located at 4q26-27. These observations, taken together with phylogenetic data, suggest that the four NDSTs evolved from a common ancestral gene, which diverged to give rise to two subtypes, NDST3/4 and NDST1/2. Reverse transcription-polymerase chain reaction analysis of various mouse tissues revealed a restricted pattern of NDST4 mRNA expression when compared with NDST1 and NDST2, which are abundantly and ubiquitously expressed. Comparison of the enzymatic properties of the four murine NDSTs revealed striking differences in N-deacetylation and N-sulfation activities; NDST4 had weak deacetylase activity but high sulfotransferase, whereas NDST3 had the opposite properties. Molecular modeling of the sulfotransferase domains of the murine and human NDSTs showed varying surface charge distributions within the substrate binding cleft, suggesting that the differences in activity may reflect preferences for different substrates. An iterative model of heparan sulfate biosynthesis is suggested in which some NDST isozymes initiate the N-deacetylation and N-sulfation of the chains, whereas others bind to previously modified segments to fill in or extend the section of modified residues.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Heparan sulfate and heparin bind a variety of growth factors, enzymes, and extracellular matrix proteins (1). These interactions depend on specific arrangements of variably sulfated glucosaminyl residues (GlcNAc, GlcN,1 and GlcNS) and glucuronic (GlcA) and iduronic acids. The assembly of these sequences proceeds in a stepwise manner as follows. (i) The chains initiate by formation of the linkage tetrasaccharide, GlcAbeta 1,3Galbeta 1,3Galbeta 1,4Xylbeta , on serine residues of core proteins; (ii) the chains elongate by alternating the additions of GlcNAcalpha 1,4 and GlcAbeta 1,4 residues; (iii) the chains are modified initially by N-deacetylation and N-sulfation of subsets of GlcNAc residues, (iv) adjacent D-GlcA residues undergo C5-epimerization to L-iduronic acid, and (v) sulfation occurs at C2 of the uronic acid residues and at C6 and C3 of glucosaminyl residues. In this scheme, GlcNAc N-deacetylation and N-sulfation creates the prerequisite substrate needed for the later modification reactions (reviewed by Rodén (2)). These reactions are catalyzed by a family of enzymes designated the GlcNAc N-deacetylase/N-sulfotransferases (NDSTs).

Three NDST isozymes have been identified in vertebrates, whereas only single orthologs are known in Drosophila melanogaster and Caenorhabditis elegans (3-11). Mutations in these genes can have profound effects on development. In D. melanogaster, loss of NDST (sulfateless) results in unsulfated chains and defective signaling by multiple growth factors and morphogens (12-14). In mice, targeted disruption of NDST1 results in neonatal lethality due to pulmonary hypoplasia and respiratory distress (15, 16). Liver heparan sulfate is also affected in composition, consistent with the widespread expression of NDST1 (10, 11). NDST2 mRNA is also highly expressed in most tissues (10, 11). However, mice lacking NDST2 develop and reproduce normally but show fewer connective tissue-type mast cells. Those cells that remain have an altered morphology and severely reduced amounts of stored histamine and mast cell proteases (17, 18). In contrast to NDST1 null mice, tissue heparan sulfates of NDST2 null mice appear to be unaffected. Murine mutants in NDST3 have not yet been described but are currently under development.2

Studies of a Chinese hamster ovary cell mutant defective in NDST1 (pgsE-606) showed that this isozyme accounts for ~50% of the N-deacetylated/N-sulfated glucosaminyl residues in this system (19-22). The remainder arise from the action of NDST2, the only other isozyme expressed by Chinese hamster ovary cells (11). The observation that NDST1 null mice accumulate partially undersulfated chains in tissues supports the idea that multiple isozymes contribute to the formation of heparan sulfate chains (16). The quantitative contribution of NDST3 is unknown but its more restricted pattern of expression in mice suggests it may play an important role in the development or physiology of specific organs (e.g. brain, liver, and kidney) (11).

Extensive analysis of public genome data bases suggested the existence of a fourth member of the NDST family. Subsequent cDNA cloning led to the identification of a new member of the vertebrate NDST gene family, designated NDST4. A preliminary report of the clone has appeared in abstract form (23). We now report the full-length sequence of NDST4 and show that the four members of the NDST family have clear differences in distribution and enzymatic properties. Different combinations of the isozymes may account for some of the differences seen in heparan sulfates from various tissues.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Identification of the Genomic Clone-- The nucleotide sequence of human NDST3 was used as a probe to search The Institute of Genome Research (TIGR) library of bacterial artificial chromosomes (BAC) end sequencing data base. BAC clone 2608.A.5, 2177.H.9, and 183.D.15 for hNDST3, hNDST4, and mNDST4, respectively, were obtained from Genome Systems Inc. or Research Genetics, Inc. DNA was prepared using the Plasmid DNA Isolation kit (Qiagen) according to the company's protocol.

Preparation of Human NDST4 cDNA by 5'- and 3'-Rapid Amplification of cDNA Ends (RACE)-- 5'- and 3'-RACE was performed using an adaptor-ligated library of human fetal brain cDNA (CLONTECH) essentially as described previously (11). Briefly, the 5' portion was prepared by cDNA amplification using a primer consisting of nucleotides 2368-2392 (hNDST4-1R, 5'-tagagggtctttctgctcagggaag-3'), anchor primer 1 (AP1), and Advantage2 polymerase (CLONTECH) according to the touch-down PCR protocol (30 cycles). An aliquot (one-fourth) of this PCR was subjected to a subsequent nested PCR (20 cycles) using a primer consisting of nucleotides 2341-2365 (NDST4-1R-GSP1, 5'-ctcaaaatactggtgggccagttgc-3') and AP1 followed by a second nested PCR (20 cycles) using a primer consisting of nucleotides 2281-2306 (NDST4-1R-GSP2, 5'-gctctgcacaaagttgaccaagttc-3') and anchor primer 2 (AP2). For the 3' portion, cDNA was first amplified using a primer consisting of nucleotides 1894-1917 (NDST4-1F-GSP3, 5'-tgcactggaacatggaataccaatc-3') and AP1 followed by a nested PCR (20 cycles) using a primer consisting of nucleotides 2088-2133 (NDST4-1F-GSP4, 5'-tcctccctcgacagacttgtgggttg-3') and anchor primer AP2 (CLONTECH). The DNA products were gel-purified and cloned into pGEM-T (Promega). The nucleotide sequence was determined using Applied Biosystems, Inc. DNA sequencers 370 and 377-18.

Preparation of Mouse cDNA for NDST1-4 by PCR-- To prepare mouse NDST4, a PCR (60 cycles) was performed using a mouse brain cDNA library as the template (CLONTECH). cDNA was amplified using a primer consisting of nucleotides 1-27 (mNDST4-1F, 5'- acatgaatgttgggaaggaaaactgg-3') and 2706-2732 (mNDST4-2R, 5'- tctccagtgctatctcactttctgcag-3') with Advantage2 polymerase (CLONTECH). The DNA products were gel-purified and cloned into pGEM-T (Promega). Preparation of cDNA for mouse NDST1, NDST2, and NDST3 were performed by PCR and will be described elsewhere.3

Nucleotide sequences of NDSTs were aligned using ClustalW and edited manually. Phylogenetic trees were built employing the Neighborhood Joining method of the program PAUP v4.00.

RT-PCR Analysis of mRNA Expression-- For RT-PCR analysis in mouse tissues, normalized cDNA was obtained from CLONTECH (Mouse Multiple Tissue cDNA Panel). PCR was performed as described above, running 29 and 35 cycles for mNDST1-4. For the specific amplification of each mNDST, specific primers were used as follows: mNDST1-1F (5'-accacagccagccagaacgcttgtg-3') and -1R (5'-agctgcgctcctctcccttactgtc-3'), mNDST2-1F (5'-tgcaacattccagtgtgggctgatggc-3') and -1R (5'-atgacattagagaggccaggccacagg-3'), mNDST3-2F (5'-tgtgtttcctgtgagtccagatgtgtg-3') and -2R (5'-attgtcctcctcacttccatcagcctg-3'), and mNDST4-1F (5'-aacaggaaatgacacttattgaaacg-3') and-1R (5'-actttggggcctttggtaatatg-3') corresponding to mNDST1 (nucleotides 3104-3128 and 3520-3544 of M92042 (rat NDST1), mNDST2 (nucleotides 2630-2656 and 3142-3168 of U02304), mNDST3 (nucleotides 1-27 and 335-361 of AF221095), and mNDST4 (nucleotides 216-242 and 711-733 of AB036838), respectively.

Enzyme Assays-- Murine NDST1-4 DNA fragments coding for the soluble form of the respective enzyme (starting at residue 43 from the N terminus) were ligated into pRK5F10PROTA (Glycomed, Inc.) and transfected into COS7 cells (LipofectAMINE, Life Technologies, Inc.). The soluble NDSTs were secreted as protein A fusion proteins into the medium. After purification on IgG-Sepharose (Sigma), protein integrity and expression levels were confirmed by SDS-polyacrylamide gel electrophoresis. Protein concentrations were estimated by silver staining samples and comparing the intensity of the chimeras to standard amounts of bovine serum albumin. NDST1-4 protein or protein A (500 ng) was used for each assay. Duplicate measurements were performed for each enzyme, and the signal obtained in the protein A control was subtracted from the values.

GlcNAc N-deacetylase activity was measured using [acetyl-3H]heparosan prepared from Escherichia coli K5 capsular polysaccharide (19, 21). The assay was performed in 50 mM MES, pH 6.5, containing 1% Triton X-100, 10 mM MnCl2, and 1 × 105 cpm [3H]heparosan. After 30 min at 37 °C, the reaction was stopped by the addition of 0.5 volumes of 0.2 M HCl, 1 volume of 0.1 M acetic acid, and 1 volume of water. [3H]Acetic acid was recovered by extracting the sample three times with 1 volume of ethyl acetate. An aliquot of the pooled fractions (0.5 ml) was analyzed by liquid scintillation counting.

GlcN N-sulfotransferase activity was measured by the incorporation of 35SO4 from PAPS to heparosan or N-desulfated heparin (19, 21). Approximately 105 cpm [35S]PAPS were incubated for 1 h at 37 °C in 50 mM HEPES, pH 7.0, 1% Triton X-100, 10 mM MgCl2, 1 mM MnCl2, 25 µg of heparosan or N-desulfated heparin, and 10 µM nonradioactive PAPS in a total volume of 50 µl. The reaction was stopped by the addition of 2 µl of 0.5 M EDTA, and 2 mg of chondroitin sulfate A was added to the reaction as carrier. The sample was applied to a small column (0.5 ml) of DEAE-Sephacel prepared in a disposable pipette tip and washed 3 times with 10 ml of 20 mM sodium acetate, pH 6.0, containing 0.2 M NaCl. The labeled chains were eluted using the same buffer containing 1 M NaCl, and the amount of radioactivity incorporated was measured by liquid scintillation counting. The concentration of acceptor was slightly greater than the Km value determined previously for NDST1 (22).

Chromosomal Location of Human NDST3 and -4-- The chromosomal location of human NDST3 and -4 were determined by in situ fluorescent hybridization (FISH, Genome Systems Inc.). BAC DNA tagged with digoxigenin-labeled dUTP by nick translation was hybridized to normal metaphase chromosomes derived from phytohaemagglutinin-stimulated peripheral blood lymphocytes in a solution containing 50% formamide, 10% dextran sulfate, and 2× SSC (1× SSC = 0.15 M NaCl and 0.015 M sodium citrate). Specific hybridization signals were detected by fluoresceinated anti-digoxigenin antibodies.

Molecular Modeling-- The N-sulfotransferase domain of human NDST1 (PDB:1NST) was used as a template for modeling the corresponding domains of the human and mouse isozymes (24, 25) using the Swiss PDBviewer v.3.5. and the program POV-Ray. The following nucleotide sequences were employed: mNDST1 (AF074926), hNDST1 (U36600), mNDST2 (AF074925), hNDST2 (U36601), mNDST3 (AF221095), hNDST3 (AF074924), mNDST4 (AB036838), hNDST4 (AB036429). Accession numbers refer to the GenBankTM/DDBJ/EMBL data bases.


    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Cloning of Human NDST4-- Using the nucleotide sequence of human NDST1 (26), NDST2 (27), and NDST3 (11), we examined a human genome data base prepared in BAC. This led to the identification of BAC clone 2177.H.9 (Fig. 1), in which a segment of 183 nucleotides showed 87% identity to human NDST3. The deduced amino acid sequence of the region was 92% identical to exon 6 in human NDST1 and NDST2 (27). The region was flanked by possible intron-exon junctions (ttcagATCAG--GGCAGgtaac; nucleotides of the putative exon are printed in capital letters), supporting the idea that this sequence represented an authentic exon (28).



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Fig. 1.   Cloning strategy for human and mouse NDST4. A, cloning of human NDST4. The nucleotide sequence of human NDST4 was reconstituted from 5'- and 3'-RACE products using the exon sequence in BAC clone 2177.H.9 homologous to other NDSTs (bi-directional arrow). B, cloning of mouse NDST4. Mouse cDNA was amplified using two primers, mNDST-1F and -2R. The nucleotide sequence of mouse NDST4 was reconstituted from 10 individual clones. The nucleotide sequences are described in AB036429 (human) and AB036838 (mouse) (GenBankTM/DDBJ/EMBL data bases). Open and solid boxes indicate translated and untranslated regions, respectively.

Based on these observations, RT-PCR was performed using two primers, hNDNST4-1F and -1R, which bounded this region (Fig. 1). DNA products of the predicted length (~180 base pairs) were produced, and the identity of nine independent clones was confirmed by sequence analysis (data not shown), suggesting that the gene was transcribed into mRNA. To obtain the full-length cDNA, we first isolated the 5' portion employing 5'-RACE. Five independent clones were selected from the products, and their sequence indicated a possible initiation codon at nucleotide 679. The 5'-ends of the RACE products were identical in two clones (pCR34-1, clone 1 and 5), whereas the nucleotide sequences of the other three started 312 nucleotides downstream. In addition, two clones (pCR34-1, clone 5 and 6) possessed a deletion of 129 nucleotides (nucleotides 1813-1941). These sequences were not bounded by nucleotides ordinarily seen at splice junctions (TTTCCtcaca-ggcccCACAT and TGGCAgaatc-acaggAACAA), suggesting that they were produced by artificial deletion during PCR. The alternative 5'-end at nucleotide 312, however, may be relevant since a CAAT and TATA box exist at nucleotides 217 and 226 in the human sequence, and comparable promoter elements exist in the mouse. In contrast, the more upstream start site does not apparently contain a nearby TATA sequence in the genomic clone. Additional studies will be required to show if these two transcription start sites are separately regulated, but the presence of a TATA-box for the shorter transcript and a TATA-less promoter for the longer transcript point toward possible differential regulation of expression.

The 3'-coding region of NDST4 was produced by 3'-RACE. DNA products were obtained after two rounds of PCR, and three individual clones were analyzed. Combining the nucleotide sequences of 5'- and 3'-RACE products yielded the full-length human cDNA containing an open reading frame of 2625 nucleotides coding for a 872-amino acid residue protein, which was designated NDST4. The isolation of a cDNA coding for mouse NDST4 was performed by PCR (Fig. 1). The forward primer was designed according to the genomic sequence of exon 1, including the start codon of mNDST4, whereas the sequence of the reverse primer mNDST4-2R corresponded to nucleotides 3281-3307 of human NDST4.3 The full-length cDNA revealed an open reading frame of 872 amino acids, showing 95.9% identity to that of hNDST4. Murine cDNAs for NDST1, NDST2, and NDST3 were also obtained in a similar manner.3

Alignment of NDST4 with murine NDST1-3 demonstrated 70.4, 66.4, and 80.3% amino acid identity, respectively (Fig. 2). A hydropathy profile indicated that, like the other NDSTs, NDST4 is a type II transmembrane protein with a short cytoplasmic tail of 12-13 amino acids and a putative transmembrane region of 22 amino acids, which is followed by a region that varies significantly among all four NDSTs. The NDST4 protein sequence also contained the two highly conserved PAPS binding motifs present in other sulfotransferases (KTGTT beginning at residue 603 and the segment bounded by residues 690-710 (11).



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Fig. 2.   Homology of amino acid sequences of murine NDST1-4 and human NDST4. The amino acid sequences of the four murine NDSTs are aligned with the human NDST4 isozyme. Identical amino acids are bordered; similar amino acids are shaded. Gaps indicated by dashes were introduced to maximize the alignment. The putative N-terminal transmembrane domain is underlined, and the variable amino acids in the substrate binding cleft are indicated by the number sign (#).

Chromosomal Localization of Human NDST3 and NDST4-- The chromosomal location of hNDST4 and hNDST3 was examined by fluorescence in situ hybridization using their respective BAC DNA clones as probes. On the basis of size, morphology, and banding pattern, specific labeling of the long arm of chromosome 4 was observed. A subsequent experiment cohybridizing a biotin-labeled probe specific for the centromere of chromosome 4 with genomic DNA of either NDST3 or NDST4 was performed. Eighty metaphase cells were analyzed, of which 68 and 74 exhibited specific labeling for NDST3 and NDST4, respectively. Measurement of 10 specifically labeled chromosomes demonstrated that NDST4 and NDST3 were located at positions that were 44 and 49% of the centromere-to-telomere total distance of chromosome 4q, an area corresponding to band 4q25-26 and 4q26-27, respectively (Fig. 3). Analysis of the human genome indicates that NDST3 and NDST4 are ~300 kilobases apart. Human NDST1 and NDST2 were previously shown to map to chromosome 5q32-33 and 10q22, respectively (27).



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Fig. 3.   Chromosomal location of human NDST3 and NDST4 by fluorescent in situ hybridization. A biotin-labeled probe specific for the centromere of chromosome 4 was cohybridized with digoxigenin dUTP-labeled DNA from BAC clone 2608.A.5 and 2177.H.9, encoding human NDST3 and NDST4, respectively. Specific hybridization signals were detected by Texas red avidin for the centromere (red) and fluoresceinated anti-digoxigenin antibodies for NDST3 or NDST4 (green).

RT-PCR Analysis in Various Tissues-- Expression of NDST4 and the other NDSTs was examined in various mouse tissues by RT-PCR (Fig. 4). The relative level of expression of NDST4 was modest in comparison to the other NDSTs, with virtually no PCR products detectable under conditions where NDST1 and NDST2 products were abundant (11). With higher amplification, NDST4 was found in adult brain and throughout embryonic development. NDST3 was expressed strongly in brain and more so at embryonic day 11 than at other stages of development. With greater amplification, message was detected in adult heart, kidney, muscle, and testis but not in other tissues. The significance of these different expression patterns is difficult to evaluate given that message levels may not necessarily correspond to levels of protein expression. Selective tissue-specific effects have been observed after ablating NDST1 (pulmonary insufficiency) and NDST2 (effects restricted to connective tissue mast cells) in the mouse, but these transcripts are broadly expressed (15-18). Therefore, understanding the contribution of NDST3 and NDST4 to heparan sulfate synthesis in particular tissues will require similar genetic experiments, which are currently under way.2



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Fig. 4.   Expression of NDSTs in mouse tissues. Forward and reverse oligonucleotides unique to each NDST clone were used to detect mRNA expression in adult and embryonic mouse tissues ("Materials and Methods"). The number of PCR cycles was varied to appreciate the difference in relative levels of expression of the four genes. Marker, 100-base pair-ladder.

Sequence Comparison and Phylogenetic Relationship-- The contiguous chromosomal location of NDST3 and NDST4 and their high sequence homology suggested that they might have diverged by duplication after separation of the ancestral gene from another that gave rise to NDST1 and NDST2. A similar explanation has been provided to explain the relationship of GlcN 3-O-sulfotransferases (29) and fucosyltransferases, FUC5 and FUC6, which are located in tandem orientation on chromosome 19, only 13 kilobases apart from each other (30). To gain insight into the origins of the four NDSTs, ClustalW analysis of the enzymes of C. elegans, D. melanogaster, mouse, and human was used to predict their evolutionary relationship (Fig. 5). Based on the dendrogram, three gene duplication events occurred in vertebrates, giving rise to four isozymes common to both mouse and human without any further radiation. The first duplication gave rise to two branches, followed by another gene duplication in each branch. NDST3 and -4 are more closely related to each other than to NDST1 or -2, which cluster together somewhat more weakly. Therefore, the duplication of the common ancestral gene of NDST1 and -2 may have preceded the duplication event that gave rise to NDST3 and -4. The NDST locus appears to be ancient since heparan sulfate of similar design is present in organisms as old as C. elegans4 and Hydra (32), which emerged more than 500 million years ago. The phylogenetic relationships remain the same when amino acid sequences are used for constructing the tree, which points toward a restriction on the evolution of these enzymes possibly due to their essential functions.



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Fig. 5.   Unrooted analysis of the nucleotide sequences of NDSTs. The following nucleotide sequences were used to calculate the dendrogram; mNDST1 (AF074926), hNDST1 (U36600), mNDST2 (AF074925), hNDST2 (U36601), mNDST3 (AF221095), hNDST3 (AF074924), mNDST4 (AB036838), hNDST4 (AB036429), D. melanogaster NDST (AF175689), and C. elegans NDST (AB038044) (GenBankTM/DDBJ/EMBL data bases). The distance between isozymes is proportional to the number of nucleotide differences and reflects evolutionary time since their divergence.

Molecular Modeling of NDSTs-- Recently the crystal structure for the sulfotransferase domain of NDST1 was solved (24). The high degree of sequence similarity of the NDSTs allowed us to model the structure of the sulfotransferase domains of NDST2-4 using NDST1 as a template. Modeling each murine and human isozyme revealed a very similar overall pattern of surface charge and shape in the domain that constitutes the substrate binding cleft and the active site. However, this pattern differed significantly across the four isozymes in both species (Fig. 7). Amino acids in the putative substrate binding cleft were responsible for many of the differences, and most of these residues were located in a random coil defined by amino acids 633-649 (human NDST1). The relevant variable residues include 638 (E/S/K/K for NDST1, -2, -3, and -4, respectively), 648 (G/S/R/G), 649 (H/P/N/N), 682 (D/D/H/H), 721 (D/G/E/E), and 743 (K/A/E/D) (Fig. 6). Fig. 7 shows more clearly that the variable residues line the putative binding cleft for the acceptor oligosaccharide. Many of the substitutions are nonconservative and change charged residues that might interact with the substrate. Since the crystal structure for the N-deacetylase domain is not yet available, we cannot make any conclusions about variation of its active site.



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Fig. 6.   Molecular modeling of the human NDST-1 sulfotransferase domain. Ribbon diagram showing the substrate binding cleft of the sulfotransferase domain of hNDST1 (NCBI protein data base: PDB:1NST). Relevant variable amino acids between the four isozymes are shown, which might determine different substrate binding preferences among the NDSTs.



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Fig. 7.   Molecular modeling of the mouse and human sulfotransferase domains of NDST1-4. Front view into the substrate binding cleft where basic amino acids are colored in blue, and acidic amino acids are colored in red (electrostatic potential -4.8 to +4.8). The structure reported by Kakuta et al. (24) served as a template. Notably, clear differences in the distribution of basic and acidic amino acids can be seen among the isozymes, which are conserved between species. Variable amino acids that might contribute to substrate binding characteristics of the four isozymes are labeled.

Enzymatic Activity of Murine NDST1-4-- Based on the above findings, we predicted that NDST4 should have both GlcNAc N-deacetylase and N-sulfotransferase activities, but that the characteristics of the isozyme might differ significantly from the other NDSTs. To test this possibility, a soluble chimera of murine NDST4 and protein A was expressed in COS cells and assayed for each activity. Like NDST1-3, NDST4 is a bifunctional enzyme possessing both N-deacetylase and N-sulfotransferase activity (Table I). However, NDST4 was peculiar in that the N-deacetylase activity was very low when compared with recombinant murine NDST1-3 produced under identical conditions. In contrast, the sulfotransferase activity was similar to NDST1 and NDST2. NDST3 had relatively higher N-deacetylase activity and the lowest sulfotransferase activity of all four isozymes. Similar results were obtained when the deacetylation assay was performed in the presence of PAPS (data not shown). The ratio of N-deacetylase to N-sulfotransferase activities differed dramatically among the four isozymes (Fig. 8). As shown previously, the ratio was greater in NDST2 than in NDST1, but the differences were most dramatic in NDST3 and NDST4, which appeared to have complementary ratios.


                              
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Table I
Murine NDST4 N-deacetylase/N-sulfotransferase activity



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Fig. 8.   Comparison of the deacetylase/sulfotransferase ratios of mNDST1-4. GlcNAc N-deacetylase activity was measured by the release of acetyl groups from [3H]acetylheparosan, and N-sulfotransferase activity was measured by the incorporation of radioactivity from [35S]PAPS into N-desulfoheparin ("Materials and Methods"). The height of each bar reflects the ratio of N-deacetylase to N-sulfotransferase activities.

These data show that NDST3 and NDST4 differ dramatically from NDST1 and NDST2 in activity and expression pattern. Based on these findings and modeling data, we propose that substrate recognition by the various isozymes may vary significantly, giving rise to the different catalytic efficiencies depending on the degree of sulfation/epimerization of the substrate. All four NDSTs apparently have the capacity to work on unmodified chains (i.e. N-acetylheparosan), but the relatively poor deacetylation activity of NDST4 would suggest that this is not the normal substrate. One possibility is that the deacetylase domain of NDST4 may prefer chains that have already undergone some degree of sulfation and uronic epimerization, and therefore greater activity would be observed with more heparin-like substrates. The presence of additional basic residues in the binding cleft of NDST4 is consistent with this possibility. Alternatively, NDST4 may add sulfate groups to glucosaminyl residues that were deacetylated by one of the other NDSTs (e.g. NDST3) but left unmodified. Heparan sulfates and heparin are known to contain a small proportion of N-deacetylated glucosamine units (33-35), and some of these units serve as substrates for selective modifying enzymes, such as the 3-O-sulfotransferases (36, 37). Additional studies with defined, chemically modified substrates are needed to determine which of these possibilities are correct. A more detailed analysis of substrate specificity and saturability is clearly needed as well.

Previous models for the biosynthesis of heparan sulfate suggest that modification of the chains initiates by the action of one or more NDSTs working on newly made, unmodified chains composed of alternating GlcNAcalpha 1,4 and GlcAbeta 1,4 units (reviewed in Ref. 2). The formation of the N-deacetylated/N-sulfated glucosaminyl residues creates the preferred substrate for the C5 epimerase that converts adjacent D-GlcA residues to L-iduronic acid. O-Sulfation reactions then occur at C2 of the uronic acids and C6 and C3 of the glucosaminyl residues. Studies of mutant cell lines confirm that this sequence of events most likely occurs in vivo as well (19-22), although some differences have been noted (e.g. failure to add sulfate to C2 of uronic acids results in accentuated N-deacetylation/N-sulfation (31)). The striking differences in activity of NDST3 and -4 are consistent with a model in which some of the isozymes act on previously modified sequences. Thus, N-deacetylation of certain residues may be catalyzed by one enzyme and then acted upon by another. An iterative process like this could guide the formation of unique sequences in heparan sulfates with specific ligand binding properties. Exploring the function of these new NDSTs will ultimately require a combination of biochemistry and genetics to alter the expression of these isozymes in animals and cells and to assess how the changes affect the composition of the chains. These studies are currently under way.2


    ACKNOWLEDGEMENTS

We thank Drs. Xiaomei Bai and Ge Wei, Jan Castagnola, and all other members of Esko group for their helpful suggestions. We also thank Dr. Tomoya Ogawa (RIKEN, Japan) for promoting this international collaboration.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants R37GM33063 and Program Project Grant HL53745 (to J. D. E.), a RIKEN President's Special Research Grant and the Human Frontier Science Program (to J. A.), and Deutsche Forschungsgemeinschaft Grant GR1748 (to K. G.).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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF074926, AF074925, AF221095, AB036429, and AB036838 for mNDST1, mNDST2, mNDST3, hNDST4, and mNDST4, respectively.

These authors contributed equally to this work.

|| To whom correspondence should be addressed: Dept. of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Dr., CMM-East 1055, La Jolla, CA 92093-0687. Tel.: 858-822-1100; Fax: 858-534-5611; E-mail: jesko@ucsd.edu.

Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M009606200

2 K. Grobe, J. Aikawa, and J. D. Esko, unpublished results.

3 J. Aikawa and J. D. Esko, unpublished results.

4 Bulik, D. A., Wei, G., Toyoda, H., Konishita-Toyoda, A., Waldrip, W. R., Esko, J. D., Robbins, P. W., and Selleck, S. B. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 10838-10843


    ABBREVIATIONS

The abbreviations used are: GlcN, unsubstituted glucosamine; GlcA, D-glucuronic acid; PAPS, adenosine 3'-phosphate 5'-phosphosulfate; NDST, heparan sulfate/heparin GlcNAc N-deacetylase/GlcN N-sulfotransferase; hNDST and mNDST, human and mouse NDST, respectively; MES, 2-(N-morpholino)ethanesulfonic acid; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription-polymerase chain reaction; BAC, bacterial artificial chromosomes.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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