From the 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
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ABSTRACT |
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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.
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, GlcA 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.
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.
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).
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).
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).
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
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.
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.
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.
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
GlcNAc
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
1,3Gal
1,3Gal
1,4Xyl
, on serine residues of core
proteins; (ii) the chains elongate by alternating the additions of
GlcNAc
1,4 and GlcA
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).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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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.
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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
(#).
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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).
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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.
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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.
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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.
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.
1,4 and GlcA
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
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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.
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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
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ABBREVIATIONS |
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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.
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