From the Department of Biology, Tokyo Metropolitan
University, Hachioji-shi, Tokyo 192-0397, Japan, the ¶ Department
of Molecular and Cellular Biology and Division of Neurobiology,
University of Arizona, Tucson, Arizona 85721, and the
Institute
for Molecular Science of Medicine, Aichi Medical University, Nagakute,
Aichi 480-1195, Japan
Received for publication, December 18, 2000, and in revised form, March 6, 2001
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ABSTRACT |
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Heparan sulfate, one of the most abundant
components of the cell surface and the extracellular matrix, is
involved in a variety of biological processes such as growth factor
signaling, cell adhesion, and enzymatic catalysis. The heparan sulfate
chains have markedly heterogeneous structures in which distinct
sequences of sulfate groups determine specific binding properties.
Sulfation at each different position of heparan sulfate is catalyzed by distinct enzymes, sulfotransferases. In this study, we identified and
characterized Drosophila heparan sulfate
6-O-sulfotransferase (dHS6ST). The deduced
primary structure of dHS6ST exhibited several common features found in
those of mammalian HS6STs. We confirmed that, when the protein encoded
by the cDNA was expressed in COS-7 cells, it showed HS6ST activity.
Whole mount in situ hybridization revealed highly specific
expression of dHS6ST mRNA in embryonic tracheal cells.
The spatial and temporal pattern of dHS6ST expression in
these cells clearly resembles that of the Drosophila
fibroblast growth factor (FGF) receptor, breathless
(btl). RNA interference experiments demonstrated that
reduced dHS6ST activity caused embryonic lethality and
disruption of the primary branching of the tracheal system. These
phenotypes were reminiscent of the defects observed in mutants of FGF
signaling components. We also show that FGF-dependent mitogen-activated protein kinase activation is significantly reduced in
dHS6ST double-stranded RNA-injected embryos. These findings indicate that dHS6ST is required for tracheal development
in Drosophila and suggest the evolutionally conserved roles
of 6-O-sulfated heparan sulfate in FGF signaling.
Heparan sulfate proteoglycans are ubiquitously present on the cell
surface and in the extracellular matrix and are known to be involved in
a variety of biological phenomena, including cell proliferation,
differentiation, cell adhesion, angiogenesis, blood coagulation, lipid
metabolism, and viral and bacterial infections. These diverse
mechanisms of action are achieved by interactions between the specific
structures of heparan sulfate and the binding proteins. The backbone of
heparan sulfate is a polysaccharide chain composed of alternating
D-glucuronic acid (GlcA) and
N-acetyl-D-glucosamine units, and some GlcA
residues are converted into L-iduronic acids. The
microheterogeneity of the heparan sulfate structures is mainly produced
by the nonrandom introduction of N-, 2-O-,
6-O-, and 3-O-sulfate groups (1, 2). Thus, the
biological functions of heparan sulfate proteoglycans are controlled by
biosynthetic events, which define the fine structures of heparan sulfate.
For many years, functional studies of structurally complex heparan
sulfate have focused on vertebrate tissues and cultured cells. Recent
studies, however, have shown that heparan sulfate is also found in
simple model organisms such as Drosophila melanogaster and
Caenorhabditis elegans (3). The structural features of heparan sulfate in Drosophila are similar to those found in
vertebrates. In addition, the analyses of heparan sulfate in
Drosophila mutants have shown that the biosynthesis
apparatus is also conserved in this organism (4). Therefore, on the
basis of their homology to vertebrate heparan sulfate-modifying genes,
it is now possible to analyze the function of modified heparan sulfate
in vivo using these genetically tractable organisms.
Recently, genetic studies on mutants for heparan sulfate
sulfotransferases indicated that the regulated synthesis of heparan sulfate affects morphogenesis during development. Bullock et
al. (5) reported that heparan sulfate
2-O-sulfotransferase
(HS2ST)1 knock-out mice
showed renal agenesis as well as defects in eyes and skeleton. In
Drosophila, pipe encodes a protein that is
structurally similar to HS2ST, and the ventral expression of
pipe in the somatic cells of ovary is required for the
formation of embryonic dorsoventral polarity (6). Mutations in
sulfateless (sfl), which encodes a heparan
sulfate N-deacetylase/N-sulfotransferase, affect
Wingless, FGF, and Hedgehog signaling pathways during development
(7-9). These findings have shown that differentially modified heparan sulfate can regulate growth factor signaling.
It is well established that heparan sulfate binds to bFGF and is
required for the activation of the signal transduction pathway. However, the mechanism by which heparan sulfate promotes bFGF action is
not clearly understood. It has been variously proposed that heparan
sulfate induces a conformational change in bFGF (10), serves as a
bridge between bFGF and FGFR (11), or promotes dimerization of FGFRs
(12). It has been reported that specific fine structures of heparan
sulfate have a critical role for binding to bFGF. Biochemical studies
have shown that N- and 2-O-sulfation is required
for binding of bFGF to heparan sulfate (13). On the other hand, studies using biological assays have shown that, in addition to
2-O-sulfation, 6-O-sulfation is responsible for
the activation of bFGF signaling (14, 15). This may reflect the fact
that heparan sulfate interacts with not only FGFs but also their
receptors. Recent crystallographic analysis also supported the idea
that 6-O-sulfation promotes the dimerization of FGFRs
(16).
In Drosophila, one FGF ligand, Branchless (Bnl), and two
FGFRs, Heartless (Htl) and Breathless (Btl) have been identified and
characterized (17-19). btl and bnl are required
for the normal development of the Drosophila respiratory
system, branching trachea that deliver air to all tissues (19, 20). The
Drosophila tracheal system shares many features with the
vertebrate vascular system and is therefore regarded as a simple model
to study tubulogenesis (21). Branching morphogenesis in both
Drosophila and mammals is regulated by growth factors FGF
and vascular endothelial growth factor (22, 23), cadherins (24, 25),
and hypoxia-inducible factor-1-like basic helix-loop-helix
transcription factors (26-28). Heparan sulfate is also critical for
both Drosophila tracheogenesis and vertebrate angiogenesis
(7, 29).
In this study, we examined the structure and function of
Drosophila 6-O-sulfotransferase (dHS6ST). We
present here the cDNA and deduced amino acid sequences of dHS6ST
and the characterization of its enzymatic activity. In situ
hybridization of dHS6ST mRNA revealed that
dHS6ST is expressed in specific patterns in several tissues.
During embryogenesis, dHS6ST mRNA was detected in
developing tracheal precursor cells that also express a
Drosophila FGF receptor, btl. Blocking the
dHS6ST activity by double-stranded RNA interference caused
embryonic lethality and defects in the migration of the tracheal cells.
In addition, Btl-dependent activation of MAPK, which is
seen normally in tracheal cells, is significantly decreased in these
embryos. These results suggested the possible involvement of
6-O-sulfated heparan sulfate in the modulation of FGF
signaling during tracheogenesis in Drosophila.
Fly Strains and Chemicals--
Oregon-R was used as a wild type
of Drosophila melanogaster. 1-eve-1 is an enhancer trap line
at the trachealess (trh) locus (30). A
Drosophila cDNA library was obtained from
CLONTECH. A rabbit anti- Cloning of dHS6ST--
The genomic sequence for a putative
Drosophila 6-O-sulfotransferase
(dHS6ST) was identified in the Drosophila genome
data base of the Berkeley Drosophila Genome Project (BDGP).
Based on the sequence, the 938-base pairs PCR-amplified genomic DNA was prepared using the 5'-primer (TCTGCCCATCCAAAAAGACG) and the 3'-primer (CTGAAAGAGCAAAGTCTTGG). Approximately 2 × 105 plaques
from a Drosophila larval cDNA library were screened
using the above PCR product from dHS6ST genomic DNA as a
probe. Hybridization was carried out as described by Sambrook et
al. (33). Four positive clones were isolated, and the inserted
cDNAs were sequenced using the BigDyeTM Thermal Cycle Sequencing
Ready Reaction Kit and the ABI PRISM 310 analyzer. Nucleotide and amino
acid sequences were analyzed using a DNASIS program version 3.5 (Hitachi).
Assay for Sulfotransferase Activities and Analysis of the
Enzymatic Reaction Products--
To express dHS6ST in tissue culture
cells, pFLAG-CMV2-dHS6ST was constructed by insertion of the
PCR product containing the open reading frame of 1374 base pairs from
positions 589-1962 for dHS6ST into the
HindIII/ClaI site of the pFLAG-CMV2 expression vector (Eastman Kodak Co.). COS-7 cells (5.5 × 105)
precultured for 48 h in a 60-mm culture dish were transfected with
5 µg of pFLAG-CMV2-dHS6ST or pFLAG-CMV2 alone. The
transfection was performed using a liposome-mediated method according
to the manufacturer's recommended protocol (TransFast, Promega). The enzyme activities of HS6ST and HS2ST in the cell extract were determined using CDSNS-heparin as a substrate. In this experiment, HS2ST activity was specifically determined in the presence of 10 mM dithiothreitol as described previously (34).
Dithiothreitol at this concentration had little effect on HS2ST
activity, whereas HS6ST activity was substantially decreased. FLAG
fusion proteins in the cell extract were isolated by anti-FLAG M2
(Kodak) affinity chromatography according to the method described by
the manufacturer, and the activities of purified dHS6ST in the
FLAG-bound fractions were measured using 25 nmol (as hexosamine, 0.5 mM) of acceptor glycosaminoglycans in the 50-µl reaction
mixture as described previously (35). The amounts of enzymes added to
the reaction mixture were chosen to obtain the linear incorporation of
[35S]sulfate into CDSNS-heparin, which was 0.028 pmol/min
(35). We measured 35S incorporation activities into various
glycosaminoglycans of the purified dHS6ST under those condition.
Analysis of the reaction products derived from CDSNS-heparin and
heparan sulfate (from a mouse EHS tumor) by expressed enzymes was
performed as described previously (35, 36).
Whole Mount in Situ Hybridization--
Fixation and pretreatment
of embryos were performed as described by Tautz and Pfeifle (37). RNA
probes were prepared as follows. dHS6ST cDNA was
inserted into pBluescript, and purified plasmid DNA was linearized by
appropriate restriction enzymes. Sense and antisense RNA probes were
prepared by in vitro transcription from linearized cDNA
templates using digoxigenin-11-UTP (Roche Molecular Biochemicals) and
T7 or T3 RNA polymerase. After fragmentation into an average size of
400 nucleotides by heating at 60 °C in a buffer containing 40 mM NaHCO3, 60 mM
Na2CO3, and 50 mM dithiothreitol, labeled RNAs were used as probes for hybridization. Hybridization and
detection of hybridized probes were carried out as described by
Hauptmann and Gerster (38) and Tautz and Pfeifle (37), respectively.
RNA Interference--
RNA interference was performed as
previously described (39). RNAs were synthesized using T3 or T7 RNA
polymerase and linearized plasmids as templates. For the annealing
reaction, equimolar quantities of sense and antisense RNAs were mixed
to a final concentration of 5 µM each and incubated at
67 °C for 10 min, followed by incubation at 37 °C for 30 min. RNA
aliquots were ethanol-precipitated and dissolved in an injection buffer
(40) to a final concentration of 5 µM. The average
injection volume was 85 pl, ranging from 65 to 110 pl, as determined by
measuring the diameter of droplets injected into halocarbon oil. Early
embryos of 1-eve-1, the trachealess (trh)-lacZ line, were collected within 2 h
of egg laying and injected with dHS6ST double-stranded RNA
(dsRNA) or a buffer only. Injected embryos were allowed to develop at
25 °C for 11 h to observe tracheal phenotypes or for 7 h
to detect MAPK activation, and subjected to the staining procedure.
Immunohistochemistry--
Cloning of dHS6ST cDNA and Genomic Organization of the
dHS6ST--
A genomic sequence, AE003728, which has been mapped to 92C
on the third chromosome (BDGP), was identified as a clone bearing sequences with significant homology to mouse heparan sulfate
6-O-sulfotransferase-1 (mHS6ST-1) (35). We named this gene
presumptive Drosophila heparan sulfate
6-O-sulfotransferase (dHS6ST). To isolate a
dHS6ST cDNA, a Drosophila larval cDNA library
was screened with the PCR-amplified genomic fragment as a probe. From
~2 × 105 plaques, four independent positive clones
were obtained and sequenced.
Comparison of the dHS6ST cDNA and the AE003728 genomic
sequences revealed that dHS6ST spans 79.0 kilobase pairs of
the genome and contains six exons separated by five introns, with the
second intron being more than 67 kilobase pairs in size (Fig. 1A). Around the genomic region
corresponding to the 5' end of the cloned dHS6ST cDNA,
we identified the sequence TCAGTT, which matches the
Drosophila consensus sequence for an initiator
"TCA(G/T)T(T/C)" (+1 site is underlined, Fig.
1B) (41-43). Although a typical TATA box was not found in
this region, the sequence GGACGTT is highly similar to the downstream
promoter element (DPE), which is contained in many TATA-less promoters
in Drosophila, at a typical position from the initiator
sequence (+28 to +34 in Fig. 1B) (44). The occurrence
of these sequences predicted the transcription start site as the A
residue shown by +1 in Fig. 1B. The cDNA clone that we
have sequenced therefore appears to be nearly full-length. Northern
blot analysis showed that dHS6ST encodes a single major transcript of ~3.1 kilobase pairs (data not shown).
BDGP suggested the existence of several genes around the
dHS6ST locus (Fig. 1A). miranda, which
encodes a cytoskeletal structural protein affecting the asymmetric
division of neuroblasts, was contained within in the fifth intron of
dHS6ST (45, 46). An EST clone, GH26202, derived from an
adult head cDNA library, was mapped to the second intron of the
dHS6ST gene. Furthermore, the 5' sequence of an EST clone,
LD07688, was found to start at 499 base pairs upstream of the 5' end of
dHS6ST in a reverse direction.
Primary Structure of dHS6ST--
The complete cDNA and
predicted amino acid sequences for dHS6ST are presented in
Fig. 2A. The amino-terminal
sequence contains three in-frame ATG codons. Out of three ATGs, the
third one appears to be the most likely initiation codon, judging from
the Drosophila consensus translation initiation sequence
(47). The pattern of the hydropathy plot for the amino acid
sequence started from the third ATG also matches those for mammalian
homologues of dHS6ST (Fig. 2B) (35, 48). The predicted
protein was composed of 432 amino acid residues with two putative
N-linked glycosylation sites. A hydropathy plot of dHS6ST
showed the type II transmembrane structure of a Golgi resident protein
with one prominent hydrophobic segment in the amino-terminal region
that extends from amino acid residue 9 to 20.
A comparison of the primary structures of mammalian and
Drosophila HS6STs revealed that the amino acid sequence of
dHS6ST is 53%, 54%, 46%, and 53% identical to the sequences of
human HS6ST (hHS6ST), mouse HS6ST-1, -2, and -3 (mHS6STs),
respectively. Although relatively low levels of similarity were
observed in both the amino- and carboxyl-terminal regions among HS6STs,
the amino acid sequences of the central region are highly conserved (Fig. 3). Putative PAPS binding sites
were present in these regions of all these proteins (49, 50).
Furthermore, all HS6STs bear two N-glycosylation sites at
conserved positions. The predicted amino acid sequence of dHS6ST
therefore shows all the structural features of HS6STs reported
previously.
Characterization of Heparan Sulfate 6-O-Sulfotransferase Activity
of dHS6ST--
To determine whether the isolated dHS6ST
cDNA encodes a protein with HS6ST activity, we examined the
activity of dHS6ST expressed in tissue culture cells. dHS6ST
cDNA was inserted in a mammalian expression vector, pFLAG-CMV2, and
COS-7 cells were transfected with this construct or with pFLAG-CMV2
alone as a control. HS6ST activity in the cells transfected with
dHS6ST was approximately twice as high as that with the
control vector, whereas the dHS6ST cDNA insert did not
increase the HS2ST activity (Table I).
These results confirmed that recombinant dHS6ST produces HS6ST
activity.
To further analyze this enzyme, we purified the epitope-tagged dHS6ST
protein and examined the sulfotransferase activity using several
heparin derivatives, heparan sulfates, and chondroitin as substrates
(Table II). The dHS6ST was able to
transfer sulfate to CDSNS-heparin, NS-heparosan, heparin, and heparan
sulfate but was marginally active using CDSNAc-heparin and chondroitin
as substrates. The ratio of the activity of dHS6ST toward NS-heparosan to that toward CDSNS-heparin was 0.36. Since NS-heparosan contains only
glucuronic acid and not its epimer iduronic acid, this result suggests
a preference of dHS6ST for iduronic acid adjacent to the targeted
N-sulfoglucosamine.
We analyzed the structures of the 35S-labeled products
obtained by incubation with purified dHS6ST and CDSNS-heparin or
heparan sulfate from a mouse EHS tumor (Fig.
4). In both products, most of the
radioactivity in the disaccharide fractions was at the position of
Expression Patterns of dHS6ST mRNA in Embryos and Imaginal
Discs--
The expression patterns of dHS6ST mRNA were
determined by in situ hybridization of whole-mount embryos
and imaginal discs (Fig. 5). We observed
high levels of accumulation of dHS6ST mRNA in syncytial
blastoderm stage embryos indicating that dHS6ST is a
maternally supplied product (Fig. 5A). During early
gastrulation, zygotic expression of dHS6ST was detected in
many tissues, including the invaginating mesoderm (Fig. 5B).
In the stage 10 embryos, we detected highly specific expression of
dHS6ST mRNA in tracheal precursor cells (Fig.
5C). dHS6ST expression in the invaginating tracheal precursor cells is maintained through stage 12 (Fig. 5,
D and E). This spatial and temporal expression
pattern of dHS6ST in tracheal precursor cells was strikingly
similar to that of breathless (btl), a
Drosophila FGF receptor (18). By stage 16, however,
dHS6ST became uniformly expressed in various tissues (Fig.
5F). The restricted expression pattern of dHS6ST
may reflect the requirements of the dHS6ST function in
tracheal development.
In Drosophila, another FGF receptor homologue is encoded by
heartless (htl) (17). htl is expressed
in the invaginating mesoderm during embryogenesis, in the morphogenetic
furrow of the eye disc and surrounding the outer proliferative center
(OPC) of the larval CNS (51). The expression pattern of
dHS6ST partially overlapped with that of htl in
these tissues (Fig. 5, B, H, and I).
In third instar larvae, dHS6ST mRNA was detected in
actively dividing neuroblasts, including cells anterior to the
morphogenetic furrow of the eye disc (Fig. 5H) and OPC of
the CNS (Fig. 5I). This observation suggests the possible
involvement of dHS6ST in htl-mediated neuronal development. We also observed high levels of expression of
dHS6ST mRNA throughout the wing disc (Fig.
5G) and in the lobula of the CNS (Fig. 5I). The
similarity of expression patterns between dHS6ST and
Drosophila FGFRs raises the possibility that
6-O-sulfated heparan sulfate is required for all FGF
signaling during Drosophila development.
dHS6ST Double-stranded RNA Inhibits FGF Signaling during Tracheal
Formation--
To elucidate the function of dHS6ST, we
performed RNA interference (RNAi), a gene silencing strategy mediated
by dsRNA (39). Approximately 75% of the control embryos, which had
been injected with buffer only, hatched normally, while the remaining
control embryos died owing to mechanical trauma (Table
III). Most of the dHS6ST
dsRNA-injected embryos (94%) died prior to the point immediately before hatching, although they survived to stage 17, the final stage of
embryogenesis (Table III). This observation suggested that
dHS6ST is essential for viability at late embryonic stages. Since whole mount in situ hybridization revealed expression
of dHS6ST in the tracheal precursor cells, we tested whether
disruption of dHS6ST activity affects tracheogenesis. The
tracheal system of the Drosophila embryo form by a
sequential series of branching steps that can be visualized by
following the expression of 1-eve-1, an enhancer trap line
for the trachealess (trh) gene (26, 52). We
injected dsRNA into the 1-eve-1 embryos at the syncytial
blastoderm stage and collected embryos at stage 13, when primary
branches continue to grow toward their targets. In 58% of
dHS6ST dsRNA-injected embryos, tracheal branch formation was
disordered while the levels of 1-eve-1 expression seemed to
be comparable to the control embryos (Table III and Fig.
6). Twenty-five percent of these embryos
exhibited stalled branches and disconnected dorsal trunks in all
segments. The remaining 33% of dHS6ST dsRNA-injected
embryos showed migration defects of the dorsal branch or lateral
trunk in one to several segments. In buffer-injected embryos, 21%
showed minor tracheal defects, and no control embryos showed severe
tracheal disruption as described for dHS6ST dsRNA-injected
animals (Table III). These results indicated that blocking
dHS6ST activity disrupted the branching of tracheal
precursor cells without affecting tracheal cell differentiation.
Expression patterns of dHS6ST mRNA and the tracheal
phenotypes of dHS6ST dsRNA-injected embryos suggested that
dHS6ST is involved in btl-mediated signaling. To
test this possibility, we examined the effects of injection of
dHS6ST dsRNA on the activity of MAPK, a downstream
transducer of FGF signaling. Activated form of MAPK can be visualized
by staining with anti-dpMAPK antibody (53, 54). When buffer was
injected as a control, most of the embryos showed normal MAPK
activation in tracheal precursor cells, while a modest reduction of
MAPK activation was observed in 26% of embryos (Table III and Fig.
6C). In contrast, in 58% of dHS6ST
dsRNA-injected embryos, anti-diphospho-MAPK immunoreactivity around
tracheal pit was below the level of detection (Fig. 6D). Our
findings strongly suggest that 6-O-sulfated heparan sulfate
is required for normal Drosophila FGF signaling during
tracheal development.
In this study, we cloned and characterized the dHS6ST
gene. dHS6ST shows similarity in amino acid sequence with human and mouse HS6STs, and the substrate specificity of dHS6ST revealed that
dHS6ST is comparable to mHS6ST-1; dHS6ST preferred the iduronosyl N-sulfoglucosamine unit as a substrate. Recent biochemical
studies of heparan sulfate from Drosophila revealed
disaccharides found in vertebrates; We have shown here that dHS6ST is expressed in embryos and
imaginal tissues. The expression of dHS6ST mRNA during
development coincides with FGF-responsive cells known to synthesize
Drosophila FGFRs, breathless (btl) and
heartless (htl). Particularly in embryonic stages, the pattern of dHS6ST expression in the tracheal
cells is analogous to those of btl. Activation of several
genes required for tracheal tubular formation, including
btl, is regulated by trachealess
(trh), which encodes a transcription factor belonging to the
basic helix-loop-helix-PAS (Per-ARNT-sim) protein family (26,
55). The heterodimers of Trachealess and Drosophila aryl hydrocarbon receptor nuclear translocator (dARNT) bind to the specific
sequence, TACGTG, in the btl regulatory region and regulate its expression in tracheal cells as well as midline precursor cells
(55). We identified a Trachealess/dARNT binding site in the first
intron of dHS6ST, suggesting that the expression of dHS6ST might be regulated in the same fashion as that of
btl by these transcription factors (Fig.
7).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase was
purchased from Cappel. A mouse anti-dpMAPK was purchased from
Sigma. [35S]H2SO4 was purchased
from PerkinElmer Life Sciences. [35S]PAPS was prepared as
described previously (31). Unlabeled PAPS was from Sigma, Hiload
Superdex 30 HR 16/60 and a fast desalting column were from Whatman
(Clifton, NJ), and a pack polyamine column was from YMC (Kyoto, Japan).
Heparitinase I (EC 4.2.2.8), II, and III (EC 4.2.2.7), chondroitin
sulfate A, completely desulfated N-sulfated heparin
(CDSNS-heparin), completely desulfated N-acetylated heparin
(CDSNAc-heparin), heparan sulfate from pig aorta, and an unsaturated
glycosaminoglycan disaccharide kit were obtained from Seikagaku Corp.
(Tokyo, Japan). Heparin from porcine intestinal mucosa was purchased
from Sigma. Heparan sulfate from mouse Engelbreth-Holm-Swarm (EHS)
tumor was a gift from Dr. T. Harada (Seikagaku Corp.). Deacetylated N-sulfated heparosan (NS-heparosan) was prepared by chemical
deacetylation and N-sulfation of N-acetyl
heparosan, which was prepared from Escherichia coli K5 by
Dr. T.Saito (Kanagawa University). Chondroitin (squid skin) was
prepared as described previously (32). Hybond N+ was from
Amersham Pharmacia Biotech (Tokyo, Japan).
-Galactosidase expressed in
1-eve-1 enhancer trap embryos was detected using rabbit
polyclonal antiserum (Cappel) at a 1:500 dilution in PBT (5% goat
serum, 0.3% Triton X-100 in phosphate-buffered saline). Anti-dpMAPK
(Sigma), which recognizes activated diphospho-MAPK was used at 1: 200. After repeated washes with PBT, the primary antibody was visualized
using the Vectastain ABC kit (Vector Laboratories) and horseradish
peroxidase immunochemistry following the protocol recommended by the
manufactures. Embryos were mounted in 80% glycerol in
phosphate-buffered saline and photographed using Olympus BX50 microscopy.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of the
dHS6ST gene locus. A, boxes
and shaded area represent exons and the protein
coding region of dHS6ST, respectively. The
numbers above the exons represent the length of each exon.
Proximal and distal show the orientation to the
centromere and telomere, respectively. miranda and two EST
clones (GH26202 and LD07688) were mapped around dHS6ST
locus. The transcriptional directions of these genes are shown by
arrows. B, genomic sequence around the putative
transcription initiation site. The transcription initiation site was
estimated at the A residue shown as +1. The putative initiator and DPE
are indicated by underline and double underline,
respectively. Consensus sequences of the initiator and DPE are shown
below the corresponding regions. The asterisk represents the
position of 5' end of the obtained dHS6ST cDNA.
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Fig. 2.
Structure of dHS6ST
cDNA. A, complete nucleotide sequence of
dHS6ST cDNA and the primary structure of dHS6ST. Three
NH2-terminal ATG codons and the presumptive polyadenylation
signal are underlined, and the deduced amino acid sequence
from the third ATG codon is represented with single
letters below the cDNA sequence. B,
hydropathic analysis of the predicted dHS6ST protein. Hydrophobicity
values were obtained according to the algorithm of Kyte and Doolitle
with a window of 6 amino acid residues (56). Positive values represent
increased hydrophobicity.
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Fig. 3.
Comparison of the amino acid sequences of
human, mouse, and Drosophila HS6STs.
Drosophila and mammalian HS6STs are aligned using the
program ClustalW (57). Indicated regions (5'PSB and
3'PB) are highly conserved among virtually all HS6STs that
are predicted to serve in 3'-phosphoadenosine 5'-phosphosulfate (PAPS)
binding (49, 50). Arrowheads indicate conserved potential
N-glycosylation signals. Consensus residues (*) are
indicated for each position where five HS6STs exhibit identical amino
acids. 5'PSB, 5'-phosphosulfate binding site;
3'PB, 3'-phosphate binding site; hHS6ST, human
HS6ST (48); mHS6ST, mouse HS6ST (35); dHS6ST,
Drosophila HS6ST.
Overexpression of dHS6ST in COS-7 cells
Acceptor substrate specificities of the recombinant dHS6ST purified by
anti-FLAG antibody affinity column chromatography
UA-GlcNS6S. We also found a small peak at the position of
UA2S-GlcNS6S from heparan sulfate. These results showed that dHS6ST
transfer sulfate preferentially to position 6 of GlcNSO3 residues. Since it has been reported that the substrate specificity of
mHS6ST-2 is affected by the concentration of the substrates (35), we
analyzed the dependence of the substrate preference of dHS6ST on
substrate concentration. The ratio of the sulfotransferase activity
toward NS-heparosan to that toward CDSNS-heparin was low at all
concentrations tested (data not shown), suggesting the substrate
specificity of dHS6ST is insensitive to the concentrations of acceptor
substrate. Taken together, our findings indicated that dHS6ST shows
HS6ST activity with preference for specific substrates.
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Fig. 4.
HPLC analysis of 35S-labeled
products of the purified dHS6ST. The purified recombinant enzyme
was prepared as described under "Experimental Procedures."
CDSNS-heparin and heparan sulfate were incubated with
[35S]PAPS and the recombinant purified dHS6ST. The
35S-labeled products were digested with a mixture of
heparitinases, and the disaccharides fractions were subjected to a pack
polyamine column as described previously (35). The dotted
lines indicate the concentrations of
KH2PO4 for elution. The open
circles show the elution patterns of the products derived
from CDSNS-heparin (A) and heparan sulfate (B).
The arrows indicate the elution positions of UA-GlcNAc6S
(1),
UA-GlcNS (2),
UA-GlcNS6S
(3),
UA2S-GlcNS (4), and
UA2S-GlcNS6S
(5).
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Fig. 5.
dHS6ST mRNA expression in
embryos and developing imaginal tissues. dHS6ST
mRNA expression was detected by in situ hybridization.
A-F, dHS6ST expression patterns in embryos.
Anterior is left. A, B, C,
D, and F, lateral views; E, ventral
view. High level accumulation of dHS6ST mRNA was
observed in syncytial blastodermal stages (A). During early
gastrulation, zygotic expression of dHS6ST was widely
detectable (B). dHS6ST is selectively expressed
in tracheal precursor cells at stage 10 (C) and stage 11 (D and E). At stage 16, dHS6ST
expression was ubiquitously detected again (F).
G-I, expression patterns in wing disc, eye disc, and CNS
from the third instar larvae. dHS6ST is detectable uniformly
in the wing disc (G). Specific expression patterns were
observed in cells anterior to the morphogenetic furrow of the eye disc
(H) and OPC and lobula of the CNS (I).
Arrowheads indicate tracheal placode (C),
tracheal pit (D), and morphogenetic furrow (H).
TPL, tracheal placode; TP, tracheal pit;
MF, morphogenetic furrow.
Effects of dHS6ST dsRNA injection on lethality, tracheal morphology,
and MAPK activation
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Fig. 6.
Disruption of dHS6ST
function by RNA interference caused the branching defects of the
tracheal system. 1-eve-1 embryos, which bear an
enhancer trap P element insertion in the trh gene, were
injected with buffer (A) or dHS6ST dsRNA
(B). Tracheal phenotypes were observed at stage 13 by
staining with anti- -galactosidase antibody. Arrows in
B point to sites of misguided or stalled branching.
C and D, the effect of injection of
dHS6ST dsRNA on the activity of MAPK at the tips of the
migrating tracheal precursor cells of stage 11 embryos was examined.
When buffer was injected as a control, most embryos showed normal
diphospho-MAPK signals in tracheal precursor cells around tracheal pit
(C). Injection of dHS6ST dsRNA disrupted
activation of MAPK (D). Arrowheads in
C and D indicate tracheal pits. Anterior of the
embryos are oriented to the left, and embryos are shown in
lateral views.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
UA-GlcNAc,
UA-GlcNS,
UA-GlcNAc6S,
UA-GlcNS6S,
UA2S-GlcNS, and
UA2S-GlcNS6S
disaccharides (3). Consistent with this biochemical evidence, our
finding of an enzymatically active HS6ST in this organism suggests an
equivalent function of 6-O-sulfated heparan sulfate in
Drosophila to that of vertebrates.
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Fig. 7.
A consensus sequence for the Trachealess
(TRH)/dARNT binding site in the dHS6ST gene. The
central midline element (CME) is a putative binding site of TRH/dARNT
heterodimers in the breathless (btl)
cis-regulatory region (55). Three btl CMEs (CME1, CME2, and
CME3) and the corresponding sequence found in the first intron of the
dHS6ST gene are aligned.
RNAi experiments showed that dHS6ST is required for normal tracheal formation. The expressivity of the phenotypes observed in dHS6ST dsRNA-injected embryos was variable, from stalled primary branches in all segments to migration defects of the dorsal branch or lateral trunk at one segment. These phenotypes are reminiscent of the defects reported in null and hypomorphic mutants of btl and bnl (18, 19). Loss of FGF-dependent MAPK activation in dHS6ST dsRNA-injected embryos strongly suggests that 6-O-sulfated heparan sulfate functions in FGF signaling, guiding tracheal cell migration. RNAi inhibition of dHS6ST function resulted in failure of hatching for 96% of injected embryos, while 58% of the embryos showed visible defects of the tracheal formation. This observation implies that dHS6ST may have functions in other developmental events essential for embryonic viability.
Previously, several biochemical studies of vertebrate cells/tissues have suggested the possibility that 6-O-sulfated heparan sulfate controls FGF signaling (14, 15, 29). Pye et al. (15) showed that there is a direct correlation between the 6-O-sulfate content of heparan sulfate oligosaccharides and their ability to promote bFGF activity. Lundin et al. (29) demonstrated that bFGF-induced angiogenesis in chick embryos was inhibited by 6-O-desulfated heparin, which binds to bFGF but fails to bind the receptor. Furthermore, the recent crystallographic analysis of two sets of FGF-FGFR-heparin complex showed that 6-O-sulfate groups interact with FGFR in the adjoining complex to promote FGFR dimerization (16). These findings suggest that 6-O-sulfation of heparan sulfate is required for a cellular response to FGF.
Our study provides direct in vivo evidence that
6-O-sulfation of heparan sulfate is critical for FGF
signaling during development. Without 6-O-sulfation, heparan
sulfate may be able to bind FGF but not mediate FGF signaling. Once
HS6ST transfers sulfate groups along the chain, the dimerization of
FGFR can occur, and cells acquire the ability to respond to FGF. Thus,
spatially and temporally regulated expression of HS6ST is may serve as
a key step for cellular responses to FGF. Since mammalian vascular
development is also dependent on FGF (22, 23), our findings support the
possibility that HS6ST will also have a critical role in vascularization.
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ACKNOWLEDGEMENTS |
---|
We thank S. Hayashi and N. Perrimon for fly stocks. We are grateful to S. Selleck for the critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by the Human Frontier Science Program and a grant-in-aid for scientific research on priority areas from the Ministry of Education, Science, Sports, and Culture of Japan.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 reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AB051856.
§ Supported by research fellowships from the Japanese Society for the Promotion of Science for Young Scientists.
** To whom correspondence should be addressed. Tel.: 81-426-77-2570; Fax: 81-426-77-2559; E-mail: nakato-hiroshi@c.metro-u.ac.jp.
Published, JBC Papers in Press, March 8, 2001, DOI 10.1074/jbc.M011354200
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ABBREVIATIONS |
---|
The abbreviations used are:
HS2ST, heparan
sulfate 2-O-sulfotransferase;
HSPG, heparan sulfate
proteoglycan;
HS6ST, heparan sulfate 6-O-sulfotransferase;
FGF, fibroblast growth factor;
dARNT, Drosophila aryl
hydrocarbon receptor nuclear translocator;
PAPS, 3'-phosphoadenosine 5'-phosphosulfate;
CDSNS-heparin, completely
desulfated and N-sulfated heparin;
NS-heparosan, deacetylated and N-sulfated heparosan;
CDSNAc-heparin, completely desulfated N-acetylated heparin;
UA-GlcNAc, 2-acetamide-2-deoxy-4-O-(4-deoxy-
-L-threo-hex-enepyranosyluronic
acid)-D-glucose;
UA-GlcNS, 2-deoxy-2-sulfamido-4-O-(4-deoxy-
-L-threo-hex-enepyranosyluronic
acid)-D-glucose;
UA-GlcNAc6S, 2-acetamide-2-deoxy-4-O-(4-deoxy-
-L-threo-hex-enepyranosyluronic
acid)-6-O-sulfo-D-glucose;
UA-GlcNS6S, 2-deoxy-2-sulfamido-4-O-(4-deoxy-2-O-sulfo-
-L-threo-hex-enepyranosyluronic
acid)-6-O-sulfo-D-glucose;
UA2S-GlcNS, 2-deoxy-2-sulfamido-4-O-(4-deoxy-2-O-sulfo-
-L-threo-hex-enepyranosyluronic
acid)-6-O-sulfo-D-glucose;
UA2S-GlcNS6S, 2-deoxy-2-sulfamido-4-O-(4-deoxy-2-O-sulfo-
-L-threo-hex-enepyranosyluronic
acid)-6-O-sulfo-D-glucose;
HPLC, high
performance liquid chromatography;
EHS, Engelbreth-Holm-Swarm;
PCR, polymerase chain reaction;
BDGP, Berkeley Drosophila Genome
Project;
DPE, downstream promoter element;
RNAi, RNA interference;
FGFR, fibroblast growth factor receptor;
bFGF, basic fibroblast growth
factor;
EST, expressed sequence tag;
CNS, central nervous system;
OPC, outer proliferative center;
dpMAPK, diphosphorylated mitogen-activated
protein kinase;
MAPK, mitogenactivated protein kinase;
CME, central midline element;
dsRNA, double-stranded RNA.
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