From the Turku Centre for Biotechnology, University
of Turku and Åbo Akademi University, FIN-20521 Turku, Finland, the
§ Department of Medical Biochemistry and Microbiology,
Uppsala University, S-75123 Uppsala, Sweden, and ¶ BioTie
Therapies Corp., FIN-20520 Turku, Finland
Received for publication, December 13, 2000, and in revised form, January 22, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fibroblast growth factors (FGFs) are
heparin-binding polypeptides that affect the growth, differentiation,
and migration of many cell types. FGFs signal by binding and activating
cell surface FGF receptors (FGFRs) with intracellular tyrosine kinase
domains. The signaling involves ligand-induced receptor dimerization
and autophosphorylation, followed by downstream transfer of the signal. The sulfated glycosaminoglycans heparin and heparan sulfate bind both
FGFs and FGFRs and enhance FGF signaling by mediating complex formation
between the growth factor and receptor components. Whereas the
heparin/heparan sulfate structures involved in FGF binding have been
studied in some detail, little information has been available on
saccharide structures mediating binding to FGFRs. We have performed
structural characterization of heparin/heparan sulfate oligosaccharides
with affinity toward FGFR4. The binding of heparin oligosaccharides to
FGFR4 increased with increasing fragment length, the minimal binding
domains being contained within eight monosaccharide units. The
FGFR4-binding saccharide domains contained both
2-O-sulfated iduronic acid and 6-O-sulfated
N-sulfoglucosamine residues, as shown by experiments
with selectively desulfated heparin, compositional disaccharide
analysis, and a novel exoenzyme-based sequence analysis of heparan
sulfate oligosaccharides. Structurally distinct heparan sulfate
octasaccharides differed in binding to FGFR4. Sequence analysis
suggested that the affinity of the interaction depended on the number
of 6-O-sulfate groups but not on their precise location.
The fibroblast growth factors
(FGFs)1 belong to a family of
about 20 related polypeptides. They display biological activity toward
cells of mesenchymal, neuronal, and epithelial origin and are involved
in processes such as cell growth, organ development, and angiogenesis
(1). The biological effects of FGFs are exerted through interactions
with FGF receptors (FGFRs). The receptor family consists of four known
members, FGFR1-4, with many isoforms (2). Upon ligand binding the
receptor is thought to be activated through dimerization and
phosphorylation by the intracellular tyrosine kinase domains (3).
Heparan sulfate proteoglycans (HSPGs), abundant components of cell
surfaces and the extracellular matrix, appear central to signaling
through FGF·FGFR complexes (for reviews, see Refs. 4-6).
Cells lacking endogenous HSPGs respond poorly to FGF, whereas the
response can be readily restored by addition of exogenous heparin (7,
8). Accumulated evidence points to formation of biologically active
complexes involving FGF, FGFR, and HSPGs, in which heparan sulfate
interacts with both the FGF and FGFR components of the complex (9-11).
A direct interaction between HSPGs and FGFRs appears critical for FGFR activation (12). A heparin-binding domain identified in the second
Ig-loop of the four FGFRs comprises sequence of about 20 amino acids
toward the NH2 terminus of the loop (12). Different splice
variants of the receptors differ in affinity for heparin, such that the
interaction may vary with the structure of the extracellular receptor
domain (13).
Heparin/HS chains are initially synthesized as polymers of
alternating glucuronic acid (GlcA) and N-acetylglucosamine
(GlcNAc) units (for reviews, see Refs. 14-16). In HS biosynthesis, the
polymer is first modified by partial
N-deacetylation/N-sulfation of GlcNAc residues.
The further modification reactions, C5-epimerization of GlcA to
iduronic acid (IdoA) units and O-sulfation at various positions (C2 of IdoA and GlcA, C3 and C6 of GlcN units), all occur in
the vicinity of previously incorporated N-sulfate groups. Heparin, a highly specialized product of mast cells, is more
extensively modified than HS and the modifications are more evenly
distributed along the polymer (14).
The heparin/HS structures required for FGFR binding are poorly defined.
However, both IdoA(2-OSO3) and
GlcNSO3(6-OSO3) residues appear to be required
for the FGF2 induced activation of FGFR1, whereas
2-O-sulfate groups alone are sufficient to mediate binding to FGF2 (11, 17, 18). While these findings suggest a role for
6-O-sulfated GlcNSO3 residues in the
interactions with FGFR1, it would seem likely that different FGFRs may
bind structurally distinct HS species. Neuroepithelial HSPG thus
preferentially bound FGFR1 at the cell surface although FGFR3 was also
present (19). The importance of polysaccharide-FGFR interaction was underpinned by the finding that heparin could alone induce
phosphorylation of FGFR4 in the absence of an FGF ligand (20).
In the present paper we describe FGF-independent binding of heparin and
HS to the extracellular domain of FGFR4. We show that the interaction
is mediated by N-sulfated octasaccharides that contain both
IdoA(2-OSO3) and GlcNSO3(6-OSO3)
residues, and provide sequence data for FGFR4-binding HS domains.
Materials--
All studies were performed using the soluble
extracellular domain of FGFR4. The expression and purification of
recombinant human FGFR4, containing the three extracellular Ig domains
(Ser25-Arg366), were as described earlier (21).
Briefly, the His-tagged protein was expressed in Sf9 insect
cells and purified directly from the culture medium by nickel and
heparin affinity chromatography. Heparin from pig intestinal mucosa
(stage 14, Inolex Pharmaceutical Division, Park Forest South, IL), was
purified as previously described (22). It was used either unlabeled or
radiolabeled by 3H-acetylation of free amino groups
(specific activity 75,300 dpm/nmol) as described (23). The selectively
desulfated heparin preparations and oligosaccharides of bovine lung
heparin (24, 25) were a kind gift from Dr. Dorothe Spillmann (Uppsala
University, Uppsala, Sweden). Heparan sulfate preparations from bovine
aorta, kidney, lung, and intestine were generously provided by Dr.
Keiichi Yoshida (Seikagaku Corp., Tokyo, Japan). N-Sulfated
HS oligosaccharides were prepared from bovine intestinal mucosa heparan
sulfate (a gift from Kabi AB, Stockholm, Sweden) and
3H-labeled as described previously (26). Briefly, HS was
N-deacetylated by hydrazinolysis followed by treatment with
nitrous acid at pH 3.9, resulting in cleavage at the
N-unsubstituted GlcN residues. The resistent
N-sulfated oligosaccharides were recovered, reduced with
NaB3H4 (28 Ci/mmol, Amersham Pharmacia Biotech,
Uppsala, Sweden), and separated by gel chromatography. The column
materials, Sephadex G-15 and CH-Sepharose-4B, were obtained from
Amersham Pharmacia Biotech, as were the PD-10 desalting and Superdex 30 columns. The Partisil-10 HPLC column (4.6 × 250 mm) was from
Whatman Inc., Clifton, NJ, and the Propac PA1 HPLC column was from
Dionex, Surrey, United Kingdom.
Binding Studies--
In the filter-trapping assay (27),
radiolabeled glycosaminoglycans were incubated with FGFR4 in 8.1 mM Na2HPO4, 1.5 mM
KH2PO4, 2.7 mM KCl, and 140 mM NaCl, pH 7.4 (PBS), containing 0.1 mg/ml bovine serum
albumin, in a total volume of 200 µl for 2 h at room temperature. The mixtures were rapidly passed through nitrocellulose filters (Sartorius, diameter 25 mm, pore size 0.45 µm) using a vacuum
suction apparatus, followed by washing with PBS. Proteins and protein
bound saccharides remain on the filter whereas unbound saccharides pass
through. The bound saccharides were released by 2 M NaCl
and quantified by a
Binding studies were also performed using a CH Sepharose-4B column,
with immobilized FGFR4, that was prepared according to the instructions
of the manufacturer. For preparation of 1 ml of the affinity matrix,
~0.5 mg of FGFR4 was used. Heparin (0.5 mg) was included in the
immobilization reaction to protect the heparin-binding site on FGFR4.
To avoid immobilization of heparin to the matrix, the heparin
preparation used had been treated with HNO2 at pH 3.9 (28)
to destroy any N-unsubstituted GlcN residues, followed by
recovery of the high molecular weight fraction by gel chromatography on
Superdex 30. Samples of 3H-labeled heparin/HS were applied
to the column in PBS, with or without CaCl2
supplementation, followed by washing with PBS and elution of the bound
material with a linear gradient of NaCl (0.14-1.0 M) in
PBS at a flow rate of 0.5 ml/min. Fractions of 1 ml were collected and
measured for radioactivity. The NaCl gradient was monitored by
measuring the conductivity of every third fraction. A control column
was prepared without immobilized FGFR4. This column did not bind any of
the tested glycosaminoglycans (data not shown). To study the
interaction of the antithrombin (AT) binding heparin domain with FGFR4,
3H-labeled heparin decasaccharides were subjected to
affinity chromatography on antithrombin-Sepharose as described (29).
Bound decasaccharides were eluted with a step gradient of NaCl (0.14, 0.5, and 2.0 M NaCl in 50 mM Tris-HCl, pH 7.4).
The high affinity (~2% of total saccharide) and non-binding
fractions recovered in the 2.0 and 0.14 M NaCl eluates,
respectively, were tested for FGFR4 binding by affinity chromatography.
Surface plasmon resonance analysis on a BiacoreX instrument (BiaCore
AB, Uppsala, Sweden) was a third means of studying saccharide:FGFR4 binding. Heparin (0.5-1 mg) was biotinylated by incubation in 0.1 M MES buffer (pH 5.5 with 50 mM biotin
hydrazide (Calbiochem, San Diego, CA) and 10 mM
N-ethyl-N'(dimethylaminopropyl)carbodiimide (Pierce Chemical Corp., Rockford, IL) for 5-6 h at room temperature. Biotinylated heparin was separated from excess reagent on a PD-10 column and immobilized to streptavidin-coated sensor chips (BiaCore AB). FGFR4 was incubated with saccharides for at least 10 min prior to
injection over the heparin-coated surface. The running buffer used was
PBS supplemented with 0.005% Tween 20. The concentrations of the
interactants and flow rates were as indicated in the figure legends. A
streptavidin surface without immobilized heparin was used as a control.
The response from this surface was subtracted from the response of the
heparin surface.
Compositional Analysis of Heparan
Sulfate--
N-Sulfated oligosaccharides from bovine
intestinal HS were fractionated by binding to the FGFR4 affinity
matrix. To deplete the unbound pool (~80% of the total saccharide)
of any remaining FGFR4 binding components, it was rechromatographed
twice on the FGFR4 column (<4% of the material was bound to the
matrix upon the second rechromatography step). The disaccharide
composition of HS samples was determined as described (30, 31).
Briefly, saccharides were treated with nitrous acid (HNO2)
at pH 1.5, leading to deaminative cleavage of the saccharide chain at
GlcNSO3 units (28). The resultant terminal anhydromannose
units were radiolabeled by reduction with
NaB3H4 (0.25-0.5 mCi/reaction) yielding
3H-labeled 2,5-anhydromannitol
([3H]aManR) residues. The labeled
disaccharides were recovered by gel chromatography and further
separated by anion-exchange HPLC on a Partisil-10 SAX column
eluted with a step gradient of KH2PO4. The disaccharide peaks were identified by comparing their elution positions to those of standard heparin disaccharides. The proportions of non-O-sulfated disaccharides were determined by high
voltage paper electrophoresis of the total labeled disaccharides in
0.83 M pyridine, 0.5 M acetic acid buffer, pH
5.3 (32).
Sequence Analysis--
FGFR4 binding HS oligosaccharides,
containing a reducing terminal [3H]aManR
residue, were prepared for sequence analysis by anion-exchange HPLC on
a Propac PA1 column in H2O, pH 3 (adjusted with HCl). The
bound oligosaccharides were eluted with a linear gradient of NaCl (up
to 1.5 M). The fractions containing the octasaccharides of
interest were pooled, desalted, dried in a centrifugal evaporator, and
sequenced through a combination of chemical and enzymatic degradation
procedures as described (33). Samples were first subjected to partial
HNO2 (pHNO2) cleavage (34) by treatment with 2 mM NaNO2 in 20 mM HCl on ice. After
incubation for various periods of time (30, 60, 90, 120, and 180 min),
aliquots were removed and the reaction was stopped by addition of 200 mM sodium acetate, pH 6. The aliquots, containing the
cleavage products from the different time points, were combined and
subjected to enzyme digestion. The exoenzymes used for sequence
analysis were iduronate-2-sulfatase (IdoA2Sase),
Binding of Heparin to the Extracellular Domain of FGFR4--
To
assess the binding of the recombinant extracellular domain of FGFR4 to
heparin, increasing amounts of [3H]heparin were incubated
with FGFR4 at physiological ionic strength. The protein-polysaccharide
complexes formed were trapped on nitrocellulose filters and the bound
saccharide was quantified by scintillation counting (27). The results
indicated that heparin bound FGFR4 in a dose-dependent and
saturable manner (Fig. 1A),
whereas the binding was completely abolished by addition of excess cold
heparin (data not shown). A KD value of 0.3-0.4
µM was determined for the FGFR-heparin interaction
(assuming an average molecular weight of 10 kDa for heparin) by fitting
a hyperbolic function using nonlinear regression analysis to the data
visualized by a Scatchard plot (inset, Fig. 1A).
The interaction was also studied by affinity chromatography of
[3H]heparin on immobilized FGFR4, as well as by surface
plasmon resonance (BiaCore) measurements of soluble FGFR4 binding to
immobilized heparin. In affinity chromatography,
[3H]heparin was found to require 0.25-0.50 M
NaCl for elution from the FGFR4 column (Fig. 1B). Surface
plasmon resonance analysis showed saturable binding of FGFR4 to the
heparin-coated surface whereas little or no binding was seen to a
control surface without heparin (data not shown). Together, these data
indicate that the FGFR4 ectodomain is capable of binding heparin in a
FGF-ligand independent fashion, in agreement with previous results (20, 21, 35).
An important role for divalent cations such as Ca2+ in the
binding of heparin to FGFR1, another member of the FGFR family, were proposed by McKeehan and co-workers (36, 37). To examine whether calcium ions affect the heparin-FGFR4 interaction, we tested the binding of [3H]heparin to the FGFR4 affinity matrix in
the presence of 1.3 mM Ca2+, which corresponds
to the physiological Ca2+ concentration of extracellular
fluids. Under these conditions, the peak elution of
[3H]heparin occurred at ~0.40 M NaCl,
as compared with a peak elution position corresponding to ~0.35
M NaCl in the absence of Ca2+ ions (Fig.
1B). These results suggest that Ca2+ ions may
slightly enhance, but are not required for, the heparin-FGFR4 interaction.
Structural Requirements for Binding of Heparin to FGFR4--
To
identify the minimal size of the FGFR4 binding heparin domain, even
numbered, 3H-end-labeled heparin oligosaccharides were
incubated with FGFR4 in solution, after which the binding was assessed
by the filter-trapping method (see "Experimental Procedures").
Octasaccharides were the shortest oligosaccharides with appreciable
FGFR4 binding capacity (Fig.
2A). The binding of
decasaccharides and longer fragments to FGFR4 increased gradually with
increasing fragment length, but without any striking differences in
binding between the consecutive fragments of the series. Surface
plasmon resonance was also used to define the minimal FGFR4-binding
heparin domain by assessing the ability of heparin oligosaccharides to
inhibit binding of FGFR4 to biotinylated full-length heparin,
immobilized on the chip surface (Fig. 2B). Octasaccharides
were the smallest fragments with substantial inhibitory capacity,
whereas decasaccharides and longer fragments had still higher
inhibitory effect. Taken together, the above data implicate a minimal
FGFR4-binding site within a sequence encompassing eight monosaccharide
units.
We next studied the importance of the N-, 2-O-,
and 6-O-sulfate groups of heparin in FGFR4 binding, by
testing the ability of selectively desulfated heparin preparations to
inhibit binding of [3H]heparin to FGFR4 in solution (Fig.
3A). Whereas low
concentrations (1-5 µg/ml) of unlabeled, native heparin blocked the
binding almost completely, corresponding amounts of the various
selectively desulfated heparin preparations showed little inhibitory
capacity. However, each of the preparations resulted in 50-75%
inhibition at high concentrations (50-100 µg/ml) (Fig.
3A). In Biacore studies (Fig. 3B), FGFR4 was
incubated with the desulfated heparin preparations prior to injection
of the mixture over the heparin-coated surface. The results were in
agreement with the data from filter trapping assays, such that each of
the selective desulfation treatments led to a dramatic decrease in
inhibitory capacity (Fig. 3B). Collectively, these results
suggest that the N-, 2-O-, and
6-O-sulfate groups of heparin all contribute to binding
FGFR4.
Recently, the AT-binding pentasaccharide motif of heparin, containing a
critical 3-O-sulfated GlcNSO3 residue was
implicated in binding to FGFRs (38). We decided to reassess this
proposal by separating 3H-labeled heparin decasaccharides
with regard to affinity for AT, and then test the resultant high and
low affinity fractions for ability to bind FGFR4. About 2% of the
starting material bound with high affinity to immobilized AT, in good
agreement with previous findings (29) and this fraction was
quantitatively retained by the FGFR4 column (Fig.
4A), in accord with the
proposal by McKeehan et al. (38). However, a major portion
of the fraction with low affinity for AT also bound to the immobilized
FGFR4, and elution of this material from the FGFR4 column required the same NaCl concentration as that needed to displace the decasaccharide with high affinity for AT (Fig. 4A). This finding is in
disagreement with the notion that the AT binding sequence is essential
for FGFR binding. Indeed, isolation of the two FGFR4-binding fractions followed by analytical AT-Sepharose chromatography confirmed that one
of the fractions, as expected, showed high affinity for AT whereas the
other did not (Fig. 4B).
Binding of Heparan Sulfate to FGFR4--
The major physiological
polysaccharide ligand for FGFR4 is presumably HS rather than heparin,
that is essentially confined to the mast cell. The interactions of
3H-labeled HS samples from bovine lung, aorta, and kidney
with FGFR4 were studied by affinity chromatography (Fig.
5). All HS species tested were retained
by the column and required 0.2-0.3 M NaCl for elution.
Generally, the binding was somewhat weaker than that of heparin,
judging from the higher NaCl concentration required to displace heparin
compared with HS. The FGFR4 binding profiles of the various HS species
differed such that a substantial portion of kidney HS emerged at NaCl
concentrations >0.35 M, whereas aorta HS contained only
minor amounts of such high-affinity material.
Disaccharide Composition of FGFR4 Binding Heparan Sulfate
Decasaccharides--
We next proceeded to characterize HS domains with
affinity toward FGFR4. The experiments with selectively desulfated
heparin suggested that the binding of HS to FGFR4 would require highly sulfated structures, of the type represented by the
N-sulfated domains of the polysaccharide. These domains are
composed of consecutive N-sulfated disaccharide units and
contain most of the O-sulfate groups of the HS chain. To
isolate such domains, we used bovine intestinal HS that bound to the
FGFR4 affinity column (data not shown) similar to the lung HS
preparation shown in Fig. 5. N-Sulfated oligosaccharides
were prepared as described under "Experimental Procedures" and
3H-end-labeled by reduction with
NaB3H4. Affinity chromatography of the
[3H]decasaccharide on the FGFR4 column yielded 20% of
bound material. After two reapplications of the unbound fraction, less
than 4% of the material bound to the matrix, indicating depletion of
FGFR4-binding species (data not shown). Analysis of the bound and
unbound decamer pools (see "Experimental Procedures") indicated
that the bound fraction was markedly enriched in 6-O-sulfate
groups, that were almost twice as abundant as in the unbound fraction
(Table I, Fig. 6). The
6-O-sulfate groups occurred
mainly in trisulfated -IdoA(2-OSO3)-GlcNSO3(6-OSO3)-
disaccharide units. Similar results were obtained upon compositional
disaccharide analysis of FGFR4 bound and unbound dodecasaccharides (not
shown). Together, these findings suggest that the FGFR4-HS interaction
is mediated by highly sulfated domains containing both
IdoA(2-OSO3) and GlcNSO3(6-OSO3) residues.
Sequence Analysis of FGFR4-binding Heparan Sulfate
Domains--
To obtain more detailed information of the FGFR4-binding
HS domain, we employed a novel method for sequencing of end-labeled, N-sulfated HS oligosaccharides (33). Affinity chromatography of [3H]octasaccharides (i.e. the smallest
fragments containing the FGFR4-binding domain; see Fig. 2) derived from
bovine intestinal HS on FGFR4, yielded a fraction (~15% of the
starting material) of labeled components that remained bound to the
receptor at physiological ionic strength (Fig.
7).
Approximately 60% of the bound octasaccharides were eluted from the
FGFR4 affinity column at 0.2 M NaCl (in the following denoted as the 0.2 M fraction) whereas ~30% required 0.3 M NaCl (0.3 M fraction) for displacement (Fig.
7). Further selection of target fractions for sequence analysis within
each affinity class aimed for components with the lowest charge
density; this approach was adopted to pinpoint the minimal structural
features required for FGFR4 binding, without redundant
O-sulfate groups. The octasaccharides thus recovered
(~10% of the labeled material) from the 0.2 M fraction
by preparative Propac PA1 chromatography (not shown) yielded a somewhat
heterogeneous peak upon analytical rechromatography (Fig.
8A), at an elution position
corresponding to an N-sulfated octasaccharide sequence with
three O-sulfate groups.
Sequence analysis involved pHNO2 treatment followed by
Propac chromatography of the resultant, labeled even-numbered
oligosaccharide fragments (di-, tetra-, and hexasaccharides from a
parental octasaccharide, along with some uncleaved material). These
oligosaccharides were then modified further, by stepwise incubations
with IdoA2Sase, IdoA2Sase/IdoAase, and IdoA2Sase/IdoAase/GlcN6Sase, the
products of each step being analyzed by Propac chromatography (33).
Sequence assignment was based on (i) the effects on each
oligosaccharide of the various enzyme treatments and (ii) the observed
elution positions of the oligosaccharides generated by
pHNO2, as related to the behavior of standard
oligosaccharides with known numbers of O-sulfate
groups.2 The effects on the oligosaccharides of the enzymes
were highly reproducible and involved large, predictable shifts in
elution position due to removal of sulfate groups, and smaller shifts after removal of nonreducing terminal IdoA residues. Notably, the
residual parental oligosaccharides terminate with a GlcA unit (adjacent
to a GlcNAc residue in the intact HS chain, hence not subject to
C5-epimerization during HS biosynthesis (39)) and therefore resist the
enzyme treatment. Even sequences of major components in mixtures could
be resolved, based on the identification of distinct equal sized
pHNO2 oligosaccharides and the known (identical) number of
sulfate groups in the corresponding intact saccharides (tri-O-sulfated octasaccharides in Fig. 8). In cases where
deduced structures were not entirely represented by resolved
degradation intermediates, the uncertainty in structural assignment has
been indicated.
Application of these procedures to the recovered
[3H]octasaccharide sample (Propac chromatography) from
the 0.2 M fraction (FGFR4 affinity chromatography) yielded
the two tri-O-sulfated sequences 8a and 8b shown in Fig. 10.
These structures were deduced as follows. A hexasaccharide fraction
(6b) appeared following pHNO2 treatment, somewhat retarded
compared with the parental octasaccharide fraction (Fig. 8B). This
increase in charge density reflected the loss of a
non-O-sulfated GlcA-GlcNSO3 disaccharide sequence (units 1 and 2 in Fig. 10;
octasaccharides 8a and 8b). The hexasaccharide
peak was eliminated by IdoA2Sase digestion, and replaced by two less
anionic, incompletely resolved peaks, thus identifying an
IdoA(2-OSO3), unit 3. One of these components lost an additional sulfate group by IdoAase/GlcN6Sase treatment, whereas the other remained unaffected (Fig. 8, D and
E). Unit 4 thus was 6-O-sulfated in
one of the sequences (octasaccharide 8a) but not in the
other (octasaccharide 8b). The elution positions of the two
major tetrasaccharide products (4a and 4b in Fig.
8B) of pHNO2 treatment suggested a difference in
composition by one sulfate group. Both tetrasaccharides were
susceptible to Ido2Sase (Fig. 8C) and IdoAase (Fig.
8D), but resistant to GlcN6Sase (Fig. 8E), and
therefore both contained a nonreducing-terminal
IdoA(2-OSO3)-GlcNSO3-disaccharide sequence
(units 5 and 6 in Fig. 10, octasaccharides
8a and 8b). The additional O-sulfate group
in octasaccharide 8b was located to the terminal aManR
residue by analysis of the labeled disaccharide fractions
(2a and 2b in Fig. 8B). None of the
disaccharides were affected by IdoA2Sase (Fig. 8C). Fraction
2a, that appeared at the elution position of nonsulfated
HexA-aManR, was partially cleaved by IdoAase, yielding
nonsulfated labeled monosaccharide; however, the major portion remained
unchanged, indicating a -GlcA-aManR- sequence for
units 7 and 8 in octasaccharide 8a (Fig. 10).
Disaccharide 2b, at the elution position of monosulfated
HexA-aManR, was quantitatively converted to labeled
aManR(6-OSO3) by digestion with IdoAase (Fig. 8D; the peak coinciding with that of the 4a'" product). This sulfated monosaccharide is not a substrate for GlcN6Sase (Fig. 8E).
Octasaccharide 8b thus terminates (units 7 and 8)
with an -IdoA-aManR(6-OSO3)- structure (Fig.
10).
An octasaccharide fraction was recovered by preparative Propac
chromatography, corresponding to ~10% of the labeled material eluted
from the FGFR4 column with 0.3 M NaCl (not shown).
Analytical Propac assessment showed a peak at an elution position
consistent with the occurrence of N-sulfated octasaccharides
containing four O-sulfate groups (Fig.
9A; cf. elution position of
analogs with three O-sulfate groups in Fig. 8A).
Of the fragments generated upon pHNO2 treatment (Fig.
9B), hexasaccharide 6c, tetrasaccharide 4c, and disaccharide
2c could be assigned to the parent octasaccharide 8c shown in Fig.
10. Briefly, the elution position of 6c
reflects the loss of a terminal mono-O-sulfated disaccharide
residue, defining units 1-2 as
GlcA-GlcNSO3(6-OSO3) (see above). Treatment
with IdoA2Sase produced major shifts in the positions of subfractions 6c and 4c, indicating loss of a 2-O-sulfate group from each
fragment (Fig. 9C). Digestion with IdoAase led to the
predicted, less prominent, shifts for 6c', 4c', and 2c, as expected for
nonreducing-terminal IdoA residues (Fig. 9D). The elution
position of the intact disaccharide 2c is that of
IdoA-aManR(6-OSO3) (Fig. 9B). None
of these three components were further affected by digestion with
GlcN6Sase (Fig. 9E).
Additional fragments created by pHNO2 treatment of the
tetra-O-sulfated octasaccharide fraction included the two
tetrasaccharides 4d and 4e, and the disaccharide 2d. Due to lack of any
detectable corresponding hexasaccharide fragments, the sequences of the
parental octasaccharides could not be directly resolved. However,
fragments 4d and 2d appeared to derive from a common parent structure,
since 4d was identified as a mono-O-sulfated tetrasaccharide
with nonreducing terminal IdoA(2-OSO3)-saccharide, and 2d
as a nonsulfated disaccharide (Fig. 9, B-E). Presumably, the
parent octasaccharide 8d contains three additional O-sulfate
groups that would be distributed among units 2-4, as shown
in Fig. 10. Fragment 4e emerged like a tri-O-sulfated heparin tetrasaccharide (Fig. 9B), and the effects of enzyme
digestions indicated a nonreducing-terminal
IdoA(2-OSO3)-GlcNSO3(6-OSO3)-disaccharide sequence (Fig. 9, C-E), thus a structure not represented by
any of the four identified octasaccharide sequences 8a-d (Fig. 10). Octasaccharide 8e thus would contain one additional
O-sulfate group at an undetermined site within units
2-4.
In this study we have resolved octasaccharide sequences (Fig. 10)
with affinity toward FGFR4. The structures represent the first HS
sequences shown to bind FGF receptors. To the best of our knowledge,
they also represent the first application of the exoenzyme-based
sequencing technology (33) to HS oligosaccharides with known protein
binding activity.
Both heparin and HS were shown to bind to the extracellular domain of
FGFR4 independently of FGF ligands. The binding of heparin was
saturable, with a KD of 0.3-0.4 µM,
thus close to the affinity proposed for binding of heparin to
FGFR2-IIIb (0.2 µM) (40). The FGFR-heparin interactions
appear to be considerably weaker than those between FGFs and heparin,
suggesting that heparan sulfate in vivo would be more prone
to bind free FGFs and FGF·FGFR complexes than free FGFR. The
KD values for binding of FGF1 and FGF2 to heparin
have been reported to be in the nanomolar range (~100 and ~2
nM, respectively) (41, 42).
The minimal heparin domain binding to FGFR4 is contained within 8 monosaccharide units. However, longer oligosaccharides displayed more
efficient binding, possibly reflecting a "ladder effect," i.e. the presence of multiple, overlapping binding sites in
the longer oligosaccharides. Heparin deca- and dodecasaccharides, perhaps even octasaccharides, appear sufficient for the induction of
FGF2 biological activity in cells expressing FGFR1 (11, 18, 43, 44),
but the corresponding requirements for FGFR4-expressing cells have not
been established. The molecular organization of the ternary complexes
between FGFRs, FGFs, and heparin/HS is still unclear. However, recent
analyses of crystallized complexes of FGFs (FGF1 or FGF2) and FGFRs
(FGFR1 or FGFR2), with or without heparin oligosaccharides, provide
novel clues as to how heparin/HS might participate in FGF signaling (9,
10, 45, 46). While the resultant models differ regarding the precise
mode of interaction or even the stoichiometry of components involved,
they all agree with previous proposals (11, 12, 18) of saccharides
interacting with both the growth factor and the receptor moieties.
These findings raise intriquing questions concerning the specificity of
the interactions with regard to carbohydrate structure.
Previous studies have shown that the minimal structural requirements
for interaction of heparin/HS chains with FGF1 and FGF2 are specific
and distinctly different (see "Introduction"). In this study we
have evaluated the requirements for FGFR4 binding. Highly stringent
structure/function relations were proposed by McKeehan et
al. (38), who claimed that a number of FGFR species bind
exclusively to the AT-binding sequence of heparin. The present results
demonstrate that heparin decasaccharides with high and low affinity for
AT can bind equally well to FGFR4 (Fig. 4). The reason for this
discrepancy is unclear. Assessment of a FGF2:FGFR1:heparin decamer
crystal suggested that the N-, 2-O-, and
6-O-sulfate groups of the saccharide are all involved in
binding to the receptor component, the 6-O-sulfate groups
appearing to be of importance (9). These findings agree with the
present compositional analysis of HS decasaccharides fractionated with
regard to affinity for FGFR4, that indicated appreciable enrichment of
6-O-sulfate groups in the bound fraction (Table I), and with
previous functional studies pointing to a role for 6-O-sulfate groups
in HS-FGFR1 binding (11). Moreover, competitive binding of
[3H]heparin and various desulfated unlabeled heparin
preparations to FGFR4 clearly implicated all three kinds of sulfate
substituents in the interaction (Fig. 3). The question then arises
whether binding to FGFR4 requires any specific disposition of these
residues. All FGFR4-binding HS octasaccharides defined with regard to
sequence (Fig. 10) were fully N-sulfated and showed the same
distribution of IdoA(2-OSO3) groups, invariably present in
both internal disaccharide units. Whether both of these
2-O-sulfate groups are indeed required for FGFR4 binding or
simply occur in most or all of the isolated octasaccharides remains
unclear. By contrast, the distribution of GlcN(6-OSO3)
residues varied conspicuously. The two octasaccharides (8a and 8b)
recovered by elution of the FGFR4 column with 0.2 M NaCl
both contained a single 6-O-sulfate group, but in different positions (units 4 and 8, respectively). If
indeed 6-O-sulfate groups contribute to the interaction
their precise location thus does not appear to be critical.
Analysis of the octasaccharide species eluted with 0.3 M
NaCl, hence with higher affinity for the receptor, reinforces this
impression. In all, three components were considered, each containing a
total of four O-sulfate groups. Even if only octasaccharide
8c could be pursued through the complete sequencing protocol, the
information regarding octasaccharides 8d and 8e sufficed to demonstrate
that the 0.3 M fraction contained octasaccharide species
with highly diverse 6-O-sulfation patterns (Fig. 10). Yet
these 6-O-sulfate groups clearly contribute to the higher
overall ligand affinity of the 0.3 M fraction. We conclude that this increase in affinity is due to charge interactions at various
locations of the saccharide:FGFR4 interface. Whether this diversity
reflects nonspecific polyelectrolyte effects or selective interactions
with distinct basic amino acid residues remains to be determined
through more refined analysis at the molecular and atomic levels.
What is the functional significance of interactions between FGFRs and
heparin-related GAGs? Notably, highly sulfated polyanions such as
heparin and synthetic polysulfonates, but not HS that is less sulfated,
can activate FGFR4 in the absence of a FGF ligand (20). This effect of
heparin is not well understood, but could conceivably be of importance
upon heparin release from degranulating mast cells in tissues with
abundant FGFR4 expression. However, the major role in vivo
of the "heparin-binding domain" of FGFR4 is presumably to mediate
the formation of ternary complexes with HS proteoglycans and FGFs,
leading to receptor activation. The interpretation of the present data
is hampered by a lack of information regarding the functional and
topological organization of HS proteoglycan species in the intact
tissue, here intestinal mucosa, used as source of HS for
oligosaccharide generation. Thus we do not know whether the HS
sequences found to bind FGFR4 were actually designed for such
interaction in vivo. The relatively modest affinities observed do not argue against an essential functional role in promoting
formation of ternary complex (47). However, for productive interactions
the FGFR-binding sequences must be contiguous with (or partially
overlap) the FGF-binding domains, hence the need for an overall deca-
to dodecamer N-sulfated region of the HS chain to
accommodate both binding sites. Such regions account for only a small
proportion, usually a few percent, of the total HS chains (48). The
structural characteristics required for productive interactions of a
particular FGF and its receptor with an N-sulfated HS domain
remain unclear. At least some of the members of the FGF family, such as
FGF1 and FGF2, show highly distinct requirements for HS ligand
structure (26, 49), but these features appear to be complemented by
less exacting terms for FGFR binding. N-Sulfated domains
that are too short, or otherwise structurally unfit to simultaneously
interact with both FGF and its receptor, may store the growth factor in
the pericellular environment and actually inhibit receptor activation
(50, 51). Heparin, frequently used as a substitute for HS in
experimental work, consists largely of trisulfated
-IdoA(2-OSO3)-GlcNSO3(6-OSO3)-
disaccharide units, and thus will basically cover most or all
combinations of N- and O-sulfate groups required
for interactions with different FGFs and FGFRs. By contrast, HS
oligosaccharides with closely similar composition can be fractionated
into species that either activate or inhibit FGF2 signaling in
HS-deficient cells (18). Moreover, the preference for growth factor
target (FGF1 or FGF2) could be shifted by subtle changes in HS
structure (52-54). These findings emphasize the need for further
analysis, not only of HS structures in relation to growth factor
action, but also of the mechanisms that control the generation of such
structures during their biosynthesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-scintillation counter.
-L-iduronidase (IdoAase), and glucosamine-6-sulfatase
(GlcN6Sase) (Oxford GlycoSciences, Abingdon, U.K.). The enzymes are
recombinant human (IdoA2Sase and IdoAase) or caprine (GlcN6Sase)
proteins produced in Chinese hamster ovary K1 cells. IdoA2Sase removes
ester sulfates at C2 of nonreducing terminal IdoA(2-OSO3)
residues of heparin/heparan sulfate whereas IdoAase cleaves the
1-4
linkage between IdoA and GlcNR in heparin/heparan sulfate (R
is -COCH3, -SO
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 1.
Interaction of heparin with the extracellular
domain of FGFR4. A, [3H]heparin was
incubated with FGFR4 (20 µg/ml) in PBS for 2 h. The formed
FGFR4-heparin complexes were recovered on nitrocellulose filters and
the filter bound radioactivity was quantified by liquid scintillation
counting (see "Experimental Procedures"). Inset shows a
Scatchard plot based on the binding data. B, affinity
chromatography of [3H]heparin on immobilized FGFR4.
Samples of [3H]heparin (10,000 dpm) were applied to the
FGFR4 column in calcium-free PBS (- - - -) or PBS containing 1.3 mM CaCl2 ( ). The bound material was eluted
with a linear gradient of NaCl. Fractions of 1 ml were collected and
analyzed for radioactivity.
View larger version (19K):
[in a new window]
Fig. 2.
Binding of heparin oligosaccharides to
FGFR4. A, the binding of even-numbered,
3H-labeled heparin oligosaccharides (20,000 dpm) to FGFR4
(15 µg/ml) was studied by the filter trapping assay (see legend to
Fig. 1 and "Experimental Procedures"). B, surface
plasmon resonance analysis of the capacity of heparin oligosaccharides
to inhibit the binding of soluble FGFR4 to immobilized heparin. Heparin
oligosaccharides (16 µg/ml) were incubated with FGFR4 (0.4 ng/ml) and
injected, at a flow rate of 30 µl/min, over biotinylated heparin
immobilized onto the streptavidin-coated sensor chip surface. The
response representing the binding of FGFR4 to heparin in the absence of
competitors was set to 100%. H, full-length heparin.
View larger version (21K):
[in a new window]
Fig. 3.
Binding of selectively desulfated heparin
preparations to FGFR4. A, unlabeled, native or
selectively N-, 2-O-, or
6-O-desulfated heparin preparations were mixed with
[3H]heparin (20 000 dpm) and FGFR4 (15 µg/ml) in PBS.
The FGFR4 bound [3H]heparin was quantified by the
filter-trapping method. The unlabeled competitors used were native
heparin ( ), N-desulfated (
), 2-O-desulfated
(
), and preferentially 6-O-desulfated (
) heparin.
B, surface plasmon resonance assay of the inhibitory
capacity of the selectively desulfated heparin preparations. The native
or desulfated saccharides (2.4 µg/ml) were mixed with FGFR4 (1.2 ng/ml) and injected over the heparin-coated surface at a flow of 10 µl/min. The data represent the average of two independent
experiments. The sensorgrams pertaining to one of the experiments are
shown in the inset (N-DS, N-desulfated
heparin; 2-O-DS, 2-O-desulfated heparin;
6-O-DS, 6-O-desulfated heparin; H,
native heparin).
View larger version (22K):
[in a new window]
Fig. 4.
Binding of antithrombin-fractionated heparin
decasaccharides to FGFR4. A, AT binding ( , 20,000 cpm) and nonbinding (- - - -, 24,000 cpm) 3H-labeled
heparin decasaccharides were affinity fractionated on the FGFR4 column.
Bound decasaccharides were eluted by a linear gradient of NaCl in PBS,
as indicated by the dotted line. B, the FGFR4
bound decasaccharides were pooled, desalted, and subjected to
chromatography on AT-Sepharose.
View larger version (22K):
[in a new window]
Fig. 5.
Binding of heparan sulfate to FGFR4.
A, [3H]heparan sulfate (10 000 dpm) from
bovine aorta ( ), kidney (
), and lung (
) were applied to the
column of immobilized FGFR4 in PBS containing 1.3 mM
CaCl2. After washing with PBS/CaCl2 the bound
material was eluted with a linear gradient of NaCl in the same buffer.
Fractions were collected and analyzed for radioactivity and NaCl
concentration (- - -).
Disaccharide composition of FGFR4 unbound and bound heparan sulfate
decasaccharides
View larger version (21K):
[in a new window]
Fig. 6.
Compositional disaccharide analysis of FGFR4
binding/nonbinding decasaccharides. N-Sulfated
decasaccharides from bovine intestinal HS were fractionated on the
FGFR4 affinity matrix. The unbound (A) and bound
(B) fractions were recovered and subjected to cleavage by
HNO2, pH 1.5, followed by radiolabeling of the resultant
disaccharides with NaB3H4. These were separated
from unincorporated radioactivity and analyzed by anion exchange HPLC
as described under "Experimental Procedures." The peaks correspond
to the following disaccharide structures in the native polysaccharide:
1) GlcA(2-OSO3)-GlcNSO3;
2) GlcA-GlcNSO3 (6-OSO3);
3) IdoA-GlcNSO3 (6-OSO3);
4) IdoA(2-OSO3)-GlcNSO3; and
5) IdoA(2-OSO3)-GlcNSO3
(6-OSO3). The asterisk (*) indicates
tetrasaccharides, in part due to "anomalous" ring contraction (28),
that were not included in the quantification of disaccharides shown in
Table I.
View larger version (17K):
[in a new window]
Fig. 7.
FGFR4 affinity fractionation of
octasaccharides for sequencing analysis.
[3H]Octasaccharides prepared from bovine intestinal HS
were fractionated on the FGFR4 affinity column in preparative fashion.
Bound octasaccharides were eluted by a stepwise gradient of NaCl
(dotted line).
View larger version (14K):
[in a new window]
Fig. 8.
Sequence analysis of minimal
FGFR4-binding octasaccharides. The sample is a fraction of HS
[3H]octasaccharides, eluted from the FGFR4 column with
0.2 M NaCl. Panels show anion-exchange chromatograms of the
material (A) before and (B) after
pHNO2 treatment. Samples (6500 dpm) of the
pHNO2-treated material were digested with IdoA2Sase
(C), IdoA2Sase + IdoAase (D), and IdoA2Sase + IdoAase + GlcN6Sase (E). Di-, tetra-, hexa-, and
octasaccharide products obtained after partial HNO2
cleavage are assigned numbers 2 to 8, the attached letter (a
and b) identifies the parent octasaccharide sequence.
Designations of enzyme digestion products by ', ", and '" indicate
removal of 2-OSO3, IdoA, and 6-OSO3,
respectively. The peaks in panel B correspond to the
following sequences: 8a,
GlcA-GlcNSO3-IdoA(2-OSO3)-GlcNSO3(6-OSO3)-IdoA(2-OSO3)-GlcNSO3-GlcA-aManR;
6a,
IdoA(2-OSO3)-GlcNSO3(6-OSO3)-IdoA(2-OSO3)-GlcNSO3-GlcA-aManR;
4a,
IdoA(2-OSO3)-GlcNSO3-GlcA-aManR;
2a, GlcA-aManR; 8b,
GlcA-GlcNSO3-IdoA(2-OSO3)-GlcNSO3-IdoA(2-OSO3)-GlcNSO3-IdoA-aManR(6-OSO3);
6b,
IdoA(2-OSO3)-GlcNSO3-IdoA(2-OSO3)-GlcNSO3-IdoA-aManR(6-OSO3);
4b,
IdoA(2-OSO3)-GlcNSO3-IdoA-aManR(6-OSO3);
and 2b, IdoA-aManR(6-OSO3). For
additional information, see text.
View larger version (14K):
[in a new window]
Fig. 9.
Sequence analysis of octasaccharides
binding FGFR4 with higher affinity. The sample is a fraction of HS
[3H]octasaccharides, eluted from the FGFR4 column with
0.3 M NaCl. The various degradation treatments illustrated
in panels B-E are the same as shown in Fig. 8.
For each enzyme digestion 10,000 dpm of pHNO2-treated
material was used. The peaks in the chromatograms correspond to the
following sequences: 8c,
GlcA-GlcNSO3(6-OSO3)-IdoA(2-OSO3)-GlcNSO3-IdoA(2-OSO3)-GlcNSO3-IdoA-aManR(6-OSO3);
6c,
IdoA(2-OSO3)-GlcNSO3-IdoA(2-OSO3)-GlcNSO3-IdoA-aManR(6-OSO3);
4c,
IdoA(2-OSO3)-GlcNSO3-IdoA-aManR(6-OSO3);
2c, IdoA-aManR(6-OSO3);
4d,
IdoA(2-OSO3)-GlcNSO3-GlcA-aManR;
2d, GlcA-aManR; 4e,
IdoA(2-OSO3)-GlcNSO3(6-OSO3)-IdoA-aManR(6-OSO3);
2e, IdoA-aManR(6-OSO3).
View larger version (37K):
[in a new window]
Fig. 10.
FGFR4 binding heparan sulfate octasaccharide
sequences. Octasaccharides 8a and 8b were eluted from the FGFR4
column with 0.2 M NaCl, octasaccharides 8c, 8d, and 8e with
0.3 M NaCl. Sequences 8a-c were completely resolved,
whereas sequences 8d and 8e were indirectly deduced from the resolved
structures of the corresponding tetrasaccharides, and the known overall
degree of O-sulfation (4 residues per molecule) of the
parent octasaccharides. The locations of the three O-sulfate
groups marked with asterisks in structure 8d (units
2-4) are tentative but highly plausible, since tetrasaccharide 4d
was shown to contain only one O-sulfate residue (unit
5). Conversely, only one of the three positions occupied by
unspecified R residues in sequence 8e carries an
O-sulfate group, the remaining three O-sulfate
residues being assigned to units 5, 6, and 8. In
some of the octasaccharides unit 7 can be either GlcA or
IdoA; the sequences shown represent the predominant structures. The
hydrogen and hydroxyl groups are not shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Jin-Ping Li (Uppsala University) for providing the antithrombin-Sepharose and Dr. Per Jemth (Uppsala University) for help with the affinity calculations.
![]() |
FOOTNOTES |
---|
* This work was supported by the European Comission programs "Biologically Active Novel Glycosaminoglycans" Grant QLK-CT-1999.00536 and "Heparan Sequencing Demonstration" Grant BIO4-CT98-0538, Swedish Medical Research Council Grants K96-03P and K99-03X, Swedish Cancer Society Grant 3919-B97, Polysackaridforskning AB (Uppsala, Sweden), the Finnish Cancer Union, the Academy of Finland (MATRA-program), and the Juselius Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Turku Centre for
Biotechnology, P.O. Box 123, FIN-20521 Turku, Finland. Tel.: 358-2-274-8964; Fax: 358-2-333-8000; E-mail:
markku.salmivirta@btk.utu.fi.
Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M011226200
2 J. Kreuger and U. Lindahl, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
FGF, fibroblast growth factor;
AT, antithrombin;
aManR, 2,5-anhydromannitol;
FGFR, fibroblast growth factor receptor;
GlcA, D-glucuronic acid;
GlcN, glucosamine;
GlcN6Sase, glucosamine 6-sulfatase;
GlcNAc, N-acetylglucosamine;
GlcNSO3, N-sulfoglucosamine;
HexA, hexuronic
acid;
HPLC, high perfomance liquid chromatography;
HSPG, heparan
sulfate proteoglycan;
IdoA, L-iduronic acid;
IdoAase, -L-iduronidase;
IdoA2Sase, iduronate 2-sulfatase;
SAX, strong anion exchange;
HS, heparan sulfate;
HSPG, heparan sulfate
proteoglycan;
PBS, phosphate-buffered saline;
MES, 2-N-(morpholino)ethanesulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Burgess, W. H., and Maciag, T. (1989) Annu. Rev. Biochem. 58, 575-606[CrossRef][Medline] [Order article via Infotrieve] |
2. | Johnson, D. E., and Williams, L. T. (1993) Adv. Cancer Res. 60, 1-41[Medline] [Order article via Infotrieve] |
3. | Ullrich, A., and Schlessinger, J. (1990) Cell 61, 203-212[Medline] [Order article via Infotrieve] |
4. |
Zimmermann, P.,
and David, G.
(1999)
FASEB J.
13,
91-100 |
5. | Rapraeger, A. C. (1993) Curr. Opin. Cell Biol. 5, 844-853[Medline] [Order article via Infotrieve] |
6. | Bernfield, M., Kokenyesi, R., Kato, M., Hinkes, M. T., Spring, J., Gallo, R. L., and Lose, E. J. (1992) Annu. Rev. Cell Biol. 8, 365-393[CrossRef] |
7. | Rapraeger, A. C., Krufka, A., and Olwin, B. B. (1991) Science 252, 1705-1708[Medline] [Order article via Infotrieve] |
8. | Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P., and Ornitz, D. M. (1991) Cell 64, 841-848[Medline] [Order article via Infotrieve] |
9. | Schlessinger, J., Plotnikov, A. N., Ibrahimi, O. A., Eliseenkova, A. V., Yeh, B. K., Yaon, A., Linhardt, R. J., and Mohammadi, M. (2000) Mol. Cell 6, 743-750[Medline] [Order article via Infotrieve] |
10. | Pellegrini, L., Burke, D. F., von Delft, F., Mulloy, B., and Blundell, T. L. (2000) Nature 407, 1029-1034[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Guimond, S.,
Maccarana, M.,
Olwin, B. B.,
Lindahl, U.,
and Rapraeger, A. C.
(1993)
J. Biol. Chem.
268,
23906-23914 |
12. | Kan, M., Wang, F., Xu, J., Crabb, J. W., Hou, J., and McKeehan, W. L. (1993) Science 259, 1918-1921[Medline] [Order article via Infotrieve] |
13. |
Wang, F.,
Kan, M.,
Yan, G.,
Xu, J.,
and McKeehan, W. L.
(1995)
J. Biol. Chem.
270,
10231-10235 |
14. | Kjellén, L., and Lindahl, U. (1991) Annu. Rev. Biochem. 60, 443-475[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Salmivirta, M.,
Lidholt, K.,
and Lindahl, U.
(1996)
FASEB. J.
10,
1270-1279 |
16. |
Rosenberg, R. D.,
Shworak, N. W.,
Liu, J.,
Schwartz, J. J.,
and Zhang, L.
(1997)
J. Clin. Invest.
99,
2062-2070 |
17. | Rusnati, M., Coltrini, D., Caccia, P., Dell'Era, P., Zoppetti, G., Oreste, P., Valasina, B., and Presta, M. (1994) Biochem. Biophys. Res. Commun. 203, 450-458[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Pye, D. A.,
Romain, R. V.,
Turnbull, J. E.,
Hyde, P.,
and Gallagher, J. T.
(1998)
J. Biol. Chem.
273,
22936-22942 |
19. |
Brickman, Y. G.,
Ford, M. D.,
Small, D. H.,
Bartlett, P. F.,
and Nurcombe, V.
(1995)
J. Biol. Chem.
270,
24941-24948 |
20. | Gao, G., and Goldfarb, M. (1995) EMBO J. 14, 2183-2190[Abstract] |
21. | Loo, B.-M., Darwish, K., Vainikka, S., Saarikettu, J., Vihko, P., Hermonen, J., Goldman, A., Alitalo, K., and Jalkanen, M. (2000) Int. J. Cell Biol. Biochem. 32, 489-497[CrossRef] |
22. |
Lindahl, U.,
Cifonelli, J. A.,
Lindahl, B.,
and Rodén, L.
(1965)
J. Biol. Chem.
240,
2817-2820 |
23. | Höök, M., Riesenfeld, J., and Lindahl, U. (1982) Anal. Biochem. 119, 236-245[Medline] [Order article via Infotrieve] |
24. |
Spillmann, D.,
Witt, D.,
and Lindahl, U.
(1998)
J. Biol. Chem.
273,
15487-15493 |
25. |
Feyzi, E.,
Lustig, F.,
Fager, G.,
Spillmann, D.,
Lindahl, U.,
and Salmivirta, M.
(1997)
J. Biol. Chem.
272,
5518-5524 |
26. |
Kreuger, J.,
Prydz, K.,
Petterson, R. F.,
Lindahl, U.,
and Salmivirta, M.
(1999)
Glycobiology
9,
723-729 |
27. | Maccarana, M., and Lindahl, U. (1993) Glycobiology 3, 271-277[Abstract] |
28. | Shively, J. E., and Conrad, H. E. (1976) Biochemistry 15, 3932-3942[Medline] [Order article via Infotrieve] |
29. | Höök, M., Björk, I., Hopwood, J., and Lindahl, U. (1976) FEBS Lett. 66, 90-93[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Kusche, M.,
Torri, G.,
Casu, B.,
and Lindahl, U.
(1990)
J. Biol. Chem.
265,
7292-7300 |
31. |
Bienkowski, M. J.,
and Conrad, H. E.
(1985)
J. Biol. Chem.
260,
356-365 |
32. | Kusche, M., Lindahl, U., Enerbäck, L., and Rodén, L. (1988) Biochem. J. 253, 885-893[Medline] [Order article via Infotrieve] |
33. | Vivès, R. R., Pye, D. A., Salmivirta, M., Hopwood, J. J., Lindahl, U., and Gallagher, J. T. (1999) Biochem. J. 339, 767-773[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Turnbull, J. E.,
Hopwood, J. J.,
and Gallagher, J. T.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
2698-2703 |
35. |
Kan, M.,
Wu, X.,
Wang, F.,
and McKeehan, W. L.
(1999)
J. Biol. Chem.
274,
15947-15952 |
36. |
Kan, M.,
Wang, F.,
Kan, M.,
To, B.,
Gabriel, J. L.,
and McKeehan, W. L.
(1996)
J. Biol. Chem.
271,
26143-26148 |
37. |
Patstone, G.,
and Maher, P.
(1995)
J. Biol. Chem.
271,
3343-3346 |
38. |
McKeehan, W. L.,
Wu, X.,
and Kan, M.
(1999)
J. Biol. Chem.
274,
21511-21514 |
39. |
Jacobsson, I.,
Lindahl, U.,
Jensen, J. W.,
Roden, L.,
Prihar, H.,
and Feingold, D. S.
(1984)
J. Biol. Chem.
259,
1056-1063 |
40. | LaRochelle, W. J., Sakaguchi, K., Atabey, N., Cheon, H.-G., Takagi, Y., Kinaia, T., Day, R. M., Miki, T., Burgess, W. H., and Bottaro, D. P. (1999) Biochemistry 38, 1765-1771[CrossRef][Medline] [Order article via Infotrieve] |
41. | Mach, H., Volkin, D. B., Burke, C. J., Middaugh, C. R., Linhardt, R. J., Fromm, J. R., Loganathan, D., and Mattsson, L. (1993) Biochemistry 32, 5480-5489[Medline] [Order article via Infotrieve] |
42. | Lee, M. K., and Lander, A. D. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2768-2772[Abstract] |
43. | Ornitz, D. M., Yayon, A., Flanagan, J. G., Svahn, C. M., Levi, E., and Leder, P. (1992) Mol. Cell. Biol. 12, 240-247[Abstract] |
44. |
Ishihara, M.,
Tyrrell, D. J.,
Stauber, G. B.,
Brown, S.,
Cousens, L. S.,
and Stack, R. J.
(1993)
J. Biol. Chem.
268,
4675-4683 |
45. | Plotnikov, A. N., Schlessinger, J., Hubbard, S. R., and Mohammadi, M. (1999) Cell 98, 641-50[Medline] [Order article via Infotrieve] |
46. | Plotnikov, A., Hubbard, S., Schlessinger, J., and Mohammadi, M. (2000) Cell 101, 413-424[Medline] [Order article via Infotrieve] |
47. | Pantoliano, M. W., Horlick, R. A., Springer, B. A., Van Dyk, D. E., Tobery, T., Wetmore, D. R., Lear, J. D., Nahapetian, A. T., Bradley, J. D., and Sisk, W. P. (1994) Biochemistry 33, 10229-10248[Medline] [Order article via Infotrieve] |
48. | Safaiyan, F., Lindahl, U., and Salmivirta, M. (2000) Biochemistry 39, 10823-10830[CrossRef][Medline] [Order article via Infotrieve] |
49. | Ishihara, M. (1994) Glycobiology 4, 817-824[Abstract] |
50. |
Lundin, L.,
Larsson, H.,
Kreuger, J.,
Kanda, S.,
Lindahl, U.,
Salmivirta, M.,
and Claesson-Welsh, L.
(2000)
J. Biol. Chem.
275,
24653-24660 |
51. | Ishihara, M., Shaklee, P., Yang, Z., Liang, W., Wei, Z., Stack, R., and Holme, K. (1994) Glycobiology 4, 452-458 |
52. | Nurcombe, V., Ford, M. D., Wildschut, J. A., and Bartlett, P. F. (1993) Science 260, 103-106[Medline] [Order article via Infotrieve] |
53. |
Brickman, Y. G.,
Ford, M. D.,
Gallagher, J. T.,
Nurcombe, V.,
Bartlett, P. F.,
and Turnbull, J. E.
(1998)
J. Biol. Chem.
273,
4350-4359 |
54. |
Nurcombe, V.,
Smart, C. E.,
Chipperfield, H.,
Cool, S. M.,
Boilly, B.,
and Hondermarck, H.
(2000)
J. Biol. Chem.
275,
30009-30018 |