 |
INTRODUCTION |
Basic fibroblast growth factor
(bFGF)1 is one of a family of
at least 18 polypeptides (reviewed in Ref. 1; also see Refs. 2-5), and
has been shown to influence a variety of cellular processes such as
proliferation, migration, and differentiation (6-8). It has been
implicated in a number of disease states, including tumor growth,
rheumatoid arthritis, and diabetic retinopathy, due to its ability to
stimulate angiogenesis (9, 10). This family of growth factors act
primarily through high affinity tyrosine kinase receptors (FGFRs) (11),
although in addition their activity is modulated by lower affinity
heparan sulfate (HS) proteoglycan receptors (12-14). However, the
mechanism by which this occurs is far from understood. The FGFR family
comprises four related molecules, which each contain a highly conserved
tyrosine kinase domain (15), further diversity being provided by the
existence of alternatively spliced isoforms (15-17). Intracellular
signaling is believed to be initiated by receptor dimerization and
receptor transphosphorylation (18, 19); however, signaling without receptor dimerization cannot be ruled out, as it has not yet been shown
whether FGFRs are activated in their monomeric forms. Indeed it has
been suggested that bFGF activates multiple signaling pathways by
utilizing either monomeric FGFRs or FGFR dimers/multimers (20).
Several HS binding sites on bFGF have been identified with the major
site comprising residues Asn-27, Lys-26, Lys-125, Lys-135, and Arg-120
(21-23). A second possible site was also identified using synthetic
di- and trisaccharides and x-ray crystallography (24). A HS binding
site has also been identified on the FGFR and was found to be located
between the receptors Ig loops I and II (25). The primary high affinity
FGFR binding domain has also been identified on bFGF and was found to
consist of three hydrophobic amino acids (Tyr-103, Leu-140, and
Met-142) and two polar amino acids (Arg-44 and Asn-101) (26, 27). A
secondary low affinity FGFR binding site was also identified (on bFGF)
and comprises amino acids Lys-110, Tyr-111, and Trp-144 (26-28).
Various groups have identified sequences within HS/heparin that
interact strongly with bFGF (29-34). These oligosaccharides were found
to be enriched in IdceA(2S)
1,4GlcNSO3(± 6S)
disaccharides, with their affinity for bFGF increasing with
2-O-sulfate content. In addition, no role was established
for 6-O-sulfation in the interaction with the primary
bFGF binding site (23). The minimum length of saccharide required to
activate bFGF in mitogenic assays has been variously reported as
ranging from di- to dodecasaccharide (14, 24, 31, 32,
35-37).
The investigations described above have led to a number of models being
proposed by which HS·bFGF·FGFR interact to bring about a biological
response. These include the simultaneous binding of two bFGF molecules
to a HS oligosaccharide sequence within the HS chain so as to present
bFGF dimers to the FGFRs, thereby enabling two high affinity FGFRs to
be brought into close proximity to one another for receptor
dimerization and transphosphorylation (14, 22, 29, 38, 39). An
alternative HS·bFGF monomer model has been suggested, in which HS
acts as a bridge by simultaneously binding single molecules of bFGF and
FGFR to form a signaling complex (25, 31). Another HS·bFGF monomeric
model has also been proposed, in which a monomeric complex of HS and
bFGF facilitates receptor dimerization through two FGFR-binding
interfaces on the growth factor (26). Other models have also been
proposed, with complexes as large as bFGF tetramers being implicated as
an active complex (40), as in cytokines such as interferon
and
platelet factor 4 (41, 42). However, conditions used to observe these complexes differ considerably from the component ratios found to be
active in cell assay systems.
One of the major issues still to be resolved in the bFGF mitogenic
signaling mechanism is the exact nature of the signaling complex and,
in particular, the stoichiometry of the HS·bFGF complex. In this
study, we have produced novel covalently cross-linked conjugates of
bFGF and HS oligosaccharides, which no longer bind any further HS but
are fully active biologically. These conjugates have enabled us to
determine the minimal bFGF·HS oligosaccharide stoichiometry required
for initiating mitogenic activity.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Horse serum and RPMI medium were obtained from
Life Technologies, Inc. (Biocult, Paisley, United Kingdom (UK)). BaF3
(clone F32) and WEHI 3b cells were supplied by the Klagsbrun Laboratory (Boston, MA). Human recombinant bFGF was supplied by R&D Systems (Abingdon, Oxon, UK). Porcine mucosal HS was obtained from Organon (Oss, The Netherlands). Heparinase III (Flavobacterium
heparinum; EC 4.2.2.8) was obtained from Grampian Enzymes (Orkney,
UK). EDC and S-NHS were supplied by Pierce (Chester, UK). Bovine serum albumin, MES, Tween 20, and rabbit anti-human bFGF antibody were supplied by Sigma-Aldrich (Poole, Dorset, UK). Horseradish
peroxidase-conjugated swine anti-rabbit IgG antibody was supplied by
Dako (Cambridge, UK). ECL reagents were obtained from Amersham
Pharmacia Biotech (Little Chalfont, Bucks, UK). [methyl
3H]Thymidine was supplied by NEN Life Science
Products (Hounslow, UK). Superose 12 columns were purchased from
Amersham Pharmacia Biotech (St. Albans, Herts, UK). Bio-Gel P6 was
supplied by Bio-Rad (Hemel Hempstead, Herts, UK). Microscint O was
obtained from Packard (Pangbourne, Berks, UK). All other reagents were
obtained from BDH-Merk Ltd (Lutterworth, Leics, UK) and were of AnalaR grade.
Preparation of HS Dodecasaccharides (dp12)--
100 mg of
porcine mucosal HS in 0.5 ml of heparinase buffer (100 mM
sodium acetate, 0.1 mM calcium acetate, pH 7.0), was
incubated initially with 0.25 IU of heparinase III followed by two
further additions after 24 and 48 h. The digest was monitored by
absorbance at 232 nm until no further increase occurred.
Oligosaccharides produced by heparinase III digestion were then
resolved using a Bio-Gel P6 column (1.5 × 170 cm) in 0.5 M NH4HCO3 at a flow rate of 6 ml/h.
Fractions of 1 ml were collected and oligosaccharides detected by
monitoring the absorbance at 232 nm. Size-defined oligosaccharides
dp2-16 were pooled, freeze-dried, and stored at
80 °C until required.
Zero Length Cross-linking--
Cross-linking was carried out
essentially as described previously by Grabarek and Gergely (43);
however, conditions were chosen so as to optimize yields of
cross-linked products. Briefly, for preparative reactions, dp12
oligosaccharides in coupling buffer (0.1 M NaCl, 0.1 M MES, pH 6.0) were incubated for 15 min at 25 °C with 6 mM EDC and 15 mM S-NHS. The reaction was
terminated by addition of
-mercaptoethanol to a final concentration
of 20 mM. Activated oligosaccharides were then added to a
50 µM solution of bFGF in coupling buffer, to give a
bFGF:dp12 molar ratio of 1:4 and incubated for 2 h at 25 °C.
Products were analyzed by standard SDS-PAGE (12% gel) and Western
blotting, followed by enhanced chemiluminescence (ECL) immunodetection
prior to purification.
Immunodetection of bFGF--
Western blots of SDS-PAGE gels on
nitrocellulose membranes were blocked overnight with 1% (w/v) bovine
serum albumin in PBS and then washed three times with PBS, 0.05% (v/v)
Tween 20. The membrane was then incubated with a 1:200 dilution of
rabbit anti human bFGF monoclonal antibody for 2 h at 4 °C. The
antibody solution was then removed and the membrane washed three times
with PBS, 0.05% (v/v) Tween 20. The membrane was then further
incubated with a 1:1000 dilution of horseradish peroxidase-conjugated
swine anti-rabbit IgG for 1 h. The membrane was then finally
washed eight times in succession with PBS, 0.05% (v/v) Tween 20 and
the presence of bFGF visualized by ECL (Amersham Pharmacia Biotech) following the manufacturer's protocol.
Purification of Cross-linked Products--
Cross-linked samples
were applied to two coupled Superose 12 columns linked to a Gilson HPLC
system, equilibrated in 50 mM phosphate buffer containing
2.0 M NaCl, pH 7.4. Samples were eluted at a flow rate of
0.5 ml/min, fractions (250 µl) were collected, and elution profile
monitored by absorbance at 280 nm. Fractions containing protein were
desalted by microdialysis (Mr cut-off 12,000)
against 5 mM Tris-HCl, pH 7.4, at a flow rate of 1.5 ml/min overnight at 4 °C. Desalted samples were analyzed for purity by SDS-PAGE, followed by Western blotting and ECL immunodetection. Fractions containing cross-linked products were pooled and reapplied to
the columns and the procedure repeated as above. Finally, purified components were lyophilized and stored at
80 °C until required.
F32 Cell Mitogenesis Assay--
BaF3 cells transfected with
FGFR1 (designated F32 cells) (14) were routinely maintained in RPMI
1640 medium, 10% horse serum supplemented with interleukin-3
conditioned medium (prepared from WEHI 3b cells) at 37 °C, 5%
CO2. For the assay system, F32 cells were plated into
96-well plates at a density of 50,000 cells/well in 100 µl of RPMI
1640 medium, 10% horse serum supplemented with cross-linked
conjugates. Cells were incubated for 46 h before addition of
[3H]thymidine (0.3 µCi/well) for another 2 h.
Incorporation of [3H]thymidine was stopped by harvesting
cells on a Filtermate-196 cell harvester. Cells were allowed to air-dry
before addition of 25 µl of Microscint O to each well, and
incorporated radioactivity counted on a top count system.
Filter Binding Assay--
The assay was performed using a
membrane filtration apparatus (Millipore, Watford, Herts, UK). Briefly,
4 µg of bFGF or bFGF·HS complex was applied to the filter in
binding buffer (10 mM Tris-HCl, pH 7.3). Filters were then
washed with 10 ml of 2.0 M NaCl to remove any
non-cross-linked oligosaccharide that may have been present, and then
re-equilibrated by washing with binding buffer. Metabolically labeled
endothelial cell [3H]HS, prepared as described previously
(44), was applied in 5 ml of binding buffer and cycled through the
filter three times. Filters were then washed twice with 5 ml of binding
buffer to remove unbound [3H]HS. Bound
[3H]HS was then removed from the growth factor by
washing, first with three 5-ml aliquots of 0.3 M NaCl,
followed by further washing with three 5-ml aliquots of 2.0 M NaCl in binding buffer. Fractions (5 ml) were collected
and eluted material quantified by scintillation counting.
 |
RESULTS |
In order to produce potentially active HS
oligosaccharide·bFGF conjugates, we have employed a specific method
of zero length cross-linking. As no spacer is introduced using this
method, cross-linking should only occur between the oligosaccharide and
amino acid side chains within the actual HS binding site of bFGF. The
cross-linking process is a two-step procedure, initially involving a
brief incubation of HS oligosaccharide with EDC in the presence of NHS.
This results in the conversion of some of the oligosaccharide carboxyls
into succinimide esters. This oligosaccharide activation reaction is then terminated by neutralizing the EDC with
-mercaptoethanol prior
to addition of growth factor. Cross-linking arises from the reaction of
the succinimide esters with the lysine
-amino groups of the growth
factor. A two-step process has the advantage that only the
oligosaccharide comes into contact with the active cross-linking
reagents; hence, only oligosaccharide protein cross-links are formed.
This eliminates possible complications arising from protein-protein
cross-linking, which could perturb further interaction of the
conjugates with FGFRs.
Analysis of Products Obtained from the Cross-linking of bFGF to
dp12 Oligosaccharides--
Preliminary experiments were carried out
using the zero length cross-linking procedure, in order to assess the
type of conjugates formed and to optimize the yield of products.
Oligosaccharides of six disaccharides in length (dp12) were chosen, as
these have previously been shown to be the minimum size saccharide
fragments that have maximal bFGF stimulating activity, when compared
with intact chains (31, 37). Fig.
1A shows an SDS-PAGE and ECL immunodetection analysis of native bFGF (approximate mass of 18 kDa)
and the products obtained by cross-linking of dp12 oligosaccharides to
bFGF. It can be seen that the products of cross-linking include both
dimer (CL-dp12·bFGF dimer) and monomer (CL-dp12·bFGF monomer) conjugates of oligosaccharide and growth factor. Increasing the ratio
of oligosaccharide to growth factor resulted in higher yields of both
CL-dp12·bFGF monomers and CL-dp12·bFGF dimers, with the most
striking increase seen with the CL-dp12·bFGF monomers. However, the
majority of the growth factor remained in the native form. This is most
likely due to a combination of the half-life of the active
oligosaccharide esters being only 1-2 h, and the slow off-rate of the
oligosaccharide when bound to the growth factor. Control experiments in
the absence of cross-linking reagents showed no conjugate bands and no
increase in disulfide-bonded bFGF dimers (Fig. 1A,
lanes 1 and 2). In order to optimize
yields of CL-dp12·bFGF dimers and CL-dp12·bFGF monomers within a
single reaction, the ratio of bFGF:dp12 oligosaccharide was varied from
4:1 to 1:32 (results not shown). Additionally, the ratio of EDC to
S-NHS has been shown to effect coupling efficiency (43); in light of
this, the ratio of EDC:S-NHS was also varied (results not shown). As a
result of these experiments, optimal cross-linking conditions for
production of both monomer and dimer conjugates were chosen to be a 1:4
ratio of bFGF:dp12 and EDC/S-NHS concentrations of 6/15 mM,
respectively. A typical distribution of reaction products is shown in
Fig. 1B. The lack of cross-linked product when denatured bFGF was used (results not shown) ruled out the possibility of the
process being the result of nonspecific random collision of active
oligosaccharide-succinimide esters and primary amines on the growth
factor.

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Fig. 1.
Analysis of dp12·bFGF cross-linking
products. A, effect of increasing oligosaccharide
concentration on cross-linking reaction products. Oligosaccharides
(dp12) in 0.1 M MES, 0.1 M NaCl, pH 6.0, were
incubated for 15 min at 25 °C with 6 mM EDC and 15 mM S-NHS. The activation of the oligosaccharide was then
terminated by the addition of -mercaptoethanol and cross-linking
initiated by the addition of bFGF. The reaction was allowed to proceed
for 2 h at 25 °C and the reaction products analyzed by
SDS-PAGE, followed by Western blotting and ECL immunodetection as
described under "Experimental Procedures." Native bFGF
(lane 1), bFGF and dp12 oligosaccharides (ratio
1:4) with no EDC or S-NHS (lane 2), bFGF plus EDC
and S-NHS with no dp12 oligosaccharides (lane 3),
bFGF:dp12 oligosaccharide ratios of 4:1, 2:1, 1:1, 1:2, and 1:4,
respectively, with EDC and S-NHS (lanes 4-8).
B, typical SDS-PAGE and ECL immunodetection analysis of the
products from a preparative dp12·bFGF cross-linking reaction.
Identity of bands is as indicated in the figure.
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Purification of bFGF·Oligosaccharide Conjugates--
Products of
the cross-linking reaction were separated by gel-filtration
chromatography using two Superose 12 columns connected in series, as
described under "Experimental Procedures," and a typical elution
profile is shown in Fig. 2A.
This clearly shows the conjugates (56-61 min) eluting prior to the
free oligosaccharide (62-65 min as identified previously by absorbance
at 232 nm), and much earlier than the native growth factor (results not
shown) (73-76 min for native bFGF disulfide dimer and 77-80 min for
the native bFGF monomer). This early elution of bFGF conjugates (just prior to free dp12 oligosaccharide) may be due to the rigid extended nature of the oligosaccharide dominating the conjugate's hydrodynamic shape. In previous work (results not shown), we have noticed that, due
to the strength of the binding of oligosaccharide to bFGF, non-cross-linked complexes of bFGF and oligosaccharide also elute at a
position just prior to the free oligosaccharide peak. This indicates
that the cross-linked conjugates resemble at least in size/shape
non-cross-linked dp12·bFGF oligosaccharide complexes. Indeed
examination of the fractions by SDS-PAGE and ECL immunodetection, showed some contamination of the conjugates by both native bFGF disulfide dimer and monomer (Fig. 2B) as a result of the
strong native bFGF·dp12 interaction described above. In order to
finally remove all traces of free growth factor from the samples,
fractions containing CL-dp12·bFGF dimers and CL-dp12·bFGF monomers
were pooled separately and reapplied to the Superose 12 column in the presence of 2 M NaCl. This resulted in the recovery of
conjugates with no free growth factor (by SDS-PAGE) or oligosaccharide
contamination (as detected by absorbance at 232 nm results not shown).
Fig. 3A shows a typical Superose 12 elution profile for the CL-dp12·bFGF monomer conjugate, with SDS-PAGE
and ECL immunodetection analysis (Fig. 3B) confirming the
absence of native growth factor. Purification of the CL-dp12·bFGF
dimer conjugates was found to require several additional cycles of gel
filtration in order to obtain an homogeneous preparation.

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Fig. 2.
Purification of cross-linking reaction
products. A, typical gel-permeation elution profile of
cross-linking reaction products. Products of the two-step cross-linking
reaction were applied to two Superose 12 columns attached in series and
eluted in 50 mM phosphate buffer, 2.0 M NaCl,
pH 7.4, at a flow rate of 0.5 ml/min. Elution was monitored by
absorbance at 280 nM and 250-µl fractions collected.
Fractions containing protein were desalted by microdialysis and
analyzed by SDS-PAGE. B, SDS-PAGE and ECL immunodetection
analysis as described under "Experimental Procedures" of alternate
fractions collected between 56 and 64 min. Fractions containing
cross-linked CL-dp12·bFGF dimer and CL-dp12·bFGF monomer conjugates
were pooled separately, freeze-dried, and stored at 80 °C until
required.
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Fig. 3.
Purification of CL-dp12·bFGF monomer
conjugates. A, typical gel-permeation elution profile
of the CL-dp12·bFGF monomer conjugates when reapplied to two Superose
12 columns attached in series and run as described in Fig. 2.
B, SDS-PAGE and ECL immunodetection analysis as described
under "Experimental Procedures" of CL-dp12·bFGF monomer
containing fractions.
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Structural Characterization of the CL-dp12·bFGF Dimer
Conjugate--
A number of possible structures were envisaged for the
CL-dp12·bFGF dimer conjugates. These were: 1) individual
cross-linking of two separate bFGF molecules to a single
oligosaccharide, as may expected in the case of the dimer activation
model which has been previously proposed (14, 22, 29, 38, 39); 2)
disulfide-bridged dimers comprising one CL-dp12·bFGF monomer
conjugate, and a molecule of native bFGF; and 3) disulfide-bridged
dimers of two CL-dp12·bFGF monomers. To clarify this, we ran
non-reduced and reduced samples of the CL-dp12·bFGF dimer conjugate
on SDS-PAGE (Fig. 4). This clearly shows
that the dimer product consists of disulfide-bridged CL-dp12·bFGF
monomers. Initial experiments also showed that small amounts of hybrid
disulfide dimers were formed, which degraded to CL-dp12·bFGF monomers
and unmodified bFGF monomers on reduction. However, more extensively
purified dimer preparations such as that used in Fig. 4 were devoid of
these hybrids. The fact that no true cross-linked dimer (two bFGFs to
one dp12 oligosaccharide) is formed provides evidence that the binding
of two bFGF molecules to a single oligosaccharide via identical growth
factor binding sites is not an accurate model for the activation of
signal transduction by HS·bFGF. As a result of this experiment, only
the CL-dp12·bFGF monomer conjugate remained as a potential minimal
mitogenic activation complex.

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Fig. 4.
SDS-PAGE analysis of CL-dp12·bFGF dimer
conjugates under reducing and non-reducing conditions. Purified
CL-dp12·bFGF dimer conjugates were analyzed by SDS-PAGE and ECL
immunodetection as described under "Experimental Procedures" under
reducing and non-reducing conditions. Non-reduced native bFGF
(lane 1), non-reduced CL-dp12·bFGF dimer
conjugate (lane 2), and -mercaptoethanol
reduced CL-dp12·bFGF dimer conjugate (lane 3)
are shown.
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Monomer Conjugate Activity in F32 Cell Assay--
The BaF3 cells
are a lymphoblastoid line that do not express FGFRs and fail to respond
to bFGF. These cells were transfected with FGFR1 to produce the F32
cell clone. The F32 cells, which are devoid of functional cell surface
heparan sulfate proteoglycans, will respond to bFGF only in the
presence of exogenous heparin or HS (14). Addition of purified
CL-dp12·bFGF monomer conjugates to the assay system showed the
complexes to be active (Fig. 5), with
activity ([3H]thymidine incorporation) reaching a plateau
at levels equivalent to maximum stimulation with intact HS and native
bFGF. It was noted, however, that a concentration in excess of 100 ng/ml CL-dp12·bFGF monomer conjugate was needed to bring about
maximum stimulation, with 50% maximum stimulation achieved at 20 ng/ml. In this assay the native bFGF and soluble dp12 oligosaccharide
give maximum activity at a concentration of only 10 ng/ml bFGF. The
lower activity of the conjugate is probably due to the presence of some
oligosaccharides in the structurally heterogeneous dp12 mixture that
bind bFGF strongly but fail to activate it (45). This would lead to a reduction in the effective concentration of active conjugate, as a
proportion of the growth factor is irreversibly linked to non-activating oligosaccharides. This is in contrast to the native form
of the growth factor, which can freely dissociate from non-activating oligosaccharides and further contribute to cell stimulation by binding
active oligosaccharides. The higher amounts of conjugate needed to
reach maximum stimulation may also indicate a level of specificity in
terms of the exact positioning of the bFGF molecule along an activating
oligosaccharide. However, the results do suggest that a bFGF·HS
oligosaccharide monomer is a sufficient minimal structural unit for the
initiation of mitogenic activity.

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Fig. 5.
[3H]Thymidine uptake of F32
cells after stimulation with CL-dp12·bFGF monomer conjugates.
Cells were plated out at a density of 50,000 cells/well in RPMI medium,
10% horse serum. CL-dp12·bFGF monomer conjugates were added over a
range of concentrations and incubated for 46 h before addition of
[3H]thymidine for another 2 h. Cells were harvested
and incorporated radioactivity determined by scintillation counting.
Controls for the experiment shown were 14,874 ± 961 cpm for 10 ng/ml bFGF and 1 µg/ml intact HS (determined optimal conditions for
stimulation by bFGF) (positive control) and 5046 ± 564 cpm for
the cell suspension in the absence of bFGF and heparin (negative
control).
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Analysis of the Affinity of Conjugates for
HS/Heparin--
Assessment of the binding of HS to the monomer
conjugate is essential in terms of validating the monomer unit as being
the minimum active structural unit of a HS·bFGF·FGFR signaling
complex. Filter binding experiments (Fig.
6) clearly show that the CL-dp12·bFGF monomer conjugates do not bind the added [3H]HS (greater
than 99% of the [3H]HS being washed directly through the
filter), as can be seen by comparison with native bFGF, which bound
approximately 50% of the [3H]HS strongly. These data
suggest that the oligosaccharide is covalently linked into the growth
factor's HS binding site, resulting in the site being completely
obscured from further HS interactions.

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Fig. 6.
Filter binding analysis of the affinity of
the CL-dp12·bFGF monomer conjugates for HS chains. Growth factor
samples (4 µg) were applied to a nitrocellulose filter in binding
buffer 10 mM Tris-HCl, pH 7.3. The immobilized growth
factor was then washed with 2 M NaCl to remove any
non-cross-linked oligosaccharide and re-equilibrated in binding buffer.
[3H]HS was applied to the filters and washed with binding
buffer; bound material was released by washing with 0.3 M
and 2.0 M NaCl in binding buffer. Fractions (5 ml) were
collected and radiolabeled material quantified by scintillation
counting. The data shown are the total [3H]HS (cpm)
eluted at each NaCl concentration. , native bFGF; ,
CL-dp12·bFGF monomers.
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 |
DISCUSSION |
The ability of bFGF to bind to its high affinity FGFR is largely
dependent on the binding of HS to the growth factor. This has led to
the proposal of a "dual receptor system" for signaling function
(46). To date much is known about the interaction between HS and bFGF,
i.e. the structural properties of HS such as the oligosaccharide length and sulfation pattern required to modulate FGF
signaling. However, a major question still to be resolved is the exact
molecular nature of the signaling complex comprising HS·bFGF·FGFR,
and in particular the minimum stoichiometry of the active HS·bFGF
complex. In this study we have addressed this problem by evaluating the
biological activity of novel covalent conjugates of HS oligosaccharides
and bFGF, which were prepared using a zero length cross-linking
procedure. Our data showed that CL-dp12·bFGF monomer conjugates were
biologically active and that any CL-dp12·bFGF dimers formed consisted
only of disulfide-bonded CL-dp12·bFGF monomers.
The evidence that these monomer conjugates are a realistic
representation of the native signaling configuration of bFGF and HS
oligosaccharide is fourfold. First, it was shown that the
CL-dp12·bFGF monomer conjugates were biologically active in a cell
assay system that is devoid of functional heparan sulfate
proteoglycans. Second, the formation of these types of zero length
cross-links is dependent on the presence of an ion-pair interaction
between a COO
group and a NH2 group, in this case
via the carboxylic acids of the oligosaccharides and amine groups of
lysines present in the growth factor. Many studies in the past have
shown the primary HS binding site of bFGF to consist of a number of
lysine residues (22, 47-51), but a more recent report of the x-ray
crystal structure of a heparin hexasaccharide bound to bFGF has
shown that a COO
group of a IdceA unit is in close
proximity (approximately 3 Å) to the nitrogen atom of the NH2
group of lysine 136 of the growth factor's primary HS binding site
(23). This distance is within the range for an ion-pair interaction to
occur and is therefore a potential cross-linking site within the growth
factor's HS binding site. Third, occupancy of this site is inferred
from filter binding experiments which showed that the growth factor (after cross-linking with dp12 oligosaccharides), had no appreciable affinity for [3H]HS chains. Finally, no conjugates were
formed when cross-linking was performed using denatured protein,
indicating that the reaction was specific for the native HS binding
site(s) in bFGF and was not the result of random molecular collisions.
Several models have been proposed by which HS·bFGF and FGFRs interact
to mediate signal transduction, with the most prevalent being the dimer
presentation model. Initially the model simply suggested that
biologically active HS oligosaccharides of at least 5-6 disaccharides
in length can simultaneously bind two FGF molecules, hence presenting a
pseudo-FGF dimer to the FGFR's (14, 22, 24, 29, 38, 39, 52), yet the
evidence for its existence was initially circumstantial. However, in
recent years, this dimer model has been more substantially investigated
using molecular modeling, NMR, and x-ray crystallography techniques.
Venkataraman and co-workers (53) suggested that bFGF preferentially
self-associates to form true dimers and larger oligomers that were
stabilized by a trisaccharide or heparin oligosaccharide, via two
independent mechanisms. Oligomerization of bFGF by HS/heparin
oligosaccharides was also implicated in a NMR and light scattering
study, in which bFGF tetramers comprising two cis-oriented
FGF dimers were formed in the presence of a decasaccharide (40). Other
growth factors such as platelet-derived growth factor, transforming
growth factor-
, and nerve growth factor have previously been shown
to form homo- and heterodimers in order to facilitate receptor
activation. In these examples, receptor dimerization occurs if each
monomer unit binds to a separate growth factor receptor, so enabling
two adjacent receptor molecules to associate (54). In the light of the
wealth of physical and chemical evidence so far described, there now seems to be little doubt that HS/heparin chains or oligosaccharides can
bring about oligomerization of bFGF under certain experimental conditions. Nevertheless, questions still remain as to the validity of
such models in activity assays, as concentrations of growth factor and
oligosaccharides used in biophysical studies, far exceed those present
under cell culture or physiological conditions.
During the course of this study, the validity of this dimeric
presentation model was put into question, as successful
-mercaptoethanol reduction of the CL-dp12·bFGF dimers formed
during the cross-linking procedure showed them to comprise two
disulfide-bonded CL-dp12·bFGF monomers. Not even small amounts of
CL-dp12·bFGF dimers were formed through direct
protein/oligosaccharide cross-linking between two bFGF molecules and a
single oligosaccharide. The latter product would be expected if HS
oligosaccharides were acting as a bridge between two bFGF molecules via
the same protein/oligosaccharide binding sites utilized in the
formation of the CL-dp12·bFGF monomer conjugates. We believe that
this result casts doubt on the likelihood of a dimeric bFGF
presentation model mediating the growth factor's mitogenic activity.
Furthermore, since we have shown that the CL-dp12·bFGF monomer
conjugates have no measurable affinity for [3H]HS chains,
it is unlikely that any association of CL-dp12·bFGF monomers to form
dimers or larger oligomers will involve further interactions with the
dp12 oligosaccharide of the conjugate. It may be that dimerization of
these monomer conjugates by mechanisms other than HS oligosaccharide
bridging is occurring. However, a strong non-covalent association seems
unlikely, as gel filtration analyses of these conjugates at
physiological salt concentrations and under reducing conditions, showed
they had no tendency for self-association (results not shown). In fact,
as previously shown, the only oligomerization of these CL-dp12·bFGF
monomer conjugates observed during this study, was the result of
disulfide bonding. It seems plausible to discount this as a method for
presenting dimeric conjugates to the receptor, as previous experimental
data have shown that disulfide bonding of bFGF is not necessary for the
growth factor's biological activity (55-57). In addition, we found
that the formation of bFGF disulfide dimers was not influenced by the
presence of HS oligosaccharides.
Additional models have also been proposed, both of which are based on
data that indicated a direct interaction between HS and FGFR1 (25).
This led to the suggestion that a short oligosaccharide (e.g. 5-6 disaccharides in length) contains two binding
sites, one for bFGF and a second for the FGFR, with the oligosaccharide acting as a template for bringing the two proteins into close proximity
to form a signaling unit. This has very strong similarities to the
catalytic action of HS/heparin on the inactivation of thrombin by
antithrombin III (58). Guimond and co-workers (31) provided further
evidence to support this theory (that HS/heparin had to bind to a
second protein) when, by the use of selectively desulfated heparins,
they showed that C-6 O-sulfation was not required for bFGF
binding, but was essential for mitogenic activity. These findings
suggest a role for 6-O-sulfation in binding to a second protein, the most likely candidate being FGFR, with signaling occurring
possibly via a single HS·bFGF·FGFR complex, or alternatively through oligomerization of these units. More recently, Pantoliano and
co-workers (26, 27) have suggested a mechanism akin to that involved in
human growth hormone action (59, 60), in which a monomeric complex of
HS oligosaccharide and bFGF facilitates receptor dimerization through
two FGFR binding interfaces on the growth factor. In essence, the
principal theme of these latter two mechanisms is the monomer
stoichiometry of the minimal active bFGF·HS complex, which is in
agreement with our experimental evidence. It should perhaps be noted
that it has previously been implied by Krufka and co-workers that
HS/heparin may act along with bFGF to initiate different signaling
pathways by employing either monomeric FGFR's or FGFR dimers/multimers
(20). Indeed, their study suggested that FGFR dimerization may not be
required for initiating a mitogenic response. Hence, both bFGF·HS
oligosaccharide monomer models described above could be utilized in
transducing different downstream signals.
In conclusion, our data suggest that the minimal bFGF·HS
oligosaccharide activating unit for mitogenic stimulation is monomeric and not a dimeric presentation of bFGF mediated by a HS bridge. The
accumulated data in this study tend to favor the two models proposed by
Guimond et al. (31) and Pantoliano et al. (26), both of which have a bFGF·HS oligosaccharide monomer as their essential structural element, although ultimately their existence and
possible roles in determining the final bFGF signaling events is yet to
be determined. The present study provides the first direct
demonstration that bioactive covalent complexes can be formed between
HS and its dependent growth factors; such complexes will in the future
facilitate further mechanistic studies and may also have considerable
therapeutic potential.