(Received for publication, January 23, 1997, and in revised form, April 21, 1997)
From the Cancer Research Campaign/University of Manchester Department of Medical Oncology, Christie Hospital National Health Service Trust, Manchester M20 4BX, United Kingdom
We have undertaken a comparative study of the
interaction of the three mammalian transforming growth factor-s
(TGF-
) with heparin and heparan sulfate. TGF-
1 and -
2, but not
-
3, bind to heparin and the highly sulfated liver heparan sulfate.
These polysaccharides potentiate the biological activity of TGF-
1
(but not the other isoforms), whereas a low sulfated mucosal heparan sulfate fails to do so. Potentiation is due to antagonism of the binding and inactivation of TGF-
1 by
2-macroglobulin, rather than by modulation of
growth factor-receptor interactions.
TGF-
2·
2-macroglobulin complexes are more refractory
to heparin/heparan sulfate, and those involving TGF-
3 cannot be
affected. Comparison of the amino acid sequences of the TGF-
isoforms strongly implicates the basic amino acid residue at position
26 of each monomer as being a vital binding determinant. A model is
proposed in which polysaccharide binding occurs at two distinct sites
on the TGF-
dimer. Interaction with heparin and liver heparan
sulfate may be most effective because of the ability of the dimer to
co-operatively engage two specific sulfated binding sequences,
separated by a distance of approximately seven disaccharides, within
the same chain.
A large number of growth factors and cytokines, belonging to structurally and biologically diverse protein families, possess affinity for heparin in vitro. Such affinity has often proved to be an experimental indicator of a physiological interaction with the heparan sulfate (HS)1 chains of heparan sulfate proteoglycans (HSPGs), a widespread and abundant family of complex glycoconjugates expressed on the surface of all adherent cells (1), and also present within basement membranes and stromal matrices (2). Binding to HSPGs in vivo may have an important role in retaining active growth factors/cytokines within a local sphere of action by protecting them from both diffusional and degradative loss. Importantly, however, in the case of an increasing number of such growth factors, e.g. various members of the fibroblast growth factor family, vascular endothelial growth factor, heparin-binding epidermal growth factor, and hepatocyte growth factor, it has been demonstrated that HSPGs have a specific co-receptor role in directly modulating growth factor activation of the respective cell surface signaling receptors (3, 4).
The transforming growth factor-s (TGF-
s) are important regulators
of the growth, differentiation, and adhesion of a wide variety of cells
(5). They are believed to have an important role in natural repair
processes, but overexpression or dysregulation can lead to the
development of various fibrotic disorders. TGF-
1 has been
demonstrated to possess strong heparin binding activity in
vitro (6). Such interaction protects TGF-
1 from proteolytic degradation in vitro (7), and also prevents the formation of inactive complexes with
2-macroglobulin
(
2M) (8). The physiological significance of an
interaction restricted to heparin, a GAG released only upon the
degranulation of activated mast cells, is unclear. Although TGF-
does bind to a number of proteoglycan species in vivo,
namely the cell surface HS-containing proteoglycan, betaglycan (the
type III TGF-
receptor) (9), as well as various members of the
family of small secreted chondroitin/dermatan sulfate (CS/DS) proteoglycans (i.e. decorin, biglycan, and fibromodulin)
(10, 11), these associations are mediated principally, if not solely, by protein-protein rather than protein-GAG interactions.
In addition to TGF-1, there are two other mammalian isoforms,
TGF-
2 and -
3, and also two distinct, but poorly characterized, non-mammalian isoforms, TGF-
4 and -
5 (identified in chick and Xenopus, respectively). TGF-
s 1-3 possess very high
levels of amino acid sequence homology within the mature bioactive
molecule (>70% amino acid identity between isoform pairs with the
majority of changes being conservative), including conservation, in
both number and position, of the eight cysteine residues that
contribute to the compact folding of the monomer, as well as the single
cysteine residue involved in disulfide-bonded dimerization.
Interestingly, however, there is little if any interspecies variation
within each individual isoform. This has led to the proposition that the individual isoforms have closely related tertiary structures, which
have diverged sufficiently to behave as functionally distinct species.
Indeed, there is substantial evidence pointing to significant differences in biological behavior between the mammalian isoforms, both
in vitro and in vivo. There are differential
patterns of isoform expression during fetal development (12, 13) and in the adult (14, 15), and indeed the existence of distinctive regulatory
elements in the promoter regions of the respective genes (16) is
suggestive of a capacity for independent isoform regulation. The
isoforms also display marked specificities or differential potencies in
various in vitro biological systems. For example, TGF-
3
is more potent than the -
1 or -
2 isoforms in inhibiting DNA
synthesis in human keratinocytes in vitro (17). While
TGF-
2, but not -
1, can stimulate mesoderm induction in Xenopus embryos (18), TGF-
2 specifically has little or no
antiproliferative effect on vascular endothelial cells in
vitro (19). Also, exogenously added TGF-
3 appears to have a
specific ability to reduce scar formation during wound healing in the
adult animal (20). These differences in biological activity may be due,
in part, to the known differences in the receptor binding properties of
the isoforms (e.g. the reduced affinity of TGF-
2 for the
type II receptor protein (21)), although other interactive
specificities may also be important.
In the light of these known isoformic differences, together with the
previously described affinity of TGF-1 for heparin, we were
interested in further elucidating the nature and extent of GAG-TGF-
interactions.
Human platelet TGF-1, recombinant human
TGF-
2, and mouse anti-human pan TGF-
monoclonal antibody were
purchased from Genzyme (West Malling, United Kingdom (UK)). Recombinant
human TGF-
3 was a generous gift from Oncogene Science (Uniondale,
NY).
2-Macroglobulin was obtained from Boehringer
Mannheim (Lewes, UK). Normal rat kidney fibroblasts (NRK 49F) and mink
lung epithelial cells (Mv1Lu CCL-64) were from the American Type
Culture Collection (ATCC). Bovine lung heparin, whale shark cartilage
CS, bovine mucosal DS, bovine kidney HS, heparin conjugated to
cross-linked 4% agarose and heparinase I (Flavobacterium
heparinum; EC 4.2.2.7) were all purchased from Sigma (Poole, UK).
Heparinase II (F. heparinum; no EC number assigned) and
heparinase III (F. heparinum; EC 4.2.2.8) were obtained from
Grampian Enzymes (Aberdeen, UK). Porcine mucosal HS was a gift from NV
Organon (Oss, The Netherlands). Decorin proteoglycan was generously
provided by Dr. H. Pearson (University of Alberta, Edmonton, Canada).
De-N-sulfated, re-N-acetylated heparin, and
selectively de-6-O-sulfated heparin were kindly provided by
Dr. B. Mulloy (National Institute for Biological Standards and Control,
Potters Bar, Herts., UK). Selectively de-2-O-sulfated heparin was a gift from Dr. B. Casu (Instituto Chimica and Biochimica "G. Ronzini," Milan, Italy).
Cell surface
HSPGs were purified from rat liver by the method of Lyon and Gallagher
(22). HS chains were liberated by alkaline elimination using 50 mM NaOH, 1 M sodium borohydride at 45 °C for
48 h. After neutralization with acetic acid the HS was
precipitated by the addition of four volumes of 95% (v/v) ethanol at
20 °C overnight. HS concentrations were quantified using an Alcian
Blue-binding microassay (23), relative to a standard curve obtained
with bovine kidney HS.
Samples were exhaustively digested with heparinases I, II, and III. The resulting disaccharide products were then resolved by strong anion-exchange chromatography on a 5-µm particle size Spherisorb SAX-HPLC column (Technicol, Stockport, UK), essentially as described by Lyon et al. (24). Disaccharides were detected by UV absorbance at 232 nm.
Assay of TGF-Batches of TGF- were routinely
assayed for their biological activity through their ability to inhibit
the incorporation of [3H]thymidine into mink lung
epithelial cells. Briefly, Mv1Lu CCL-64 cells were plated out at
104 cells/well of a 24-well plate (Costar, High Wycombe,
UK) in Dulbecco's modified Eagle's medium/F-12 medium (Life
Technologies, Inc., Paisley, UK) containing 5% (v/v) donor calf serum.
After 4 h, cultures were treated with a range of TGF-
concentrations and then pulsed with 0.5 µCi/ml
[3H]thymidine (NEN Life Science Products, Stevenage, UK)
for 2 h. The incorporation of 3H radiolabel into
trichloroacetic acid-insoluble cellular material was then
determined.
Samples of TGF-1, -
2, and -
3 (50 ng of
each) were individually applied to 0.3-ml volume columns of
heparin-agarose and Sepharose CL4B (as a control) in 0.3 ml of 0.05 M NaCl, 100 µg of bovine serum albumin/ml, 10 mM Tris-HCl, pH 7.0. The sample was recycled through the
columns five times and then left on the column for an additional 1 h at room temperature before washing with the same solution to remove
all non-binding material. Columns were then sequentially step eluted
with 2 ml each of 0.1, 0.15, 0.2, 0.25, 0.5, and 2.0 M NaCl
in 10 mM Tris-HCl, pH 7.0, containing 100 µg of bovine
serum albumin/ml. The individual eluates were assayed for their TGF-
content by immunodetection, as described below.
Samples of TGF-1, -
2, and -
3 (50 ng each) were
individually incubated with 1 µg (quantified as HS) of rat liver HSPG
in 20 µl of phosphate-buffered saline (PBS) containing 0.1% (v/v) Tween 20, for 2 h at room temperature. The mixtures were then chromatographed on a TSK-3000 PW (7.8 mm × 30 cm; Toyo Soda
Manufacturing Co. Ltd., Tokyo, Japan) HPLC size exclusion column eluted
with PBS, 0.1% (v/v) Tween 20 at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected and analyzed for their TGF-
content. The
column was calibrated using dextran blue (Vo) and sodium dichromate (VT). The interaction of
TGF-
3 (50 ng) and
2M (750 µg) was analyzed in an
identical manner.
Chromatography fractions were loaded into the wells of a
dot blot apparatus (Bio-Rad Laboratories, Hemel Hempstead, UK) and adsorbed by filtration under vacuum onto Protran nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) washed through with
PBS. The nitrocellulose was removed, blocked overnight in PBS
containing 1% (w/v) bovine serum albumin and then washed three times
with PBS, 0.05% (v/v) Tween 20. After incubation with a 1:200 dilution
of mouse anti-human pan TGF- monoclonal antibody for 2 h at
4 °C, the membrane was washed three times with PBS, 0.05% (v/v)
Tween 20, and then further incubated with a 1:1000 dilution of
horseradish peroxidase-conjugated rabbit anti-mouse IgG (Dako, High
Wycombe, UK) for 1.5 h. The membrane was finally washed six times
in succession with PBS, 0.05% (v/v) Tween 20, and the presence of
TGF-
was then visualized by enhanced chemoluminescence (ECL)
following the manufacturer's (Amersham International, Amersham, UK)
protocol. The resulting dot blot images were then scanned and analyzed
using a model GS-700 Imaging Densitometer (Bio-Rad) operating in
transmittance mode, yielding semi-quantitative measurements of TGF-
levels in arbitrary units of darkness per unit area.
Small (35 mm) bacteriological dishes (Nunc, Life
Technologies, Inc., Paisley, UK) were overlaid with 0.5 ml of molten
5% (w/v) agar (Difco Laboratories, West Molesey, UK) in water and
allowed to set. An additional 0.75 ml of molten 5% (w/v) agar in water was diluted with 4.25 ml of assay medium (Dulbecco's modified Eagle's
medium; Life Technologies, Inc.) supplemented with 5% (v/v) fetal calf
serum (Life Technologies, Inc.), 10 ng of epidermal growth factor/ml, 1 mM sodium pyruvate, and a standard concentration of
non-essential amino acids. Four milliliters of this warm agar mixture
was then added to a 1-ml suspension of 5 × 104 NRK
49F cells in assay medium (i.e. a final agar concentration of 0.6% (w/v)). In test cases the cell suspensions also contained 0.25 ng/ml (a suboptimal concentration) of individual TGF- isoforms, with
or without the addition of various glycosaminoglycans over a range of
concentrations. After thorough mixing, 1.5 ml of the agar-suspended
cells were plated out onto the agar-coated dishes. Cells were then
incubated at 37 °C in a humidified atmosphere of 5% CO2
(v/v) in air for 7 days. After this period the number of colonies
comprising greater than 50 cells were counted under a Leitz/Diavert
inverted microscope (25, 26).
For experiments performed in the absence of 2M, a
serum-free assay (27) was adopted in which the fetal calf serum in the assay medium was replaced by a mixture of 10 µg of insulin, 5 µg of
transferrin, 0.3 mg of high density lipoprotein, and 1 mg of bovine
serum albumin per ml of medium.
Interaction with heparin was assessed by affinity
chromatography on heparin-agarose columns. Because of the known ability of TGF- to bind strongly and nonspecifically to many surfaces, the
chromatography was performed in the presence of a relatively high
concentration of serum albumin. Samples were also run in parallel on
control columns of Sepharose CL-4B. The in vitro biological activity of each TGF-
isoform batch (i.e.. the ability to
inhibit the incorporation of [3H] thymidine by mink lung
epithelial cells) was tested beforehand to confirm that an essentially
active and native conformation was being analyzed (data not shown).
TGF-1 demonstrated a strong interaction with heparin-agarose
(Fig. 1A), confirming the original
observation of McCaffrey et al. (6). The bound
fraction was predominantly released by a 0.5 M NaCl step,
although a proportion required a higher ionic strength for elution. The
behavior of TGF-
2 was consistently more complex showing marked
nonspecific binding, as evidenced by its behavior on the Sepharose
CL-4B column. Nevertheless, a small but consistent fraction of the
TGF-
2 bound specifically to the heparin-agarose column requiring 0.5 M NaCl, or higher, for elution (Fig. 1B). A
significant proportion of both TGF-
1 and -
2 failed to bind to the
heparin-agarose. This was a reproducible, although quantitatively
variable, phenomenon, which has been observed with radiolabeled
TGF-
1 (6), and probably reflects a continual process of denaturation
and loss of activity with time of TGF-
stocks.
Biologically active TGF-3 displayed no discernible affinity for
heparin at physiological ionic strength or above (Fig. 1C). This was confirmed by the inability of TGF-
3, either covalently coupled to Affi-Gel 10 (Bio-Rad) or non-covalently adsorbed onto nitrocellulose, to capture [3H]heparin (NEN Life Science
Products) (data not shown), and also by heparin's apparent inability
to afford any protection to TGF-
3 from digestion by trypsin (data
not shown).
Affinity for heparan sulfate was assessed by rapid
chromatography of a preincubated mixture of TGF- and purified rat
liver HSPG (approximately 25-fold molar excess of HS chains), at
physiological ionic strength and pH, on a size-exclusion HPLC column.
Intact HSPGs, rather than HS chains, were used so as to maximize the potential size difference between free TGF-
and any complex formed. Whereas a significant fraction of both TGF-
1 and -
2 eluted as a
high molecular weight complex in the presence of the liver HSPG, there
was no evidence of any such complex formed with TGF-
3 (Fig. 2). Thus the differential binding properties of the
TGF-
isoforms were identical for both heparin and liver HSPG. This
similarity suggests that the major binding site within the HSPG resides
in the HS chains and not the core protein. Although TGF-
does bind specifically and with high affinity to the core protein of the betaglycan HSPG, the latter species is essentially absent from the
purified liver HSPG preparation employed here (28). Also, betaglycan
interacts non-discriminately with all three TGF-
s (29).
Biological Activity of TGF-
All three TGF- isoforms stimulate to a
similar extent the anchorage-independent proliferation of colonies of
NRK 49F fibroblasts in soft agar suspension, primarily through the
stimulation of the endogenous synthesis and secretion of extracellular
matrix macromolecules, especially fibronectin. Using suboptimal
concentrations of the TGF-
isoforms (0.25 ng/ml, equivalent to
approximately 10 pM), it was observed that the addition of
heparin markedly potentiated the activity of TGF-
1, with the optimal
heparin concentration of 1 µg/ml eliciting a 6.6-fold potentiation
(Fig. 3A). This effect was specific in that
there was no discernible effect on cellular proliferation induced by
either TGF-
2 or -
3 over the same range of heparin concentrations
(at a higher concentration of 10 µg/ml, a barely significant
potentiation of TGF-
2 was seen) (Fig. 3A). Heparin alone,
in the absence of TGF-
, has no independent stimulatory effect on
colony growth (Fig. 3A).
In contrast to the effects of heparin, a porcine mucosal heparan
sulfate preparation failed to potentiate any of the TGF- isoforms,
including TGF-
1 (Fig. 3B). Interestingly, however, HS
chains derived from the liver HSPG did markedly potentiate the activity
of TGF-
1 (Fig. 3C), and to an extent comparable with that
of heparin. This effect is consistent with the earlier premise that the
TGF-
binding activity of intact liver HSPG resides in the HS chains
and not the core protein. The apparent reduction in potentiation of
TGF-
1 at the highest concentrations of heparin (Fig. 3A)
and liver HS (Fig. 3C) may be due to a competing direct antagonistic effect on the cells of these GAGs, apparent only at higher
concentrations, or an inhibitory sequestration of the TGF-
at very
high molar ratios of GAG to TGF-
. Protection assays also indicate
that both heparin and liver HS protect TGF-
1 from tryptic
degradation, whereas porcine mucosal HS does not (data not shown),
implying that their ability to potentiate TGF-
1 activity is
positively correlated with direct high affinity binding.
The foregoing data indicate that of the
N-sulfated GAGs tested the most potent were found to be
heparin and rat liver HS, while the lower sulfated porcine mucosal HS
had little effect (see Table I for structural
comparisons). Compared with heparin, other classes of sulfated GAGs
were relatively ineffective. CS had no discernible effect, while a
mucosal DS, although having a potentiating effect at the highest
concentration tested (1 µg/ml), was approximately 10-fold less potent
(data not shown). However, as this DS preparation was found to be
contaminated to at least 3-4% with highly sulfated HS and/or heparin,
the specificity of this level of activity is uncertain. Indeed,
McCaffrey et al. (8) observed no apparent interaction of
TGF-1 with DS by agarose electrophoresis. However, a weak inherent
DS binding activity of heparin/HS-binding proteins is not unusual,
presumably because DS also contains both iduronate and variable
sulfation which may partially satisfy the binding requirements.
|
To try and elucidate the relative importance of the different sulfate
groups in heparin for the binding and subsequent potentiation of
TGF-1 activity, a series of selectively desulfated heparins (see
Table I for structural comparisons) were tested for their effect in the
soft agar colony growth assay. The results, although complex, suggested
that the specific loss of N-sulfates had a greater effect
than the selective loss of either 2-O- or
6-O-sulfates. At a heparin concentration of 0.1 µg/ml
(giving a 4-fold potentiation of TGF-
1 activity),
de-N-sulfation, with replacement by N-acetyl groups, resulted in a 95 ± 3% reduction in activity. Removal of 2-O- or 6-O-sulfates resulted in lesser, although
similar reductions of 48.5 ± 10.8% and 51.5 ± 4.5%
respectively (although the 2-O-sulfates had been more
selectively removed than the 6-O-sulfates; see Table I). In
all cases a 10-fold increase in concentration of the modified heparins
increased the level of potentiation. De-2-O- and
de-6-O-sulfated heparins at 1 µg/ml gave 79.9 ± 1.3% and 88.7 ± 3%, respectively, of the potentiation observed
with 0.1 µg/ml heparin, although the de-N-sulfated
derivative still only achieved 56.5 ± 9% (all % values being
the mean of triplicate determinations ± S.E.). This complex
behavior suggests that the TGF-
1 binding site in heparin/liver HS
probably involves a combination of specific structural determinants of
which N-sulfation is a major contributor.
The NRK 49F colony growth assay is
normally performed in the presence of 5% (v/v) fetal calf serum (25,
26). The requirement for serum can, however, be replaced by a defined
protein mixture (comprising insulin, transferrin, high density
lipoprotein, and serum albumin) (27) in which the cells remain
responsive to stimulation by all three TGF- isoforms (data not
shown). Interestingly, under these serum-free conditions, the
potentiating effect of exogenous heparin upon TGF-
1 is lost, and the
behavior of TGF-
1 becomes comparable to that of TGF-
2 (Fig.
4A). This suggests that the potentiating
effect is not due to a direct heparin-mediated enhancement of TGF-
1
binding to its receptor, but to a modulation by heparin of a TGF-
1
neutralizing activity present in serum. The most likely candidate
molecule is
2M, which is known to be the major
TGF-
-binding protein in serum (30) and forms non-covalent complexes
in which the TGF-
is rendered latent (30, 31). The addition of 750 µg/ml
2M to the serum-free cell system inhibited the
activities of all the TGF-
isoforms by between 43% (TGF-
3) and
68% (TGF-
1 and -
2) (Fig. 4B). Heparin alone at 1 µg/ml had no effect (Fig. 4B). However, the combination of
heparin and
2M elicited differential responses. Whereas
the activity of TGF-
3 remained completely suppressed, that of
TGF-
1 was markedly restored by heparin to 84% of control
levels, although the activity of TGF-
2 was restored to a
significantly lesser extent (57% of the control; Fig.
4B).
We have demonstrated that marked differences exist in the
interaction of the mammalian TGF- isoforms with the heparin/HS family. Both TGF-
1 and -
2 possess affinity for heparin and highly sulfated HS, whereas TGF-
3 does not. Heparin and liver HS potentiate TGF-
1 in supporting the "anchorage-independent" growth of NRK fibroblasts. In keeping with its failure to bind heparin, the activity
of TGF-
3 was unaffected by it, although, paradoxically, so also was
the activity of the heparin-binding TGF-
2. The relative lack of
effect of CS and DS on TGF-
1, coupled with the marked reduction in
activity of heparin after removal of its N-sulfate groups,
indicates a strong requirement for N-sulfated GAGs. However, even among the latter there is a marked selectivity, as liver HS (24)
was as potent as heparin, while a mucosal HS preparation was
essentially inactive. Liver HS is highly sulfated (Table I), and
occupies one extreme end of the HS spectrum (32). Additionally, its
markedly asymmetric chain structure (24), with the great majority of
both N- and O-sulfates concentrated within the
distal two-thirds, generates a localized sulfate density approaching that of heparin. The similarity in properties of heparin and liver HS
may thus reflect a TGF-
binding requirement for a specific highly
sulfated sequence, which is rare or absent in most lesser sulfated HS
species. Selective depletion of either the 2-O- or 6-O-sulfate groups in heparin brings about a more modest
reduction in activity, compared with N-sulfate removal.
Interestingly, all the selectively desulfated heparins are less
effective than liver HS, although they retain a higher overall sulfate
density (Table I). This also suggests a requirement for a complex
binding specificity, rather than just the exceeding of a particular
sulfate density.
The potentiation of TGF-1 by heparin/liver HS, but only in the
presence of serum or purified
2M, confirms that the GAGs are acting as antagonists of serum
2M, rather than as
direct modulators of receptor binding/activation. The majority of
endogenous serum TGF-
is known to be inactive and complexed to
2M in vivo (30, 31).
2M is a
proteinase inhibitor, which irreversibly traps circulating proteinases
and in the process undergoes a major conformational change. The
conformationally activated form of
2M is specifically
cleared from the circulation by the low density lipoprotein
receptor-related protein (LRP) in the liver. Both forms of
2M can also carry a wide range of cytokines. TGF-
1 possesses a higher affinity for proteolytically activated
2M than for the native form (33, 34). In contrast,
TGF-
2 possesses a higher overall affinity for
2M,
although it displays less selectivity between the two forms (34, 35).
TGF-
3 also interacts with
2M in vitro
(data not shown), although its comparative affinities are not known.
Reflecting these affinity differences,
2M is more effective at counteracting the mitoinhibitory action of TGF-
2, than
of TGF-
1, on cultured hepatocytes (36). Heparin's potentiation of
the antiproliferative effect of TGF-
1 on smooth muscle cells (8) may
be due to blocking of the
2M-TGF-
1 interaction (note that
2M is not itself a heparin-binding protein; Ref.
37). Electrophoretic analyses have also suggested that
2M·TGF-
2 complexes in vitro are less
susceptible to dissociation by heparin than those containing TGF-
1
(33). We have shown that a differential sensitivity of TGF-
1 and
-
2 complexes with
2M can be demonstrated with either
heparin or liver HS in an in vitro bioassay and,
importantly, that TGF-
3 is unusual in that, lacking a
heparin/HS-binding site, its inactivation by
2M cannot
be counteracted in this way.
McCaffrey et al. (6) identified three potential
heparin-binding sites in TGF-1, corresponding to residues 23-32,
30-41, and 92-99. Residues 23-32 were considered most likely to
constitute an in vivo binding site, being most able, as a
free peptide, to inhibit the binding of TGF-
1 to immobilized heparin
(6). In light of the extensive sequence homologies between TGF-
s,
but the specific absence of a heparin/HS-binding site in TGF-
3, we thought it possible that information on the binding site may be indirectly obtained from the conservation of basic amino acids (important for interaction with polyanionic GAGs) across the isoforms (Fig. 5). Two specific basic residues, at positions 26 and 60 in both TGF-
1 (38) and -
2 (39), are replaced by neutral ones in TGF-
3 (40). Interestingly, basic residues are also conserved
at these positions in TGF-
4 (41) and -
5 (42) and, as there are no
other such differences in TGF-
4, and only a single change at residue
110 (a conformationally buried residue) in TGF-
5, it is likely that
these isoforms will also prove to be heparin/HS-binding. TGF-
3 may
thus be the sole isoform that lacks this property.
We have not ascertained experimentally which of these two basic
residues are required for interaction, although residue 26 does
fall within the major peptide sequence implicated by
McCaffrey (6). Recent x-ray crystallographic studies of
TGF-2 (43, 44) and -
3 (45), and solution NMR studies of TGF-
1
(46), are, however, informative. These place the side chains of
residues 26 and 60 at opposite ends and sides of the monomer, and
therefore unlikely to form a single coherent binding site. TGF-
is a
disulfide-bonded dimer (see Fig. 6), and residue 60 lies
only partially solvent-exposed in the interface region, whereas residue
26 is fully solvent exposed on the periphery (44, 45). Additionally,
residue 26 and the closely apposed basic residues at positions 25, 31, 34, 37, and 94 form the most positively charged surface on the monomer
(45). In the dimer these are duplicated at opposite poles, but on the same face, approximately 60 Å apart. These may constitute two independent heparin/HS-binding sites (Fig. 6) in which residue 26 is
critical. Nevertheless, it is also possible that the highest affinity
interaction that most effectively inhibits
2M
complexation could be formed by two co-operative interactions within
the same GAG chain. As the length of a heparin disaccharide in solution is 8.6 Å (47), the minimum sequence required to span the two sites on
the TGF-
dimer would comprise approximately seven disaccharides. This suggests an alternative explanation for the apparent functional selectivity of heparin and liver HS. In addition to possessing specific
monomer recognition sequences, they may also effectively present two
such sequences at an appropriate spacing to engage both binding sites
in the dimer. Heparin, being highly sulfated in a relatively uniform
manner throughout, could accommodate dimer binding at multiple
positions. Liver HS possesses relatively large sulfated domains (7-9
disaccharides long) separated by only short N-acetylated
sequences (24), so interaction may be possible within a single large
sulfated domain or involve two adjacent ones. In contrast, the sulfated
domains in a low sulfated HS species are likely to be too short, and/or
too far apart (48), to optimally engage both sites.
Selective interaction of the TGF- isoforms with heparin/HS may have
significant biological implications. The specific structure of the HS
expressed by a particular cell type or tissue may affect the potency
and isoformic specificity of TGF-
activity. In addition, the
relatively high levels of TGF-
, mostly TGF-
1, released from activated platelets (49) in response to tissue damage must have their
activity restricted to the injury site, and be inhibited from having an
undesired systemic effect through entry, in an active state, into the
circulation. This is primarily achieved by efficient inactivation by
the high levels of serum
2M, although locally this
inactivation must be temporarily forestalled. Platelet-derived thrombospondin appears to possess a protective activity (50), and in
certain tissues rich in highly sulfated HS, e.g. liver (24)
and possibly lung (51), this function may also be served by HSPGs.
Interestingly, TGF-
is the most potent chemotactic factor so far
described for mast cells (52), which often accumulate at wound sites.
In addition to releasing heparin upon degranulation, mast cells have
recently been shown to secrete TGF-
1 in vitro (53, 54).
Coordinate release of heparin and TGF-
1 may act to maintain locally
active TGF-
1. By comparison, the release of TGF-
2 or -
3 under
similar circumstances, with little or no respective protection being
afforded by heparin, would be relatively ineffective. All the TGF-
s
can also be neutralized in the extracellular matrix by complexation
with the core proteins of decorin, biglycan and fibromodulin (11).
Preliminary evidence suggests, however, that the interaction of
TGF-
1 with decorin is not inhibited by heparin (data not shown).
Overall these issues of potential bioavailability may be particularly
important in the evaluation of the different TGF- isoforms for
potential therapeutic use in wound healing and chemoprotection (55-57). For example, in certain circumstances it may be more
effective to employ TGF-
1 in combination with an appropriate
protective heparinoid species.
TGF- bound to serum
2M was thought to be cleared from
the circulation by liver LRP, the specific receptor for activated
2M. When TGF-
1 is injected intravenously the majority
is rapidly sequestered and cleared by the liver, and to a lesser extent
the lungs (58, 59). Clearance of TGF-
1 precomplexed with
methylamine-activated
2M (which mimics
proteinase-activated
2M) is, as expected, even more
liver-specific (59). However, the serum concentration of native
2M far exceeds that of the activated form, and
consequently the former is considered to be the primary carrier of
TGF-
in vivo (60, 61). This suggests that a considerable
proportion of the rapid clearance may not be LRP-mediated. Indeed, it
has been demonstrated that clearance is not affected by preblocking of
LRP with activated
2M (61). Conceivably, HS expressed on the sinusoidal surface of hepatocytes (and possibly a highly sulfated counterpart in the lungs) may itself function as a major mediator of
TGF-
1 clearance by displacing it from native
2M.
TGF-
delivered to the liver by this endocrine mechanism may be
important in liver regulation and repair, being mito-inhibitory for
hepatocytes.
We thank Dr. John Haley (Oncogene Science)
for the generous provision of recombinant human TGF-3 and also
Suzanne Bridge for secretarial assistance.