Glycobiology vol. 2 no. 6 pp. 523-528, 1992
MINI REVIEW
Heparan sulphate in the binding and activation of basic fibroblast growth factor
J.T.Gallagher and J.E.Turnbull Departments of Medical Oncology and Clinical Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester, M20 9BX, UK
Key words: cell surfaces/fibroblast growth factor (FGF)/heparan sulphate/proteoglycans/syndecan
Introduction Heparan sulphates (HS) are A'-sulphated polysaccharide components of proteoglycans (PGs). They consist of a disaccharide repeat of a,/3-linked glucosamine and hexuronic acid Qinkage sequence [(l-*4)a-r>glucosaminyl-(l-*4)/3-D-hexuronosyl]n where n = 50—150) in which the glucosamine may be A'-acetylated or A'-sulphated, and the hexuronate is present as glucuronate (GlcA) or the C-5 epimer, iduronate (IdoA). Ester (O)sulphation, principally at C-2 of IdoA and C-6 of the glucosamine residues, but also rarely at C-2 of GlcA and C-3 of glucosamine, adds significant charge density and structural complexity to the polymer chain (Figure 1; Kjellen and Lindahl, 1991; Gallagher etal., 1992). Sulphation appears to be carefully regulated during HS synthesis and different cells can often be distinguished by the fine structural characteristics of their HS chains (Gallagher et al., 1986). Synthesis and molecular organization of HS Our knowledge of the synthesis of HS is derived mainly from studies on the synthesis of heparin (Lindahl, 1989), which is exclusively a product of connective tissue mast cells, whereas HS is synthesized by most vertebrate cells (Kolset and Gallagher, 1990). These polysaccharides have a similar disaccharide unit structure to HS, but heparin contains a significantly higher concentration of sulphate groups (see below). HS and heparin synthesis occur within the Golgi complex, and begin '- Oxford University Press
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Heparan sulphate proteoglycans (HSPGs) are widely distributed in animal tissues, but their most prominent locations are cell surface membranes and basement membranes. Their influence on various fundamental aspects of cell behaviour (e.g. cell adhesion, growth and morphogenesis) are dependent on the specific binding properties of the heparan sulphate (HS) chains. These polysaccharides are complex structures in which A'-sulphated glucosamine and ester sulphate groups tend to be clustered in discrete regions of the chain separated by sequences enriched in iV-acetylglucosamine residues, but with a low sulphate concentration. The sulphated domains contain the sugar residue sequences for interaction with specific proteins essential for HS function. In this review, we describe the plasma membrane HSPGs and their role in regulating the activity of basic fibroblast growth factor (bFGF).
with the formation of a non-sulphated precursor, heparan, composed of GlcNAc-GkA repeats assembled on core protein acceptors primed with the typical glycosaminoglycan (GAG)tetrasaccharide linkage sequence (Ser)-Xyl-Gal-Gal-GlcA-. The heparan precursor is transformed into HS (or heparin) by a stepwise series of mainly polymer-level modifications, the first being the conversion of GlcNAc residues to GlcNSO3 (Lindahl and Kjellen, 1987). Recent evidence suggests that N-sulphation of glucosamine residues may be required to drive polymerization to completion (Lidholt et al., 1989). Further modifications then take place in the vicinity of the A^-sulphate groups. GlcA can be epimerized to IdoA and the polymer is then further modified by a complex pattern of substitution with ester-(O)sulphate groups which completes the biosynthetic process. A key aspect of the mechanism of synthesis is that for each stage only a fraction of potential substrates are modified, resulting in considerable scope for sequence diversity (Gallagher and Lyon, 1989). There is an - 5 0 % conversion of GlcNAc to GlcNSO3 in HS, but in heparin the conversion frequency is much higher and 80—90% of disaccharides are A'-sulphated in the mature polymer (Gallagher and Walker, 1985). There appears to be targeting of polymer sulphation to particular regions of the HS chain and Figure 2 shows a model depicting the notion of structural domains in HS. N- and Osulphate groups are in close proximity, arranged in relatively discrete clusters (average length 5—6 disaccharides) separated by regions of low sulphation in which the disaccharides are mainly N-acetylated (Turnbull and Gallagher, 1991a). N-Su\phation and its regulation must, therefore, play a critical role in the synthesis of HS, although the precise mechanisms which determine the final detailed structure are still unknown. The domain organization is a characteristic feature of HS that distinguishes it from the more uniform sulphation of other GAGs. Figure 2 shows an A'-acetyl-rich sequence between the protein core and the most proximal A'-sulphated domain. A cleavage site for the enzyme heparinase, which only attacks linkages between GlcNSO3(± 6S) and IdoA(2S) (Linhardt etal., 1990), is located —16 disaccharides from the core protein in the majority of HS chains (Turnbull and Gallagher, 1991b). These disaccharides are found centrally within A'-sulphated domains and this first downstream heparinase site identifies the position of a cluster of sulphated residues (see Figure 2). A downstream A'-sulphate group, identified using nitrous acid hydrolysis, was encountered ~ 10 disaccharides from the protein core (range 9—12 disaccharides), but the location of this GlcNSO3 residue, and the most proximal heparinase cleavage site, showed some chain-to-chain variability, clearly indicative of sequence microheterogeneity (Lyon et al., 1987; Turnbull and Gallagher, 1991b; Lindblom etal., 1991). The findings reveal that the heparan sulphates do not have a rigidly defined primary sequence analogous to proteins and nucleic acids. Nevertheless, as illustrated in Figure 2, there appears to be a
J.T.Gallagher and J.E.Turnbull a)
significant degree of order to the polymeric structure, but more detailed analysis of different HS species, and new developments in sequencing methods, are now needed to assess the general validity of the domain model.
N-Acetvtated
so" COOH
NH
GlcNAc
OH
,o
otl->-4 GlcA
N-Sulphated
GlcNSO
a
I-
4 IdoA
Fig. 1. Disaccharides in heparan sulphate. W-Acetylated and W-sulphated disaccharides are present in HS. Ester (O)-sulphation can occur at the positions indicated
Heparan sulphates are mainly present on cell surfaces and in the extracellular matrix as proteoglycans (Gallagher, 1989; Kjellen and Lindahl, 1991; Yanagashita and Hascall, 1992). Molecular cloning and sequencing has determined the primary structure of several core proteins that bear HS chains. In basement membranes, the major heparan sulphate proteoglycan (HSPG) has a 400 kDa protein (Noonan etal., 1988; Kallunki and Tryggvason, 1992) and has been named perlecan because of the 'beads' on a string appearance of the protein when examined by electron microscopy (Paulson et al., 1987). There are 3-4 HS chains projecting from a terminal globule. A unique family of four cell surface HSPGs has been identified by molecular cloning studies. These PGs have highly homologous transmembrane and C-terminal cytoplasmic domains, and they have been collectively named the syndecan family of PGs (Bernfield et al., 1992), adopting the name given to the first member of this family (isolated from mouse mammary epithelial cells) to be fully sequenced (Saunders et al., 1989; see also Mali et al., 1990). The name syndecan has a classical rather than morphological basis being derived from the Greek syndein, meaning 'to bind together', and indicates a suggested function of syndecan to bind cells to the ECM. The ectodomains of the syndecans are not homologous, with the notable exception of the presumed 3—4 sites of HS glycanation (Figure 3) located towards the N-terminal region of the proteins. The prototypic syndecan (syndecan 1) also contains two short chondroitin sulphate chains, but the other syndecans seem to be substituted only with HS.
HS Chain Core Protein
I I
I I
III
I I JLJL
N-acetyi rich sequences
N-sulphated domains
I O
GlcNSO (+ 6S) - IdoA,2S 3 Heparinase scission Proximal N-sulphate
Fig. 2. Domain structure of heparan sulphate. Heparan sulphate is believed to have a domain structure with A'-sulphated regions interspersed with long W-aceryl-rich sequences. Heparinase cleavage sites are present in the sulphated domains. The most proximal (to the protein) heparinase site is ~ 16 disaccharides from the core protein, whereas the innermost W-sulphate is —10 disaccharides from the protein.
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b)
HS-proteoglycan
Heparan sulphate proteoglycan regulation of bFGF
by cultured keratinocytes (Brown etal., 1991). The cytoplasmic regions of CD44 and jSetaglycan are not homologous with each other nor with the syndecans.
HS and the regulation of growth factors Ectodomains
Cytoplasmic Regions
Fibroglycan
N-Syndecan
(33kDa)
(23kDa)
(35kDa)
Ryudocan or Amphiglycan (21kDa)
Fig. 3. The syndecan family of trans-membrane HSPGs. The syndecans are type 1 membrane-spanning proteins (i.e. amino terminus in the ectodomain) and are homologous in the cytoplasmic and transmembrane regions. The proteins are not homologous in the ectodomains, with the notable exception of amino acid sequences in the HS glycanalion region.
There are some interesting differences in the cellular distribution of the syndecans. In the adult, syndecan 1 is mainly found in epithelial cells, whereas syndecan 2 (alternative name fibroglycan) is most prominent in fibroblasts and other mesenchymal cells (Marynen et at., 1989; Lories et at., 1992), but it is also the main membrane HSPG in rat hepatocytes (Pierce etal., 1992). Syndecan 3 (N-syndecan) is mainly associated with nervous tissue and developing mesenchyme (Carey et at., 1992), whereas syndecan 4, which has alternative names of somewhat arcane derivation (i.e. ryudocan/amphiglycan; David et at., 1992; Kojima et al., 1992) is the most widely distributed member of the syndecan family. It seems reasonable to assume that functional differences exist between the various syndecans. Their individual properties will probably depend on the concerted actions of the HS chains which confer binding and recognition properties at the cell surface, and the cytoplasmic domains which are likely to possess related but distinct biochemical modes of action inside the cell. This suggestion implies that the syndecans function through transmembrane signalling and we currently await direct evidence that this is so. In addition to the syndecan family, the protein sequences of two more membrane-associated HSPGs have been deduced from cDNA cloning. These are a 64 kDa protein named glypican, because it contains a GPI-anchor (David et al., 1990) and a 92 kDa integral membrane protein called /Setaglycan because it binds TGF-/3, and is one of three TGF-/3 binding proteins on cell surfaces (Lopez-Casillas etal., 1991; Wang et at., 1991). However jSetaglycan is not a signal-transducing receptor for TGF-0 and its function is presently unknown. The only other membrane HSPG to be identified is an epithelial specific isoform of the CD44/Hermes glycoprotein synthesized
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Syndecan
It is well recognised that HS (and heparin) bind with appreciable affinity to a wide spectrum of cytokines that affect cell growth and differentiation. TGF-/3 (McCaffrey etal., 1992), endothelial cell growth factor (Gitay-Goren et at., 1992), IL3 and GM-CSF (Roberts et al., 1988), PDGF (Raines and Ross, 1991), interferon-7 (Lortat-Jacob and Grimaud, 1991), hepatocyte growth factor/scatter factor (Rosen etal., 1989) and the fibroblast growth factor family [FGF-1 (acidic FGF), FGF-2 (bFGF), FGF-3 (int-2), F G F ^ (Hst-l, K-FGF), FGF-5, FGF-6 (Hst-2) and FGF-7 (keratinocyte GF) (Burgess and Macaig, 1989; Klagsbrun, 1989, 1990; Baird and Klagsbrun, 1991)], can all be described as heparin/HS-binding cytokines. Heparin affinity chromatography is commonly used for their purification, although the strength of these interactions varies considerably among the different factors. Both heparin and HS have significant effects on cytokine activities. For example, heparin will release TGF-/3 from inactive complexes with a2- m a c r °gl°b u n n a n d will potentiate TGF-/3 action (McCaffrey et al., 1989). The stability in solution of acidic and basic FGF (aFGF and bFGF) is enhanced in the presence of HS/heparin, and the polysaccharides potentiate the mitogenic activity of the FGFs, especially of aFGF (Saksela et al. ,1988; Barzu et al., 1989; Sommer and Rifkin, 1989; Sudhalter et al., 1989). These effects are presumed to be due to the formation of complexes between FGF and heparin which prolong the biological lifetime of the proteins by protecting them from proteolysis and thermal denaturation. In tissues, aFGF and bFGF can be detected in the extracellular matrix and basement membranes, where they are bound to HS (Folkman et al., 1988; Flaumenhaft et al., 1989; Klagsbrun, 1990; Vlodavsky et al., 1991). It has been proposed that the action of heparanases or proteases that degrade HSPGs will release FGFs from the basement membrane, enabling them to act on nearby target cells (Bashkin etal., 1989; Presta etal., 1989; Vlodavsky et al., 1991). In addition to effects on FGF stability and tissue localization, a central role has now been described for HS in controlling the interaction of bFGF with cell signalling receptors. bFGF elicits signal responses in cells by binding to specific high-affinity cell surface tyrosine kinase receptors (K^ = 10—500 pm) such as the products of the fig and bek genes (Ruta etal., 1988; Klagsbrun, 1989; Pasquale and Singer, 1989). HSPGs constitute a second class of lower affinity cell surface binding molecules for bFGF (K^ = 5-50 nM) which now appear to be essential for the cellular response mechanism. This was first demonstrated by Yayon et al. (1991) who transfected the FGF receptor fig into mutagenized Chinese hamster ovary (CHO) cells defective in HS synthesis; FGF would not bind to these cells unless heparin or HS was also added to the culture medium. This was in contrast to FGF binding to flgtransfected wild-type CHO cells (with normal levels of cell surface HS) which occurred without the addition of polysaccharide. It was subsequently shown that the interaction of bFGF with a recombinant form of the fig ectodomain required the presence of heparin/HS, and that FGF-induced mitogenesis was also heparin dependent (Omitz et al., 1992). These results
J.T.Gallagher and J.E.TurnbuU
bFGF binding sequences in HS The effects of HS and heparin on bFGF are reminiscent of the activation of antithrombin III (AT-HI) by heparin. The AT-EQ binding sequence in heparin is a pentasaccharide that contains the rare 3-0-sulphated glucosamine residue. The sequence is GlcNAc(6S)-GlcA-GlcNSO3(3S)-IdoA(2S)-GlcNSO3 and its identification was an important milestone in our understanding of structure—activity relationships in complex polysaccharides (Lindahl etai, 1984). Recently we isolated and characterized the structure of an oligosaccharide sequence from human skin fibroblast HS which binds with high affinity to bFGF (Turnbull et al., 1992). The enzyme heparitinase (Linhardt et al., 1990) was used to excise the sulphated regions of HS from the parent molecule and bFGF-affinity chromatography led to the isolation of an oligosaccharide composed of seven disaccharide units [dp (degree of polymerization) 14] which bound as strongly to bFGF as the intact HS chain. The sequence of this oligosaccharide, named oligo-H, is: GlcA 01,4 GIcNSOjal,4[IdoA,2S al,4GIcNSO3],al,4IdoAal,4GlcNAc
in which the N-sulphates and iduronate 2-sulphate residues are essential for binding activity. The internal homotypic repeat of five IdoA(2S)-GlcNSO3 disaccharide units is a very distinctive and somewhat unexpected sequence in view of the variety of sugar sulphate isomers that are present in HS; the absence of 6-sulphate groups was particularly notable because they are quite common in HS and a major substituent of heparin. Analyses of other large heparitinase-resistant oligosaccharides indicated that the internal decamer repeat described above gave superior FGF binding to that of saccharides with an octamer 526
Cell Membrane FGF-R HSPG
QJ
- bFGF - inactive
Y7
- bFGF - active
Fig. 4. bFGF activation by heparan sulphate. bFGF is inactive (O) unless it binds to cell surface HS which induces a conformational change rendering the bFGF active (A) and able to engage the signal transducing receptors (FGF-R).
repeat of the same units (Turnbull et al., 1992). Other oligosaccharides (dp 12) with a relatively low content of IdoA(2S), but a corresponding increase in 6-sulphate groups, displayed only weak binding to bFGF, emphasizing the significance of C-2 sulphation of iduronate residues. A recent report on the bFGF affinity of HS fragments isolated from aorta HS by heparitinase scission also emphasizes the importance of IdoA(2S) (Habuchi et al, 1992). Although oligo-H may not be the minimal structure for optimal binding to bFGF, it may be significant that full activation (equivalent to heparin) of bFGF binding to the fig receptor requires heparin fragments of the same size as oligo-H (i.e. dp 14-16) (Ornitz et al., 1992). In addition, aFGF is strongly activated by heparin oligosaccharides of this size (Barzu et al., 1989; Sudhalter et al., 1989). Concluding remarks Future studies will no doubt focus on investigations of the structure of oligosaccharide sequences involved in the binding of all proteins in the FGF family and of the wider range of growth factors which interact with HS. It is now important to examine the relationship between oligosaccharide affinity and activation of bFGF. It is possible that a structure as large as oligo-H can bind two bFGF molecules (these are small compact proteins, mol. wt 18 kDa) with a resultant strong co-operative effect in binding to FGF immobilized on an affinity matrix. If cell surface HS creates a 'dimeric' FGF ligand, this could facilitate the dimerization of FGF receptors which may be necessary for the induction of receptor tyrosine kinase activity (Ornitz etai, 1992). Chemical synthesis of HS-oligosaccharides should help to define the minimal structural requirements for activation of bFGF. It will be essential to define the saccharide binding specificities of other members of the FGF family. There may be subtle differences in their sequence requirements for activation which would provide a rational explanation for the polymorphism of HS. Differential cellular responses to the FGFs could be regulated by minor
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suggest that in the plasma membrane fig recognizes a particular conformation of bFGF induced by binding to cell surface HS. HS may, therefore, be considered as a component of a dual receptor system for bFGF (Klagsbrun and Baird, 1991) which activates the bound growth factor prior to delivery to the signal transducing receptors (Figure 4). Other studies have given support to this interesting concept and indicate that it may be applicable to other members of the FGF family. Shortly after the paper by Yayon et al. (1991), Rapraeger and co-workers (1991) reported that 3T3 fibroblasts and MM 14 myoblasts were rendered unresponsive to bFGF when cultured with sodium chlorate, which suppresses GAG sulphation. Mitogenic activity of bFGF was restored when heparin was added to the culture medium. The activities of aFGF and K-FGF in the myoblast cultures are also dependent on the presence of cell surface HS (Olwin and Rapraeger, 1992). Heparan sulphate also contributes to the binding of bFGF to high-affinity receptors on adrenocortical cells (Savona et al., 1991). Furthermore, aFGF and bFGF stimulation of the growth and differentiation of myeloid cells engineered to express the fig receptor require the presence of heparin (Bernard et al., 1991; Li and Bernard, 1992). Binding of bFGF to the bek receptor is also a heparin-dependent process (Mansukhani et al., 1992). A further development in this area was the identification of a new high-affinity aFGF receptor on parathyroid cells which is actually an HSPG (Sakaguchi et al., 1991). aFGF can be chemically cross-linked to the protein core of this receptor, but binding requires the presence of the HS chain.
Heparan sulphate proteoglycan regulation of bFGF
reprogramming of polysaccharide structure. From a pharmaceutical perspective, a thorough understanding of the molecular basis of HS-FGF recognition might lead to the design of saccharide analogues that suppress or activate bFGF action. These analogues could have wide therapeutic applications in oncology, wound healing, nerve regeneration and ocular diseases (Gospodarowicz, 1991). bFGF has provided valuable insights into the functions of HS, but we are only at the beginning of a new and exciting era of research on the mechanisms of growth factor regulation by complex polysaccharides.
Acknowledgements We thank the Cancer Research Campaign and the Christie Hospital NHS Trust for their kind support.
AT-III, antithrombin III; CHO, Chinese hamster ovary; dp, degree of polymerization, FGF, fibroblast growth factor (a = acidic, b = basic), GAG, glycosaminoglycan; GlcA, glucuronate; HS, heparan sulphate; HSPG, heparan sulphate proteoglycan; IdoA, iduronate.
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Abbreviations
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Received on September 18, 1992; accepted on September 21, 1992
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Omitz.D.M., Yayon.A., Flanagan .J.G., Svahn,C.M., Levi.E. and Leder.P. (1992) Heparin is required for cell-free binding of basic fibroblast growth factor to a soluble receptor and for mitogenesis in whole cells. Mol. Cell. Biol., 12, 240-247. Pasquale.E.B. and Singer.S.J. (1989) Identification of a developmentally regulated protein-tyrosine kinase by using anti-phosphotyrosine antibodies to screen a cDNA expression library. Proc. Natl. Acad. Sd. USA, 86, 5449-5453. Paulsson.M., Yurchenko.P.D., Ruben.G.C, Engel.S. and Timple.R. (1987) Structure of a low density heparan sulphate proteoglycan isolated from a mouse tumour basement membrane. J. Mol. Biol., 197, 297—313. Presta.M., Maier.J.A.M., Rusnati.M. and Ragnotti.G. (1989) Basic fibroblast growth factor is released from endothelial extracellular matrix in a biologically active form. J. Cell. Physiol., 140, 68-74. Raines,E.W. and Ross.R. (1992) Compartmentalisation of PDGF on extracellular binding sites dependent on exon-6-encoded sequences. J. Cell Biol., 116, 533-543. Rapraeger.A.C, Krufka,A. and Olwin.B.B. (1991) Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoWast differentiation. Science, 252, 1705-1708 Roberts,R., Gallagher,J.T., Spooncer.E., Allen,T.D., Bloomfield.F. and Dexter.T.M. (1989) Heparan sulphate bound growth factors: a mechanism for stromal cell-mediated haemopoiesis. Nature, 332, 376—378. Rosen.E.M., Goldberg,I.D., Kacinslri.B.M., Buckholz.T. and Vinter.D.W. (1989) Smooth muscle releases an epithelial cell scatter factor which binds to heparin. In Vitro, 25, 163-173. Ruoslahti,E. and Yamaguchi,Y. (1991) Proteoglycans as modulation of growth factor activities. Cell, 64, 867-869. Ruta.M., Burgess.W., Givol.D., Epstein.J., Neiger.N., Crumley.G., Dionne.C, Jaye,M. and Schlessinger,J. (1988) Receptor for acidic fibroblast growth factor is related to the tyrosine kinase encoded by the fms-like gene (Fig). Proc. Nad. Acad. Sd. USA, 86, 8722-8726. Sakaguchi.K., Yanagashita.M., Takeuchi.Y. and Aurbach.G.D. (1991) Identification of heparan sulphate proteoglycans as a high affinity receptor for acidic fibroblast growth factor (aFGF) in a parathyroid cell line. J. Biol. Chem., 266, 7270-7278. Saksela.O., MoscateUi,D., Sommer.A. and Rifkin.D.B. (1988) Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation. J. Cell Biol., 107, 743-751. Savona.C, Chambaz.E.M. and Feige.J.J. (1991) Proteoheparan sulphates contribute to the binding of basic FGF to its high affinity receptors on bovine adrenocortical cells. Growth Factors, 5, 273-282. Sommer.A. and Rifkin.D.B. (1989) Interaction of heparin with human basic fibroblast growth factor: protection of the angiogenic protein from proteolytic degradation by a glycosamirjoglycan. J. Cell. Physiol., 138, 215—220. SudhalterJ., FoltananJ., Svahn.C.M., Bergendal.K. and D'Amore.P.A. (1989) Importance of size, sulphation and anticoagulant activity in the potentiation of acidic fibroblast growth factor by heparin. J. Biol. Chem., 264, 6892-6897. TurnbullJ.E. and Gallagher.J.T. (1990) Molecular organisation of heparan sulphate from human skin fibroblasts. Biochem. J., 265, 715—724. Turnbull.J.E. and Gallagher.J.T. (1991a) Distribution of iduronate-2-sulphate in heparan sulphate: evidence for an ordered polymeric structure. Biochem. J., 273, 553-559. Turnbuiy.E. and GallagherJ.T. (1991b) Sequence analysis of heparan sulphate indicates defined locations of /V-sulphated glucosamine and iduronate-2-sulphate residues proximal to the protein linkage region. Biochem. J., 277, 297-303. TurnbulU.E., Fermg.D., Ke,Y., Wilkinson,M.C. and GaUagher,J.T. (1992) Identification of the basic FGF binding sequence in fibroblast heparan sulphate. J. Biol. Chem., 267, 10337-10341. Vlodavsky.I., Bar-Shavh.R., Ishai-Michaeli,R., Bashkin.P. and Fuks,Z. (1991) Extracellular sequestration and release of fibroblast growth factor: a regulatory mechanism. Trends Biochem. Sci., 16, 268-271 Wang^X.-F., UnJ.Y., Ng-Eaton.E., Downward^., Lodish.H.F. and Weinberg.R.A. (1991) Expression cloning and characterisation of the TGF-/3 type m receptor. Cell, 67, 797-805. Yanagashita.M. and Hascall.V.C. (1992) Cell surface heparan sulphate proteoglycans. J. Biol. Chem., 267, 9451-9454. Yayon.A., Klagsbrun.M., Esko,J.D., Leder.P. and Ornitz.D.M. (1991) Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell, 64, 841-848.
MINI REVIEW
Heparan sulphate in the binding and activation of basic fibroblast growth factor
J.T.Gallagher and J.E.Turnbull Departments of Medical Oncology and Clinical Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester, M20 9BX, UK
Key words: cell surfaces/fibroblast growth factor (FGF)/heparan sulphate/proteoglycans/syndecan
Introduction Heparan sulphates (HS) are A'-sulphated polysaccharide components of proteoglycans (PGs). They consist of a disaccharide repeat of a,/3-linked glucosamine and hexuronic acid Qinkage sequence [(l-*4)a-r>glucosaminyl-(l-*4)/3-D-hexuronosyl]n where n = 50—150) in which the glucosamine may be A'-acetylated or A'-sulphated, and the hexuronate is present as glucuronate (GlcA) or the C-5 epimer, iduronate (IdoA). Ester (O)sulphation, principally at C-2 of IdoA and C-6 of the glucosamine residues, but also rarely at C-2 of GlcA and C-3 of glucosamine, adds significant charge density and structural complexity to the polymer chain (Figure 1; Kjellen and Lindahl, 1991; Gallagher etal., 1992). Sulphation appears to be carefully regulated during HS synthesis and different cells can often be distinguished by the fine structural characteristics of their HS chains (Gallagher et al., 1986). Synthesis and molecular organization of HS Our knowledge of the synthesis of HS is derived mainly from studies on the synthesis of heparin (Lindahl, 1989), which is exclusively a product of connective tissue mast cells, whereas HS is synthesized by most vertebrate cells (Kolset and Gallagher, 1990). These polysaccharides have a similar disaccharide unit structure to HS, but heparin contains a significantly higher concentration of sulphate groups (see below). HS and heparin synthesis occur within the Golgi complex, and begin '- Oxford University Press
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Heparan sulphate proteoglycans (HSPGs) are widely distributed in animal tissues, but their most prominent locations are cell surface membranes and basement membranes. Their influence on various fundamental aspects of cell behaviour (e.g. cell adhesion, growth and morphogenesis) are dependent on the specific binding properties of the heparan sulphate (HS) chains. These polysaccharides are complex structures in which A'-sulphated glucosamine and ester sulphate groups tend to be clustered in discrete regions of the chain separated by sequences enriched in iV-acetylglucosamine residues, but with a low sulphate concentration. The sulphated domains contain the sugar residue sequences for interaction with specific proteins essential for HS function. In this review, we describe the plasma membrane HSPGs and their role in regulating the activity of basic fibroblast growth factor (bFGF).
with the formation of a non-sulphated precursor, heparan, composed of GlcNAc-GkA repeats assembled on core protein acceptors primed with the typical glycosaminoglycan (GAG)tetrasaccharide linkage sequence (Ser)-Xyl-Gal-Gal-GlcA-. The heparan precursor is transformed into HS (or heparin) by a stepwise series of mainly polymer-level modifications, the first being the conversion of GlcNAc residues to GlcNSO3 (Lindahl and Kjellen, 1987). Recent evidence suggests that N-sulphation of glucosamine residues may be required to drive polymerization to completion (Lidholt et al., 1989). Further modifications then take place in the vicinity of the A^-sulphate groups. GlcA can be epimerized to IdoA and the polymer is then further modified by a complex pattern of substitution with ester-(O)sulphate groups which completes the biosynthetic process. A key aspect of the mechanism of synthesis is that for each stage only a fraction of potential substrates are modified, resulting in considerable scope for sequence diversity (Gallagher and Lyon, 1989). There is an - 5 0 % conversion of GlcNAc to GlcNSO3 in HS, but in heparin the conversion frequency is much higher and 80—90% of disaccharides are A'-sulphated in the mature polymer (Gallagher and Walker, 1985). There appears to be targeting of polymer sulphation to particular regions of the HS chain and Figure 2 shows a model depicting the notion of structural domains in HS. N- and Osulphate groups are in close proximity, arranged in relatively discrete clusters (average length 5—6 disaccharides) separated by regions of low sulphation in which the disaccharides are mainly N-acetylated (Turnbull and Gallagher, 1991a). N-Su\phation and its regulation must, therefore, play a critical role in the synthesis of HS, although the precise mechanisms which determine the final detailed structure are still unknown. The domain organization is a characteristic feature of HS that distinguishes it from the more uniform sulphation of other GAGs. Figure 2 shows an A'-acetyl-rich sequence between the protein core and the most proximal A'-sulphated domain. A cleavage site for the enzyme heparinase, which only attacks linkages between GlcNSO3(± 6S) and IdoA(2S) (Linhardt etal., 1990), is located —16 disaccharides from the core protein in the majority of HS chains (Turnbull and Gallagher, 1991b). These disaccharides are found centrally within A'-sulphated domains and this first downstream heparinase site identifies the position of a cluster of sulphated residues (see Figure 2). A downstream A'-sulphate group, identified using nitrous acid hydrolysis, was encountered ~ 10 disaccharides from the protein core (range 9—12 disaccharides), but the location of this GlcNSO3 residue, and the most proximal heparinase cleavage site, showed some chain-to-chain variability, clearly indicative of sequence microheterogeneity (Lyon et al., 1987; Turnbull and Gallagher, 1991b; Lindblom etal., 1991). The findings reveal that the heparan sulphates do not have a rigidly defined primary sequence analogous to proteins and nucleic acids. Nevertheless, as illustrated in Figure 2, there appears to be a
J.T.Gallagher and J.E.Turnbull a)
significant degree of order to the polymeric structure, but more detailed analysis of different HS species, and new developments in sequencing methods, are now needed to assess the general validity of the domain model.
N-Acetvtated
so" COOH
NH
GlcNAc
OH
,o
otl->-4 GlcA
N-Sulphated
GlcNSO
a
I-
4 IdoA
Fig. 1. Disaccharides in heparan sulphate. W-Acetylated and W-sulphated disaccharides are present in HS. Ester (O)-sulphation can occur at the positions indicated
Heparan sulphates are mainly present on cell surfaces and in the extracellular matrix as proteoglycans (Gallagher, 1989; Kjellen and Lindahl, 1991; Yanagashita and Hascall, 1992). Molecular cloning and sequencing has determined the primary structure of several core proteins that bear HS chains. In basement membranes, the major heparan sulphate proteoglycan (HSPG) has a 400 kDa protein (Noonan etal., 1988; Kallunki and Tryggvason, 1992) and has been named perlecan because of the 'beads' on a string appearance of the protein when examined by electron microscopy (Paulson et al., 1987). There are 3-4 HS chains projecting from a terminal globule. A unique family of four cell surface HSPGs has been identified by molecular cloning studies. These PGs have highly homologous transmembrane and C-terminal cytoplasmic domains, and they have been collectively named the syndecan family of PGs (Bernfield et al., 1992), adopting the name given to the first member of this family (isolated from mouse mammary epithelial cells) to be fully sequenced (Saunders et al., 1989; see also Mali et al., 1990). The name syndecan has a classical rather than morphological basis being derived from the Greek syndein, meaning 'to bind together', and indicates a suggested function of syndecan to bind cells to the ECM. The ectodomains of the syndecans are not homologous, with the notable exception of the presumed 3—4 sites of HS glycanation (Figure 3) located towards the N-terminal region of the proteins. The prototypic syndecan (syndecan 1) also contains two short chondroitin sulphate chains, but the other syndecans seem to be substituted only with HS.
HS Chain Core Protein
I I
I I
III
I I JLJL
N-acetyi rich sequences
N-sulphated domains
I O
GlcNSO (+ 6S) - IdoA,2S 3 Heparinase scission Proximal N-sulphate
Fig. 2. Domain structure of heparan sulphate. Heparan sulphate is believed to have a domain structure with A'-sulphated regions interspersed with long W-aceryl-rich sequences. Heparinase cleavage sites are present in the sulphated domains. The most proximal (to the protein) heparinase site is ~ 16 disaccharides from the core protein, whereas the innermost W-sulphate is —10 disaccharides from the protein.
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b)
HS-proteoglycan
Heparan sulphate proteoglycan regulation of bFGF
by cultured keratinocytes (Brown etal., 1991). The cytoplasmic regions of CD44 and jSetaglycan are not homologous with each other nor with the syndecans.
HS and the regulation of growth factors Ectodomains
Cytoplasmic Regions
Fibroglycan
N-Syndecan
(33kDa)
(23kDa)
(35kDa)
Ryudocan or Amphiglycan (21kDa)
Fig. 3. The syndecan family of trans-membrane HSPGs. The syndecans are type 1 membrane-spanning proteins (i.e. amino terminus in the ectodomain) and are homologous in the cytoplasmic and transmembrane regions. The proteins are not homologous in the ectodomains, with the notable exception of amino acid sequences in the HS glycanalion region.
There are some interesting differences in the cellular distribution of the syndecans. In the adult, syndecan 1 is mainly found in epithelial cells, whereas syndecan 2 (alternative name fibroglycan) is most prominent in fibroblasts and other mesenchymal cells (Marynen et at., 1989; Lories et at., 1992), but it is also the main membrane HSPG in rat hepatocytes (Pierce etal., 1992). Syndecan 3 (N-syndecan) is mainly associated with nervous tissue and developing mesenchyme (Carey et at., 1992), whereas syndecan 4, which has alternative names of somewhat arcane derivation (i.e. ryudocan/amphiglycan; David et at., 1992; Kojima et al., 1992) is the most widely distributed member of the syndecan family. It seems reasonable to assume that functional differences exist between the various syndecans. Their individual properties will probably depend on the concerted actions of the HS chains which confer binding and recognition properties at the cell surface, and the cytoplasmic domains which are likely to possess related but distinct biochemical modes of action inside the cell. This suggestion implies that the syndecans function through transmembrane signalling and we currently await direct evidence that this is so. In addition to the syndecan family, the protein sequences of two more membrane-associated HSPGs have been deduced from cDNA cloning. These are a 64 kDa protein named glypican, because it contains a GPI-anchor (David et al., 1990) and a 92 kDa integral membrane protein called /Setaglycan because it binds TGF-/3, and is one of three TGF-/3 binding proteins on cell surfaces (Lopez-Casillas etal., 1991; Wang et at., 1991). However jSetaglycan is not a signal-transducing receptor for TGF-0 and its function is presently unknown. The only other membrane HSPG to be identified is an epithelial specific isoform of the CD44/Hermes glycoprotein synthesized
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Syndecan
It is well recognised that HS (and heparin) bind with appreciable affinity to a wide spectrum of cytokines that affect cell growth and differentiation. TGF-/3 (McCaffrey etal., 1992), endothelial cell growth factor (Gitay-Goren et at., 1992), IL3 and GM-CSF (Roberts et al., 1988), PDGF (Raines and Ross, 1991), interferon-7 (Lortat-Jacob and Grimaud, 1991), hepatocyte growth factor/scatter factor (Rosen etal., 1989) and the fibroblast growth factor family [FGF-1 (acidic FGF), FGF-2 (bFGF), FGF-3 (int-2), F G F ^ (Hst-l, K-FGF), FGF-5, FGF-6 (Hst-2) and FGF-7 (keratinocyte GF) (Burgess and Macaig, 1989; Klagsbrun, 1989, 1990; Baird and Klagsbrun, 1991)], can all be described as heparin/HS-binding cytokines. Heparin affinity chromatography is commonly used for their purification, although the strength of these interactions varies considerably among the different factors. Both heparin and HS have significant effects on cytokine activities. For example, heparin will release TGF-/3 from inactive complexes with a2- m a c r °gl°b u n n a n d will potentiate TGF-/3 action (McCaffrey et al., 1989). The stability in solution of acidic and basic FGF (aFGF and bFGF) is enhanced in the presence of HS/heparin, and the polysaccharides potentiate the mitogenic activity of the FGFs, especially of aFGF (Saksela et al. ,1988; Barzu et al., 1989; Sommer and Rifkin, 1989; Sudhalter et al., 1989). These effects are presumed to be due to the formation of complexes between FGF and heparin which prolong the biological lifetime of the proteins by protecting them from proteolysis and thermal denaturation. In tissues, aFGF and bFGF can be detected in the extracellular matrix and basement membranes, where they are bound to HS (Folkman et al., 1988; Flaumenhaft et al., 1989; Klagsbrun, 1990; Vlodavsky et al., 1991). It has been proposed that the action of heparanases or proteases that degrade HSPGs will release FGFs from the basement membrane, enabling them to act on nearby target cells (Bashkin etal., 1989; Presta etal., 1989; Vlodavsky et al., 1991). In addition to effects on FGF stability and tissue localization, a central role has now been described for HS in controlling the interaction of bFGF with cell signalling receptors. bFGF elicits signal responses in cells by binding to specific high-affinity cell surface tyrosine kinase receptors (K^ = 10—500 pm) such as the products of the fig and bek genes (Ruta etal., 1988; Klagsbrun, 1989; Pasquale and Singer, 1989). HSPGs constitute a second class of lower affinity cell surface binding molecules for bFGF (K^ = 5-50 nM) which now appear to be essential for the cellular response mechanism. This was first demonstrated by Yayon et al. (1991) who transfected the FGF receptor fig into mutagenized Chinese hamster ovary (CHO) cells defective in HS synthesis; FGF would not bind to these cells unless heparin or HS was also added to the culture medium. This was in contrast to FGF binding to flgtransfected wild-type CHO cells (with normal levels of cell surface HS) which occurred without the addition of polysaccharide. It was subsequently shown that the interaction of bFGF with a recombinant form of the fig ectodomain required the presence of heparin/HS, and that FGF-induced mitogenesis was also heparin dependent (Omitz et al., 1992). These results
J.T.Gallagher and J.E.TurnbuU
bFGF binding sequences in HS The effects of HS and heparin on bFGF are reminiscent of the activation of antithrombin III (AT-HI) by heparin. The AT-EQ binding sequence in heparin is a pentasaccharide that contains the rare 3-0-sulphated glucosamine residue. The sequence is GlcNAc(6S)-GlcA-GlcNSO3(3S)-IdoA(2S)-GlcNSO3 and its identification was an important milestone in our understanding of structure—activity relationships in complex polysaccharides (Lindahl etai, 1984). Recently we isolated and characterized the structure of an oligosaccharide sequence from human skin fibroblast HS which binds with high affinity to bFGF (Turnbull et al., 1992). The enzyme heparitinase (Linhardt et al., 1990) was used to excise the sulphated regions of HS from the parent molecule and bFGF-affinity chromatography led to the isolation of an oligosaccharide composed of seven disaccharide units [dp (degree of polymerization) 14] which bound as strongly to bFGF as the intact HS chain. The sequence of this oligosaccharide, named oligo-H, is: GlcA 01,4 GIcNSOjal,4[IdoA,2S al,4GIcNSO3],al,4IdoAal,4GlcNAc
in which the N-sulphates and iduronate 2-sulphate residues are essential for binding activity. The internal homotypic repeat of five IdoA(2S)-GlcNSO3 disaccharide units is a very distinctive and somewhat unexpected sequence in view of the variety of sugar sulphate isomers that are present in HS; the absence of 6-sulphate groups was particularly notable because they are quite common in HS and a major substituent of heparin. Analyses of other large heparitinase-resistant oligosaccharides indicated that the internal decamer repeat described above gave superior FGF binding to that of saccharides with an octamer 526
Cell Membrane FGF-R HSPG
QJ
- bFGF - inactive
Y7
- bFGF - active
Fig. 4. bFGF activation by heparan sulphate. bFGF is inactive (O) unless it binds to cell surface HS which induces a conformational change rendering the bFGF active (A) and able to engage the signal transducing receptors (FGF-R).
repeat of the same units (Turnbull et al., 1992). Other oligosaccharides (dp 12) with a relatively low content of IdoA(2S), but a corresponding increase in 6-sulphate groups, displayed only weak binding to bFGF, emphasizing the significance of C-2 sulphation of iduronate residues. A recent report on the bFGF affinity of HS fragments isolated from aorta HS by heparitinase scission also emphasizes the importance of IdoA(2S) (Habuchi et al, 1992). Although oligo-H may not be the minimal structure for optimal binding to bFGF, it may be significant that full activation (equivalent to heparin) of bFGF binding to the fig receptor requires heparin fragments of the same size as oligo-H (i.e. dp 14-16) (Ornitz et al., 1992). In addition, aFGF is strongly activated by heparin oligosaccharides of this size (Barzu et al., 1989; Sudhalter et al., 1989). Concluding remarks Future studies will no doubt focus on investigations of the structure of oligosaccharide sequences involved in the binding of all proteins in the FGF family and of the wider range of growth factors which interact with HS. It is now important to examine the relationship between oligosaccharide affinity and activation of bFGF. It is possible that a structure as large as oligo-H can bind two bFGF molecules (these are small compact proteins, mol. wt 18 kDa) with a resultant strong co-operative effect in binding to FGF immobilized on an affinity matrix. If cell surface HS creates a 'dimeric' FGF ligand, this could facilitate the dimerization of FGF receptors which may be necessary for the induction of receptor tyrosine kinase activity (Ornitz etai, 1992). Chemical synthesis of HS-oligosaccharides should help to define the minimal structural requirements for activation of bFGF. It will be essential to define the saccharide binding specificities of other members of the FGF family. There may be subtle differences in their sequence requirements for activation which would provide a rational explanation for the polymorphism of HS. Differential cellular responses to the FGFs could be regulated by minor
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suggest that in the plasma membrane fig recognizes a particular conformation of bFGF induced by binding to cell surface HS. HS may, therefore, be considered as a component of a dual receptor system for bFGF (Klagsbrun and Baird, 1991) which activates the bound growth factor prior to delivery to the signal transducing receptors (Figure 4). Other studies have given support to this interesting concept and indicate that it may be applicable to other members of the FGF family. Shortly after the paper by Yayon et al. (1991), Rapraeger and co-workers (1991) reported that 3T3 fibroblasts and MM 14 myoblasts were rendered unresponsive to bFGF when cultured with sodium chlorate, which suppresses GAG sulphation. Mitogenic activity of bFGF was restored when heparin was added to the culture medium. The activities of aFGF and K-FGF in the myoblast cultures are also dependent on the presence of cell surface HS (Olwin and Rapraeger, 1992). Heparan sulphate also contributes to the binding of bFGF to high-affinity receptors on adrenocortical cells (Savona et al., 1991). Furthermore, aFGF and bFGF stimulation of the growth and differentiation of myeloid cells engineered to express the fig receptor require the presence of heparin (Bernard et al., 1991; Li and Bernard, 1992). Binding of bFGF to the bek receptor is also a heparin-dependent process (Mansukhani et al., 1992). A further development in this area was the identification of a new high-affinity aFGF receptor on parathyroid cells which is actually an HSPG (Sakaguchi et al., 1991). aFGF can be chemically cross-linked to the protein core of this receptor, but binding requires the presence of the HS chain.
Heparan sulphate proteoglycan regulation of bFGF
reprogramming of polysaccharide structure. From a pharmaceutical perspective, a thorough understanding of the molecular basis of HS-FGF recognition might lead to the design of saccharide analogues that suppress or activate bFGF action. These analogues could have wide therapeutic applications in oncology, wound healing, nerve regeneration and ocular diseases (Gospodarowicz, 1991). bFGF has provided valuable insights into the functions of HS, but we are only at the beginning of a new and exciting era of research on the mechanisms of growth factor regulation by complex polysaccharides.
Acknowledgements We thank the Cancer Research Campaign and the Christie Hospital NHS Trust for their kind support.
AT-III, antithrombin III; CHO, Chinese hamster ovary; dp, degree of polymerization, FGF, fibroblast growth factor (a = acidic, b = basic), GAG, glycosaminoglycan; GlcA, glucuronate; HS, heparan sulphate; HSPG, heparan sulphate proteoglycan; IdoA, iduronate.
References Baird.A. and Klagsbrun,M. (1991) The fibroblast growth family factor. Cancer Cells, 3, 239-243. Barzu.T., Lormeau,J.-C., Petitou.M., Michelson,S. and Choay.T. (1989) Hepann-derived oligosaccharides: affinity for acidic fibroblast growth factor and effect on its growth promoting activity for human endothelial cells. J. Cell Physiol. 140, 538-548. Bashkin.P., Doctrow.S., Klagsbrun.M., Svahn.C.M., Folkman.J. and Vlodavsky.I. (1989) Basic fibroblast growth factor binds to subendothelial extracellular matnx and is released by heparitinase and heparin-like molecules. Biochemistry, 28, 1737-1743. Bemfield.M., Kokenyesi,R., Kato,M. Hinkes.M.T., Spring.S., Gallo.R.L. and Lose,E. (1992) Biology of the syndecans: A family of transmembrane heparan sulphate proteoglycans. Arum. Rev. Cell Biol., 8, 365-393. Brown.T.A., Bouchard,T., St John,T., Wayner.E. and Carter,W.G. (1991) Human keratinocytes express a new CD44 core protein (CD44E) as a heparan sulphate intrinsic membrane proteoglycan with additional exons. J. Cell Biol., 113, 207-221. Burgess.W.H. and Maciag,T (1989) The heparin-binding (fibroblast) growth factor family of proteins. Annu. Rev. Biochem., 58, 575—606. David.G., van de Schueren.B., Marynen.P., Cassiman,J.-T. and Van den Berghe.H. (1992) Molecular cloning of amphiglycan; a novel integral membrane heparan sulphate proteoglycan expressed by epithelial and fibroblastic cells. / Cell Biol, 118, 961-969. Flaumenhaft.R., Moscatelli.D., Saksela.O. and Rifkin.D.B. (1989) Role of extracellular matrix in the action of basic fibroblast growth factor: matrix as a source of growth factor for long-term stimulation of plasminogen activator production and DNA synthesis. J. Cell. Physiol, 140, 75-81. FolkmanJ., Klagsbrun,M., SasseJ., Wadzinski.M , lngber.D. and Vlodavsky, I. (1988) A heparin-binding angiogenic protein—basic fibroblast growth factor —is stored within basement membrane. Am. J. Pathol, 130, 393^100. Gallagher J.T. (1989) The extended family of proteoglycans: social residents of the pericellular zone. Curr. Opin. Cell Biol, 1, 1201-1298. Gallagher^.T. and Lyon,M. (1989) Molecular organisation and functions of heparan sulphate. In Lane,D.A. and Lindahl,U. (eds), Heparin. Edward Arnold, London, pp. 135-158. GallagherJ.T. and Walker.A.W. (1985) Molecular distinctions between heparan sulphate and heparin. Analysis of sulphation patterns indicate that heparin sulphate and heparin are separate families of JV-sulphated polysaccharides. Biochem. J., 230, 665-675. GallagherJ.T., Lyon.M. and Steward.W.P. (1986) Structure and function of heparan sulphate proteoglycans. Biochem. J., 236, 313—325. Gallagher J X , TurnbullJ.E. and Lyon.M. (1992) Patterns of sulphation in heparan sulphate: polymorphism based on a common structural theme. Int. J. Biochem., 24, 553-556.
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Abbreviations
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J.T.Gallagher and J.E.Tumbull
Received on September 18, 1992; accepted on September 21, 1992
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