Regulated Diversity of Heparan Sulfate

heparan sulfate N-acetylated N-sulfated fibroblast growth factor platelet-derived growth factor N-deacetylase/N-sulfotransferase GlcN 6-O-sulfotransferase GlcN 3-O-sulfotransferase IdoA 2-O-sulfotransferase 3′-phosphoadenosine 5′-phosphosulfate. Large numbers of proteins in animal tissues occur immobilized in the extracellular space, at cell surfaces or in the extracellular matrix. Some are anchored through interactions with other proteins. However, current research increasingly implicates proteoglycans as scaffold structures, designed to accommodate proteins through noncovalent binding to their glycosaminoglycan side chains (1Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1660) Google Scholar). In particular, heparan sulfate (HS)1 proteoglycans are recognized as ubiquitous protein ligands. Binding of proteins to HS chains may serve a variety of functional purposes, from simple immobilization or protection against proteolytic degradation to distinct modulation of biological activity. Because of such interactions HS proteoglycans are critically involved in a variety of biological phenomena at various levels of complexity, including organogenesis in embryonic development, angiogenesis, regulation of blood coagulation and growth factor/cytokine action, cell adhesion, lipid metabolism, etc. (2Lindahl U. Lidholt K. Spillmann D. Kjellén L. Thromb. Res. 1994; 75: 1-32Abstract Full Text PDF PubMed Scopus (341) Google Scholar, 3Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 10: 1270-1279Crossref PubMed Scopus (392) Google Scholar, 4Rosenberg R.D. Shworak N.W. Liu J. Schwartz J.J. Zhang L. J. Clin. Invest. 1997; 99: 2062-2070Crossref PubMed Scopus (253) Google Scholar). In this review we outline the evidence for regulated expression of specific domains in HS chains and its relation to selective protein binding. Further, we consider the current information on HS biosynthesis, with the aim of understanding the mechanisms in control of generating specific saccharide sequences. Contrary to heparin, which is synthesized exclusively as serglycin proteoglycan in connective tissue-type mast cells (1Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1660) Google Scholar), the structurally related HS is produced by most mammalian (and many other) cell types. A variety of core proteins have been shown to carry HS chains, including agrin and perlecan in the extracellular matrix (5Iozzo R.V. Matrix Biol. 1994; 14: 203-208Crossref PubMed Scopus (126) Google Scholar) and the syndecan- and glypican-type species at the cell surface (6David G. FASEB J. 1993; 7: 1023-1030Crossref PubMed Scopus (373) Google Scholar). There is so far no evidence of any correlation between the structures of core proteins and those of the attached HS chains, whereas, conversely, the fine structure of the HS chains on a given proteoglycan species may differ between cell types (7Kato M. Wang H. Bernfield M. Gallagher J.T. Turnbull J.E. J. Biol. Chem. 1994; 269: 18881-18890Abstract Full Text PDF PubMed Google Scholar). The carbohydrate backbone in heparin and HS consists of alternating hexuronic acid (d-glucuronic acid (GlcA) or l-iduronic acid (IdoA)) and d-glucosamine (GlcN) units. The GlcN residues in heparin are predominantly N-sulfated, whereas those in HS show a more varied N-substitution pattern with appreciable proportions of both N-sulfated and N-acetylated and a smaller amount of N-unsubstituted GlcN units (3Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 10: 1270-1279Crossref PubMed Scopus (392) Google Scholar, 8Gallagher J.T. Walker A. Biochem. J. 1985; 230: 665-674Crossref PubMed Scopus (190) Google Scholar,9van den Born J. Gunnarsson K. Bakker M.A.H. Kjellén L. Kusche-Gullberg M. Maccarana M. Berden J.H.M. Lindahl U. J. Biol. Chem. 1995; 270: 31303-31309Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). These structures are generated through the formation of a GlcA-GlcNAcn polymer that is subsequently modified by partial N-deacetylation/N-sulfation of GlcNAc units, C-5 epimerization of GlcA to IdoA residues, and, finally, incorporation of O-sulfate groups at various positions (Fig. 1). Compositional analysis of several HS preparations derived from different bovine organs revealed certain domain characteristics of unexpected regularity (10Maccarana M. Sakura Y. Tawada A. Yoshida K. Lindahl U. J. Biol. Chem. 1996; 271: 17804-17810Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Thus 20–30% of the disaccharide units in all preparations were consistently arranged in alternatingN-acetylated and N-sulfated sequence (NA/NS domain in Fig. 1). Moreover, although the proportions of consecutiveN-sulfated disaccharide units (NS domain in Fig. 1) varied between preparations, those of IdoA residues within the NS domains and the extent of 2-O-sulfation of these units were remarkably constant. Very few 2-O-sulfate groups were located outside the NS domains. By contrast, more than half of the other majorO-sulfate substituent, at C-6 of GlcN units, along with an appreciable proportion of IdoA residues occurred in NA/NS domains (Fig. 1). The trisulfated disaccharide unit, -IdoA(2-OSO3)-GlcNSO3(6-OSO3)-, abundant in heparin, is restricted to NS domains in HS (10Maccarana M. Sakura Y. Tawada A. Yoshida K. Lindahl U. J. Biol. Chem. 1996; 271: 17804-17810Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 11Turnbull J.E. Gallagher J.T. Biochem. J. 1991; 273: 553-559Crossref PubMed Scopus (122) Google Scholar), where it provides a key element in protein recognition. One route toward sequence variability in HS obviously involves modulation of 6-O-sulfation. What are the potentials and limitations in the design of specific protein-binding domains in a HS chain? Clearly, major constraint is because of the substrate specificities of the enzymes that catalyze the various polymer modification reactions in HS biosynthesis (12Lindahl U. Lane D.A. Lindahl U. Heparin—Chemical and Biological Properties, Clinical Applications. Edward Arnold, London1989: 159-189Google Scholar). The internal portions of extended NA domains thus are devoid of IdoA units and sulfate groups and so far have not been implicated in direct interactions with proteins. Conversely, the seemingly nonselective interaction of many proteins with the heavily sulfated NS domains of heparin has tended to obscure the notion of selectivity in protein binding to HS (3Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 10: 1270-1279Crossref PubMed Scopus (392) Google Scholar). Such selectivity may be accomplished in various ways. The involvement of a “rare” component is conceptually appealing, because it accounts for the “unique” quality that may be recognized by some proteins but not by others. In fact, the early recognition of the specific AT-binding region in heparin and HS may be largely ascribed to the key position in this domain of a “unique”O-sulfate group (at C-3 of a single N-sulfated GlcN unit) (3Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 10: 1270-1279Crossref PubMed Scopus (392) Google Scholar, 4Rosenberg R.D. Shworak N.W. Liu J. Schwartz J.J. Zhang L. J. Clin. Invest. 1997; 99: 2062-2070Crossref PubMed Scopus (253) Google Scholar) (see Fig. 1). However, less conspicuous structures may have equal potential for selective ligand binding. The generation of a unique binding epitope may be based on theN-substituent pattern, in that the overall length or relative positioning of individual NS, NA/NS, or NA domains may be of importance, or on unusual combinations of N-substituents and other modifications, such as the occasional occurrence of 2-O-sulfated IdoA units in NA/NS domains (see Ref. 10Maccarana M. Sakura Y. Tawada A. Yoshida K. Lindahl U. J. Biol. Chem. 1996; 271: 17804-17810Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Indeed, selective protein binding may depend on the precise distribution of sugar units and O-sulfate groups within, for example, extended N-sulfated but otherwise sparingly modified domains, features not readily detectable upon routine structural analysis of HS preparations. To what extent does the structural variability of HS preparations reflect regulated rather than random biosynthetic polymer modification? Some insight has been gained through compositional analysis of HS preparations from different bovine (10Maccarana M. Sakura Y. Tawada A. Yoshida K. Lindahl U. J. Biol. Chem. 1996; 271: 17804-17810Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) or human (13Lindahl B. Eriksson L. Lindahl U. Biochem. J. 1995; 306: 177-184Crossref PubMed Scopus (98) Google Scholar) organs (see also Ref. 14Lyon M. Deakin J.A. Gallagher J.T. J. Biol. Chem. 1994; 269: 11208-11215Abstract Full Text PDF PubMed Google Scholar). Remarkably, although HS from human cerebral cortex clearly differed in composition from HSs of several other tissues (liver, aorta, kidney), cerebral HS samples from different individuals appeared indistinguishable (13Lindahl B. Eriksson L. Lindahl U. Biochem. J. 1995; 306: 177-184Crossref PubMed Scopus (98) Google Scholar). The conclusion from these findings, that the structures of HSs from different sources are strictly regulated, was underlined by the observation that 2-O-sulfated GlcA units were abundant in HS from adult brain but lacking in HS from neonatal cerebral cortex (13Lindahl B. Eriksson L. Lindahl U. Biochem. J. 1995; 306: 177-184Crossref PubMed Scopus (98) Google Scholar). Immunohistochemical application of monoclonal anti-HS antibodies revealed additional complexity (9van den Born J. Gunnarsson K. Bakker M.A.H. Kjellén L. Kusche-Gullberg M. Maccarana M. Berden J.H.M. Lindahl U. J. Biol. Chem. 1995; 270: 31303-31309Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 15van den Born J. Jann K. Assmann K.J.M. Lindahl U. Berden J.H.M. J. Biol. Chem. 1996; 271: 22802-22809Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 16van Kuppevelt T.H. Dennissen M.A.B.A. van Venrooij W.J. Hoet R.M.A. Veerkamp J.H. J. Biol. Chem. 1998; 273: 12960-12966Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). A panel of such antibodies that recognized different, albeit yet poorly defined, HS epitopes thus yielded markedly different staining patterns when applied to sections of rat kidney. Apparently, the composite kidney tissue displays several HS species, with distinct topology in relation to different cell types. The pronounced interindividual similarity in overall composition of HS from a given organ (13Lindahl B. Eriksson L. Lindahl U. Biochem. J. 1995; 306: 177-184Crossref PubMed Scopus (98) Google Scholar, 17Lindahl B. Lindahl U. J. Biol. Chem. 1997; 272: 26091-26094Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) suggests that any HS subspecies present must be expressed in remarkably constant proportion from one individual to another. Changes in HS composition have been observed in association with development/aging as well as with certain pathological processes. During development of the embryonic brain, HS-mediated growth factor activity is switched between members of the fibroblast growth factor (FGF) family, from FGF-2 to FGF-1 (18Brickman Y.G. Ford M.D. Gallagher J.T. Nurcombe V. Bartlett P.F. Turnbull J.E. J. Biol. Chem. 1998; 273: 4350-4359Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). This change, which correlates with a transition from proliferation of neural precursor cells to neuronal differentiation, is accompanied by alterations in patterns of 6-O-sulfation, total chain length, and the number of sulfated domains of the predominant HS species. A somewhat related phenomenon, albeit on a different time scale, was observed on analysis of HS from human aorta (19Feyzi E. Saldeen T. Larsson E. Lindahl U. Salmivirta M. J. Biol. Chem. 1998; 273: 13395-13398Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). An age-dependent increase in GlcN 6-O-sulfation was demonstrated, resulting in increased abundance of the trisulfated -IdoA(2-OSO3)-GlcNSO3(6-OSO3)- disaccharide unit. Concomitantly enhanced binding of the HS was noted to isoforms of platelet-derived growth factor (PDGF) A and B chains containing polybasic cell retention sequences. By contrast, the binding to FGF-2 was affected to a much lesser extent. Several reports have described changes in the sulfation pattern of HS in response to “differentiation” or “transformation” of cells in culture (see e.g. Refs. 20Jayson G.C. Lyon M. Paraskeva C. Turnbull J.E. Deakin J.A. Gallagher J.T. J. Biol. Chem. 1998; 273: 51-57Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar and 21Safaiyan F. Lindahl U. Salmivirta M. Eur. J. Biochem. 1998; 252: 576-582Crossref PubMed Scopus (45) Google Scholar, and references therein). These data do not provide any unified picture; for instance, the expression of a given sulfate substituent may be up- or down-regulated because of transformation, depending on cell type. Nevertheless, the data demonstrate that the structure of HS produced by a single cell line may be modulated because of distinct stimuli. Also other diseases may be associated with changes in HS structure. Experimentally induced diabetes in rats thus resulted in decreasedN-sulfation of hepatic HS (22Kjellén L. Bielefeld D. Hook M. Diabetes. 1983; 32: 337-342Crossref PubMed Scopus (0) Google Scholar). Further, recent analysis of the HS accumulated in internal organs in AA amyloidosis indicated a switch in O-sulfation, from the distinct distributions characteristic of each individual organ (liver and spleen) to a novel pattern that was common to the amyloid-associated HS irrespective of organ source (17Lindahl B. Lindahl U. J. Biol. Chem. 1997; 272: 26091-26094Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Although a few protein-binding domains in HS chains have been defined, information has been accumulated to some of the potential domain types including composite binding such as H. Turnbull J.E. Biochem. J. 1995; PubMed Scopus Google Scholar), factor Gallagher J.T. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), or D. D. Lindahl U. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) thus to HS through interactions of the with different NS domains within a single In the region such NS domains may be by an NA domain of to units. The internal NA domain of the region may to sugar units. However, most proteins so far to with saccharide of units in single domains. The AT-binding region is one of such a sequence that is on the occurrence of a “rare” in this the (Fig. 1). In the AT-binding region the is at to a GlcA (3Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 10: 1270-1279Crossref PubMed Scopus (392) Google Scholar). HS J. Biol. Chem. Full Text PDF PubMed Google Scholar) and, from a G. Lindahl U. J. Biol. Chem. Full Text PDF PubMed Google Scholar), also the GlcN at to for specific binding to proteins. unusual in both heparin and HS are the 2-O-sulfated GlcA and N-unsubstituted GlcN units. The residues are expressed in certain HS subspecies (13Lindahl B. Eriksson L. Lindahl U. Biochem. J. 1995; 306: 177-184Crossref PubMed Scopus (98) Google Scholar, J. Biol. PubMed Scopus Google Scholar) but have not been implicated with any functional GlcN residues are in HS species of binding and and thus involved in A. K. A. J. Clin. Invest. 1998; PubMed Scopus Google Scholar). protein-binding HS so far do not to depend on the of unique but are of the major HS Such an does not selectivity in binding. instance, FGF-2 sequence with a single IdoA 2-O-sulfate group for binding to HS M. B. Lindahl U. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, 1996; 271: PubMed Scopus Google Scholar). GlcN residues are but not for binding. By contrast, interactions with E. F. F. Spillmann D. Lindahl U. Salmivirta M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), growth factor M. Deakin J.A. K. T. Gallagher J.T. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), B. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), and E. E. T. Lindahl U. Spillmann D. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) all depend on the of one or more GlcN This was expressed through the binding of containing the trisulfated disaccharide unit, Although of interaction be it is noted that the implicated disaccharide the of heparin chains but is in HS species, where it in internal portions of NS domains (10Maccarana M. Sakura Y. Tawada A. Yoshida K. Lindahl U. J. Biol. Chem. 1996; 271: 17804-17810Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 11Turnbull J.E. Gallagher J.T. Biochem. J. 1991; 273: 553-559Crossref PubMed Scopus (122) Google Scholar). the formation of the trisulfated disaccharide units in HS by regulated 6-O-sulfation of 2-O-sulfated NS (Fig. 1). Such regulation the of on binding of FGF-2 to human as described certain biological of HS chains may binding of e.g. FGF-2 and its Maccarana M. Lindahl U. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar) or and R.D. Shworak N.W. Liu J. Schwartz J.J. Zhang L. J. Clin. Invest. 1997; 99: 2062-2070Crossref PubMed Scopus (253) Google Scholar), to but structurally different saccharide domains. Such a on regulation of saccharide The of information the of NA/NS domains in protein binding the associated with the of containing such sequences. A sequence the chain to a in the core one of the enzymes in the of this the GlcA has been H. Y. T. T. K. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). This is distinct from the T. Lindahl U. Lidholt K. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar) that the of the as the of the GlcNAc the toward HS biosynthesis, a 1997; 7: PubMed Scopus Google Scholar). Although the precise relation between chain and modification has not yet been defined, the to be at (3Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 10: 1270-1279Crossref PubMed Scopus (392) Google Scholar). A development of the research in this may be because the enzymes implicated with polymer modification in HS biosynthesis were all a number of these enzymes to occur as isoforms of different or of the The modification which the group of the GlcNAc with a sulfate at distinct isoforms Y. A. G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. M. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, D. B. Lindahl U. Kjellén L. J. Biol. 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The of different isoforms in a given as by expression thus may be of to the structure of the in this with including the generation of with in HS biosynthesis and its regulation insight the of the the of individual The of interaction of the various biosynthetic enzymes with each other as well as their positioning within the also to be in that substrate recognition by most of the enzymes on structural in reactions (3Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 10: 1270-1279Crossref PubMed Scopus (392) Google Scholar, U. Lane D.A. Lindahl U. Heparin—Chemical and Biological Properties, Clinical Applications. Edward Arnold, London1989: 159-189Google Scholar). The the C-5 the and the enzymes all to be to the that a sequence of length N.W. Liu J. Schwartz J.J. Zhang L. D. R.D. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). and may be located in different because their including the and the show M. Eriksson D. Kjellén L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), and this region is for protein retention and Biol. 1991; Full Text PDF PubMed Scopus Google Scholar). of potential the of the sulfate as by the of the and the C. Biochem. 1997; Full Text PDF PubMed Scopus Google Scholar). The sulfation patterns may because of of the as by of the various sulfation have been indicated in Fig. in recognition of such and may the enzymes of the biosynthetic the structure of the HS thus on a protein for expression of activity D. B. Lindahl U. Kjellén L. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), whereas does not M. A. U. A. 1993; PubMed Scopus Google Scholar). The overall of HS as by the of the biosynthetic poorly The of this may be by to the GlcA C-5 epimerization is that this in the is by at of the IdoA However, a that extended chains were within less than a each of GlcA to IdoA was in because no epimerization be (12Lindahl U. Lane D.A. Lindahl U. Heparin—Chemical and Biological Properties, Clinical Applications. Edward Arnold, London1989: 159-189Google Scholar, G. M. Lindahl U. A. L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). to current notion of HS structure this information to the generation of an extended NS within a single the formation of HS chains, with their various domains, different in of a single The to this on in as well as in the structures for the biosynthetic enzymes has evidence for the of HS in embryonic development, as revealed through in of these on growth to the and the of HS chains attached to a core protein as the in their interaction with associated with distinct references 1997; PubMed Scopus Google Scholar) have been to in key enzymes in HS biosynthesis, including to the and Moreover, protein B. 1991; PubMed Google Scholar) appreciable sequence with the mammalian IdoA M. H. M. K. J. Biol. 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