Biosynthesis and structure of heparin/HS
Structures of HS-related GAGs are best understood in terms of their biosynthesis in the Golgi compartment of cells (Fig. 1a) [11, 12]. Selected core proteins are used as primers for GAG polymerization and modification. The products, proteoglycans (PGs), carry HS or heparin GAG chains (Fig. 1b) . The heparin PG serglycin occurs in the granules of connective-tissue-type mast cells, where the high negative charge of the heparin chains facilitates packaging of positively charged mast-cell proteases, histamine and other inflammatory mediators . HSPGs are found on the surface of virtually all cells and in the extracellular matrix/basement membranes outside cells. The two major families of cell-surface HSPGs are the syndecans and the glypicans. The core proteins of syndecans (four distinct species) span the plasma membrane, the cytoplasmic parts interacting with intracellular proteins, whereas the glypicans (six species) are attached to the outer surface of cell membranes through a glycosyl-phosphatidylinositol anchor. The main secreted extracellular HSPGs are perlecan, agrin and collagen XVIII. Compared with the vast number of proteins carrying N-linked or O-linked oligosaccharides, only a few proteins serve as PG core proteins. Nonetheless, all cells investigated, including red blood cells , contain HS (or heparin) PGs.
Figure 1. (a) Biosynthesis of HS. The process is initiated by the formation of a nonsulphated polymer of alternating glucuronic acid and N-acetylglucosamine units attached to the serine residue of a PG core protein through a galactose–galactose–xylose trisaccharide sequence. This precursor polysaccharide undergoes sequential modification steps involving substitution of N-sulphate for N-acetyl groups (N-sulphation, indicated by grey ellipsoids), C5 epimerization of D-glucuronic to L-iduronic acid residues, and sulphation of hydroxyl groups at C2 of iduronic acid (and some glucuronic acid) units, and C6 and C3 of glucosamine units (O-sulphation, indicated by yellow ellipsoids). The modifications are generally incomplete, such that only a fraction of potential substrate sites are actually targeted. Owing to the substrate specificity of the epimerase and O-sulphotransferases, modifications are essentially restricted to the N-sulphated regions of the chains (NS domains, indicated by a zig-zag line under the bottom sequence). Other regions remain largely unmodified (NA domains, straight line). The pentasaccharide structure defined by the horizontal bracket above the bottom sequence binds with high affinity to AT and accounts for the anticoagulant activity of heparin/HS molecules. The various domains rarely exceed 10 sugar units in length, except for the NA domain adjacent to the linkage to protein (16–18 units). (b) Illustration of cell-surface HSPGs (syndecan and glypican) and extracellular HSPG (perlecan), with distribution of NS and NA domains indicated according to the symbols in panel (a). The intracellular serglycin proteoglycans in mast cells carry heparin chains, best envisaged as extended, highly sulphated NS domains.
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The formation of HS chains is initiated by the formation of a tetrasaccharide linkage region, synthesized by stepwise addition of xylose, followed by two galactose units and a glucuronic acid residue, from their respective UDP-sugars to a serine residue in the core protein. A fifth enzyme then transfers the first N-acetylglucosamine residue to the linkage region, followed by extensive addition of glucuronic acid and N-acetylglucosamine units in alternating sequence, which is catalysed by HS-copolymerase (EXT1/EXT2) (Fig. 1a). The nascent GAG chain is modified by sulphotransferases and an epimerase. First, some of the N-acetylglucosamine residues are N-deacetylated and N-sulphated by a dual-activity N-deacetylase/N-sulphotransferase (NDST) enzyme, using PAPS (3′-phosphoadenosine-5′-phosphosulphate) as sulphate donor. The partially N-sulphated polysaccharide is a substrate for the next modification enzyme, glucuronyl C5-epimerase, which converts glucuronic into iduronic acid residues. Iduronic acid provides flexibility to the polysaccharide chain, facilitating interactions between the GAG and proteins . 2-O-Sulphation locks iduronic acid residues in the interaction-prone configuration. The modification process is completed through 6-O- and 3-O-sulphation of glucosamine units. 2-O-Sulphated or nonsulphated iduronic acid residues, and 6-O-sulphated glucosamines are abundant modification products, whereas 2-O-sulfated glucuronic acid and 3-O-sulphated or N-unsubstituted glucosamine units are scarce . Notably, completed chains may be further edited through Sulph-catalysed 6-O-desulphation, preferentially of N-, 2-O-, 6-O-trisulphated disaccharide units . Whilst C5-epimerase and 2-O-sulphotransferase are transcribed from single genes, the other modification enzymes occur as multiple isoforms .
Structural complexity and heterogeneity, the hallmarks of HS-related GAGs, are introduced at various levels of the biosynthetic process. Because epimerization and O-sulphation occur preferentially in close vicinity to N-sulphate groups, N-sulphation is a key regulatory step that sets a limit for overall subsequent modification [11, 12, 20, 21]. Heparin features 80–90% N-sulphated glucosamine units and a total (N- and O-) average sulphate content typically greater than two residues per disaccharide unit, whereas HS is 30–60% N-sulphated with a total sulphate content of approximately one residue per disaccharide unit. HS has a characteristic domain structure comprising highly sulphated regions (NS domains) alternating with nonsulphated (NA domains) and mixed (NA/NS) domains . Heparin is more uniformly sulphated and resembles a continuous NS domain (Fig. 1). However, HS species from different sources differ not only regarding size and distribution of the various domains, but also with respect to levels of subsequent modifications within these domains. The modification patterns are cell-specific, such that HS samples from a given murine tissue differ from those of other tissues, but are indistinguishable from corresponding samples isolated from other syngenic mice . Notably, these patterns appear to be largely independent of the core protein type . Obviously, polymer modification is not a random process; the question that arises is how it is regulated. Factors of potential importance include expression levels of different enzymes, modulation of their catalytic activities, and the availability of precursor molecules. The activity of an enzyme in a HS-synthesizing Golgi compartment may thus depend on both transcriptional and translational control of expression [23, 24], as well as post-translational modification of the enzyme. Interactions between biosynthesis enzymes have been demonstrated [25-29] and it has been suggested that both elongation and modification of the HS chain take place in so-called GAGosomes , molecular complexes of enzymes and other (as yet unidentified) regulatory molecules.
Finally, polysaccharide chain length is another source of heterogeneity. Newly synthesized heparin chains typically fall within the range 60–100 kDa , whilst HS chains are shorter (Mr = 14–45 kDa) [32, 33]. However, even highly purified HS preparations are polydisperse, with a size distribution that varies with the source. Chain size may be established during biosynthesis, but may also reflect endolytic heparanase action. Native heparin chains are generally processed in mast cells by heparanase cleavage, which yields 5 to 25-kDa fragments that are recovered as unfractionated commercial heparin . Notably, the so-called low-molecular-weight heparins (4–7 kDa) that currently dominate clinical practice are generated by chemical or enzymatic cleavage of unfractionated heparin . The smallest and most recently available heparin-related antithrombotic compound is the antithrombin (AT)-binding pentasaccharide (Fig. 1a), obtained through chemical synthesis .
Biological functions of HSPGs generally depend on interactions, mainly electrostatic in nature, with proteins that bind to the sulphated domains of HS chains . The first protein-binding site in a HS chain to be characterized in detail was the AT-binding pentasaccharide sequence in heparin (Fig. 1a). Each of four distinct sulphate groups is essential for productive AT binding, and hence blood anticoagulant activity [16, 37]. One of these, a GlcN 3-O-sulphate group, is a rare constituent that was initially believed to be unique to the AT-binding sequence. However, subsequent work showed that 3-O-sulphate groups may be selectively expressed in various HS species and identified a family of 3-O-sulphotransferase isoforms all capable of catalysing specific incorporation of this residue . These findings and other reports of rare sequences raised the notion of selective protein binding dependent on specific sulphation patterns, or a so-called sulphation code . Recent comprehensive studies, however, revealed interactions of highly variable specificity, involving functionally important proteins capable of binding to several polyanionic sequences, provided that these were of sufficient overall charge density [40, 41]. The highly charged heparin molecule binds a multitude of proteins. However, there are reasons not to simply dismiss such interactions as being merely nonspecific. The strict regulation of HS biosynthesis described previously is likely to control the extent of the various polymer-modification reactions rather than the formation of precisely tailored, minimal-binding sequences towards selected protein ligands. Nevertheless, a sulphation code may apply, in a sense, whenever functional binding requires that selected (preferentially basic) amino-acid residues are targeted by critical sulphate groups (indicated by blue arcs in Fig. 2). A protein-binding sequence would thus contain critical sulphate groups either alone (which is rare) or along with other sulphate groups not essential for (but not interfering with) the interaction. The highly sulphated heparin molecule will therefore express abundant potential protein-binding sequences. This concept is illustrated in simplified form in Fig. 2. Adding to the complexity is the conformational flexibility of a GAG chain; a defined three-dimensional pattern of critical sulphate groups may conceivably be attained by more than one linear sequence . A 2-O-sulphate group could thus substitute for neighbouring 6-O- or N-sulphate groups, given appropriate adjustments of the chain conformation. Perhaps the 3-O-sulphate group, which is rare in general but more frequent in sequences participating in specific interactions, plays a special role; recent NMR results show that a hydrogen bond between the N-sulphate and the adjacent 3-O-sulphate group in the AT-binding pentasaccharide preorganizes the secondary structure in a way that facilitates binding .
Figure 2. Schematic illustration of hidden sequence specificity, depending on the defined location of critical sulphate groups (indicated by blue arcs). Only HS domains that accommodate the critical sulphate constellation (enclosed in ellipsoids) promote a functional response in the target protein. The probability of generating such an assemblage increases with the level of sulphation, even given a stochastic distribution of sulphate residues. Fully sulphated heparin-like structures (bottom models) yield maximal productive interactions. For references, see the text.
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Several ‘heparin-binding’ proteins were initially identified without any clue to their functional significance. Further research revealed that the endogenous polysaccharide implicated is generally HS rather than heparin, and many such interactions have now been ascribed functions and physiological roles . From a mechanistic standpoint, they generally fall into either of two categories . HSPGs may provide scaffolds at cell surfaces or in the extracellular matrix to ensure that proteins committed to specific processes in development or homoeostasis are presented at given sites and times in the body (Fig. 3a). The diversity of protein ligands is striking. HSPGs serve as carriers for enzymes, lipoproteins, chemokines, growth factors and morphogens, with important roles in lipid metabolism, inflammatory processes, angiogenesis and various morphogenetic developmental events. Transient capture of growth factors or morphogens may help to stabilize protein gradients, control the range of signalling, or simply protect the proteins against degradation. Interactions of HSPGs in basement membranes with matrix proteins, such as fibronectin and laminin, provide support, resistance to mechanical stress and filtration barrier properties.
Figure 3. HS–protein interactions and their inhibition. (a) Proteins bind to HS chains of HSPGs at the cell surface (syndecan depicted) or in the extracellular matrix (perlecan), mainly through electrostatic interaction. (d) HS chains serve as coreceptors for many proteins; the example shown here promotes growth factor action. Interactions may be disrupted by (b, e) competitive saccharides (or mimetics) that block HS-binding sites on target proteins or (c, f) decoy peptides (mimetics) that block protein-binding HS sequences. (g) Authentic or modified HS/heparin fragments may replace endogenous HS and augment protein responses. Fragmentation of HS chains by heparanase may release a ligand, either as (h) a free protein or (i) bound to an oligosaccharide fragment. (j,k) Perturbation of HS–protein interactions resulting from interference with HS biosynthesis. For further information and references, see the text.
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The other major type of HS–protein interaction contributes more directly to biological activities or signalling processes (Fig. 3d) . A well-studied example is the anticoagulant activity of heparin (and some HS species), which is mediated by binding of a specific pentasaccharide species (Fig. 1a) to the protease inhibitor AT. The resultant subtle conformational change turns the AT molecule into a more efficient inhibitor of procoagulant serine proteases . Interactions between HSPGs and a variety of growth factor or morphogens modulate FGF, Hedgehog, TGF-β, BMP and Wnt signalling . Whereas the precise nature of HS involvement in some of these pathways remains unclear, direct participation of polysaccharide chains in signalling complexes with some cell-surface receptors has been demonstrated [46, 47]. Further details in connection with specific pathophysiological aspects are discussed below.