The classification of GAGs is based on the structure of the repeating disaccharide units present in their chain and the species shown in Fig. 1 have been recognized.
 

Fig. 1: Schematic structures of GAGs. Heparin/HS and hyaluronic acid (HA) are glycosaminoglycans; chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S) and dermatan sulfate (DS) are galactosaminoglycans; keratan sulfate (KS) is a sulfated polylactosamine. Since heparin/HS structures are highly heterogeneous, only their most abundant disaccharide unit IdoA(2-OSO₃)-GlcNSO₃(6-OSO₃) is shown here.

PGs can be classified on the basis of their localization, GAG-chain composition, and on the type of the core protein. As shown in Tab. 1, HSPGs are localized mainly in the basement membrane and on the cell surface (see below).
 
 

Tab. 1: Classification of proteoglycans on the basis of their localization and type of core protein

Localization	GAG-chain	Mr of the core protein (kD)	Principal members
ECM	HA, CS, KS	225-250	aggrecan, versican
Collagen-associated	CS, DS, KS	40	decorin, biglycan fibromodulin
Basement membrane	HS	120	perlecan
Cell-surface	HS, CS	33[a]-60[b]-92[c]	syndecans[a], glypican[b], betaglycan[c], CD44E, cerebroglycan
Intracellular granules	heparin, CS	17-19	serglycin

CS, chondroitin sulfate; DS, dermatan sulfate; KS, keratan sulfate; HA, hyaluronic acid; HS, heparan sulfate

The biosynthesis of HSPGs is a process that leads to the production of molecules characterized by great structural heterogeneity with respect to the size of the polysaccharide chain, the ratio of iduronic (IdoA) to glucuronic acid (GlcA) units, and the amount and distribution of sulfate groups along the carbohydrate backbone.

The biosynthesis of heparin/HS can be conveniently separated into three steps:

• formation of the linkage region to the core protein;
• chain elongation;
• chain modifications (see ref. 1 for a detailed description of the process).

ii) The non reducing end of the neutral trisaccharide xylosyl-galactosyl-galactose becomes the primer for the elongation of the polysaccharide. In the case of HS and heparin, polymer formation occurs through an alternating transfer of GlcA units and N-acetylglucosamine (GlcNAc) units to the growing chain. The mechanisms that control the length of the fully growth polysaccharides are not fully elucidated. In general, the length of the final chain increases with the availability of the UDP-sugar precursor and decreases with the availability of the core protein.

iii) Subsequent to polymer formation, the repeating GlcA-GlcNAc disaccharide chain undergoes a number of enzymatic modifications that occur with the following order:

1) N-deacetylation of GlcNAc units that originates glucosamine (GlcN) residues.
2) N-sulfation of newly formed GlcN residues.
3) C5 epimerization of GlcA residues; this reaction leads to the formation of IdoA units.
4) 2-O-sulfation of newly originated IdoA residues.
5) 6-O-sulfation of GlcN residues.
6) Sulfation can also occur at C3 of GlcN units and at C2 or C3 of GlcA units to a limited extent.

All these polymer modifications are incomplete in vivo. In other words, not all the sugar residues that are potential substrate for the various enzymes are transformed into their relative products. Since 2-O- and 6-O-sulfation occur only after C5 epimerization that, in turn, needs the preceding N-deacetylation/N-sulfation reaction, the distribution of 2-O- and 6-O-sulfate groups is restricted to N-sulfated regions.

This partial modification process is the biosynthetic basis for the structural heterogeneity of heparin/HS, but its fine regulation remains unexplained. At this regard it has been demonstrated that the product of the preceding biosynthetic step is the substrate for the next enzyme. This makes the first enzyme of the biosynthetic pathway, namely the deacetilase, the key molecule for regulating the whole process. This and the following enzymes may be regulated in their activity by cellular factors such as ions, pH, cofactors, and other metabolites and by the epigenetic control of the expression of the corresponding genes.

The regulation of the chain modification process leads to cell- or organ-specific HS structures that may allow a fine modulation of the biological functions of HSPGs.

As stated above, the final structure of heparin/HS depends upon the incompleteness of the reactions that occur during the biosynthetic process. The modification process is more complete in heparin where the final disaccharide IdoA(2-OSO₃)-GlcNSO₃(6-OSO₃) represents up to 70% of the chain, leading to a heavily O-sulfated polysaccharide with a high IdoA/GlcA ratio.

In contrast, the modifications that occur during the biosynthesis of HS are less extensive, leading to HS molecules characterized by lower IdoA content and a lower overall degree of O-sulfation and resulting in high heterogeneity of distribution of the sulfate groups along the chain.

Eventually, disaccharides containing GlcNAc or GlcNSO₃ may form clusters ranging from 2 to 20 adjacent GlcNAc-containing disaccharides and from 2 to 10 adjacent GlcNSO₃-containing disaccharides. However, about 20-30% of the chain contains alternate GlcNAc- and GlcNSO₃-disaccharides units (2).

Typical concentrations of HSPGs on the cell surface are in the range of 10⁵-10⁶ molecules/cells as measured in various cell culture systems.

HSPGs can link to plasma membrane through a hydrophobic transmembrane domain of their core protein or through a glycosyl-phosphatidylinositol (GPI) anchor covalently bound to the core protein (transmembrane HSPGs). Also, HSPGs can interact with the cell by non-covalent linkage to different cell-surface macromolecules (peripheral membrane HSPGs) (Fig. 2).
 

Fig. 2: Association of HSPGs with the cell surface. 1) HSPG linked to plasma membrane through a hydrophobic transmembrane domain of the core protein; 2) HSPG associated to plasma membrane through a glycosyl-phosphatidylinositol (GPI) anchor covalently bound to the core protein; 3) non-covalent linkage between the core protein of HSPG and a cell-surface receptor (e.g. integrin receptor).

Transmembrane HSPGs are glypican (3), cerebroglycan (4), betaglycan (5), CD44 (6), and the members of the syndecan family (7): syndecan 1, fibroglycan (syndecan 2), N-syndecan (syndecan 3) and ryudocan (syndecan 4).

Glypican and cerebroglycan are typical GPI-anchored HSPGs. Syndecans and betaglycan are typical transmembrane HSPGs characterized by a core protein composed of an extracellular domain, a single membrane-spanning domain and a short cytoplasmic domain (28 to 34 amino acid residues). In the extracellular domain are present the consensus sequences for glycosylation and a conserved putative proteolytic cleavage site. The cytoplasmic domain of syndecans can interact with the cytoskeleton and contains four conserved tyrosine residues, one of them hypothesized to be substrate for enzymatic phosphorylation.

Perlecan is a typical peripheral membrane HSPG that interacts with the cell surface through its core protein (8). The cell-adhesion motif Arg-Gly-Asp within the core protein of perlecan binds integrins b₁ or b₃ present on endothelial cell surface (9). However, HSPGs may associate to the cell surface and/or ECM also through their GAG-chain, as demonstrated by the observation that half of the total content of HSPGs in endothelial cells can be released after incubation with soluble heparin (10).

HSPGs exist also in soluble form following their mobilization from the cell surface. Transmembrane HSPGs are released after proteolytic digestion of their core protein (11). GAG-chain-associated HSPGs are released by exogenous GAGs by a simple law mass action (10) or by enzymatic digestion of their polysaccharidic backbone (12). GPI-anchored HSPGs can be released by action of endogenous phospholipase (13).

Finally, it is important to recall that cell-associated HSPGs can be internalized via endocytosis and metabolized in the lysosomal compartment (1). In some cell types oligosaccharides originated during intracellular degradation appears to be delivered specifically to the nucleus (14).

HSPGs are necessary for the structural and functional integrity of the endothelium. HSPGs present at the basal site of blood vessels act as matrix receptors by interacting with a variety of basement membrane proteins (9). Moreover, basal HSPGs are responsible for the charge selectivity of filtration in endothelium (15) and inhibit smooth muscle cell proliferation and migration (16). HSPGs are also present at the luminal surface of endothelium (17) where they are involved in the binding and internalization of lipoprotein lipase (18). Also, luminal HSPGs play a major role in determining the anticoagulative properties of the vessel surface by binding to proteases of the intrinsic coagulation cascade, thrombin, and protease inhibitors, including antithrombin III (19). Finally, endothelial cell-surface HSPGs act as co-receptors for a wide spectrum of angiogenic growth factors, being involved in the control of angiogenesis (see below).

Both macro- and microvascular endothelial cells synthesize HSPGs (20-22). Different HSPGs have been identified in endothelial cells in culture (23-25) where they account for the majority of extracellular sulfated GAGs (20).

Endothelial HSPGs may be found at intracellular level (7), associated to plasma membrane and ECM, or in a soluble form (10, 26). Syndecan 1 is the most represented HSPG in microvascular endothelial cells (24). It is mainly stored inside the cell, a small portion being present at the basal surface (27). Syndecan 4 is also expressed on the surface of endothelial cells (25), while perlecan is abundant in endothelial ECM (28). Finally, a variety of poorly characterized soluble HSPGs with molecular weight spanning from 28 to 800 kD have been isolated from cultured media of endothelial cells (11, 29).

The observation that the levels of HSPGs in endothelial cells derived from the microvasculature, where the angiogenic process takes place, are 10-15 times higher than those found in macrovascular endothelial cells (30) is in keeping with the role played by endothelial HSPGs in the modulation of angiogenesis. Indeed, depletion of HSPGs from endothelial cell surface inhibits neovascularization (31).

The HSPG-dependent regulation of angiogenesis is due, at least in part, to their capacity to bind to and modulate the activity of angiogenic growth factors (see below), and contrasting effects can be obtained depending on the types of HSPGs and/or on the experimental conditions adopted.

For instance, purified perlecan enhances FGF-2-dependent angiogenesis while other purified HSPGs are ineffective (28). Overexpression of syndecan 1 on the surface of NIH 3T3 cells inhibits FGF-2-induced cell proliferation (32). In contrast, syndecan 1 stimulates FGF-2-mediated cell growth when immobilized to matrix (33). However, soluble syndecan, glypican, and fibroglycan block the restoration of FGF-2 receptor binding induced by heparin/HS in cell mutants deficient in cell surface HSPGs (34) and soluble heparin/HS inhibit the binding of FGF-2 to its receptors and to HSPGs present on the surface of endothelial cells (35).

These data suggest that negative effects on angiogenesis may be exerted by the binding of angiogenic growth factors to soluble HSPGs or GAGs rather than to cell-associated HSPGs.

Besides their capacity to modulate receptor binding, HSPGs may affect angiogenesis also by protecting angiogenic growth factors from heat (36) and proteolytic degradation (11, 37) and by increasing their radius of diffusion (38).

Finally, HSPGs present in the ECM may act as a reservoir for angiogenic growth factors that will reach higher local concentrations and will sustain the long-term stimulation of endothelial cells (12, 39-41).

Given the above considerations, the expression of endothelial HSPGs during the angiogenic process must be tightly enforced. This kind of control may take place at different levels:

• quantitative and qualitative differential expression of the various HSPG species;
• modulation of the composition of GAG chains;
• induction of HSPG mobilization from cell surface.