The functional diversity of HS began to emerge in the 1970s when the first of numerous heparin/HS-binding proteins were discovered. The groups of Klagsbrun and Folkman, in the early 1980s, championed the use of heparin affinity chromatography to purify EC mitogens.14 This application was a major trigger of investigations into HS-mediated signaling by heparin-binding growth factors, which continue to the present.

3.The Structure, Synthesis, and Post-Synthetic Modification of HSPGs

3.1. The HSPG core proteins

There are multiple HSPG core proteins which contribute to the broad functional repertoire of these hybrid molecules (Fig.1A). Table 2 lists the major vertebrate HSPGs and indicates which are known to exhibit expression in ECs. Some core proteins carry two types of GAG chains — HS and chondroitin sulfate (CS); whereas others exclusively bear HS chains. The major carriers of non-surface bound HS in the extracellular matrix appear to be agrin, perlecan, and collagen XVIII (reviewed in Refs. 11 and 15). Most cell surface HS is carried by glypicans, a family of at least six homologous glycophosphatidyl-inositol (GPI)- anchored proteins, syndecans and four related transmembrane proteins. However, a variety of additional integral membrane proteins, such as betaglycan and splice variants of CD44, are considered as “part-time” proteoglycans because they occasionally bear HS chains. ECs are presently known to express at least 11 distinct HSPG core proteins (Table 2). Specialized features of a given HSPG core protein can: (1) provide direct interactions with cytoskeletal and/or signaling components, (2) define localization to intracellular or extracellular compartments, and (3) allow for cellular internalization, recycling, or transcellular transport (reviewed in Refs. 11, 16 and 17). Thus, the multiplicity of core proteins does not merely reflect redundancy but rather amplifies functional diversity. The core protein together with its associated cellular machinery constitute a platform of such profound utility

124 N. W. Shworak

Fig. 1. Major HSPGs. (A) Major HSPGs of ECs. The EC cell surface and basement membrane are schematically depicted showing a major representative of an integral membrane (syndecan-4), a GPI-linked (glypican-1), and an extracellular (perlecan) HSPG. Syndecan-4 is presented as carrying two HS (unbroken line) and one CS (dotted line) chains, but can bear multiple permutations of these GAGs. (B) The structure of an HS disaccharide repeat. The same repeated disaccharide occurs in both HS and heparin. Remodeling of the glucosamine can involve addition of sulfates to the C3 or C6 positions. Furthermore, the N-acetyl group can be replaced with either a sulfate group or a proton (which generates a free amino group). The uronic acid can be remodeled by adding a sulfate at C2, or by epimerization at C5, which converts glucuronic acid (only C5 region shown) to iduronic acid.

that almost all eukaryotic tissues and cells employ HSPGs in multiple roles.

3.2. The structure of the HS chain

Apart from the multiplicity of core proteins, the HS component of HSPGs further amplifies their complexity and functional diversity. HS is a heterogeneous linear polysaccharide consisting of a repeated disaccharide unit of glucuronic or iduronic acid alternating with glucosamine [hexuronic acid β1→4 N-acetylglucosamine (GlcNAc) α1→4], that is partially decorated with N- and various O-sulfate groups (Fig.1B). The specific arrangement of the sulfate moieties, in large part, gives rise to distinct binding motifs that interact with an increasingly expansive list

Table 2.. HSPG core proteins (derived from Refs. 11 and 15).

of protein effectors (Table 1).12,18,19 The HS chains are quite long ( 40– 80 nm) and range from 100 to 200 disaccharide units, respectively. Each chain is internally repetitive, containing short blocks of minimal sulfation alternating with blocks of highly sulfated motifs (reviewed in Refs. 11, 16 and 17). Multiple copies of different ligand binding motifs can occur on a single HS chain;20 consequently, HS is exquisitely suited to

positioned sulfate groups by transferring a sulfuryl group from the universal “sulfate” donor 3 phosphoadensosine-5 -phosophosulfate (PAPS) (reviewed in Ref. 15). Although many functions are conveyed by core proteins, they do not play a substantial role in defining the types of HS motifs that occur upon their HS chains.21 Rather, the sequencespecific properties of the various modification enzymes is the critical factor that dictates the production of specific motifs.

Synthesis begins by the assembly of a short linkage tetrasaccharide at defined serine residues of the core proteins (Fig. 2). This step initiates both HS and CS synthesis, which explains why many HSPGs can also carry CS chains. CS exhibits different biological activities than HS due to its distinct structure; CS chains, compared to HS, contain galactosamine instead of GlcNAc, are sulfated in some distinct positions and are shorter. Commitment of the primed structure specifically down the HS pathway is determined by the addition of a GlcNAc residue by either EXTL2 or EXTL3, which are distinct isoforms of GlcNAc transferase I. Conversely, addition of a galactosamine residue triggers CS synthesis. The HS backbone is then polymerized by the simultaneous action of enzymes from the genes EXT1 and EXT2 (reviewed in Refs. 12 and 13).

The copolymer backbone is then remodeled by a semi-ordered series of reactions, which generate distinct HS motifs. First, the bifunctional enzyme HS N-deacetylase/N-sulfotransferase (NDST) deacetylates and N-sulfates subsets of N-acetylglucosamine residues.22,23 Occasional residues escape sulfation, which results in a free amino group. Blocks containing a high density of N-sulfated glucosamine are then preferential substrates for the subsequent, less frequent modification reactions. The HS C5 epimerase transforms occasional glucuronic acid residues into iduronic acid.24,25 The HS 2-sulfotransferase (HS2ST) next produces 2-O-sulfated iduronic acid or to a much lesser degree 2-O-sulfated glucuronic acid.26,27 Occasional glucosamine residues undergo 6-O- sulfation by HS 6-O-sulfotransferase (HS6ST).28 “Late” modification also includes the generation of 3-O-sulfated glucosamine by HS 3-O- sulfotransferase (HS3ST, also known as 3OST).21,29−31

The NDST, HS6ST and HS3ST enzymes have multiple isozymes encoded by distinct genes. This multiplicity serves two purposes. First, multiple isoforms allow for cell type-specific production of distinct HS