Molecular diversity of heparan sulfate (original) (raw)

The fine structure of the chains ultimately depends on the regulated expression and action of multiple glycosyltransferases, sulfotransferases, and an epimerase, which are arrayed in the lumen of the Golgi apparatus (Figure 1). In addition, a series of cytoplasmic enzymes are needed to catalyze nucleotide sugar (UDP-Xyl, UDP-Gal, UDP-GlcA, UDP-GlcNAc) and nucleotide sulfate (PAPS) formation, and multiple membrane transporters to import the nucleotides from the cytosol to the lumen of Golgi apparatus (24, 25). The assembly process may also depend on the availability of GlcN or other sugars in the diet. Synthesis initiates through the assembly of a linkage tetrasaccharide, GlcAβ1,3Galβ1,3Galβ1,4Xylβ1-, on serine residues in the proteoglycan core polypeptide (26). This process is catalyzed by four enzymes that add individual sugar residues sequentially to the nonreducing end of the growing chain. The initiating reaction, catalyzed by xylosyltransferase, occurs at specific sites, defined by Ser-Gly residues flanked by one or more acidic residues (27). The linkage region also undergoes phosphorylation at C2 of xylose and sulfation at C4 or C6 of the galactose residues, but the functions of these modifications remain unclear (26). These linkage region modifications tend to be substoichiometric and are absent in some proteoglycans. Phosphorylation may be transient, suggesting a role in secretion or in regulation of the assembly process.

After assembly of the linkage region, one or more α-GlcNAc transferases add a single α1,4-linked GlcNAc unit to the chain, which commits the intermediate to the assembly of HS. Competition exists between this reaction and the addition of a β1,4-linked GalNAc residue catalyzed by a separate enzyme. When the latter reaction occurs, the intermediate serves as a primer for chondroitin sulfate formation. Evidence suggests that amino acid determinants lying close to the glycosaminoglycan attachment site or structural domains at some distance away regulate this process, with chondroitin sulfate representing the default pathway (27). Polymerization of HS then takes place by the alternating addition of GlcAβ1,4 and GlcNAcα1,4 residues, catalyzed by proteins now recognized as members of the exostosin family of tumor suppressors (see Duncan et al., this Perspective series, ref. 28). As the chain polymerizes, it undergoes a series of modifications that include GlcNAc N-deacetylation and N-sulfation, C5 epimerization of GlcA to IdoA, and variable O-sulfation at C2 of IdoA and GlcA, at C6 of GlcNAc and GlcNS units, and occasionally at C3 of GlcN residues (Figure 1). The concerted action of the enzymes catalyzing these reactions results in the formation and organization of NA, NS, and NA/NS domains (16).

Most of the enzymes involved in modifying the chain have now been purified and molecularly cloned. The GlcNAc N-deacetylase/N-sulfotransferase (NDST), the glucosaminyl 6-O-sulfotransferases (6OST), and the glucosaminyl 3-O-sulfotransferases (3OST) each represent a gene family whose members appear to be expressed in a tissue-specific and developmentally regulated pattern. Four NDSTs, five 3OSTs, and three 6OSTs are known. Substrate specificity studies performed in vitro indicate that the individual members of each enzyme subfamily catalyze the same reaction, but in different chemical contexts. For example, 3OST-1 is the only 3OST isozyme that can form the antithrombin binding sequence (i.e., domains containing GlcA-GlcNS3S). In contrast, 3OST-2 transfers sulfate to GlcA2S-GlcNS and IdoA2S-GlcNS, whereas 3OST-3A transfers sulfate to IdoA2S-GlcN, where the GlcN has an unsubstituted amino group, thus generating the binding site for the viral gD glycoprotein (29, 30). The three 6-O-sulfotransferases add sulfate to the C6 of GlcN units, but the preferred location of the target relative to GlcA and IdoA varies (31). The four NDST isozymes show variation in relative ratios of N-deacetylase and N-sulfotransferase activity. Modeling studies of the NDSTs against the crystal structure of the sulfotransferase domain of NDST1 (32) suggest that modulations of the binding cleft for the sugar chain may confer different substrate specificities for the enzymes (33).

In contrast to these sulfotransferases, only one 2-O-sulfotransferase (2OST) (34, 35) and one epimerase (36, 37) appear to exist in vertebrates. A survey of lower organisms has shown only single isozymes for the other sulfotransferases, suggesting that the ancestral forms of these enzymes perform all the basic reactions of HS biosynthesis required to generate the diversity of structure necessary for the various biological activities essential to the organisms.

In spite of the detailed information available about primary sequence of the isozymes and the evolving information about their substrate preferences, the formation of binding sites for antithrombin and glycoprotein gD remain the only known examples in which formation of a biologically significant structure can be correlated with expression of a specific isozyme (3OST-1 and 3OST-3A, respectively) (14, 30). Notably, these examples both involve a “rare” component, i.e. the 3-O-sulfated GlcN residue, and are therefore, in a sense, conceptually simple compared with ligands that depend on the differential topology of major building blocks that are present in most HS species. Examples of such ligands include members of the FGF family, FGF1 and FGF2, with different binding requirements for GlcN 6-O-sulfate and IdoA 2-O-sulfate groups that are expressed in a selective fashion on distinct HS species (see Gallagher, this Perspective series, ref. 20). The precise structures of the corresponding binding sites and their distribution in HS chains are unknown. Clearly, a major effort in the field should aim at understanding the functional properties of the biosynthetic enzymes, as required to generate specific (or sometimes overlapping) binding sites in HS chains for a variety of protein ligands.

New insights into this problem should emerge from multiple directions. First, it should be possible to refine our understanding of the catalytic specificities of the various isozymes using recombinant enzymes with chemically defined substrates. Recent advances in carbohydrate synthesis (38, 39) represent a step in the right direction, but ultimately large scale synthesis of oligosaccharide libraries will be needed to elucidate substrate specificities in depth. Secondly, we need to understand the topographic organization of the biosynthetic apparatus, including the localization of enzymes in the Golgi membrane, their interaction with each other and with any auxiliary proteins, and their mode of processing the polysaccharide substrate. This objective can be approached in a variety of ways, ranging from immunochemical localization of native and mutated proteins in the cell (M.A.S. Pinhal and J.D. Esko, unpublished work) to the not yet realized assembly of model biosynthetic systems, using artificial membranes and recombinant proteins.