Fig. 2. Heparan sulfate chain biosynthesis. Heparan sulfate (HS) glycosaminoglycan (GAG) chains are synthesized on a core protein by the sequential action of individual glycosyltransferases and modification enzymes, in a three-step process involving chain initiation, polymerization and modification. HS chain synthesis begins with the assembly of a linkage tetrasaccharide on serine residues in the core polypeptide. This process is catalyzed by four enzymes (Xyl transferase, Gal transferase I-II and GlcA transferase I), which add individual sugar residues sequentially to the non-reducing end of the growing chain. After the assembly of the linkage region, one or more -GlcNAc transferases add a single 1,4-linked GlcNAc unit to the chain, which initiates the HS polymerization process. HS chain polymerization then takes place by the addition of alternating GlcA and GlcNAc residues, which is catalyzed by the EXT family proteins. 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. The HS GAG chains are 100 or more sugar units long and have numerous structural heterogeneities. Four Drosophila enzymes, including Botv, Ttv, Sotv and Sfl, which are homologs of vertebrate EXTL3, EXT1, EXT2 and N-deacetylase/N-sulfotransferase, respectively, are highlighted in red. Gal, galactose; GlcNAc, N-acetylglucosamine; GlcA, glucuronic acid; GlcNS, N-sulfoglucosamine; IdoA, iduronic acid.