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motifs (reviewed in Refs. 12 and 13). Second, individual isoforms can exhibit distinct sequence specificities, which further expands the structural diversity of HS.31−33 These concepts are best illustrated by the largest multigene family of HS modification enzymes, the seven distinct isoforms of HS3ST. Despite the family’s large size, 3-O-sulfates are the rarest HS modification (usually comprising < 0.5% of total sulfate moieties). Their rarity make 3-O-sulfates ideal for serving key regulatory roles.21,34 The enzymes can be categorized as either AT-type or gD-type, based on their enzymatic specificities.35 HS3ST1 preferentially modifies a specific pentasaccharide precursor structure to create HS with high affinity binding motifs for antithrombin (HSAT+), and so is an AT-type enzyme. HS3ST1 is clearly regulatory in ECs, as its expression level governs the level of HSAT+ synthesis.29 The interaction of antithrombin with this motif is thought to convey anti-inflammatory and anticoagulant properties to the endothelium. In contrast, HS3ST2, HS3ST3A, HS3ST3B, HS3ST4 and HS3ST6 are gD-type enzymes; they preferentially recognize a distinct precursor structure to create another 3-O-sulfated motif (HSgD+), known to bind glycoprotein gD of herpes simplex virus-1. These isoforms are also regulatory, as cellular entry of this herpes simplex virus-1 can be dependent on cellular expression of gD-type enzymes.36 Although the endogenous ligand for HSgD+ is not yet known, this 3-O-sulfated motif has recently been implicated in Notch signaling, described below. HS3ST5 is able to generate both motifs with equal efficiency, so falls into both classes.37 Although this system appears to show redundancy, most isoforms exhibits unique cell typeand tissue-specific expression patterns. Sequence specificity and cell type-specific expression are also properties of the isoforms of NDST (NDST1 to NDST4) and HS6ST (HS6ST1 to HS6ST3) (reviewed in Refs. 12 and 13). Thus, individual cells produce distinct arrays of HS motifs through the expression of distinct combinations of HS sulfotransferases.

3.4. The post-synthetic processing of HSPGs

Four types of post-biosynthetic processing are known to further modify the properties of HSPGs. First, extracellular heparanases derived