*2.2. Heparan Sulfate Biochemical and Structural Features*

HSPGs are composed by a core protein with covalently linked HS chains, whose length ranges between 50–400 disaccharide units [25]. The glycan portion directly attached to the protein is termed tetrasaccharide linker and is composed by a residue of xylose (Xyl) directly attached to the core protein, two galactose (Gal) residues and one GlcA residue. This region is followed by repeating disaccharide units of glucosamine and uronic acid residues. It is the sulfation pattern of these repeating units that generate large structural and functional diversity. The glucosamine residues can either be *N*-sulfated (GlcNS) or *N*-acetylated (GlcNAc), both of which can suffer 6-*O*-sulfation (GlcNS(6S) and GlcNAc(6S)). GlcNS and GlcNS (6S) can also be further 3-*O*-sulfated (GlcNS(3S) and GlcNS(3,6S)). The uronic acid residues that can either be GlcA or its epimer Iduronic Acid (IdoA), can also be 2-*O*-

sulfated (IdoA(2S) and GlcA(2S)) [6,25,26]. These sulfation and epimerisation reactions give rise to, at least, 23 different HS disaccharide structures that constitute the sulfated (S)-domains subsequently repeated through the chains. The *S*-domains are intercalated by *N*-acetylated (NA)-domains, which are enriched in less modified disaccharides, providing great variability within HS polysaccharides [25].

HS biosynthesis occurs in Golgi apparatus or at the endoplasmic reticulum (ER)-Golgi interface, and is organised in three major events: (i) GAG-protein tetrasaccharide linker assembly, through which HS are covalently attached to particular serine residues in the PG core protein; (ii) HS chains polymerisation; and (iii) structural modifications of the elongated chains [25]. The first two stages include a series of sequential steps catalysed by different glycosyltransferases. It starts with the transfer of a Xyl residue, catalysed by two *O*-xylosyltransferases (XYLT1 and XYLT2), followed by the addition of a Gal residue, by Galactosyltransferase-I/β4-Galactosyltransferase 7 (β4Gal-T7) and subsequent transient phosphorylation of the Xyl residue mediated by the kinase FAM20B. This last step is essential for the following reactions of assembly, as it enhances the activity of subsequent glycosyltransferases, namely, the Galactosyltransferase-II/β3-Galactosyltransferase 6 (β3Gal-T6), which will then add the second residue of Gal to the nascent polysaccharide chain [27,28]. The biosynthesis of the tetrasaccharide linker (GlcAβ1-3Gal-β1-3Gal-β1- 4Xyl-β1-*O*-Ser) is completed once the Glucuronyltransferase I (GlcAT-I) adds a GlcA residue to the extremity of the chain, in a reaction step that occurs simultaneously with the dephosphorylation of the Xyl residue by the 2-Phosphoxylose phosphatase (XYLP) [29].

Knock-out (KO) cellular glycoengineering showed that abrogation of XYLT2 in CHO cells that do not express XYLT1, abolished HS biosynthesis. Additionally, elimination of *B4galt7* and *B3gat3* (GlcAT-I) gene expression also fully impaired GAGs biosynthesis, while the KO of the genes coding for the enzymes β3Gal-T6 and FAM20B only reduced its synthesis [30]. Koike et al. conducted silencing experiments in HeLa cells and observed great reduction of the levels of HS chains in lower XYLP expressing cells, suggesting that the dephosphorylation of xylose residues is necessary for correct tetrasaccharide linker assembly [29]. However, more recently, it was determined that the KO of *Pxylp1*, performed on CHO cells, did not alter the levels of GAGs [30]. These results indicate that the role of XYLP in the maturation of the tetrasaccharide linker might be dependent of the cellular context.

The above-mentioned enzymatic steps are common to the biosynthesis of heparin/HS and CS/DS GAG chains, while the following events dictate the biosynthesis of a particular type of GAG chains. Focusing on HS, the addition of a GlcNAc residue to the linkage tetrasaccharide initiates the polymerisation of these chains (in detriment of the polymerisation of CS chains). This reaction involves the catalytic activity of two members of the Exostosin (EXT) family, EXT-like proteins 2 and 3 (EXTL2 and EXTL3), and is followed by further elongation promoted by a hetero-oligomeric complex formed by EXT1 and EXT2 that mediates the intercalated transfer of GlcNAc and GlcA residues [31–34].

EXTL3 acts as a highly efficient α1,4-GlcNAc transferase towards mature tetrasaccharide linkers by adding the first GlcNAc to the HS chains [32]. Different in vitro and in vivo models have revealed that KO of *EXTL3* results in the abolition of HS biosynthesis, uncovering the crucial role of this enzyme in initiating the elongation of HS chains [30,35,36]. The regulatory activity of EXTL2 in this step stills raises significant doubt. EXTL2 is characterised as an α1,4-*N*-acetylhexosaminyltransferase, displaying dual in vitro catalytic activity by adding both GlcNAc and GalNAc residues to linker mimetics. It has been demonstrated that this glycosyltransferase cannot add GlcNAc residues to mature tetrasaccharide linker substrates [33], however it exhibits significant *N*-acetylglucosamine-transferase activity towards phosphorylated forms of the tetrasaccharide linker. By adding a GlcNAc residue to immature linker structures (GlcAβ1–3Galβ1–3Galβ1-4Xyl(2-*O*-phosphate)-β1- *O*-Ser), EXTL2 promotes the synthesis of phosphorylated pentasaccharides (GlcNAcα1- 4GlcUAβ1–3Galβ1–3Galβ1-4Xyl(2-*O*-phosphate)-β1-*O*-Ser) that neither EXT1 nor EXT2 can further polymerise, ultimately resulting in premature HS chains termination [37]. This is in accordance with the increased HS content reported in EXTL2 KO cell models [30] and EXTL2 deficient mice [37,38].

Once polymerised, HS chains are matured by HS modifying enzymes, including *N*-Deacetylase/*N*-Sulfotransferases (NDST1-4), C5-epimerase and different Sulfotransferases (2OST, 6OSTs, 3OSTs) and sulfatases (Sulf-1 and Sulf-2) [6,39]. HS chain features are not directly encoded by the genome, showing a high level of heterogeneity and large structural diversity in terms of monomer sequence, chain length and sulfation profile, all due to post-translational modifications regulated in the Golgi [25]. Therefore, the resulting HS chains are involved in multiple biological processes, varying over different organs [40,41], stages of development [42–44] and pathologies [45,46]. HS chains sulfation and length are crucial to the roles displayed by HSPGs, as these determine the binding affinity to the respective targets. The HS sulfation degree, in particular, confers high negative charge to GAGs, prompting HSPGs to interact, in a non-covalent ionic manner, with several proteins [12].
