*2.4. Hyaluronan*

Transmembrane enzymes denominated HA synthases (HAS) produce HA chains. The three HAS isoforms, HAS1, HAS2, and HAS3, use cytoplasmic UDP-glucuronic acid

and UDP-N-acetylglucosamine as substrates. Their active site is localized intracellularly, whereas the synthesized HA chain extrudes into the ECM [13]. This non-sulfated GAG is composed of repeating units of GlcNAc and GlcA combined by β-1.3 and β-1.4 linkages, with an average mass of 100–2000 kDa [13]. HAS1 and HAS2 synthesize a high molecular weight polymer, whereas HAS3 produces shorter chains (~2 × 10<sup>6</sup> Da vs. ~2 × 10<sup>5</sup> Da, respectively) [69]. HA's biological information is translated to the length of its polymers and defines its effects [70]. The UDPsugar precursors and holistic cell metabolism responsible for producing HAS substrates critically regulate HASs activities [71]. HA-mediated effects are executed through various mechanisms that involve the binding of HA to surface receptors such as CD44 and RHAMM [72–74] and the internalization of HA through receptor-mediated endosomal pathways [75].

The human genome contains five active hyaluronidases (Hyals) (Hyal1–Hyal4 and PH-20) and the non-transcribed Hyal pseudogene (HyalP1). Hyal 2 and 3 exhibit degrading activity, exclusively for HA [76]. Some human Hyals exhibit degrees of CS-degrading activity. Thus, PH20 shows high activity for HA and low CS-degrading activity. On the other hand, Hyal1 degrades CS-A more swiftly than HA [77]. Hyal-4 is misnamed, as it shows specificity for CS and no ability to degrade HA [78].

Hyal1 is widely expressed and localized to lysosomes or trafficking vesicles [79]. However, Hyal 1 can also be secreted to the ECM by tumor cells [80]. Hyal1 is upregulated in many human cancers and has been correlated with tumorigenesis [81].

In contrast, Hyal2 is bound onto the cell membrane via a GPI anchor and is usually associated with lipid rafts [82], wherein, in common with CD44 and Hyal1, it promotes HA cellular uptake and endocytic internalization [75].
