title: Modulation of hyaluronan synthase activity in cellular membrane fractions.
abstract: Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in eukaryotic cells and addressed the question of HAS activity during intracellular protein trafficking. We prepared three cellular fractions: plasma membrane, cytosol (containing membrane proteins mainly from the endoplasmic reticulum and Golgi), and nuclei. After incubation with UDP-sugar precursors, newly synthesized HA was quantified by polyacrylamide gel electrophoresis of fluorophore-labeled saccharides and high performance liquid chromatography. This new method measured HAS activity not only in the plasma membrane fraction but also in the cytosolic membranes. This new technique was used to evaluate the effects of 4-methylumbeliferone, phorbol 12-myristate 13-acetate, interleukin 1beta, platelet-derived growth factor BB, and tunicamycin on HAS activities. We found that HAS activity can be modulated by post-translational modification, such as phosphorylation and N-glycosylation. Interestingly, we detected a significant increase in HAS activity in the cytosolic membrane fraction after tunicamycin treatment. Since this compound is known to induce HA cable structures, this result links HAS activity alteration with the capability of the cell to promote HA cable formation.
authors.affiliation: Dipartimento di Scienze Biomediche Sperimentali e Cliniche, Università degli Studi dell'Insubria, 21100 Varese, Italy.
authors.forename: Davide
authors.initials: D
authors.lastname: Vigetti
authors.forename: Anna
authors.initials: A
authors.lastname: Genasetti
authors.forename: Evgenia
authors.initials: E
authors.lastname: Karousou
authors.forename: Manuela
authors.initials: M
authors.lastname: Viola
authors.forename: Moira
authors.initials: M
authors.lastname: Clerici
authors.forename: Barbara
authors.initials: B
authors.lastname: Bartolini
authors.forename: Paola
authors.initials: P
authors.lastname: Moretto
authors.forename: Giancarlo
authors.initials: G
authors.lastname: De Luca
authors.forename: Vincent C
authors.initials: VC
authors.lastname: Hascall
authors.forename: Alberto
authors.initials: A
authors.lastname: Passi
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D014453:Glucuronosyltransferase / Q000032:analysis / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D000076002:Hyaluronan Synthases; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D014415:Tunicamycin; D014453:Glucuronosyltransferase; C523794:HAS1 protein, human; C101754:HAS2 protein, human; C105458:HAS3 protein, human; D000076002:Hyaluronan Synthases