Modulation of hyaluronan synthase activity in cellular membrane fractions.
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.
Vigetti
Davide
D
Dipartimento di Scienze Biomediche Sperimentali e Cliniche, Università degli Studi dell'Insubria, 21100 Varese, Italy.
Genasetti
Anna
A
Karousou
Evgenia
E
Viola
Manuela
M
Clerici
Moira
M
Bartolini
Barbara
B
Moretto
Paola
P
De Luca
Giancarlo
G
Hascall
Vincent C
VC
Passi
Alberto
A
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
D000900:Anti-Bacterial Agents; D014415:Tunicamycin; D014453:Glucuronosyltransferase; C523794:HAS1 protein, human; C101754:HAS2 protein, human; C105458:HAS3 protein, human; D000076002:Hyaluronan Synthases