| Title | Modulation of hyaluronan synthase activity in cellular membrane fractions. | | Author(s) | Vigetti D, Genasetti A, Karousou E, Viola M, Clerici M, Bartolini B, Moretto P, De Luca G, Hascall VC, Passi A | | Institution | DSBSC - University of Insubria, Italy; | | Source | J Biol Chem 2009 Sep 8. | | 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. As the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of 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 (ER) and Golgi), and nuclei. After incubation with UDP-sugar precursors, newly synthesized HA was quantified by polyacrylamide gel electrophoresis of fluorophore-labeled saccharides (PAGEFS) and HPLC. 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 (PMA), interleukin 1beta (IL-1b), platelet-derived growth factor BB (PDGF-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. As 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. | | Language | ENG | | Pub Type(s) | JOURNAL ARTICLE
| | PubMed ID | 19737932 |
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