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 1β, 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. 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 1β, 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. Hyaluronan (HA) 3The abbreviations used are: HAhyaluronan4-MU4-methylumbeliferonAMAC2-aminoacridoneCSchondroitin sulfateERendoplasmic reticulumDTTdithiothreitolGAGglycosaminoglycanHAShyaluronan synthaseIL-1βinterleukin 1βPAGEFSpolyacrylamide gel electrophoresis of fluorophore-labeled saccharidesPDGF-BBplatelet-derived growth factor-BBPMAphorbol 12-myristate 13-acetateSAPshrimp alkaline phosphataseFBSfetal bovine serumPBSphosphate-buffered salineRTreverse transcriptionANOVAanalysis of variancePMplasma membraneCMcytosolic membrane(s)NMnuclear membrane(s)Δdi-nonSHA and Δ-HAΔ-disaccharides of HA (non-sulfated)Δdi-nonScsdisaccharides of CS (non-sulfated)Δdi-mono6cschondroitin 6, sulfate disaccharideΔdi-mono4cschondroitin 4, sulfate disaccharideΔdi-mono2cschondroitin 2, sulfate disaccharide. is the only non-sulfated linear polymer belonging to the family of glycosaminoglycans (GAGs). HA is an unbranched polymer of alternating GlcNAc and GlcUA residues linked by alternate β(1→4) and β(1→3) bonds. Native HA is typically larger than other GAGs, reaching molecular mass values between 106 and 107 Da. hyaluronan 4-methylumbeliferon 2-aminoacridone chondroitin sulfate endoplasmic reticulum dithiothreitol glycosaminoglycan hyaluronan synthase interleukin 1β polyacrylamide gel electrophoresis of fluorophore-labeled saccharides platelet-derived growth factor-BB phorbol 12-myristate 13-acetate shrimp alkaline phosphatase fetal bovine serum phosphate-buffered saline reverse transcription analysis of variance plasma membrane cytosolic membrane(s) nuclear membrane(s) Δ-disaccharides of HA (non-sulfated) disaccharides of CS (non-sulfated) chondroitin 6, sulfate disaccharide chondroitin 4, sulfate disaccharide chondroitin 2, sulfate disaccharide. 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M. P. Y. M. T. M. Y. Y. S. Spicer A.P. McDonald J.A. J. Biol. PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar, A.P. Biol. Google Scholar, L. P. J. PubMed Scopus (39) Google Scholar, T. F. S. R. M. J. Biol. 2004; PubMed Scopus Google and HAS activity by in newly synthesized on the other involved with of the that this In to the newly synthesized HA in non-radioactive we an method to Δ-disaccharides from of newly synthesized HA M. G. De Luca G. Hascall V.C. Passi A. 2004; PubMed Scopus Google Scholar). This method with a and the has to Moreover, the of the not the of UDP-sugar precursors, is an with the we on this method to assay HAS activity using was developed and PubMed Scopus Google Scholar). we prepared three the and the fraction and for plasma membrane and the of CD44 in the membrane to the cytosolic HA only to and in the of the of HAS activity in cytosol membrane the question of HA the cells in In J. 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M. 2004; PubMed Scopus Google Scholar). after tunicamycin that HAS proteins In the of in proteins from cells HAS activity to to in proteins prepared from cells in was by the treatment. is to be as for other not of incubation to a protein we used protein and that with tunicamycin from tunicamycin that new HAS not to the HA Moreover, a increase of HAS activity in was an of plasma membrane was used in with that of HAS from plasma membrane is to to the HA activity in the membranes. has been to of the involving HA be that CD44 was on the plasma membrane that of HA is but we the of HA an alteration of the HA to treatment. HA described after by with in and in Hascall V.C. T. 2006; PubMed Scopus Google Scholar). HA for cells and have a in inflammation and for Hascall V.C. J. J. 2003; PubMed Scopus Google Scholar). that HA to have not only with the surface of the cells HAS but also with intracellular that to the nuclear V.C. De A. J.A. 2004; PubMed Scopus Google Scholar). Since tunicamycin has been to induce HA cable we the in intracellular HA be involved in HA cable formation. of HA cable we the to cells but found that the was not This that cells not In a cell from a after tunicamycin that this cell was to HA cable in of HA cable was by that cells cells HA that the of and cells to HA cable increase HA in to tunicamycin not known but for the is that alteration of HAS activity have a in cable and we found that cell HA activity in in and membranes. Interestingly, HAS activity in the plasma membrane in to tunicamycin in to HA but not in a the other the of HAS in the cell in cells and in have a in this Interestingly, and on the that have analysis that only has residues and in the in the with that be residues in the cytosolic of the for this This the that such not directly on the HAS but in other not that and HAS of HAS on cells that of HA cable of Hascall V.C. T. 2006; PubMed Scopus Google Scholar, C. T. Hascall V.C. J. 2006; 17: PubMed Scopus Google Scholar). In by using the new non-radioactive method to HAS activity in we to the question the HAS activity in and the of and phosphorylation to HAS activity and the of intracellular in the HA cable formation. We to of for for the cell for and for We the for with