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1.
Mitsutaka Ogawa Shogo Sawaguchi Takami Kawai Daita Nadano Tsukasa Matsuda Hirokazu Yagi Koichi Kato Koichi Furukawa Tetsuya Okajima 《The Journal of biological chemistry》2015,290(4):2137-2149
Epidermal growth factor (EGF) domain-specific O-linked N-acetylglucosamine (EOGT) is an endoplasmic reticulum (ER)-resident O-linked N-acetylglucosamine (O-GlcNAc) transferase that acts on EGF domain-containing proteins such as Notch receptors. Recently, mutations in EOGT have been reported in patients with Adams-Oliver syndrome (AOS). Here, we have characterized enzymatic properties of mouse EOGT and EOGT mutants associated with AOS. Simultaneous expression of EOGT with Notch1 EGF repeats in human embryonic kidney 293T (HEK293T) cells led to immunoreactivity with the CTD110.6 antibody in the ER. Consistent with the GlcNAc modification in the ER, the enzymatic properties of EOGT are distinct from those of Golgi-resident GlcNAc transferases; the pH optimum of EOGT ranges from 7.0 to 7.5, and the Km value for UDP N-acetylglucosamine (UDP-GlcNAc) is 25 μm. Despite the relatively low Km value for UDP-GlcNAc, EOGT-catalyzed GlcNAcylation depends on the hexosamine pathway, as revealed by the increased O-GlcNAcylation of Notch1 EGF repeats upon supplementation with hexosamine, suggesting differential regulation of the luminal UDP-GlcNAc concentration in the ER and Golgi. As compared with wild-type EOGT, O-GlcNAcylation in the ER is nearly abolished in HEK293T cells exogenously expressing EOGT variants associated with AOS. Introduction of the W207S mutation resulted in degradation of the protein via the ubiquitin-proteasome pathway, although the stability and ER localization of EOGTR377Q were not affected. Importantly, the interaction between UDP-GlcNAc and EOGTR377Q was impaired without adversely affecting the acceptor substrate interaction. These results suggest that impaired glycosyltransferase activity in mutant EOGT proteins and the consequent defective O-GlcNAcylation in the ER constitute the molecular basis for AOS. 相似文献
2.
Davide Vigetti Sara Deleonibus Paola Moretto Timothy Bowen Jens W. Fischer Maria Grandoch Alexander Oberhuber Dona C. Love John A. Hanover Raffaella Cinquetti Eugenia Karousou Manuela Viola Maria Luisa D'Angelo Vincent C. Hascall Giancarlo De Luca Alberto Passi 《The Journal of biological chemistry》2014,289(42):28816-28826
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Kirsi Rilla Sanna Oikari Tiina A. Jokela Juha M. T. Hyttinen Riikka K?rn? Raija H. Tammi Markku I. Tammi 《The Journal of biological chemistry》2013,288(8):5973-5983
Mammals have three homologous genes encoding proteins with hyaluronan synthase activity (Has1–3), all producing an identical polymer from UDP-N-acetylglucosamine and UDP-glucuronic acid. To compare the properties of these isoenzymes, COS-1 cells, with minor endogenous hyaluronan synthesis, were transfected with human Has1–3 isoenzymes. HAS1 was almost unable to secrete hyaluronan or form a hyaluronan coat, in contrast to HAS2 and HAS3. This failure of HAS1 to synthesize hyaluronan was compensated by increasing the cellular content of UDP-N-acetyl glucosamine by ∼10-fold with 1 mm glucosamine in the growth medium. Hyaluronan synthesis driven by HAS2 was less affected by glucosamine addition, and HAS3 was not affected at all. Glucose-free medium, leading to depletion of the UDP-sugars, markedly reduced hyaluronan synthesis by all HAS isoenzymes while raising its concentration from 5 to 25 mm had a moderate stimulatory effect. The results indicate that HAS1 is almost inactive in cells with low UDP-sugar supply, HAS2 activity increases with UDP-sugars, and HAS3 produces hyaluronan at high speed even with minimum substrate content. Transfected Has2 and particularly Has3 consumed enough UDP-sugars to reduce their content in COS-1 cells. Comparison of different human cell types revealed ∼50-fold differences in the content of UDP-N-acetylhexosamines and UDP-glucuronic acid, correlating with the expression level of Has1, suggesting cellular coordination between Has1 expression and the content of UDP-sugars. 相似文献
6.
Xiaogang Gu John Glushka Sung G. Lee Maor Bar-Peled 《The Journal of biological chemistry》2010,285(32):24825-24833
We have identified an operon and characterized the functions of two genes from the severe food-poisoning bacterium, Bacillus cereus subsp. cytotoxis NVH 391-98, that are involved in the synthesis of a unique UDP-sugar, UDP-2-acetamido-2-deoxyxylose (UDP-N-acetyl-xylosamine, UDP-XylNAc). UGlcNAcDH encodes a UDP-N-acetyl-glucosamine 6-dehydrogenase, converting UDP-N-acetylglucosamine (UDP-GlcNAc) to UDP-N-acetyl-glucosaminuronic acid (UDP-GlcNAcA). The second gene in the operon, UXNAcS, encodes a distinct decarboxylase not previously described in the literature, which catalyzes the formation of UDP-XylNAc from UDP-GlcNAcA in the presence of exogenous NAD+. UXNAcS is specific and cannot utilize UDP-glucuronic acid and UDP-galacturonic acid as substrates. UXNAcS is active as a dimer with catalytic efficiency of 7 mm−1 s−1. The activity of UXNAcS is completely abolished by NADH but unaffected by UDP-xylose. A real-time NMR-based assay showed unambiguously the dual enzymatic conversions of UDP-GlcNAc to UDP-GlcNAcA and subsequently to UDP-XylNAc. From the analyses of all publicly available sequenced genomes, it appears that UXNAcS is restricted to pathogenic Bacillus species, including Bacillus anthracis and Bacillus thuringiensis. The identification of UXNAcS provides insight into the formation of UDP-XylNAc. Understanding the metabolic pathways involved in the utilization of this amino-sugar may allow the development of drugs to combat and eradicate the disease. 相似文献
7.
Glycosylation Provides Both Stimulatory and Inhibitory Effects on Cell Surface and Soluble CD44 Binding to Hyaluronan 总被引:11,自引:0,他引:11
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Timothy P. Skelton Chunxun Zeng Aaron Nocks Ivan Stamenkovic 《The Journal of cell biology》1998,140(2):431-446
Glycosylation has been implicated in the regulation of CD44-mediated cell binding of hyaluronan (HA). However, neither the relative contribution of N- and O-linked glycans nor the oligosaccharide structures that alter CD44 affinity for HA have been elucidated. To determine the effect of selective alteration of CD44 oligosaccharide composition on the affinity of CD44 for HA, we developed a novel strategy based on the use of affinity capillary electrophoresis (ACE). Soluble recombinant CD44–immunoglobulin fusion proteins were overproduced in the mutant CHO cell line ldl-D, which has reversible defects in both N- and O-linked oligosaccharide synthesis. Using this cell line, a panel of recombinant glycosidases, and metabolic glycosidase inhibitors, CD44 glycoforms with defined oligosaccharide structures were generated and tested for HA affinity by ACE. Because ldl-D cells express endogenous cell surface CD44, the effect of any given glycosylation change on the ability of cell surface and soluble CD44 to bind HA could be compared. Four distinct oligosaccharide structures were found to effect CD44-mediated HA binding: (a) the terminal α2,3-linked sialic acid on N-linked oligosaccharides inhibited binding; (b) the first N-linked N-acetylglucosamine residue enhanced binding; (c) O-linked glycans on N-deglycosylated CD44 enhanced binding; and (d) N-acetylgalactosamine incorporation into non–N-linked glycans augmented HA binding by cell surface CD44. The first three structures induced up to a 30-fold alteration in the intrinsic CD44 affinity for HA (Kd = 5 to >150 μM). The fourth augmented CD44-mediated cellular HA avidity without changing the intrinsic HA affinity of soluble CD44. 相似文献
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Anne Kultti Riikka K?rn? Kirsi Rilla Pertti Nurminen Elina Koli Katri M. Makkonen Jutong Si Markku I. Tammi Raija H. Tammi 《The Journal of biological chemistry》2010,285(30):22901-22910
Hyaluronan synthases (HAS1–3) are integral plasma membrane proteins that synthesize hyaluronan, a cell surface and extracellular matrix polysaccharide necessary for many biological processes. It has been shown that HAS is partly localized in cholesterol-rich lipid rafts of MCF-7 cells, and cholesterol depletion with methyl-β-cyclodextrin (MβCD) suppresses hyaluronan secretion in smooth muscle cells. However, the mechanism by which cholesterol depletion inhibits hyaluronan production has remained unknown. We found that cholesterol depletion from MCF-7 cells by MβCD inhibits synthesis but does not decrease the molecular mass of hyaluronan, suggesting no major influence on HAS stability in the membrane. The inhibition of hyaluronan synthesis was not due to the availability of HAS substrates UDP-GlcUA and UDP-GlcNAc. Instead, MβCD specifically down-regulated the expression of HAS2 but not HAS1 or HAS3. Screening of signaling proteins after MβCD treatment revealed that phosphorylation of Akt and its downstream target p70S6 kinase, both members of phosphoinositide 3-kinase-Akt pathway, were inhibited. Inhibitors of this pathway suppressed hyaluronan synthesis and HAS2 expression in MCF-7 cells, suggesting that the reduced hyaluronan synthesis by MβCD is due to down-regulation of HAS2, mediated by the phosphoinositide 3-kinase-Akt-mTOR-p70S6K pathway. 相似文献
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Mark E. Lauer Georgiana Cheng Shadi Swaidani Mark A. Aronica Paul H. Weigel Vincent C. Hascall 《The Journal of biological chemistry》2013,288(1):423-431
We tested the hypothesis that the artificial addition of heavy chains from inter-α-inhibitor to hyaluronan (HA), by adding recombinant TSG-6 (TNF-stimulated gene-6) to the culture medium of murine airway smooth muscle (MASM) cells, would enhance leukocyte binding to HA cables produced in response to poly(I:C). As predicted, the addition of heavy chains to HA cables enhanced leukocyte adhesion to these cables, but it also had several unexpected effects. (i) It produced thicker, more pronounced HA cables. (ii) It increased the accumulation of HA in the cell-associated matrix. (iii) It decreased the amount of HA in the conditioned medium. Importantly, these effects were observed only when TSG-6 was administered in the presence of poly(I:C), and TSG-6 did not exert any effect on its own. Increased HA synthesis occurred during active, poly(I:C)-induced HA synthesis and did not occur when TSG-6 was added after poly(I:C)-induced HA synthesis was complete. MASM cells derived from TSG-6−/−, HAS1/3−/−, and CD44−/− mice amplified HA synthesis in response to poly(I:C) + TSG-6 in a manner similar to WT MASM cells, demonstrating that they are expendable in this process. We conclude that TSG-6 increases the accumulation of HA in the cell-associated matrix, partially by preventing its dissolution from the cell-associated matrix into the conditioned medium, but primarily by inducing HA synthesis. 相似文献
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Ashik Jawahar Deen Kirsi Rilla Sanna Oikari Riikka K?rn? Genevieve Bart Jukka H?yrinen Avinash Rahul Bathina Antti Ropponen Katri Makkonen Raija H. Tammi Markku I. Tammi 《The Journal of biological chemistry》2014,289(12):8375-8389
Hyaluronan synthases (HAS1–3) are unique in that they are active only when located in the plasma membrane, where they extrude the growing hyaluronan (HA) directly into cell surface and extracellular space. Therefore, traffic of HAS to/from the plasma membrane is crucial for the synthesis of HA. In this study, we have identified Rab10 GTPase as the first protein known to be involved in the control of this traffic. Rab10 colocalized with HAS3 in intracellular vesicular structures and was co-immunoprecipitated with HAS3 from isolated endosomal vesicles. Rab10 silencing increased the plasma membrane residence of HAS3, resulting in a significant increase of HA secretion and an enlarged cell surface HA coat, whereas Rab10 overexpression suppressed HA synthesis. Rab10 silencing blocked the retrograde traffic of HAS3 from the plasma membrane to early endosomes. The cell surface HA coat impaired cell adhesion to type I collagen, as indicated by recovery of adhesion following hyaluronidase treatment. The data indicate a novel function for Rab10 in reducing cell surface HAS3, suppressing HA synthesis, and facilitating cell adhesion to type I collagen. These are processes important in tissue injury, inflammation, and malignant growth. 相似文献
11.
Geneviève Bart Nuria Ortega Vico Antti Hassinen Francois M. Pujol Ashik Jawahar Deen Aino Ruusala Raija H. Tammi Anthony Squire Paraskevi Heldin Sakari Kellokumpu Markku I. Tammi 《The Journal of biological chemistry》2015,290(18):11479-11490
In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1–3 (HAS1–3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647–23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis. 相似文献
12.
Yi-Kai Niou Wan-Ling Wu Ling-Chun Lin Hung-Yu Shu Guang-Huey Lin 《Biochemical and biophysical research communications》2009,390(2):313-1863
Thermus thermophilus and Thermus aquaticus are thermophilic bacteria that are frequently found to attach to solid surfaces in hot springs to form biofilms. Uridine diphosphate (UDP)-galactose-4′-epimerase (GalE) is an enzyme that catalyzes the conversion of UDP-galactose to UDP-glucose, an important biochemical step in exopolysaccharide synthesis. We expressed GalE obtained from T. thermophilus HB8 in Escherichia coli and found that the enzyme is stable at 80 °C and can epimerize UDP-galactose to UDP-glucose and UDP-N-acetylgalactosamine (UDP-GalNAc) to UDP-N-acetylglucosamine (UDP-GlcNAc). Enzyme overexpression in T. thermophilus HB27 led to an increased capacity of biofilm production. Therefore, the galE gene is important to biofilm formation because of its involvement in epimerizing UDP-galactose and UDP-N-acetylgalactosamine for exopolysaccharide biosynthesis. 相似文献
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Preparation of Uridine Diphosphate-N-Acetylgalactosamine from Uridine Diphosphate-N-Acetylglucosamine by Using Microbial Enzymes
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A method was developed for the large scale preparation of uridine diphosphate-N-acetylgalactosamine (UDP-GalNAc) from uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) by means of microbial enzymes. With Bacillus subtilis cell-free extract as a source of UDP-GlcNAc 4-epimerase, about 35% of the UDP-GlcNAc added was converted to UDP-GalNAc. After the residual UDP-GlcNAc was degraded to uridine triphosphate and N-acetylglucosamine-1-phosphate with a protamine-treated extract of bakers' yeast as a source of UDP-GlcNAc pyrophosphorylase, UDP-GalNAc was separated by anion-exchange column chromatography. The nucleotide was recovered by adsorption on charcoal and elution with ammoniacal ethanol. The final yield was about 100 μmol. 相似文献
15.
Hua-Dong Liu Yu-Fen Zhao Yan-Mei Li 《International journal of peptide research and therapeutics》2006,12(3):237-241
The dynamic modification of many nuclear and cytoplasmic proteins with O-linked beta-N-acetylglucosamine (O-GlcNAc) on serine or threonine is catalyzed by O-GlcNAc transferase (OGT). The conserved GPGTF (glycogen phosphorylase/glycosyl transferase) motif, one of the α-helices of the second domain in OGT, was identified as a putative UDP-GlcNAc binding site. A miniature protein was designed which contains all of the conserved residues of GPGTF motif in the O-GlcNAc transferase, and was shown to adopt an alpha helix in 10% trifluoroethanol. It was anticipated that the miniature protein could shed light on the mechanism of dynamic O-GlcNAc modification and provide a potential drug for the diabetes and neurodegenerative diseases. 相似文献
16.
Yuichiro Kashima Masafumi Takahashi Yuji Shiba Naoki Itano Atsushi Izawa Jun Koyama Jun Nakayama Shun'ichiro Taniguchi Koji Kimata Uichi Ikeda 《PloS one》2013,8(3)
Background
Hyaluronan (HA) is a primary component of the extracellular matrix of cells, and it is involved in the pathogenesis of atherosclerosis. The purpose of this study was to investigate the role of HA in neointimal formation after vascular injury and determine its tissue-specific role in vascular smooth muscle cells (VSMCs) by using a cre-lox conditional transgenic (cTg) strategy.Methods and Results
HA was found to be expressed in neointimal lesions in humans with atherosclerosis and after wire-mediated vascular injury in mice. Inhibition of HA synthesis using 4-methylumbelliferone markedly inhibited neointimal formation after injury. In vitro experiments revealed that low-molecular-weight HA (LMW-HA) induced VSMC activation, including migration, proliferation, and production of inflammatory cytokines, and reactive oxygen species (ROS). The migration and proliferation of VSMCs were mediated by the CD44/RhoA and CD44/ERK1/2 pathways, respectively. Because HA synthase 2 (HAS2) is predominantly expressed in injured arteries, we generated cTg mice that overexpress the murine HAS2 gene specifically in VSMCs (cHAS2/CreSM22α mice) and showed that HA overexpression markedly enhanced neointimal formation after cuff-mediated vascular injury. Further, HA-overexpressing VSMCs isolated from cHAS2/CreSM22α mice showed augmented migration, proliferation, and production of inflammatory cytokines and ROS.Conclusion
VSMC-derived HA promotes neointimal formation after vascular injury, and HA may be a potential therapeutic target for cardiovascular disease. 相似文献17.
Jessica Andrés-Bergós Lidia Tardio Ane Larranaga-Vera Rodolfo Gómez Gabriel Herrero-Beaumont Raquel Largo 《The Journal of biological chemistry》2012,287(40):33615-33628
Insulin is an inducer of chondrocyte hypertrophy and growth plate chondrogenesis, although the specific molecular mechanisms behind these effects are mostly unknown. Our aim was to investigate whether insulin-induced chondrocyte hypertrophy occurs through a modification in the amount of O-linked N-acetylglucosamine (O-GlcNAc)-modified proteins and in the expression of the key enzymes of this pathway, O-GlcNAc transferase and O-GlcNAcase (OGA). We also studied if O-GlcNAc accumulation per se, induced by an OGA inhibitor, was able to induce pre-hypertrophic chondrocyte differentiation both in vitro and in vivo. Insulin-induced differentiation of ATDC5 pre-chondrocytes occurred alongside a gradual increase in the accumulation of O-GlcNac-modified proteins (O-GlcNAcylated proteins), as well as an increase in the expression of O-GlcNAc transferase and OGA. In the absence of insulin, O-GlcNAc accumulation induced by thiamet-G, a specific OGA inhibitor, was able to increase the gene expression of differentiation markers, as well as the activity of MMP-2 and -9. Thiamet-G also activated pERK, p-JNK, and p-p38 and the O-GlcNAcylation of Akt. Thiamet-G administration to C57/bl mice induced a significant expansion in the growth plate height and in the hypertrophic zone height. Therefore, our results show that O-GlcNAc glycosylation has chondromodulating activity. 相似文献
18.
Dorota Maszczak-Seneczko Paulina Sosicka Teresa Olczak Piotr Jakimowicz Micha? Majkowski Mariusz Olczak 《The Journal of biological chemistry》2013,288(30):21850-21860
SLC35A3 is considered the main UDP-N-acetylglucosamine transporter (NGT) in mammals. Detailed analysis of NGT is restricted because mammalian mutant cells defective in this activity have not been isolated. Therefore, using the siRNA approach, we developed and characterized several NGT-deficient mammalian cell lines. CHO, CHO-Lec8, and HeLa cells deficient in NGT activity displayed a decrease in the amount of highly branched tri- and tetraantennary N-glycans, whereas monoantennary and diantennary ones remained unchanged or even were accumulated. Silencing the expression of NGT in Madin-Darby canine kidney II cells resulted in a dramatic decrease in the keratan sulfate content, whereas no changes in biosynthesis of heparan sulfate were observed. We also demonstrated for the first time close proximity between NGT and mannosyl (α-1,6-)-glycoprotein β-1,6-N-acetylglucosaminyltransferase (Mgat5) in the Golgi membrane. We conclude that NGT may be important for the biosynthesis of highly branched, multiantennary complex N-glycans and keratan sulfate. We hypothesize that NGT may specifically supply β-1,3-N-acetylglucosaminyl-transferase 7 (β3GnT7), Mgat5, and possibly mannosyl (α-1,3-)-glycoprotein β-1,4-N-acetylglucosaminyltransferase (Mgat4) with UDP-GlcNAc. 相似文献
19.
《Matrix biology》2014
Hyaluronan (HA) is a glycosaminoglycan composed by repeating units of D-glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) that is ubiquitously present in the extracellular matrix (ECM) where it has a critical role in the physiology and pathology of several mammalian tissues. HA represents a perfect environment in which cells can migrate and proliferate. Moreover, several receptors can interact with HA at cellular level triggering multiple signal transduction responses. The control of the HA synthesis is therefore critical in ECM assembly and cell biology; in this review we address the metabolic regulation of HA synthesis. In contrast with other glycosaminoglycans, which are synthesized in the Golgi apparatus, HA is produced at the plasma membrane by HA synthases (HAS1-3), which use cytoplasmic UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. UDP-GlcUA and UDP-hexosamine availability is critical for the synthesis of GAGs, which is an energy consuming process. AMP activated protein kinase (AMPK), which is considered a sensor of the energy status of the cell and is activated by low ATP:AMP ratio, leads to the inhibition of HA secretion by HAS2 phosphorylation at threonine 110. However, the most general sensor of cellular nutritional status is the hexosamine biosynthetic pathway that brings to the formation of UDP-GlcNAc and intracellular protein glycosylation by O-linked attachment of the monosaccharide β-N-acetylglucosamine (O-GlcNAcylation) to specific aminoacid residues. Such highly dynamic and ubiquitous protein modification affects serine 221 residue of HAS2 that lead to a dramatic stabilization of the enzyme in the membranes. 相似文献
20.
Davide Vigetti Anna Genasetti Evgenia Karousou Manuela Viola Moira Clerici Barbara Bartolini Paola Moretto Giancarlo De Luca Vincent C. Hascall Alberto Passi 《The Journal of biological chemistry》2009,284(44):30684-30694
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)3 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.HA is a major component of extracellular matrices and in pericellular spaces, particularly in tissues with rapid cell proliferation and cell migration (1). Through interactions with cell surface receptors, notably CD44 and RHAMM (receptor for HA-mediated motility), HA has important roles in regulating cell behavior, including signal transduction, cell adhesion, proliferation, migration, and differentiation (2). Recently, novel interactions involving HA and Toll-like receptors 4 and 2 have been described that have important roles in inflammation (3, 4). Moreover, HA has been implicated in morphogenesis (5–8), wound healing (9), angiogenesis (10), malignancies, cancer growth, and tumor invasion (11).In mammals, HA is normally synthesized at the plasma membrane and extruded directly into the extracellular space by three isoforms of HA synthases (HASs), HAS1, -2, and -3. The three HAS isoforms differ in tissue distribution, regulation, and enzymatic properties (12); nevertheless, they are similar in amino acid sequences and molecular structures.HA biosynthesis is under the control of a wide variety of cytokines and growth factors (13). The changes in HA synthesis can be related to HAS mRNA expression (14), to availability of the UDP-sugar precursors (15, 16), or to modulation by phosphorylation of HAS (17–19) in response to cytokines and growth factors. Moreover, HA chain synthesis can be controlled by additional mechanisms, such as cell type, intracellular environment, or HAS accessory proteins (20). Cultures of smooth muscle cells isolated from human colon increase synthesis of HA after treatment with a viral mimetic molecule (poly(I-C)) (21). The HA is organized into novel cable-like structures, and their synthesis may be initiated in the perinuclear and/or the endoplasmic reticulum (ER) membranes (22). Furthermore, HA interstitial deposition is correlated with inflammatory processes (23, 24) in which HA-CD44 interactions stimulate leukocyte adhesion in order to generate an inflammatory response (25).Cytokines and growth factors, such as IL-1β and platelet-derived growth factor BB (PDGF-BB), as well as 4-methylumbeliferone (4-MU) and the tumor promoter phorbol 12-myristate 13-acetate (PMA), also modulate HA synthesis (26–29). In order to elucidate how these different effectors affect HAS activity, it is important to purify and solubilize the HAS enzymes as previously underlined in studies on eukaryotic cell lines (30). In this context, Itano and Kimata (31) used a mammalian transient expression system to characterize the three different HAS isoforms in either cells or cellular membrane extracts. On the other hand, Spicer (32) described three relatively simple procedures for the detection of HA synthase activity in cultured mammalian cell lines. In all of these studies, the enzyme activity was measured by incubating cellular membrane extracts with radiolabeled UDP-sugar precursors, and the final analysis of the products was done by liquid scintillation counting. Various other strategies and methods can be used to determine the HA biosynthetic capacity of cells, although they are always based on the use of radiolabeled UDP-sugar precursors (33–35).In our previous studies, we described methods of polyacrylamide gel electrophoresis of fluorophore-labeled saccharides (PAGEFS) and high performance liquid chromatography (HPLC) for the analysis of disaccharides derived from HA and chondroitin sulfate (33–38). In order to improve the sensitivity of this method, a derivatization with 2-aminoacridone (AMAC) was done, followed by fluorescence detection (39). In this study, we modified this method to address the question of localization of HAS activity during intracellular trafficking, since HA has been detected inside cells in previous studies (40–42). This new non-radioactive method was used to quantify HAS activity on cell membranes fractionated by sucrose gradient methods. To test the robustness of our approach, we analyzed the effect of 4-MU, PMA, IL-1β, PDGF-BB, and tunicamycin on cell cultures. In particular, we found that tunicamycin induced an increase of HA synthesis in both plasma and internal cell membranes in EVC cells, whereas it increased HA synthesis only in the internal cell membranes in the OVCAR-3 cells. The results suggest that post-translational modulation of HAS activity is responsible for the increased HA synthesis inside the cells. Moreover, since tunicamycin induced HA cable structures in the OVCAR-3 cells, we correlated the altered intracellular HAS activity with the capability to promote HA cable formation. 相似文献