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Cellular content of UDP-N-acetylhexosamines controls hyaluronan synthase 2 expression and correlates with O-linked N-acetylglucosamine modification of transcription factors YY1 and SP1.

ABSTRACT: Hyaluronan, a high molecular mass polysaccharide on the vertebrate cell surface and extracellular matrix, is produced at the plasma membrane by hyaluronan synthases using UDP-GlcNAc and UDP-GlcUA as substrates. The availability of these UDP-sugar substrates can limit the synthesis rate of hyaluronan. In this study, we show that the cellular level of UDP-HexNAc also controls hyaluronan synthesis by modulating the expression of HAS2 (hyaluronan synthase 2). Increasing UDP-HexNAc in HaCaT keratinocytes by adding glucosamine down-regulated HAS2 gene expression, whereas a decrease in UDP-HexNAc, realized by mannose treatment or siRNA for GFAT1 (glutamine:fructose-6-phosphate amidotransferase 1), enhanced expression of the gene. Tracing the UDP-HexNAc-initiated signal to the HAS2 promoter revealed no change in the binding of STAT3, NF-κB, and cAMP response element-binding protein, shown previously to mediate growth factor and cytokine signals on HAS2 expression. Instead, altered binding of SP1 and YY1 to the promoter correlated with cellular UDP-HexNAc content and inhibition of HAS2 expression. siRNA silencing of YY1 and SP1 confirmed their inhibitory effects on HAS2 expression. Reduced and increased levels of O-GlcNAc-modified SP1 and YY1 proteins were associated with stimulation or inhibition of HAS2 expression, respectively. Our data are consistent with the hypothesis that, by regulating the level of protein O-GlcNAc modifications, cellular UDP-HexNAc content controls HAS2 transcription and decreases the effects on hyaluronan synthesis that would result from cellular fluctuations of this substrate.

SUBMITTER: Jokela TA

PROVIDER: S-EPMC3190925 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Role of UDP-N-acetylglucosamine (GlcNAc) and O-GlcNAcylation of hyaluronan synthase 2 in the control of chondroitin sulfate and hyaluronan synthesis.

Project description:Hyaluronan (HA) is a glycosaminoglycan present in most tissue microenvironments that can modulate many cell behaviors, including proliferation, migration, and adhesive proprieties. In contrast with other glycosaminoglycans, which are synthesized in the Golgi, HA is synthesized at the plasma membrane by one or more of the three HA synthases (HAS1-3), which use cytoplasmic UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. Previous studies revealed the importance of UDP-sugars for regulating HA synthesis. Therefore, we analyzed the effect of UDP-GlcNAc availability and protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAcylation) on HA and chondroitin sulfate synthesis in primary human aortic smooth muscle cells. Glucosamine treatment, which increases UDP-GlcNAc availability and protein O-GlcNAcylation, increased synthesis of both HA and chondroitin sulfate. However, increasing O-GlcNAcylation by stimulation with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate without a concomitant increase of UDP-GlcNAc increased only HA synthesis. We found that HAS2, the main synthase in aortic smooth muscle cells, can be O-GlcNAcylated on serine 221, which strongly increased its activity and its stability (t(½) >5 h versus ∼17 min without O-GlcNAcylation). S221A mutation prevented HAS2 O-GlcNAcylation, which maintained the rapid turnover rate even in the presence of GlcN and increased UDP-GlcNAc. These findings could explain the elevated matrix HA observed in diabetic vessels that, in turn, could mediate cell dedifferentiation processes critical in vascular pathologies.

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Project description: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.

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Project description: Not available

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Plant UDP-N-acetylglucosamine (UDP-GlcNAc) transporter (UGNT1) glycopeptide analysis

Project description:Glycosylation reactions require activated glycosyl donors in form of nucleotide sugars to drive processes such as post-translational protein modifications, glycolipid and polysaccharide biosynthesis. Most of these reactions occur in the Golgi requiring cytosolic-derived nucleotide sugars, which are actively transferred into the Golgi lumen by nucleotide sugar transporters. Here we present the identification of the plant UDP-N-acetylglucosamine (UDP-GlcNAc) transporter (UGNT1) indispensable for the delivery of a substrate for maturation of N-glycans and glycosyl inositol phosphorylceramides (GIPCs). Profiles of N-glycopeptides revealed that UGNT1 loss-of-function mutants are devoid of complex and hybrid N-glycans. Instead, most of the glycol-N-peptide population contained high mannose structures, representing the structure prior to the addition of the first GlcNAc in the Golgi. Our findings emphasize that the reference plant Arabidopsis contains a single UDP-GlcNAc transporter responsible for the maturation of complex N-glycans in the Golgi lumen.

2018-09-24 | PXD006635 | Pride

Structure of MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) from Vibrio fischeri in complex with substrate UDP-N-acetylglucosamine and the drug fosfomycin.

Project description:The development of new antibiotics is necessitated by the rapid development of resistance to current therapies. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), which catalyzes the first committed step of bacterial peptidoglycan biosynthesis, is a prime candidate for therapeutic intervention. MurA is the target of the antibiotic fosfomycin, a natural product produced by Streptomyces. Despite possessing a high degree of sequence conservation with MurA enzymes from fosfomycin-susceptible organisms, recent microbiological studies suggest that MurA from Vibrio fischeri (VfiMurA) may confer fosfomycin resistance via a mechanism that is not yet understood. The crystal structure of VfiMurA in a ternary complex with the substrate UDP-N-acetylglucosamine (UNAG) and fosfomycin has been solved to a resolution of 1.93 Å. Fosfomycin is known to inhibit MurA by covalently binding to a highly conserved cysteine in the active site of the enzyme. A comparison of the title structure with the structure of fosfomycin-susceptible Haemophilus influenzae MurA (PDB entry 2rl2) revealed strikingly similar conformations of the mobile substrate-binding loop and clear electron density for a fosfomycin-cysteine adduct. Based on these results, there are no distinguishing sequence/structural features in VfiMurA that would translate to a diminished sensitivity to fosfomycin. However, VfiMurA is a robust crystallizer and shares high sequence identity with many clinically relevant bacterial pathogens. Thus, it would serve as an ideal system for use in the structure-guided optimization of new antibacterial agents.

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Project description:UDP-N-acetylglucosamine pyrophosphorylase (UAP1) catalyses the last step in eukaryotic biosynthesis of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), converting UTP and GlcNAc-1P to the sugar nucleotide. Gene disruption studies have shown that this gene is essential in eukaryotes and a possible antifungal target, yet no inhibitors of fungal UAP1 have so far been reported. Here we describe the crystal structures of substrate/product complexes of UAP1 from Aspergillus fumigatus that together provide snapshots of catalysis. A structure with UDP-GlcNAc, pyrophosphate and Mg2+ provides the first Michaelis complex trapped for this class of enzyme, revealing the structural basis of the previously reported Mg2+ dependence and direct observation of pyrophosphorolysis. We also show that a highly conserved lysine mimics the role of a second metal observed in structures of bacterial orthologues. A mechanism-inspired UTP α,β-methylenebisphosphonate analogue (meUTP) was designed and synthesized and was shown to be a micromolar inhibitor of the enzyme. The mechanistic insights and inhibitor described here will facilitate future studies towards the discovery of small molecule inhibitors of this currently unexploited potential antifungal drug target.

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Structure of UDP-N-acetylglucosamine acyltransferase with a bound antibacterial pentadecapeptide.

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