N-Acetylglucosamine

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William M. Canfield - One of the best experts on this subject based on the ideXlab platform.

  • structural requirements for efficient processing and activation of recombinant human udp n acetylglucosamine lysosomal enzyme n acetylglucosamine 1 phosphotransferase
    Journal of Biological Chemistry, 2006
    Co-Authors: Mariko Kudo, William M. Canfield
    Abstract:

    Mannose 6-phosphate-modified N-glycans are the determinant for intracellular targeting of newly synthesized lysosomal hydrolases to the lysosome. The enzyme responsible for the initial step in the synthesis of mannose 6-phosphate is UDP-N-Acetylglucosamine:lysosomal-enzyme-N-acetylglucosmine-1-phosphotransferase(GlcNAc-phosphotransferase). GlcNAc-phosphotransferase is a multisubunit enzyme with an alpha2beta2gamma2 arrangement that requires a detergent for solubilization. Recent cloning of cDNAs and genes encoding these subunits revealed that the alpha- and beta-subunits are encoded by a single gene as a precursor, whereas the gamma-subunit is encoded by a second gene. The hydropathy plots of the deduced amino acid sequences suggested that the alpha- and beta-subunits but not the gamma-subunit contain transmembrane domains. Access to these cDNAs allowed us to express a soluble form of human recombinant GlcNAc-phosphotransferase by removing the putative transmembrane and cytoplasmic domains from the alpha- and beta-subunits. Because this modification prevented precursor processing to mature alpha- and beta-subunits, the native cleavage sequence was replaced by a cleavage site for furin. When the modified alpha/beta-subunits (alpha'/beta'-subunits) precursor and wild type gamma-subunit cDNAs were co-expressed in 293T or CHO-K1 cells, a furin-like protease activity in these cells cleaved the precursor and produced an active and processed soluble GlcNAc-phosphotransferase with an alpha'2beta'2gamma2-subunits arrangement. Recombinant soluble GlcNAc-phosphotransferase exhibited specific activity and substrate preferences similar to the wild type bovine GlcNAc-phosphotransferase and was able to phosphorylate a lysosomal hydrolase, acid alpha-glucosidase in vitro.

  • bovine udp n acetylglucosamine lysosomal enzyme n acetylglucosamine 1 phosphotransferase ii enzymatic characterization and identification of the catalytic subunit
    Journal of Biological Chemistry, 1996
    Co-Authors: B J Elmendorf, J L Booth, R R Drake, William M. Canfield
    Abstract:

    Abstract The kinetic properties of UDP-N-Acetylglucosamine:lysosomal-enzyme N-Acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) purified to homogeneity from lactating bovine mammary gland have been investigated. GlcNAc-phosphotransferase transferred GlcNAc 1-phosphate from UDP-GlcNAc to the synthetic acceptor α-methylmannoside, generating GlcNAc-1-phospho-6-mannose α-methyl, the structure of which was confirmed by mass spectroscopy. GlcNAc-phosphotransferase was active between pH 5.7 and 9.3, with optimal activity between pH 6.6 and 7.5. Activity was strictly dependent on Mg2+ or Mn2+. The Km for Mn2+ was 185 μM. The Km for UDP-GlcNAc was 30 μM, and that for α-methylmannoside was 63 mM. The enzyme was competitively inhibited by UDP-Glc, with a Ki of 733 μM. The 166-kDa subunit was identified as the catalytic subunit by photoaffinity labeling with azido-[β-32P]UDP-Glc. Purified GlcNAc-phosphotransferase utilizes the lysosomal enzyme uteroferrin ∼163-fold more effectively than the non-lysosomal glycoprotein ribonuclease B. Antibodies to GlcNAc-phosphotransferase blocked the transfer to cathepsin D, but not to α-methylmannoside, suggesting that protein-protein interactions are required for the efficient utilization of glycoprotein acceptors. These results indicate that the purified bovine GlcNAc-phosphotransferase retains the specificity for lysosomal enzymes as acceptors previously observed with crude preparations.

  • bovine udp n acetylglucosamine lysosomal enzyme n acetylglucosamine 1 phosphotransferase i purification and subunit structure
    Journal of Biological Chemistry, 1996
    Co-Authors: Ming Bao, J L Booth, B J Elmendorf, William M. Canfield
    Abstract:

    UDP-N-Acetylglucosamine:lysosomal-enzyme N-Acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) catalyzes the initial step in the synthesis of the mannose 6-phosphate determinant required for efficient intracellular targeting of newly synthesized lysosomal hydrolases to the lysosome. The enzyme was partially purified approximately 30,000-fold by chromatography of solubilized membrane proteins from lactating bovine mammary glands on DEAE-Sepharose, reactive green 19-agarose, and Superose 6. The partially purified enzyme was used to generate a panel of murine monoclonal antibodies. The anti-GlcNAc-phosphotransferase monoclonal antibody PT18 was coupled to a solid support and used to immunopurify the enzyme approximately 480,000-fold to apparent homogeneity with an overall yield of 29%. The purified enzyme has a specific activity of 10-12 micromol of GlcNAc phosphate transferred per h/mg using 100 mM alpha-methylmannoside as acceptor. The subunit structure of the enzyme was determined using a combination of analytical gel filtration chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino-terminal sequencing. The data indicate that bovine GlcNAc-phosphotransferase is a 540,000-Da complex composed of disulfide-linked homodimers of 166,000- and 51,000-Da subunits and two identical, noncovalently associated 56,000-Da subunits.

  • bovine udp n acetylglucosamine lysosomal enzyme n acetylglucosamine 1 phosphotransferase i purification and subunit structure
    Journal of Biological Chemistry, 1996
    Co-Authors: J L Booth, B J Elmendorf, William M. Canfield
    Abstract:

    Abstract UDP-N-Acetylglucosamine:lysosomal-enzyme N-Acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) catalyzes the initial step in the synthesis of the mannose 6-phosphate determinant required for efficient intracellular targeting of newly synthesized lysosomal hydrolases to the lysosome. The enzyme was partially purified ∼30,000-fold by chromatography of solubilized membrane proteins from lactating bovine mammary glands on DEAE-Sepharose, reactive green 19-agarose, and Superose 6. The partially purified enzyme was used to generate a panel of murine monoclonal antibodies. The anti-GlcNAc-phosphotransferase monoclonal antibody PT18 was coupled to a solid support and used to immunopurify the enzyme ∼480,000-fold to apparent homogeneity with an overall yield of 29%. The purified enzyme has a specific activity of 10-12 μmol of GlcNAc phosphate transferred per h/mg using 100 mM α-methylmannoside as acceptor. The subunit structure of the enzyme was determined using a combination of analytical gel filtration chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino-terminal sequencing. The data indicate that bovine GlcNAc-phosphotransferase is a 540,000-Da complex composed of disulfide-linked homodimers of 166,000- and 51,000-Da subunits and two identical, noncovalently associated 56,000-Da subunits.

Stephan Hinderlich - One of the best experts on this subject based on the ideXlab platform.

Alan D Grund - One of the best experts on this subject based on the ideXlab platform.

  • engineering a new pathway for n acetylglucosamine production coupling a catabolic enzyme glucosamine 6 phosphate deaminase with a biosynthetic enzyme glucosamine 6 phosphate n acetyltransferase
    Enzyme and Microbial Technology, 2006
    Co-Authors: Mingde Deng, Sarah L Wassink, Alan D Grund
    Abstract:

    A metabolic pathway for high level production of N-Acetylglucosamine has been engineered in Escherichia coli by overexpressing E. coli glucosamine synthase (GlmS) and Saccharomyces cerevisiae glucosamine-6-phosphate acetyltransferase (GNA1). GlmS catalyzes the synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. GNA1 converts glucosamine-6-phosphate into N-Acetylglucosamine, which is dephosphorylated and secreted into the growth medium. In the present work, E. coli glucosamine-6-phosphate deaminase (NagB) was evaluated as an alternative to GlmS for the production of glucosamine and N-Acetylglucosamine. NagB is a catabolic enzyme that converts glucosamine-6-phosphate to fructose-6-phosphate and ammonia. The reverse biosynthetic reaction forming glucosamine-6-phosphate is kinetically unfavorable. In a glmS deletion strain requiring glucosamine supplement to survive and grow, NagB overexpression resulted in the synthesis of glucosamine-6-phosphate. This supported cell growth, but little or no glucosamine accumulated in the medium. Overexpression of both NagB and GNA1 resulted in production of N-Acetylglucosamine at levels comparable to strains overexpressing both GlmS and GNA1. This indicates that the overexpression of GNA1 played a critical role in determining the direction and efficiency of the reaction catalyzed by NagB. These data demonstrate that a catabolic enzyme can be utilized in a biosynthetic pathway by coupling with an efficient downstream reaction.

  • metabolic engineering of escherichia coli for industrial production of glucosamine and n acetylglucosamine
    Metabolic Engineering, 2005
    Co-Authors: Mingde Deng, Sarah L Wassink, Alan D Grund, David K Severson, Richard P Burlingame, Alan Berry, Jeffrey A Running, Candice A Kunesh, Linsheng Song, Thomas A Jerrell
    Abstract:

    Abstract Glucosamine and N -acetylglucosamine are currently produced by extraction and acid hydrolysis of chitin from shellfish waste. Production could be limited by the amount of raw material available and the product potentially carries the risk of shellfish protein contamination. Escherichia coli was modified by metabolic engineering to develop a fermentation process. Over-expression of glucosamine synthase (GlmS) and inactivation of catabolic genes increased glucosamine production by 15 fold, reaching 60 mg l −1 . Since GlmS is strongly inhibited by glucosamine-6-P, GlmS variants were generated via error-prone PCR and screened. Over-expression of an improved enzyme led to a glucosamine titer of 17 g l −1 . Rapid degradation of glucosamine and inhibitory effects of glucosamine and its degradation products on host cells limited further improvement. An alternative fermentation product, N -acetylglucosamine, is stable, non-inhibitory to the host and readily hydrolyzed to glucosamine under acidic conditions. Therefore, the glucosamine pathway was extended to N -acetylglucosamine by over-expressing a heterologous glucosamine-6-P N -acetyltransferase. Using a simple and low-cost fermentation process developed for this strain, over 110 g l −1 of N -acetylglucosamine was produced.

Isao Nagaoka - One of the best experts on this subject based on the ideXlab platform.

  • glucosamine a naturally occurring amino monosaccharide modulates ll 37 induced endothelial cell activation
    International Journal of Molecular Medicine, 1998
    Co-Authors: Yinghua Ju, Koji Sakamoto, Hideoki Ogawa, Isao Nagaoka
    Abstract:

    Atheroscleros is now considered as a chronic inflammatory disease, and glucosamine has a potential to exhibit anti-inflammatory action. Thus, we investigated the effect of glucosamine on LL-37-induced endothelial cell activation. HUVEC (human umbilical vein endothelial cells) were stimulated by LL-37 in the presence or absence of glucosamine (0.01-1 mM) or its analogue, N-Acetylglucosamine (0.1-1 mM). mRNA expression of MCP-1 (monocyte chemoattractant protein- 1) and ICAM-1 (intercellular adhesion molecule-1) was evaluated by real-time RT-PCR, and their protein levels were analyzed by ELISA and Western blotting, respectively. Furthermore, the effect of glucosamine on O -N-Acetylglucosamine (O-G1cNAc) modification was evaluated by Western blotting. Glucosamine but not N-Acetylglucosamine suppressed the LL-37-induced expression of MCP-1 and ICAM-1 at both mRNA (p<0.05 at 0.1 mM) and protein levels (p<0.05 at 1 mM). Of interest, O-G1cNAc modification was induced by incubating HUVEC with glucosamine (p<0.05 at 1 mM) but not N-Acetylglucosamine. Of note, alloxan, an O -N-Acetylglucosamine transferase inhibitor, which prevented the glucosamine-induced O-G1cNAc modification, abrogated the suppressive effect of glucosamine on MCP-1 and ICAM-1 expression (p<0.05 at 0.5 mM). These observations suggest that glucosamine modulates endothelial cell activation possibly via O-G1cNAc modification, and may exhibit an anti-inflammatory action on atherosclerosis.

  • modulation of tnf α induced endothelial cell activation by glucosamine a naturally occurring amino monosaccharide
    International Journal of Molecular Medicine, 1998
    Co-Authors: Yinghua Ju, Koji Sakamoto, Hideoki Ogawa, Isao Nagaoka
    Abstract:

    : Atherosclerosis is now considered a chronic inflammatory disease, and glucosamine has the potential to exhibit an anti-inflammatory action. Thus, we investigated the effect of glucosamine on tumor necrosis factor alpha (TNF-alpha)-induced endothelial cell activation. Human umbilical vein endothelial cells (HUVECs) were stimulated by TNF-alpha in the presence or absence of glucosamine or its analogue, N-Acetylglucosamine. mRNA expression of MCP-1 (a chemoattractant protein) and ICAM-1 (an adhesion molecule) was evaluated by real-time RT-PCR, and their protein levels were analyzed by ELISA and Western blotting, respectively. Furthermore, the effects of glucosamine on the phosphorylation of p38MAPK and NF-kappaB, and O-N-Acetylglucosamine (O-GlcNAc) modification were evaluated by Western blotting. The results demonstrated that glucosamine but not N-Acetylglucosamine suppressed TNF-alpha-induced expression of MCP-1 and ICAM-1 at both the mRNA and protein levels. Furthermore, glucosamine abrogated the phosphorylation of p38MAPK and NF-kappaB. To note, glucosamine induced O-GlcNAc modification, which was negatively correlated with the expression of MCP-1 and ICAM-1, and phosphorylation of p38MAPK and NF-kappaB. Thus, glucosamine is likely to suppress endothelial cell activation (TNF-alpha-induced ICAM-1 and MCP-1 expression) possibly by affecting p38MAPK and NF-kappaB signaling via O-GlcNAc modification.

Sebastien Rigali - One of the best experts on this subject based on the ideXlab platform.

  • the permease gene nage2 is the key to n acetylglucosamine sensing and utilization in streptomyces coelicolor and is subject to multi level control
    Molecular Microbiology, 2010
    Co-Authors: Harald Nothaft, Sebastien Rigali, Bart Boomsma, Magdalena Swiatek, Kenneth J Mcdowall, Gilles P Van Wezel, Fritz Titgemeyer
    Abstract:

    Summary The availability of nutrients is a major determinant for the timing of morphogenesis and antibiotic production in the soil-dwelling bacterium Streptomyces coelicolor. Here we show that N-Acetylglucosamine transport, the first step of an important nutrient signalling cascade, is mediated by the NagE2 permease of the phosphotransferase system, and that the activity of this permease is linked to nutritional control of development and antibiotic production. The permease serves as a high-affinity transporter for N-Acetylglucosamine (Km of 2.6 µM). The permease complex was reconstituted with individually purified components. This showed that uptake of N-Acetylglucosamine requires a phosphoryl group transfer from phosphoenolpyruvate via the phosphotransferases EI, HPr and IIACrr to NagF, which in turn phosphorylates N-Acetylglucosamine during transport. Transcription of the nagF and nagE2 genes is induced by N-Acetylglucosamine. Nutrient signalling by N-Acetylglucosamine that triggers the onset of development was abolished in the nagE2 and nagF mutants. nagE2 is subject to multi-level control by the global transcription factor DasR and the activator AtrA that also stimulates genes for antibiotic actinorhodin biosynthesis. Hence, it is apparent that streptomycetes tightly control the nutritional state in a complex manner to ensure the correct timing for the developmental programme.

  • feast or famine the global regulator dasr links nutrient stress to antibiotic production by streptomyces
    EMBO Reports, 2008
    Co-Authors: Sebastien Rigali, Fritz Titgemeyer, David A Hopwood, Sharief Barends, Suzanne Mulder, Andreas W Thomae, Gilles P Van Wezel
    Abstract:

    Members of the soil‐dwelling prokaryotic genus Streptomyces produce many secondary metabolites, including antibiotics and anti‐tumour agents. Their formation is coupled with the onset of development, which is triggered by the nutrient status of the habitat. We propose the first complete signalling cascade from nutrient sensing to development and antibiotic biosynthesis. We show that a high concentration of N ‐acetylglucosamine—perhaps mimicking the accumulation of N ‐acetylglucosamine after autolytic degradation of the vegetative mycelium—is a major checkpoint for the onset of secondary metabolism. The response is transmitted to antibiotic pathway‐specific activators through the pleiotropic transcriptional repressor DasR, the regulon of which also includes all N ‐acetylglucosamine‐related catabolic genes. The results allowed us to devise a new strategy for activating pathways for secondary metabolite biosynthesis. Such ‘cryptic’ pathways are abundant in actinomycete genomes, thereby offering new prospects in the fight against multiple drug‐resistant pathogens and cancers.

  • the sugar phosphotransferase system of streptomyces coelicolor is regulated by the gntr family regulator dasr and links n acetylglucosamine metabolism to the control of development
    Molecular Microbiology, 2006
    Co-Authors: Sebastien Rigali, Harald Nothaft, Elke E E Noens, Maximilian Schlicht, Sevrine Colson, Marisa Muller, Bernard Joris, Henk K Koerten, David A Hopwood
    Abstract:

    Summary Members of the soil-dwelling, sporulating prokaryotic genus Streptomyces are indispensable for the recy- cling of the most abundant polysaccharides on earth (cellulose and chitin), and produce a wide range of antibiotics and industrial enzymes. How do these organisms sense the nutritional state of the environ- ment, and what controls the signal for the switch to antibiotic production and morphological develop- ment? Here we show that high extracellular concen- trations of N-Acetylglucosamine, the monomer of chitin, prevent Streptomyces coelicolor progressing beyond the vegetative state, and that this effect is absent in a mutant defective of N-Acetylglucosamine transport. We provide evidence that the signal is transmitted through the GntR-family regulator DasR, which controls the N-Acetylglucosamine regulon, including the pts genes ptsH, ptsI and crr needed for uptake of N-Acetylglucosamine. Deletion of dasR or the pts genes resulted in a bald phenotype. Binding of DasR to its target genes is abolished by glucosamine 6-phosphate, a central molecule in N-Acetylglucosamine metabolism. Extracellular com- plementation experiments with many bld mutants showed that the dasR mutant is arrested at an early stage of the developmental programme, and does not fit in the previously described bld signalling cascade. Thus, for the first time we are able to directly link carbon (and nitrogen) metabolism to development, highlighting a novel type of metabolic regulator, which senses the nutritional state of the habitat, maintaining vegetative growth until changing circum- stances trigger the switch to sporulation. Our work, and the model it suggests, provide new leads towards understanding how microorganisms time develop- mental commitment.