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PubMed1995

Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 A resolution.

BACKGROUND: Glucosamine 6-phosphate deaminase from Escherichia coli is an allosteric hexameric enzyme which catalyzes the reversible conversion of D-glucosamine 6-phosphate into D-fructose 6-phosphate and ammonium ion and is activated by N-acetyl-D-glucosamine 6-phosphate. Mechanistically, it belongs to the group of aldoseketose isomerases, but its reaction also accomplishes a simultaneous amination/deamination. The determination of the structure of this protein provides fundamental knowledge for understanding its mode of action and the nature of allosteric conformational changes that regulate its function. RESULTS: The crystal structure of glucosamine 6-phosphate deaminase with bound phosphate ions is presented at 2.1 A resolution together with the refined structures of the enzyme in complexes with its allosteric activator and with a competitive inhibitor. The protein fold can be described as a modified NAD-binding domain. CONCLUSIONS: From the similarities between the three presented structures, it is concluded that these represent the enzymatically active R state conformer. A mechanism for the deaminase reaction is proposed. It comprises steps to open the pyranose ring of the substrate and a sequence of general base-catalyzed reactions to bring about isomerization and deamination, with Asp72 playing a key role as a proton exchanger.

Aldose-Ketose Isomerases↗

PubMed2001

Effects of oral glucosamine and chondroitin sulfate alone and in combination on the metabolism of SHR and SD rats.

Glucosamine (G), often combined with chondroitin sulfate (CS), is a popular natural supplement used widely to treat osteoarthritis. However, use of glucosamine has been linked to development of insulin resistance. To assess the association between glucosamine and insulin resistance more closely, we challenged two rat strains highly sensitive to sugar-induced insulin resistance-Sprague-Dawley (SD) and Spontaneously Hypertensive (SHR) rats. Since elevations of systolic blood pressure (SBP) have been found to be an early and highly sensitive sign of insulin resistance in these two rat strains, we used this parameter as our primary endpoint. Four groups of both rat strains received either no agent (control), G, CS, or a combination of both for 9 weeks. The intake of each agent was calculated to be approximately 3-7 times comparable to human dose. Throughout the study, SBP of both strains consuming the two ingredients alone and in combination were not elevated. Rather, they were significantly lower than control, contrary to what is found in glucose-induced insulin resistance in rats. Over the study period, body weights of the four groups of SD and SHR did not vary significantly. Furthermore, no consistent trends in circulating glucose concentrations were found among the four different groups in the two strains after oral challenge with glucose. Finally, no significant histological differences were found in hearts, kidneys, and livers among the various groups of SHR and SD. From the above result, we conclude that glucosamine and chondroitin sulfate given alone or together do not produce insulin resistance or other related perturbations in two rat strains highly sensitive to sugar-induced insulin resistance.

Administration, Oral↗

PubMed2002

From Lobry de Bruyn to enzyme-catalyzed ammonia channelling: molecular studies of D-glucosamine-6P synthase.

This review summarizes the state of knowledge on D-glucosamine-6P synthesis catalyzed by glucosamine-6P synthase. The mechanisms of L-glutamine hydrolysis, ammonia transfer and fructose-6P conversion into D-glucosamine-6P are analyzed with the E. coli enzyme in light of recent X-ray structures. With 92 references this paper covers the literature up to June 2001 and emphasizes the potential implication of the mammalian glucosamine-6P synthase in type 2 diabetes.

Ammonia↗

PubMed1970

The role of the phosphoenolpyruvate phosphotransferase system in the transport of N-acetyl-D-glucosamine by Escherichia coli.

The properties of an N-acetyl-d-glucosamine-transport system have been studied by following the intracellular accumulation of methyl 2-acetamido-2-deoxy-alpha-d-[1-(14)C]glucoside by Escherichia coli. The same analogue was used to assay phosphoenolpyruvate phosphotransferase activity of toluene-treated cells. Transport and phosphorylation are induced by growth on d-glucosamine or N-acetyl-d-glucosamine. Mutants resistant to N-iodoacetyl-d-glucosamine are defective in the uptake and phosphorylation of the labelled glycoside.

Biological Transport, Active↗

PubMed1971

The formation of glycoproteins in tissues of higher plants. Specific labelling with D-(1- 14 C)glucosamine.

1. Radioactivity from d-[l-(14)C]glucosamine is incorporated into ethanol-insoluble compounds of high molecular weight in a number of plant tissues, including roots of corn (Zea mays), callus cells of sycamore (Acer pseudoplatanus), axenic cultures of duckweed (Lemna minor) and germinating seedlings of corn, broad bean (Vicia faba) and barley (Hordeum vulgare). 2. Except in the case of Lemna, where some of the radioactivity was recovered in glucose, hydrolysis of these ethanol-insoluble materials with acid released [(14)C]glucosamine as the major radioactive product. 3. The labelled compounds isolated from Zea roots and the Acer cells are believed to be glycoproteins rather than polysaccharides on the basis of their solubility properties, their charge characteristics and their susceptibility to hydrolysis by 0.5m-potassium hydroxide and by the proteases trypsin and Pronase. Further, radioactive peptides were isolated and purified after Pronase treatment and shown to contain glucosamine as well as a number of amino acids. 4. The experiments therefore indicate that d-[(14)C]glucosamine can be used as a specific precursor of the amino sugar units of plant as well as animal glycoproteins.

Alkalies↗

PubMed1973

Studies on bone matrix glycoproteins. Incorporation of (1-14C)glucosamine and plasma (14C)glycoprotein into rabbit cortical bone.

The radioactively labelled constituents present in bone matrix were compared 12 days after injection of either [(14)C]glucosamine or plasma [(14)C]glycoprotein. Both precursors are utilized in the synthesis of organic matrix by bone tissue. Cortical bone from animals injected with [(14)C]glucosamine contains radioactivity derived from glucosamine and plasma glycoproteins and all glycoprotein fractions are labelled. Plasma [(14)C]glycoprotein labels the less acidic glycoproteins to a greater extent than the more acidic components. An antibody has been raised against the less-acidic-glycoprotein fraction of bone. The latter contains a glycoprotein of alpha-mobility that appears to be concentrated specifically in bone tissue and which is present also in plasma. This alpha-glycoprotein accounts for a large proportion of the components labelled and retained in bone matrix after [(14)C]glucosamine injection.

Albumins↗

PubMed2003

Correlation between radiographic severity of knee osteoarthritis and future disease progression. Results from a 3-year prospective, placebo-controlled study evaluating the effect of glucosamine sulfate.

OBJECTIVE: To investigate the relationship between baseline radiographic severity of knee osteoarthritis (OA) and the importance of long-term joint space narrowing. DESIGN: Sub-analysis from a three-year randomized, placebo-controlled, prospective study, of 212 patients with knee OA, recruited in an osteoarthritic outpatient clinic and having been part of a study evaluating the effect of glucosamine sulfate on symptom and structure modification in knee OA. MATERIAL AND METHODS: Measurements of mean joint space width (JSW), assessed by a computer-assisted method, were performed at baseline and after 3 years, on weightbearing anteroposterior knee radiographs. RESULTS: In the placebo group, baseline JSW was significantly and negatively correlated with the joint space narrowing observed after 3 years (r=-0.34, P=0.003). In the lowest quartile of baseline mean JSW (<4.5mm), the JSW increased after 3 years by (mean (S.D.)) 3.8% (23.8) in the placebo group and 6.2% (17.5) in the glucosamine sulfate group. The difference between the two groups in these patients with the most severe OA at baseline was not statistically significant (P=0.70). In the highest quartile of baseline mean JSW (>6.2mm), a joint space narrowing of 14.9% (17.9) occurred in the placebo group after 3 years while patients from the glucosamine sulfate group only experienced a narrowing of 6.0% (15.1). Patients with the most severe OA at baseline had a RR of 0.42 (0.17-1.01) to experience a 0.5mm joint space narrowing over 3 years, compared to those with the less affected joint. In patients with mild OA, i.e. in the highest quartile of baseline mean JSW, glucosamine sulfate use was associated with a trend (P=0.10) towards a significant reduction in joint space narrowing. CONCLUSION: These results suggest that patients with the less severe radiographic knee OA will experience, over 3 years, the most dramatic disease progression in terms of joint space narrowing. Such patients may be particularly responsive to structure-modifying drugs.

Aged↗

PubMed1998

Glucosamine induces translocation of protein kinase C isoenzymes in mesangial cells.

Activation of protein kinase C (PKC) has been implicated in the high glucose-induced stimulation of matrix protein production in mesangial cells. Since we have found (Kolm-Litty et al., 1998) that glucosamine, similar to the PKC activator phorbol myristate acetate (PMA), mimicks high glucose-induced TGF-beta1 overexpression and subsequent matrix overproduction, the action of these agents on the translocation of PKC isoenzymes was studied in cultured mesangial cells. Exposure to 12 mM glucosamine resulted in rapid and specific translocation of PKC-isoenzymes in mesangial cells i.e. glucosamine caused an increased and sustained translocation of PKC-alpha, -beta and -epsilon while PKC-zeta was essentially unaffected. Comparison with PMA-induced translocation exhibited distinct differences. Exposure to high glucose concentrations of mesangial cells induced translocation of PKC-beta and down-regulation of PKC-epsilon while PKC-alpha and -zeta were essentially unaltered. Presence of azaserine an inhibitor of glutamine: fructose-6-phosphate amidotransferase, the key enzyme of the hexosamine pathway, attenuated the high glucose-induced effects on the membrane fraction of PKC-beta. Our results indicate that i) glucosamine is a potent stimulator of PKC-translocation exhibiting an isoenzyme specific translocation kinetic which is different from PMA-induced PKC-isoenzyme translocation ii) the hexosamine pathway may be possibly involved in the high glucose-induced activation of PKC.

Animals↗

PubMed2004

Recent advances in glucosamine and chondroitin supplementation.

Glucosamine and chondroitin are alternative solutions to previous pharmaceutical options for the treatment of osteoarthritis. This article describes the mechanisms of action, pharmacokinetics, recent findings, and upcoming studies of these two natural remedies. The majority of studies on the mechanisms behind glucosamine and chondroitin have been performed in vitro or on animal models; however, the results have shown favorable effects on the balance between cartilage matrix synthesis and degradation. The pharmacokinetics of the three main forms of glucosamine were compared, and glucosamine hydrochloride displayed the greatest compound purity, despite the compounds having equal oral absorption rates of 90%. Chondroitin sulfate has been the principal clinical formulation with a slightly lower oral absorption of 70%. Clinical trials were evaluated based on two categories-radiographic changes and symptom improvement of pain and function. Although adverse effects of these two remedies were minor, the quality and labeled quantity of these relatively unregulated products must be considered. More randomized controlled studies on humans in vivo need to evaluate the efficacy, long-term effects, and quality of these compounds.

Cartilage, Articular↗

PubMed1969

Immunoglobulin synthesis and secretion. 3. Incorporation of glucosamine into immunoglobulin on polyribosomes.

Mouse myeloma cells were pulse-labeled in vitro with (3)H-D-glucosamine and (14)C-L-leucine. Analysis on sucrose gradients revealed incorporation of both isotopes into polyribosomes and release of most of such radioactivity after treatment of labeled cells in vitro with puromycin. A mixing experiment excluded the in vitro binding to unlabeled polyribosomes of (3)H-glucosamine in labeled post-ribosomal material. Polyribosomes labeled with (3)H-glucosamine were precipitated with antiserum specific for mouse immunoglobulin. By chromatography and gel filtration, the precipitable radioactivity was shown to be glucosamine covalently bound to heavy and light chains.

Animals↗

PubMed2000

Inhibition of Escherichia coli glucosamine-6-phosphate synthase by reactive intermediate analogues. The role of the 2-amino function in catalysis.

Glucosamine-6-phosphate synthase (GlmS) catalyzes the formation of D-glucosamine 6-phosphate from D-fructose 6-phosphate using L-glutamine as the ammonia source. Because N-acetylglucosamine is an essential building block of both bacterial cell walls and fungal cell wall chitin, the enzyme is a potential target for antibacterial and antifungal agents. The most potent carbohydrate-based inhibitor of GlmS reported to date is 2-amino-2-deoxy-D-glucitol 6-phosphate, an analogue of the putative cis-enolamine intermediate formed during catalysis. The interaction of a series of structurally related cis-enolamine intermediate analogues with GlmS is described. Although arabinose oxime 5-phosphate is identified as a good competitive inhibitor of GlmS with an inhibition constant equal to 1. 2 (+/-0.3) mM, the presence of the amino function at the 2-position is shown to be important for potent inhibition. Comparison of the binding affinities of 2-deoxy-D-glucitol 6-phosphate and 2-amino-2-deoxy-D-glucitol 6-phosphate indicates that the amino function contributes -4.1 (+/-0.1) kcal/mol to the free energy of inhibitor binding. Similarly, comparison of the binding affinities of 2-deoxy-D-glucose 6-phosphate and D-glucosamine 6-phosphate indicates that the amino function contributes -3.0 (+/-0.1) kcal/mol to the free energy of product binding. Interactions between GlmS and the 2-amino function of its ligands contribute to the uniform binding of the product and the cis-enolamine intermediate as evidenced by the similar contribution of the amino group to the free energy of binding of D-glucosamine 6-phosphate and 2-amino-2-deoxy-D-glucitol 6-phosphate, respectively.

Catalytic Domain↗

PubMed2003

Role of hexosamine biosynthesis in glucose-mediated up-regulation of lipogenic enzyme mRNA levels: effects of glucose, glutamine, and glucosamine on glycerophosphate dehydrogenase, fatty acid synthase, and acetyl-CoA carboxylase mRNA levels.

Glucose uptake into adipose and liver cells is known to up-regulate mRNA levels for various lipogenic enzymes such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC). To determine whether the hexosamine biosynthesis pathway (HBP) mediates glucose regulation of mRNA expression, we treated primary cultured adipocytes for 18 h with insulin (25 ng/ml) and either glucose (20 mm) or glucosamine (2 mm). A ribonuclease protection assay was used to quantitate mRNA levels for FAS, ACC, and glycerol-3-P dehydrogenase (GPDH). Treatment with insulin and various concentrations of d-glucose increased mRNA levels for FAS (280%), ACC (93%), and GPDH (633%) in a dose-dependent manner (ED50 8-16 mm). Mannose similarly elevated mRNA levels, but galactose and fructose were only partially effective. l-glucose had no effect. Omission of glutamine from the culture medium markedly diminished the stimulatory effect of glucose on mRNA expression. Since glutamine is a crucial amide donor in hexosamine biosynthesis, we interpret these data to mean that glucose flux through the HBP is linked to regulation of lipogenesis through control of gene expression. Further evidence for hexosamine regulation was obtained using glucosamine, which is readily transported into adipocytes where it directly enters the HBP. Glucosamine was 15-30 times more potent than glucose in elevating FAS, ACC, and GPDH mRNA levels (ED50 approximately 0.5 mm). In summary: 1) GPDH, FAS, and ACC mRNA levels are upregulated by glucose; 2) glucose-induced up-regulation requires glutamine; and 3) mRNA levels for lipogenic enzymes are up-regulated by glucosamine. Hyperglycemia is the hallmark of diabetes mellitus and leads to insulin resistance, impaired glucose metabolism, and dyslipidemia. We postulate that disease pathophysiology may have a common underlying factor, excessive glucose flux through the HBP.

Acetyl-CoA Carboxylase↗

PubMedDate not supplied

An analysis of high glucose and glucosamine-induced gene expression and oxidative stress in renal mesangial cells.

Renal mesangial cells play an important role in the development of diabetic kidney disease. We have previously demonstrated that some of the effects of high glucose on mesangial extracellular matrix (ECM) protein expression are mediated by the hexosamine biosynthesis pathway (HBP) in which fructose-6-phosphate is converted to glucosamine-6-phosphate by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT). Using Affymetrix murine expression U430 2.0 oligochips, we examined the global effects of high glucose (HG) and glucosamine (GlcN) on mRNA expression of a mouse mesangial cell line (MES-13). We sought to determine the portion of mRNA expression in MES-13 cells, which is mediated both by high glucose and glucosamine, i.e., via the HBP. Of the 34,000 genes on the chip, approximately 55.7 - 60.8% genes are detected in MES-13 cells. Culturing MES-13 cells for 48 h with HG alters the expression of approximately 389 genes at our preset threshold levels (at least 2-fold change) where 263 genes are up-regulated and 126 genes are down-regulated. GlcN also increases the expression of 106 genes and decreases 94 genes during the same period of incubation. Seventy-two genes in the chip are commonly regulated by HG and GlcN, in which 33 genes are up and 39 genes are down. The mRNA level of thioredoxin interacting protein (TXNIP), an inhibitor of thioredoxin activity, is maximally increased approximately 18.8 and 9.9-fold respectively by HG and GlcN. The differential expression of several genes found in the microarray analysis is further validated by real-time quantitative PCR. Significant biological processes commonly targeted by HG and GlcN are the TXNIP-thioredoxin system, oxidative stress, endoplasmic reticulum (ER) stress, extracellular matrix genes, and interferon-inducible genes. Stable overexpression of TXNIP in MES-13 cells increases glucose and glucosamine-mediated ECM gene expression and oxidative stress. We conclude from these results that the HBP mediates several effects of high glucose on mesangial cell metabolism, which promotes reactive oxygen species generation to cause cellular oxidative stress, ECM gene expression and apoptosis.

Animals↗

PubMed1972

Antigen-induced stimulation of glucosamine incorporation by guinea pig peritoneal macrophages in delayed hypersensitivity.

The interaction between sensitized lymphocytes and specific antigen occurring in delayed hypersensitivity causes bystander macrophages to undergo a variety of light-microscopic, ultrastructural, and biochemical changes, which are reflected in alterations in cell movement and intercellular contacts. Since such alterations involve functions of the cell periphery, we postulated that metabolic changes in this polysaccharide-rich zone would accompany the expression of delayed hypersensitivity. We here demonstrate that the incorporation of radioactive glucosamine by peritoneal macrophages into TCA-precipitable, membrane-associated material is regularly enhanced when these are cultured in the presence of specific antigen and nonadherent cells (lymphocytes) primed for delayed hypersensitivity. Lymphocytes from unsensitized animals, or from animals immunized so as to form antibody but not delayed hypersensitivity, do not stimulate such incorporation. Antigen-induced glucosamine incorporation is maximal at 2 or 3 days of culture and is not observed earlier; it may be elicited with as little as 0.1 microg/ml PPD, and affords an exceedingly reproducible and sensitive index of delayed hypersensitivity. Radioautographic studies indicate that nearly all plastic adherent cells (90% macrophages) incorporate glucosamine and that grains are concentrated in the regions of the perinuclear zone and cell membrane. Subcellular fractionation indicates that nearly 30% of counts and the highest specific activity are associated with the membrane-rich microsomal fraction; the microsomal distribution of counts increases in both absolute and relative terms when macrophages are cultured in the presence of specific antigen and sensitized lymphocytes. Taken together, these data indicate that a sizable fraction of incorporated glucosamine is localized to the vicinity of the cell periphery but lack sufficient resolution to determine whether this material is associated with the cell membrane itself or with the extramembranous cell coat. This last possibility is of particular interest since we have previously shown that macrophage cell coat material is lost or altered as a consequence of an interaction between sensitized lymphocytes and specific antigen.

Animals↗

PubMed1975

Phosphorylation of D-glucosamine by rat liver glucokinase.

D-Glucosamine was found to be phosphorylated by a rat liver extract in the presence of a high concentration of glucose, which was formerly believed to be a strong competitive inhibitor of this reaction. Results suggested that glucosamine may be phosphorylated by high Km hexokinase, i.e. glucokinase [EC 2.7.1.2]. The enzyme involved was separated from specific N-acetyl-D-glucosamine kinase [EC 2.7.1.59]. The phosphorylation was not inhibited by a physiological level of glucose or glucose 6-phosphate, which strongly inhibited low Km hexokinase. The apparent Km of glucokinase for glucosamine was estimated as 8 mM, which is ten times that of low Km hexokinase.

Animals↗

PubMed2002

A randomized, double-blind, placebo-controlled trial of glucosamine sulphate as an analgesic in osteoarthritis of the knee.

OBJECTIVES: A randomized, placebo-controlled, double-blind trial of the relative effectiveness of glucosamine sulphate and placebo in managing pain in osteoarthritis (OA) of the knee. METHODS: Eighty patients with OA of the knee were recruited from a rheumatology out-patient clinic and received either glucosamine sulphate 1500 mg daily for 6 months or dummy placebo. The primary outcome measure was patients' global assessment of pain in the affected knee. RESULTS: Area under the curve analysis for the primary outcome measure showed no difference between placebo and glucosamine [mean difference 0.15 mm, 95% confidence interval (CI) -8.78 to 9.07]. The placebo response was 33%. There was a statistically significant difference between groups in knee flexion (mean difference 13 degrees, 95% CI -23.13 to -1.97), but this difference was small and could have been due to measurement error. CONCLUSIONS: As a symptom modifier in OA patients with a wide range of pain severities, glucosamine sulphate was no more effective than placebo.

Adult↗

PubMed2005

Inhibitory effects of glucosamine on lipopolysaccharide-induced activation in microglial cells.

The aim of the present study was to investigate the effects of glucosamine on lipopolysaccharide (LPS)-induced cellular activation in microglia and to evaluate the inhibitory mechanisms involved. Lipopolysaccharide (100 ng/mL) was used for the activation of primary cultured rat microglial or BV2 microglial cells. Changes in intracellular Ca2+ levels and outward K+ currents were measured using fura-2/AM and whole-cell patch-clamp methods, respectively. Lipopolysaccharide-induced expression of tumour necrosis factor (TNF)-alpha mRNA was analysed by reverse transcription-polymerase chain reaction. Lipopolysaccharide transformed cell morphology into an amoeboid shape in vitro and induced microglial activation in vivo, as measured by immunohistochemical staining, but glucosamine inhibited this activation. Glucosamine also inhibited LPS-induced Ca2+ influx, outward K+ currents and TNF-alpha mRNA expression, which are typically representative of microglial activation. 4. The results suggest that the inhibitory mechanisms of glucosamine on LPS-induced microglial activation include inhibition of Ca2+ influx and outward K+ currents, as well as downregulation of the microglial activator gene TNF-alpha.

Animals↗

PubMed1973

Occurrence of glucosamine residues with free amino groups in cell wall peptidoglycan from bacilli as a factor responsible for resistance to lysozyme.

Analysis by dinitrophenylation techniques revealed the occurrence of significant amounts of glucosamine residues with free amino groups in the peptidoglycan component of cell walls isolated from Bacillus cereus, Bacillus subtilis, and Bacillus megaterium. A close correlation was demonstrated between the content of N-unacetylated glucosamine residues in the peptidoglycan component and the resistance of the cell walls to lysozyme. These lysozyme-resistant cell walls and peptidoglycan were converted into a lysozyme-sensitive form by means of N-acetylation with acetic anhydride. Thus, the occurrence of the N-unacetylated glucosamine residues in the peptidoglycan component accounts for the resistance of these cell walls to lysozyme. The N-unacetylated glucosamine residues were not found in a significant amount in the cell walls of Micrococcus lysodeikticus, Staphylococcus aureus, Streptococcus faecalis, Lactobacillus casei, or Lactobacillus arabinosus.

Alanine↗