Article

Biochemical characterization of human and murine isoforms of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE)

Institute of Biochemistry and Molecular Biology, Charité-University Medicine Berlin, Campus Benjamin Franklin, Arnimallee 22, 14195, Berlin-Dahlem, Germany.
Glycoconjugate Journal (Impact Factor: 2.52). 10/2008; 26(4):415-22. DOI: 10.1007/s10719-008-9189-6
Source: PubMed

ABSTRACT

The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is the key enzyme for the biosynthesis of sialic acids, terminal components of glycoconjugates associated with a variety of physiological and pathological processes. Different protein isoforms of human and mouse GNE, deriving from splice variants, were predicted recently: GNE1 represents the GNE protein described in several studies before, GNE2 and GNE3 are proteins with extended and deleted N-termini, respectively. hGNE2, recombinantly expressed in insect and mamalian cells, displayed selective reduction of UDP-GlcNAc 2-epimerase activity by the loss of its tetrameric state, which is essential for full enzyme activity. hGNE3, which had to be expressed in Escherichia coli, only possessed kinase activity, whereas mGNE1 and mGNE2 showed no significant differences. Our data therefore suggest a role of GNE1 in basic supply of cells with sialic acids, whereas GNE2 and GNE3 may have a function in fine-tuning of the sialic acid pathway.

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Available from: Stefan O Reinke, Mar 09, 2015
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    • "UDP-GlcNAc2-epimerase catalyzes the rate-limiting step in sialic acid biosynthesis and MNK catalyzes the subsequent step. Sialic acids, which are N-acetylated derivatives of neuraminic acid, are the most abundant terminal monosaccharides on the glycoconjugates of eukaryotic cell surfaces, and they play important roles in development, regeneration, and biomedical functioning.2,3 "
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    ABSTRACT: GNE myopathy is characterized by early-adult-onset distal myopathy sparing quadriceps caused by mutations in the GNE gene encoding UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, an enzyme in the sialic-acid synthesis pathway. A 27-year-old Korean woman presented a rapid deterioration in strength of the distal lower limbs during her first pregnancy. She was diagnosed with GNE myopathy and carrying the compound heterozygous mutations of the GNE gene (D208N/M29T). This is a representative case implying that an increased requirement of sialic acid during pregnancy might trigger a clinical worsening of GNE myopathy.
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    • "Uridine diphosphate (UDP)-GlcNAc is initially epimerized to N-acetyl mannosamine (ManNAc) by UDP-GlcNAc 2-epimerase (GNE), and ManNAc is phosphorylated to ManNAc-6-phosphate by ManNAc kinase (MNK). These two enzymes are integrated into a single bifunctional enzyme, GNE/MNK (EK), in mammalian cells (Stäsche et al. 1997; Reinke et al. 2009). ManNAc-6-phosphate is condensed with phosphoenolpyruvate to synthesize N-acetyl neuraminic acid (NeuNAc)-9-phosphate, catalyzed by sialic acid-9-phosphate synthase (Roseman et al. 1961; Warren and Felsenfeld 1961; Viswanathan et al. 2005), and NeuNAc (sialic acid) is synthesized by dephosphorylation. "
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    ABSTRACT: Therapeutic glycoproteins with exposed galactose (Gal) residues are cleared rapidly from the bloodstream by asialoglycoprotein receptors in hepatocytes. Various approaches have been used to increase the content of sialic acid, which occupies terminal sites of N- or O-linked glycans and thereby increases the half-life of therapeutic glycoproteins. We enhanced sialylation of human erythropoietin (EPO) by genetic engineering of the sialylation pathway in Chinese hamster ovary (CHO) cells. The enzyme GNE (uridine diphosphate-N-acetyl glucosamine 2-epimerase)/MNK (N-acetyl mannosamine kinase), which plays a key role in the initial two steps of sialic acid biosynthesis, is regulated by cytidine monophosphate (CMP)-sialic acid through a feedback mechanism. Since sialuria patient cells fail in regulating sialic acid biosynthesis by feedback mechanism, various sialuria-like mutated rat GNEs were established and subjected to in vitro activity assay. GNE/MNK-R263L-R266Q mutant showed 93.6% relative activity compared with wild type and did not display feedback inhibition. Genes for sialuria-mutated rat GNE/MNK, Chinese hamster CMP-sialic acid transporter and human α2,3-sialyltransferase (α2,3-ST) were transfected simultaneously into recombinant human (rh) EPO-producing CHO cells. CMP-sialic acid concentration of engineered cells was significantly (>10-fold) increased by sialuria-mutated GNE/MNK (R263L-R266Q) expression. The sialic acid content of rhEPO produced from engineered cells was 43% higher than that of control cells. Ratio of tetra-sialylated glycan of rhEPO produced from engineered cells was increased ∼32%, but ratios of asialo- and mono-sialylated glycans were decreased ∼50%, compared with control. These findings indicate that sialuria-mutated rat GNE/MNK effectively increases the intracellular CMP-sialic acid level. The newly constructed host CHO cell lines produced more highly sialylated therapeutic glycoproteins through overexpression of sialuria-mutated GNE/MNK, CMP-SAT and α2,3-ST.
    Preview · Article · Mar 2011 · Glycobiology
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    • "Novel protein isoforms of human GNE were predicted from the two splice variants including exon A1 (Reinke and Hinderlich, 2007). Consequently, human GNE exist in three different isoforms, namely hGNE1, hGNE2, and hGNE3 (Reinke et al., 2009). hGNE2 and hGNE3 possess extended and deleted N-termini, respectively (Figure 3). "
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    ABSTRACT: The key enzyme for the biosynthesis of N-acetylneuraminic acid, from which all other sialic acids are formed, is the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). GNE is a highly conserved protein found throughout the animal kingdom. Its highest expression is seen in the liver and placenta. GNE is regulated by a variety of biochemical means, including tetramerization promoted by the substrate UDP-GlcNAc, phosphorylation by protein kinase C and feedback inhibition by CMP-Neu5Ac, which is defect in the human disease sialuria. GNE knock-out in mice leads to embryonic lethality, emphasizing the crucial role of this key enzyme for sialic acid biosynthesis. The metabolic capacity to synthesize sialic acid and CMP-sialic acid upon ManNAc loads is amazingly high. An additional characteristic of GNE is its interaction with proteins involved in the regulation of development, which might play a crucial role in the hereditary inclusion body myopathy. Due to the importance of increased concentrations of tumor-surface sialic acid, first attempts to find inhibitors of GNE have been successful.
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