Protein kinase C phosphorylates and regulates UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase

UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (UDP-GlcNAc 2-epimerase) is the key enzyme in the de novo synthesis pathway of neuraminic acid, which is widely expressed as a terminal carbohydrate residue on glycoconjugates. UDP-GlcNAc 2-epimerase is a bifunctional enzyme and catalyzes the first two steps of neuraminic acid synthesis in the cytosol, the conversion of UDP-N-acetylglucosamine to ManAc and the phosphorylation to ManAc-6-phosphate. So far, regulation of this essential enzyme by posttranslational modification has not been shown. Since UDP-N-acetylglucosamine is a cytosolic protein containing eight conserved motifs for protein kinase C (PKC), we investigated whether its enzymatic activity might be regulated by phosphorylation by PKC. We showed that UDP-GlcNAc 2-epimerase interacts with several isoforms of PKC in mouse liver and is phosphorylated in vivo. Furthermore, PKC phosphorylates UDP-GlcNAc 2-epimerase and this phosphorylation results in an upregulation of the UDP-GlcNAc 2-epimerase enzyme activity.     

Efficient biochemical engineering of cellular sialic acids using an unphysiological sialic acid precursor in cells lacking UDP-N-acetylglucosamine 2-epimerase

Sialic acids comprise a family of terminal sugars essential for a variety of biological recognition systems. N-Propanoylmannosamine, an unphysiological sialic acid precursor, is taken up and metabolized by mammalian cells resulting in oligosaccharide-bound N-propanoylneuraminic acid. N-Propanoylmannosamine, applied to endogenously hyposialylated subclones of the myeloid leukemia HL60 and of the B-cell lymphoma BJA-B, both deficient in UDP-N-acetylglucosamine 2-epimerase, is efficiently metabolized to CMP-N-propanoylneuraminic acid resulting in up to 85% of glycoconjugate-associated sialic acids being unphysiological N-propanoylneuraminic acid. Thus, UDP-N-acetylglucosamine 2-epimerase-deficient cell lines provide an important experimental progress in engineering cells to display an almost homogeneous population of defined, structurally altered sialic acids.     

Prediction of three different isoforms of the human UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase

The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is the key enzyme of the biosynthesis of sialic acids, terminal components of glycoconjugates associated with a variety of cellular processes. Two novel isoforms of human GNE, namely GNE2 and GNE3, which possess extended and deleted N-termini, respectively, were characterized. GNE2 was also found in other species like apes, rodents, chicken or fish, whereas GNE3 seems to be restricted to primates. Both, GNE2 and GNE3, displayed tissue specific expression patterns, therefore may contribute to the complex regulation of sialic acid metabolism.     

The analysis of N-glycans of cell membrane proteins from human hematopoietic cell lines reveals distinctions in their pattern

Abstract Human cell lines are often different in their features and present variations in the glycosylation patterns of cell membrane proteins. Protein glycosylation is the most common posttranslational modification and plays a particular role in functionality and bioactivity. The key approach of this study is the comparative analysis of five hematopoietic cell lines for their N-glycosylation pattern. The N-glycans of membrane proteins were elucidated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and MALDI-TOF/TOF-MS analyses. Furthermore, the expression of a set of glycosyltransferases was determined via RT-PCR. The B-lymphoma BJA-B and promyelocytic HL-60 cell lines distinguish in levels and linkages of glycan-bound sialic acids. Furthermore, subclones of BJA-B and HL-60 cells, which completely lack UDP-N-acetylglucosamine 2-ēpimerase/N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis, contained almost no sialylated N-glycans. Compared to wild-type cells, the GNE-deficient cells pres\xadented a similar cell surface N-glycosylation pattern in terms of antennarity and fucosylation. The Jurkat T-cell line revealed only partially sialylated N-glycans. Additionally, the different hematopoietic cell lines vary in their level of bisecting GlcNAcylation and antennary fucosylation with the quantities of bisecting N-acetylglucosamine (GlcNAc) and core fucose coinciding with the expression of GnT-III and FucT-VIII. Of note is the occurrence of N-acetyllactosamine (LacNAc) extensions on tetraantennary structures in GNE-deficient cell lines.     

Induction of N-acetylmannosamine catabolic pathway in yeast.

N-Acetylmannosamine kinase activity is absent from yeast cells grown on N-acetylmannosamine. However, other enzymes of the catabolic pathway, namely, N-acetylmannosamine-2-epimerase, N-acetylglucosamine kinase and glucosamine-6-phosphate deaminase are induced. In addition, a high affinity uptake system (permease) for the uptake of N-acetylglucosamine is synthesized under these conditions. The presence of either N-acetylmannosamine or N-acetylglucosamine as inducer is essential for the induced synthesis of these enzymes. The enzyme synthesis stops and their concentration in the cells declines rapidly as soon as inducer is removed from the medium. N-Acetyl-D-galactosamine can also induce all these enzymes except for N-acetylmannosamine-2-epimerase, suggesting the convergence of catabolic pathways for both the aminosugars at N-acetyl-D-glycosamine. Experiments with inhibitors of macromolecule synthesis suggest that the snythesis of RNA and protein is necessary for the induction of these cyzymes whereas the synthesis of DNA is not.     

Evidence for dynamic interplay of different oligomeric states of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase by biophysical methods

The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is a key enzyme for the biosynthesis of sialic acids, the terminal sugars of glycoconjugates associated with a variety of physiological and pathological processes such as cell adhesion, development, inflammation and cancer. In this study, we characterized rat GNE by different biophysical methods, analytical ultracentrifugation, dynamic light-scattering and size-exclusion chromatography, all revealing the native hydrodynamic behavior and molar mass of the protein. We show that GNE is able to reversibly self-associate into different oligomeric states including monomers, dimers and tetramers. Additionally, it forms non-specific aggregates of high molecular mass, which cannot be unequivocally assigned a distinct size. Our results also indicate that ligands of the epimerase domain of the bifunctional enzyme, namely UDP-N-acetylglucosamine and CMP-N-acetylneuraminic acid, stabilize the protein against aggregation and are capable of modulating the quaternary structure of the protein. The presence of UDP-N-acetylglucosamine strongly favors the tetrameric state, which therefore likely represents the active state of the enzyme in cells.     

Tissue expression and amino acid sequence of murine UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase.

Neuraminic acids are widely expressed as terminal carbohydrates on glycoconjugates and are involved in a variety of biological functions. The key enzyme of N-acetylneuraminic acid synthesis is UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase, which catalyses the first two steps of neuraminic acid biosynthesis in the cytosol. In this study we report the complete amino acid sequence of the mouse UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase. The ORF of 2166 bp encodes 722 amino acids and a protein with a predicted molecular mass of 79.2 kDa. Northern blot analysis and in situ hybridization revealed that UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase is expressed at early stages during development and in all tissues investigated with a maximal expression in the liver.     

Efficient metabolic oligosaccharide engineering of glycoproteins by UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) knock-down

Improving the accessibility and functions of therapeutic and diagnostic glycoproteins is one of the major goals of glycobiotechnology. Here we present that stable knock-down of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme in the sialic acid biosynthetic pathway, dramatically increases incorporation of N-acetylmannosamine analogues into glycoproteins of HEK293 cells. By means of these GNE-deficient cells highly sialylated glycoproteins can efficiently be decorated with reactive functional groups, which can be employed in bioorthogonal functionalization strategies for fluorescence labelling or biotinylation.     

The collapsin response mediator protein 1 (CRMP-1) and the promyelocytic leukemia zinc finger protein (PLZF) bind to UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis

Sialic acids (Sia) are expressed as terminal sugars in many glycoconjugates. They are involved in a variety of cell-cell interactions and therefore play an important role during development and regeneration. UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is the key enzyme in the de novo synthesis of Sia and it is a regulator of cell surface sialylation. Inactivation of GNE in mice results in early embryonic lethality. Mutations in the GNE gene are of clinical relevance in hereditary inclusion body myopathy, but these mutations do not necessarily decrease the enzymatic activity of GNE. In this study, we searched for novel function of the GNE protein beside its enzymatic function in the Sia biosynthesis. We here report the identification of novel GNE-interacting proteins. Using a human prey matrix we identified four proteins interacting with GNE in a yeast two-hybrid assay. For two of them, the collapsin response mediator protein 1 and the promyelocytic leukemia zinc finger protein, we could verify protein-protein interaction with GNE.     

Identification of N-acetylhexosamines produced by enzymes of the N-acetylneuraminic acid metabolic pathway by borate complex anion-exchange chromatography of the corresponding N-acetylhexosaminitols

A mixture of hexosaminitols obtained by reducing N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine with sodium borohydride was resolved by borate complex anion-exchange chromatography. This procedure yielded a complete separation of N-acetylglucosaminitol, N-acetylgalactosaminitol, and N-acetylmannosaminitol and provided a rapid and accurate means for identifying and measuring N-acetylhexosamines in biological samples. This method was applied to studies on N-acetylneuraminic acid metabolism in human skin fibroblasts. It was used to identify reaction products in two enzymatic reactions: the conversion of UDP-N-acetylglucosamine to N-acetylmannosamine and UDP by UDP-N-acetylglucosamine 2-epimerase and the conversion of N-acetylneuraminic acid to N-acetylmannosamine and pyruvate by N-acetylneuraminate pyruvate-lyase. It was also used to identify the free 3H-labeled N-acetylhexosamines found in fibroblasts cultured in the presence of N-[3H]acetylmannosamine.     

Differential sialylation of cell surface glycoconjugates in a human B lymphoma cell line regulates susceptibility for CD95 (APO-1/Fas)-mediated apoptosis and for infection by a lymphotropic virus.

Sialic acid, as a terminal saccharide residue on cell surface glycoconjugates, plays an important role in a variety of biological processes. In this study, we investigated subclones of the human B lymphoma cell line BJA-B for differences in the glycosylation of cell surface glycoconjugates, and studied the functional implications of such differences. With respect to the expression level of most of the tested B cell-associated antigens, as well as the presence of penultimate saccharide moieties on oligosaccharide chains, subclones were phenotypically indistinguishable. Marked differences among subclones, however, were found in the overall level of glycoconjugate sialylation, involving both alpha-2,6 and alpha-2,3-linked sialic acid residues. Accordingly, subclones were classified as highly- (group I) or hyposialylated (group II). The function of two sialic acid-dependent receptor-mediated processes is correlated with the sialylation status of BJA-B subclones. Susceptibility to and binding of the B lymphotropic papovavirus (LPV) was dependent on a high sialylation status of host cells, suggesting that differential sialylation in BJA-B cells can modulate LPV infection via its alpha-2,6-sialylated glycoprotein receptor. CD95-mediated apoptosis, induced by either the human CD95 ligand or a cytotoxic anti-CD95 monoclonal antibody, was drastically enhanced in hyposialylated group II cells. An increase in endogenous sialylation may be one antiapoptotic mechanism that converts tumor cells to a more malignant phenotype. To our knowledge, this is the first report demonstrating that differential sialylation in a clonal cell line may regulate the function of virus and signal-transducing receptors.     

Influence of UDP-GlcNAc 2-epimerase/ManNAc kinase mutant proteins on hereditary inclusion body myopathy

Hereditary inclusion body myopathy (HIBM), a neuromuscular disorder, is caused by mutations in UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis. To date, more than 40 different mutations in the GNE gene have been reported to cause the disease. Ten of them, representing mutations in both functional domains of GNE, were recombinantly expressed in insect cells (Sf9). Each of the mutants that was analyzed displayed a reduction in the two known GNE activities, thus revealing that mutations may also influence the function of the domain not harboring them. The extent of reduction strongly differs among the point mutants, ranging from only 20% reduction found for A631T and A631V to almost 80% reduction of at least one activity in D378Y and N519S mutants and more than 80% reduction of both activities of G576E, underlined by structural changes of N519S and G576E, as observed in CD spectroscopy and gel filtration analysis, respectively. We therefore generated models of the three-dimensional structures of the epimerase and the kinase domains of GNE, based on Escherichia coli UDP-N-acetylglucosamine 2-epimerase and glucokinase, respectively, and determined the localization of the HIBM mutations within these proteins. Whereas in the kinase domain most of the mutations are localized inside the enzyme, mutations in the epimerase domain are mostly located at the protein surface. Otherwise, the different mutations result in different enzymatic activities but not in different disease phenotypes and, therefore, do not suggest a direct role of the enzymatic function of GNE in the disease mechanism.     

Molecular cloning and characterization of murine and human N-acetylglucosamine kinase.

N-Acetylglucosamine is produced by the endogenous degradation of glycoconjugates and by the degradation of dietary glycoconjugates by glycosidases. It enters the pathways of aminosugar metabolism by the action of N-acetylglucosamine kinase. In this study we report the isolation and characterization of a cDNA clone encoding the murine enzyme. An open reading frame of 1029 base pairs encodes 343 amino acids with a predicted molecular mass of 37.3 kDa. The deduced amino-acid sequence contains matches of the sequences of eight peptides derived from tryptic cleavage of rat N-acetylglucosamine kinase. The recombinant murine enzyme was functionally expressed in Escherichia coli BL21 cells, where it displays N-acetylglucosamine kinase activity as well as N-acetylmannosamine kinase activity. The complete cDNA sequence of human N-acetylglucosamine kinase was derived from the nucleotide sequences of several expressed sequence tags. An open reading frame of 1032 base pairs encodes 344 amino acids and a protein with a predicted molecular mass of 37.4 kDa. Similarities between human and murine N-acetylglucosamine kinase were 86.6% on the nucleotide level and 91.6% on the amino-acid level. Amino-acid sequences of murine and human N-acetylglucosamine kinase show sequence similarities to other sugar kinases, and all five sequence motifs necessary for the binding of ATP by sugar kinases are present. Tissue distribution of murine N-acetylglucosamine kinase revealed an ubiquitous occurrence of the enzyme and a very high expression in testis. The size of the murine mRNA was 1.35 kb in all tissues investigated, with the exception of testis, where it was 1.45 kb mRNA of the murine enzyme was continuously expressed during mouse development. mRNA of the human enzyme was expressed in all investigated human tissues, as well as in cancer cell lines. In both the tissues and the cancer cell lines, the human mRNA was 1.35 kb in size.     

Experimental approaches to interfere with the polysialylation of the neural cell adhesion molecule in vitro and in vivo

Sialic acid (Sia) is expressed as terminal sugar in many glycoconjugates and plays an important role during development and regeneration. Addition of homopolymers of Sia (polysialic acid; polySia/PSA) is a unique and highly regulated post-translational modification of the neural cell adhesion molecule (NCAM). The presence of polySia affects NCAM-dependent cell adhesion and plays an important role during brain development, neural regeneration, and plastic processes including learning and memory. PolySia-NCAM is expressed on several neuroendocrine tumors of high malignancy and correlates with poor prognosis. Two closely related enzymes, the polysialyltransferases ST8SiaII and ST8SiaIV, catalyze the biosynthesis of polySia. This review summarizes recent knowledge on Sia biosynthesis and the correlation between Sia biosynthesis and polysialylation of NCAM and report on approaches to modify the degree of polySia on NCAM in vitro and in vivo. First, we describe the inhibition of polysialylation of NCAM in ST8SiaII-expressing cells using synthetic Sia precursors. Second, we demonstrate that the key enzyme of the Sia biosynthesis (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase) regulates and limits the synthesis of polySia by controlling the cellular Sia concentration.     

No overall hyposialylation in hereditary inclusion body myopathy myoblasts carrying the homozygous M712T GNE mutation

Hereditary inclusion body myopathy (HIBM) is a unique group of neuromuscular disorders characterized by adult-onset, slowly progressive distal and proximal muscle weakness, which is caused by mutations in UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme in the biosynthetic pathway of sialic acid. In order to investigate the consequences of the mutated GNE enzyme in muscle cells, we have established cell cultures from muscle biopsies carrying either kinase or epimerase mutations. While all myoblasts carrying a mutated GNE gene show a reduction in their epimerase activity, only the cells derived from the patient carrying a homozygous epimerase mutation present also a significant reduction in the overall membrane bound sialic acid. These results indicate that although mutations in each of the two GNE domains result in an impaired enzymatic activity and the same HIBM phenotype, they do not equally affect the overall sialylation of muscle cells. This lack of correlation suggests that the pathological mechanism of the disease may not be linked solely to the well-characterized sialic acid pathway.     

Characterization of ligand binding to the bifunctional key enzyme in the sialic acid biosynthesis by NMR: I. Investigation of the UDP-GlcNAc 2-epimerase functionality

The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase is the key enzyme for the biosynthesis of sialic acids. As terminal components of glycoconjugates, sialic acids are associated with a variety of pathological processes such as inflammation and cancer. For the first time, this study reveals characteristics of the interaction of the epimerase site of the enzyme with its natural substrate, UDP-N-acetylglucosamine (UDP-GlcNAc) and derivatives thereof at atomic resolution. Saturation transfer difference NMR experiments were crucial in obtaining ligand binding epitopes and to rank ligands according to their binding affinities. Employing a fragment based approach, it was possible to assign the major component of substrate recognition to the UDP moiety. In particular, the binding epitopes of the uridine moieties of UMP, UDP, UDP-GalNAc, and UDP-GlcNAc are rather similar, suggesting that the binding mode of the UDP moiety is the same in all cases. In contrast, the hexopyranose units of UDP-GlcNAc and UDP-GalNAc display small differences reflecting the inability of the enzyme to process UDP-GalNAc. Surprisingly, saturation transfer difference NMR titrations show that UDP has the largest binding affinity to the epimerase site and that at least one phosphate group is required for binding. Consequently, this study provides important new data for rational drug design.     

2-Acetamidoglucal, a new metabolite isolated from the urine of a patient with sialuria.

A new metabolite, namely 2-acetamidoglucal, has been found in the urine of a patient with sialuria in addition to the metabolites N-acetylneuraminic acid, N-acetylmannosamine, N-acetylglucosamine and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid reported earlier. the structure has been identified by mass spectrometry and 360 MHz proton nuclear magnetic resonance spectroscopy and verified by synthesis. All accumulated compounds fit into the metabolic pathway for the biosynthesis of CMP-N-acetylneuraminic acid. Sialuria is discussed in terms of a failure of regulation of UDP-N-acetylglucosamine 2-epimerase.     

Metal ion requirement of bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase from rat liver

The metal ion requirement for both enzymatic activitiesof the bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosaminekinase (E.C. 5.1.3.14/ 2.7.1.60), the key enzyme of N-acetylneuraminic acidbiosynthesis in ratliver, was investigated. UDP-N-acetylglucosamine 2-epimerase was active inimida-zole/HCl buffer in the complete absence of any metal ion. 200 mM Na + , K + , Rb + and Cs +activated enzymeactivity up to five-fold, whereas lower concentrations of thesemonovalent metal ions showed only a small effect on UDP-N-acetylglucosamine 2-epimeraseactivity. In sodium phosphate buffer the enzyme activitywas increased by 0.5 mM Mg , Sr , Ba and Mn , while in the presence of 200 mM NaCl UDP-N-acetyl-glucosamine2-epimerase activity showed astronger activation by these divalent metal ions. In imidazole/HClbuffer, UDP-N-acetylglucosamine2-epimerase activity was partially inhibited by 0.5 mM Be , Mg , Ba ,Mn , Sn and Fe , and completely inhibited by 0.5 mM Zn and Cd . Divalent metal ions were essen-tialforN-acetylmannosamine kinase activity, the most effective being Mg , followed byMn and Co .The optimal concentration of these metal ions was 3 mM. Less effective were Ni and Cd , whereas Ca ,Ba , Cu , Fe and Zn showed no effect on enzyme activity.     

The intracellular concentration of sialic acid regulates the polysialylation of the neural cell adhesion molecule

Sialic acids are expressed as terminal sugars in many glycoconjugates and play an important role during development and regeneration, as they are involved as polysialic acid in a variety of cell-cell interactions mediated by the neural cell adhesion molecule NCAM. The key enzyme for the biosynthesis of sialic acid is the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE). Mutations in the binding site of the feedback inhibitor CMP-sialic acid of the GNE leads to sialuria, a disease in which patients produce sialic acid in gram scale. Here, we report on the consequences after expression of a sialuria-mutated GNE. Expression of the sialuria-mutated GNE leads to a dramatic increase of both cellular sialic acid and polysialic acid on NCAM. This could also be achieved by application of the sialic acid precursor N-acetylmannosamine. Our data suggest that biosynthesis of sialic acid regulates and limits the synthesis of polysialic acid.