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.     

2',3'-Dialdehydo-UDP-N-acetylglucosamine inhibits UDP-N-acetylglucosamine 2-epimerase,the key enzyme of sialic acid biosynthesis

Chromogranin B (CgB) is a member of the granin family of neuroendocrine secretory proteins, which has been proposed to play a role in secretory granule biogenesis and as a precursor to bioactive peptides. The cloning of CgB in a phylogenetically distant vertebrate, the frog Rana ridibunda, reveals a modest overall homology (35-40%) with mammalian CgB. However, the sequences of the N- and C-terminal regions are more highly conserved (57-65% amino acid identity) and may give rise to novel regulatory peptides. In frog, intense expression of CgB mRNA was observed in particular structures of the brain and in the distal lobe of the pituitary.     

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.     

Roles for UDP-GlcNAc 2-epimerase/ManNAc 6-kinase outside of sialic acid biosynthesis: modulation of sialyltransferase and BiP expression, GM3 and GD3 biosynthesis, proliferation, and apoptosis, and ERK1/2 phosphorylation

Roles for UDP-GlcNAc 2-epimerase/ManNAc 6-kinase (GNE) beyond controlling flux into the sialic acid biosynthetic pathway by converting UDP-GlcNAc to N-acetylmannosamine are described in this report. Overexpression of recombinant GNE in human embryonic kidney (HEK AD293) cells led to an increase in mRNA levels for ST3Gal5 (GM3 synthase) and ST8Sia1 (GD3 synthase) as well as the biosynthetic products of these sialyltransferases, the GM3 and GD3 gangliosides. Conversely, down-regulation of GNE by RNA interference methods had the opposite, but consistent, effect of lowering ST3Gal5 and ST8Sia1 mRNAs and reducing GM3 and GD3 levels. Control experiments ensured that GNE-mediated changes in sialyltransferase expression and ganglioside biosynthesis were not the result of altered flux through the sialic acid pathway. Interestingly, exogenous GM3 and GD3 also changed the expression of GNE and led to reduced ST3Gal5 and ST8Sia1 mRNA levels, demonstrating a reciprocating feedback mechanism where gangliosides regulate upstream biosynthetic enzymes. Cellular responses to the GNE-mediated changes in ST3Gal5 and ST8Sia1 expression and GM3 and GD3 levels were investigated next. Conditions that led to reduced ganglioside production (e.g. short hairpin RNA exposure) stimulated proliferation, whereas conditions that resulted in increased ganglioside levels (e.g. recombinant GNE and exogenous gangliosides) led to reduced proliferation with a concomitant increase in apoptosis. Finally, changes to BiP expression and ERK1/2 phosphorylation consistent with apoptosis and proliferation, respectively, were observed. These results provide examples of specific biochemical pathways, other than sialic acid metabolism, that are influenced by GNE.     

Characterization of ligand binding to the bifunctional key enzyme in the sialic acid biosynthesis by NMR: II. Investigation of the ManNAc kinase functionality

N-Acetylmannosamine (ManNAc) is the physiological precursors to all sialic acids that occur in nature. As variations in the sialic acid decoration of cell surfaces can profoundly affect cell-cell, pathogen-cell, or drug-cell interactions, the enzymes that convert ManNAc into sialic acid are attractive targets for the development of drugs that specifically interrupt sialic acid biosynthesis or lead to modified sialic acids on the surface of cells. The first step in the enzymatic conversion of ManNAc into sialic acid is phosphorylation, yielding N-acetylmannosamine-6-phosphate. The enzyme that catalyzes this conversion is the N-acetylmannosamine kinase (ManNAc kinase) as part of the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Here, we employed saturation transfer difference (STD) NMR experiments to study the binding of ManNAc and related ligands to the ManNAc kinase. It is shown that the configuration of C1 and C4 of ManNAc is crucial for binding to the enzyme, whereas the C2 position not only accepts variations in the attached N-acyl side chain but also tolerates inversion of configuration. Our experiments also show that ManNAc kinase maintains its functionality, even in the absence of Mg(2+). From the analysis of the STD NMR-derived binding epitopes, it is concluded that the binding mode of the N-acylmannosamines critically depends on the N-acyl side chain. In conjunction with the relative binding affinities of the ligands obtained from STD NMR titrations, it is possible to derive a structure-binding affinity relationship. This provides a cornerstone for the rational design of drugs for novel therapeutic applications by altering the sialic acid decorations of cell walls.     

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.     

Localization of UDP-GlcNAc 2-epimerase/ManAc kinase (GNE) in the Golgi complex and the nucleus of mammalian cells

The bifunctional enzyme UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) is essential for early embryonic development and catalyzes the rate limiting step in sialic acid biosynthesis. Although epimerase and kinase activities have been attributed to GNE, little is known about the regulation, differential expression, and subcellular localization of GNE in vivo. Mutations in GNE cause a rare inherited muscle disorder in humans called hereditary inclusion body myopathy (HIBM). However, the role of GNE in HIBM pathogenesis has not been defined yet. Here, we show that the GNE protein is expressed in various mammalian cells and tissues with highest levels found in cancer cells and liver. In human skeletal muscle, GNE protein is developmentally regulated: high levels are found in immature myoblasts but low levels in mature skeletal muscle. The GNE protein colocalizes with resident proteins of the Golgi compartment in a variety of human cells including muscle. Drug-induced disruption of the Golgi and subsequent recovery reveals co-distribution of GNE along with Golgi-targeted proteins. This subcellular localization of GNE is in good agreement with its established role as the key enzyme of sialic acid biosynthesis, since the sialylation of glycoconjugates takes place in the Golgi complex. Surprisingly, GNE is also detected in the nucleus. Upon nocodazole treatment, GNE redistributes to the cytoplasm suggesting that GNE may act as a nucleocytoplasmic shuttling protein. A regulatory role for GNE shifting between the nuclear and the Golgi compartment is proposed. Further insight into GNE regulation may promote the understanding of HIBM pathogenesis.     

Enhanced sialylation of EPO by overexpression of UDP-GlcNAc 2-epimerase/ManAc kinase containing a sialuria mutation in CHO cells

Sialylation (e.g. expression of sialic acid) plays a crucial role for function and stability of most glycoproteins. 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 use in biotechnology of sialuria-mutated GNE. Expression of the sialuria-mutated GNE in CHO-cells leads to increased sialylation of recombinant expressed erythropoietin (EPO). Our data show that sialuria-mutated-GNE over-expressing cells are the perfect platform to express highly sialylated therapeutic proteins, such as EPO.     

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.     

Biosynthesis of N-acetylneuraminic acid in cells lacking UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase

The first two steps in mammalian biosynthesis of N-acetylneuraminic acid, an important carbohydrate moiety in biological recognition systems, are performed by the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. A subclone of the human B lymphoma cell line BJA-B K20, lacking UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase mRNA as well as epimerase activity, displayed hyposialylated, functionally impaired cell surface glycoconjugates. Here we show that this cell line surprisingly still retains N-acetylmannosamine kinase activity. A gel filtration analysis of BJA-B K88 control cells, which express UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, revealed two N-acetylmannosamine kinase activity peaks, one co-eluting with UDP-N-acetylglucosamine 2-epimerase activity and one co-eluting with N-acetylglucosamine kinase. For this enzyme previous studies already showed a ManNAc kinase activity in vitro. In contrast, the hyposialylated BJA-B K20 subclone displayed only the N-acetylmannosamine kinase peak, co-migrating with N-acetylglucosamine kinase. The CMP-N-acetylneuraminic acid content of both K88 and K20 cells and the sialylation of cell surface glycoconjugates of K20 cells could be significantly increased by supplementing the medium with N-acetylmannosamine. This N-acetylmannosamine-induced increase was drastically reduced by co-supplementation with N-acetylglucosamine only in K20 cells. We therefore propose the phosphorylation of N-acetylmannosamine as a hitherto unrecognized role of N-acetylglucosamine kinase in living cells.     

Identification,expression, and tissue distribution of the three rat lysyl hydroxylase isoforms

Lysyl hydroxylases (LH) (procollagen-lysine 2-oxoglutarate 5-dioxygenase; PLOD) catalyse the hydroxylation of lysine residues during the post-translational modification of collagenous proteins. In this paper, we describe the first identification and cloning of LH isoforms 2 and 3 from the rat, including both LH2 splice variants (LH2a and LH2b). The rat LHs are expressed in almost all tissue and cell types examined, indicating a probable lack of tissue specificity for LH function. All LH isoforms were stably transfected into CHO-K1 cells and this represents the first example of recombinant LH production in a eukaryotic cell line. Expression and production of all LH isoforms led to an increase in total collagen synthesis. LH1 and LH2a expression and production led to an increase in total pyridinium cross-link production. Evidence that LH2a possesses telopeptide lysyl hydroxylase activity, previously thought to be a novel enzyme, is presented.     

A single bifunctional UDP-GlcNAc/Glc 4-epimerase supports the synthesis of three cell surface glycoconjugates in Campylobacter jejuni

The major cell-surface carbohydrates (lipooligosaccharide, capsule, and glycoprotein N-linked heptasaccharide) of Campylobacter jejuni NCTC 11168 contain Gal and/or GalNAc residues. GalE is the sole annotated UDP-glucose 4-epimerase in this bacterium. The presence of GalNAc residues in these carbohydrates suggested that GalE might be a UDP-GlcNAc 4-epimerase. GalE was shown to epimerize UDP-Glc and UDP-GlcNAc in coupled assays with C. jejuni glycosyltransferases and in sugar nucleotide epimerization equilibria studies. Thus, GalE possesses UDP-GlcNAc 4-epimerase activity and was renamed Gne. The Km(app) values of a purified MalE-Gne fusion protein for UDP-GlcNAc and UDP-GalNAc are 1087 and 1070 microm, whereas those for UDP-Glc and UDP-Gal are 780 and 784 microm. The kcat and kcat/Km(app) values were three to four times higher for UDP-GalNAc and UDP-Gal than for UDP-GlcNAc and UDP-Glc. The comparison of the kinetic parameters of MalE-Gne to those of other characterized bacterial UDP-GlcNAc 4-epimerases indicated that Gne is a bifunctional UDP-GlcNAc/Glc 4-epimerase. The UDP sugar-binding site of Gne was modeled by using the structure of the UDP-GlcNAc 4-epimerase WbpP from Pseudomonas aeruginosa. Small differences were noted, and these may explain the bifunctional character of the C. jejuni Gne. In a gne mutant of C. jejuni, the lipooligosaccharide was shown by capillary electrophoresis-mass spectrometry to be truncated by at least five sugars. Furthermore, both the glycoprotein N-linked heptasaccharide and capsule were no longer detectable by high resolution magic angle spinning NMR. These data indicate that Gne is the enzyme providing Gal and GalNAc residues with the synthesis of all three cell-surface carbohydrates in C. jejuni NCTC 11168.     

Sialyltransferase, sialic acid and sialoglycoconjugates in metastatic tumor and human liver tissue

1. The specific activities of sialytransferase in metastatic tumor sites were 5-37% of those of uninvolved non-cancerous tissue. The non-cancerous host tissues had a slightly lower average sialyltransferase activity than that of non-pathological control livers (986 vs 1194 dpm/min/mg protein). 2. The levels of total and bound sialic acid are increased 1.4 to 7.2-fold in homogenates, supernatants and resuspended pellets of metastatic tumor sites compared to non-cancerous and non-pathological control livers. For all these tissues, 24-29% of the bound sialic acid is found on soluble components (in the 16,300 g supernatant). 3. Soluble sialoglycoconjugates from most metastatic tumor sites give gel filtration profiles different from those of non-cancerous and non-pathological control livers.     

Identification of functionally important cysteine residues of the human renin-binding protein as the enzyme N-acetyl-D-glucosamine 2-epimerase

Renin-binding protein (RnBP) is an endogenous renin inhibitor originally isolated from porcine kidney. It was recently identified as the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase [Takahashi, S. et al. (1999) J. Biochem. 125, 348-353] and its active site residue was determined to be cysteine 380 by site-directed mutagenesis [Takahashi, S. et al. (1999) J. Biochem. 126, 639-642]. To further investigate the relationship between structure and function of recombinant human (rh) RnBP as a GlcNAc 2-epimerase, we have constructed several C-terminal deletion and multi-cysteine/serine mutants of rhGlcNAc 2-epimerase and expressed them in Escherichia coli cells. The expression was detected by Western blotting using anti-rhRnBP antiserum. The C-terminal deletion mutant, Delta400-417, had approximately 50% activity relative to the wild-type enzyme, but other C-terminal deletion mutants, Delta380-417, Delta386-417, and Delta390-417, had no enzymatic activity. Mutational analysis of multi-cysteine/serine mutants revealed that cysteines 41 and 390 were critical for the activity or stabilization of the enzyme, while cysteine residues in the middle of the enzyme, cysteines 125, 210, 239, and 302, had no essential function in relation to the activity.     

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.     

Identification of the mitochondrial glutamate transporter. Bacterial expression, reconstitution, functional characterization, and tissue distribution of two human isoforms

The mitochondrial carriers are a family of transport proteins in the inner membranes of mitochondria. They shuttle substrates, metabolites, and cofactors through this membrane and connect cytoplasm functions with others in the matrix. Glutamate is co-transported with H(+) (or exchanged for OH(-)), but no protein has ever been associated with this activity. Two human expressed sequence tags encode proteins of 323 and 315 amino acids with 63% identity that are related to the aspartate-glutamate carrier, a member of the carrier family. They have been overexpressed in Escherichia coli and reconstituted into phospholipid vesicles. Their transport properties demonstrate that the two proteins are isoforms of the glutamate/H(+) symporter described in the past in whole mitochondria. Isoform 1 is expressed at higher levels than isoform 2 in all the tissues except in brain, where the two isoforms are expressed at comparable levels. The differences in expression levels and kinetic parameters of the two isoforms suggest that isoform 2 matches the basic requirement of all tissues especially with respect to amino acid degradation, and isoform 1 becomes operative to accommodate higher demands associated with specific metabolic functions such as ureogenesis.