Stephan Hinderlich

Sialylation is essential for early development in mice

Sialic acids are widely expressed as terminal carbohydrates on glycoconjugates of eukaryotic cells. Sialylation is crucial for a variety of cellular functions, such as cell adhesion or signal recognition, and regulates the biological stability of glycoproteins. The key enzyme of sialic acid biosynthesis is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (UDP-GlcNAc 2-epimerase), which catalyzes the first two steps of sialic acid biosynthesis in the cytosol. We report that inactivation of the UDP-GlcNAc 2-epimerase by gene targeting causes early embryonic lethality in mice, thereby emphasizing the fundamental role of this bifunctional enzyme and sialylation during development. The need of UDP-GlcNAc 2-epimerase for a defined sialylation process is exemplified with the polysialylation of the neural cell adhesion molecule in embryonic stem cells.

A Bifunctional Enzyme Catalyzes the First Two Steps in N-Acetylneuraminic Acid Biosynthesis of Rat Liver PURIFICATION AND CHARACTERIZATION OF UDP-N-ACETYLGLUCOSAMINE 2-EPIMERASE/N-ACETYLMANNOSAMINE KINASE

Biosynthesis ofN-acetylneuraminic acid (Neu5Ac), a prominent component of glycoconjugates, is initiated by the action of UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase, EC 5.1.3.14) and N-acetylmannosamine kinase (ManNAc kinase, EC 2.7.1.60). We demonstrate for the first time that the two activities are parts of one bifunctional enzyme in rat liver. The enzyme was purified to homogeneity from rat liver cytosol using salmine sulfate precipitation and chromatography on phenyl-Sepharose, ATP-agarose, and Mono Q. The purification resulted in one polypeptide with an apparent molecular mass of 75 kDa. Immunoprecipitation with a polyclonal antibody against the polypeptide reduced both enzyme activities in equal amounts. Gel filtration analysis of purified UDP-GlcNAc 2-epimerase/ManNAc kinase showed that the polypeptide self-associates as a dimer and as a hexamer with apparent molecular masses of 150 and 450 kDa, respectively. The hexamer was fully active for both enzyme activities, whereas the dimer catalyzed only the phosphorylation ofN-acetylmannosamine (ManNAc). Incubation of the dimer with UDP-N-acetylglucosamine led to reassembly of the fully active hexamer; maximal quantities of the hexamer were produced after incubation for 3 h. Kinetic analysis of purified hexameric and dimeric enzyme revealed significantly lower Michaelis constants (93 ± 3 to 121 ± 15 μm for ManNAc and 1.18 ± 0.13 to 1.67 ± 0.20 mm for ATP) and higher cooperativity (Hill coefficients of 1.42 ± 0.16 to 1.17 ± 0.06 for ManNAc and 1.30 ± 0.09 to 1.05 ± 0.14 for ATP) for the hexamer for both substrates of ManNAc kinase. The Michaelis constant of UDP-GlcNAc 2-epimerase for its substrate was 11 ± 2 μm. The Hill coefficient of 0.45 ± 0.07 represents strongly negative cooperativity in substrate binding. UDP-GlcNAc 2-epimerase was feedback inhibited by CMP-Neu5Ac. Complete inhibition was achieved with 60 μmCMP-Neu5Ac, and highly positive cooperativity (Hill coefficient of 4.1) was found for inhibitor binding.

A Bifunctional Enzyme Catalyzes the First Two Steps in N-Acetylneuraminic Acid Biosynthesis of Rat Liver MOLECULAR CLONING AND FUNCTIONAL EXPRESSION OF UDP-N-ACETYL-GLUCOSAMINE 2-EPIMERASE/N-ACETYLMANNOSAMINE KINASE

N-Acetylneuraminic acid (Neu5Ac) is the precursor of sialic acids, a group of important molecules in biological recognition systems. Biosynthesis of Neu5Ac is initiated and regulated by its key enzyme, UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase, EC5.1.3.14)/N-acetylmannosamine kinase (ManNAc kinase, EC2.7.1.60) in rat liver (Hinderlich, S., Stäsche, R., Zeitler, R., and Reutter, W. (1997) J. Biol. Chem. 272, 24313–24318). In the present paper we report the isolation and characterization of a cDNA clone encoding this bifunctional enzyme. An open reading frame of 2166 base pairs encodes 722 amino acids with a predicted molecular mass of 79 kDa. The deduced amino acid sequence contains exact matches of the sequences of five peptides derived from tryptic cleavage of the enzyme. The recombinant bifunctional enzyme was expressed in COS7 cells, where it displayed both epimerase and kinase activity. Distribution of UDP-GlcNAc 2-epimerase/ManNAc kinase in the cytosol of several rat tissues was investigated by determining both specific enzyme activities. Secreting organs (liver, salivary glands, and intestinal mucosa) showed high specific activities of UDP-GlcNAc 2-epimerase/ManNAc kinase, whereas significant levels of these activities were absent from other organs (lung, kidney, spleen, brain, heart, skeletal muscle, and testis). Northern blot analysis revealed no UDP-GlcNAc 2-epimerase/ManNAc kinase mRNA in the non-secreting tissues.

Selective Loss of either the Epimerase or Kinase Activity of UDP-N-acetylglucosamine 2-Epimerase/N-Acetylmannosamine Kinase due to Site-directed Mutagenesis Based on Sequence Alignments

N-Acetylneuraminic acid is the most common naturally occurring sialic acid, as well as being the biosynthetic precursor of this group of compounds. UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase has been shown to be the key enzyme of N-acetylneuraminic acid biosynthesis in rat liver, and it is a regulator of cell surface sialylation. The N-terminal region of this bifunctional enzyme displays sequence similarities with prokaryotic UDP-GlcNAc 2-epimerases, whereas the sequence of its C-terminal region is similar to sequences of members of the sugar kinase superfamily. High level overexpression of active enzyme was established by using the baculovirus/Sf9 system. For functional characterization, site-directed mutagenesis was performed on different conserved amino acid residues. The histidine mutants H45A, H110A, H132A, H155A, and H157A showed a drastic loss of epimerase activity with almost unchanged kinase activity. Conversely, the mutants D413N, D413K, and R420M in the putative kinase active site lost their kinase activity but retained their epimerase activity. To estimate the structural perturbation effect due to site-directed mutagenesis, the oligomeric state of all mutants was determined by gel filtration analysis. The mutants D413N, D413K, and R420M as well as H45A were shown to form a hexamer like the wild-type enzyme, indicating little influence of mutation on protein folding. Histidine mutants H155A and H157A formed mainly trimeric enzyme with small amounts of hexamer. Oligomerization of mutants H110A and H132A was also significantly different from that of the wild-type enzyme. Therefore the loss of epimerase activity in mutants H110A, H132A, H155A, and H157A can largely be attributed to incorrect protein folding. In contrast, the mutation site of mutant H45A seems to be involved directly in the epimerization process, and the amino acids Asp-413 and Arg-420 of UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase are essential for the phosphorylation process. The fact that either epimerase or kinase activity are lost selectively provides evidence for the existence of two active sites working quite independently.

The homozygous M712T mutation of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase results in reduced enzyme activities but not in altered overall cellular sialylation in hereditary inclusion body myopathy

Hereditary inclusion body myopathy (HIBM) is a neuromuscular disorder, caused by mutations in UDP‐N‐acetylglucosamine 2‐epimerase/N‐acetylmannosamine kinase, the key enzyme of sialic acid biosynthesis. In Middle Eastern patients a single homozygous mutation occurs, converting methionine‐712 to threonine. Recombinant expression of the mutated enzyme revealed slightly reduced N‐acetylmannosamine kinase activity, in agreement with the localization of the mutation within the kinase domain. B lymphoblastoid cell lines derived from patients expressing the mutated enzyme also display reduced UDP‐N‐acetylglucosamine 2‐epimerase activity. Nevertheless, no reduced cellular sialylation was found in those cells by colorimetric assays and lectin analysis, indicating that HIBM is not directly caused by an altered overall expression of sialic acids.

UDP-N-Acetylglucosamine 2-Epimerase/N-Acetylmannosamine Kinase (GNE) Binds to Alpha-Actinin 1: Novel Pathways in Skeletal Muscle?

Background Hereditary inclusion body myopathy (HIBM) is a rare neuromuscular disorder caused by mutations in GNE, the key enzyme in the biosynthetic pathway of sialic acid. While the mechanism leading from GNE mutations to the HIBM phenotype is not yet understood, we searched for proteins potentially interacting with GNE, which could give some insights about novel putative biological functions of GNE in muscle. Methodology/Principal Findings We used a Surface Plasmon Resonance (SPR)-Biosensor based assay to search for potential GNE interactors in anion exchanged fractions of human skeletal muscle primary culture cell lysate. Analysis of the positive fractions by in vitro binding assay revealed α-actinin 1 as a potential interactor of GNE. The direct interaction of the two proteins was assessed in vitro by SPR-Biosensor based kinetics analysis and in a cellular environment by a co-immunoprecipitation assay in GNE overexpressing 293T cells. Furthermore, immunohistochemistry on stretched mouse muscle suggest that both GNE and α-actinin 1 localize to an overlapping but not identical region of the myofibrillar apparatus centered on the Z line. Conclusions/Significance The interaction of GNE with α-actinin 1 might point to its involvement in α-actinin mediated processes. In addition these studies illustrate for the first time the expression of the non-muscle form of α-actinin, α-actinin 1, in mature skeletal muscle tissue, opening novel avenues for its specific function in the sarcomere. Although no significant difference could be detected in the binding kinetics of α-actinin 1 with either wild type or mutant GNE in our SPR biosensor based analysis, further investigation is needed to determine whether and how the interaction of GNE with α-actinin 1 in skeletal muscle is relevant to the putative muscle-specific function of α-actinin 1, and to the muscle-restricted pathology of HIBM.

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

Abstract The first two steps in mammalian biosynthesis of Nacetylneuraminic acid, an important carbohydrate moiety in biological recognition systems, are performed by the bifunctional enzyme UDPNacetylglucosamine 2-epimerase/Nacetylmannosamine kinase. A subclone of the human B lymphoma cell line BJAB K20, lacking UDPNacetylglucosamine 2- epimerase/Nacetylmannosamine kinase mRNA as well as epimerase activity, displayed hyposialylated, functionally impaired cell surface glycoconjugates. Here we show that this cell line surprisingly still retains Nacetylmannosamine kinase activity. A gel filtration analysis of BJAB K88 control cells, which express UDPNacetylglucosamine 2-epimerase/Nacetylmannosamine kinase, revealed two Nacetylmannosamine kinase activity peaks, one coeluting with UDPNacetylglucosamine 2-epimerase activity and one coeluting with Nacetylglucosamine kinase. For this enzyme previous studies already showed ManNAc kinase activity in vitro. In contrast, the hyposialylated BJAB K20 subclone displayed only the Nacetylmannosamine kinase peak, comigrating with Nacetylglucosamine kinase. The CMPNacetylneuraminic acid content of both K88 and K20 cells and the sialylation of cell surface glycoconjugates of K20 cells could be significantly increased by supple menting the medium with Nacetylmannosamine. This Nacetylmannosamineinduced increase was drastically reduced by cosupplementation with Nacetylglucosamine only in K20 cells. We therefore propose the phosphorylation of Nacetylmannosamine as a hitherto unrecognized role of Nacetylglucosamine kinase in living cells.

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.

Regulation and pathophysiological implications of UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) as the key enzyme of sialic acid biosynthesis

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.

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.