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

AbstractThe 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

Betriebliche Mitbestimmung im Wandel: ein britisch-deutscher Vergleich

"Die Welt befindet sich in einer Phase des strukturellen Wandels. Durch das Zusammenwachsen der Wirtschaftsräume erhöht sich der Wettbewerbsdruck auf die deutschen Unternehmen. Die deutschen Arbeitsbeziehungen - und damit auch das Mitbestimmungssystem - werden dabei in Konkurrenz zu den Systemen anderer Länder gesehen. Bedenken über die internationale Wettbewerbsfähigkeit der deutschen Arbeitsbeziehungen sind allgegenwärtig in der öffentlichen Diskussion. Die Analyse der betrieblichen und überbetrieblichen Mitbestimmung in Deutschland steht im Mittelpunkt dieser Arbeit. Dabei soll zum einen untersucht werden, wie Betriebsräte und Tarifverträge die betriebliche Produktivität, die Prozessinnovationen und die Eigenkündigungen beeinflussen. Zum anderen soll aufgezeigt werden, wie sich der Einfluss beider Mitbestimmungsinstitutionen in den letzten Jahren durch den institutionellen Wandel verändert hat. Herausgehobene Bedeutung kommt in diesem Kontext der Novellierung des Betriebsverfassungsgesetzes im Jahr 2001 zu. Wenn Kritik an dem deutschen Mitbestimmungssystem geäußert wird, wird häufig auf das britische Mitbestimmungssystem als positives Gegenbeispiel verwiesen. In diesem Kontext wird regelmäßig die britische Reformpolitik hervorgehoben, die das britische Mitbestimmungssystem - insbesondere das Gewerkschaftssystem - in den 80er und 90er Jahren grundlegend verändert hat. Eine eventuelle Vorteilhaftigkeit des britischen Systems im Allgemeinen sowie eine mögliche Übertragbarkeit der britischen Reformpolitik auf die deutschen Gegebenheiten im Speziellen ist ebenfalls Gegenstand dieser Arbeit." (Autorenreferat

Purification and Characterization of Antibodies Directed against the α-Gal Epitope

The α-Gal epitope is an immunogen trisaccharide structure consisting of N-acetylglucosamine (GlcNAc)β1,4-galactose (Gal)α1,3-Gal. It is presented as part of complex-type glycans on glycoproteins or glycolipids on cell surfaces of non-primate mammalians. About 1% of all antibodies in human sera are specific toward α1,3-Gal and are therefore named as anti-α-Gal antibodies. This work comprises the purification and characterization of anti-α-Gal antibodies from human immunoglobulin G (IgG). A synthetically manufactured α Gal epitope affinity resin was used to enrich anti-α-Gal antibodies. Selectivity experiments with purified antibodies were carried out using enzyme-linked immunosorbent assays (ELISA), Western blotting, and erythrocyte agglutination. Furthermore, binding affinities toward α-Gal were determined by surface plasmon resonance (SPR) and the IgG distribution of anti α Gal antibodies (83% IgG2, 14% IgG1, 2% IgG3, 1% IgG4) was calculated applying ELISA and immunodiffusion. A range of isoelectric points from pH 6 to pH 8 was observed in 2D gel electrophoresis. Glycan profiling of anti α Gal antibodies revealed complex biantennary structures with high fucosylation grades (86%). Additionally, low amounts of bisecting GlcNAc (15%) and sialic acids (13%) were detected. The purification of anti-α-Gal antibodies from human IgG was successful, and their use as detection antibodies for α Gal-containing structures was evaluated

Bioengineering of rFVIIa Biopharmaceutical and Structure Characterization for Biosimilarity Assessment

Eptacog alfa (NovoSeven®) is a vitamin K-dependent recombinant Factor VIIa produced by genetic engineering from baby hamster kidney (BHK) cells as a single peptide chain of 406 residues. After activation, it consists of a light chain (LC) of 152 amino and a heavy chain (HC) of 254 amino acids. Recombinant FVIIa undergoes many post-translational modifications (PTMs). The first ten glutamic acids of the N-terminal moiety are γ-carboxylated, Asn145 and Asn322 are N-glycosylated, and Ser52 and Ser60 are O-glycosylated. A head-to-head biosimilarity study was conducted for the originator and the first biosimilar AryoSeven™ to evaluate comparable bioengineering. Physicochemical properties were analyzed based on mass spectrometry, including intact mass, PTMs and higher-order structure. Both biotherapeutics exhibit a batch-to-batch variability in their N-glycan profiles. N-Glycopeptide analysis with UHPLC- QTOF-MSE confirmed N-glycosylation sites as well as two different O-glycopeptide sites. Ser60 was found to be O-fucosylated and Ser52 had O-glucose or O-glucose-(xylose)1,2 motifs as glycan variants. Ion mobility spectrometry (TWIMS) and NMR spectroscopy data affirm close similarity of the higher-order structure of both biologicals. Potency of the biodrugs was analyzed by a coagulation assay demonstrating comparable bioactivity. Consequently, careful process optimization led to a stable production process of the biopharmaceuticals

GNE Is Involved in the Early Development of Skeletal and Cardiac Muscle

UDP-N-acetylglucosamine 2 epimerase/N-acetylmannosamime kinase (GNE) is a bifunctional enzyme which catalyzes the two key sequential steps in the biosynthetic pathway of sialic acid, the most abundant terminal monosaccharide on glycoconjugates of eukaryotic cells. GNE knock out (GNE KO) mice are embryonically lethal at day E8.5. Although the role of GNE in the sialic pathway has been well established as well as the importance of sialylation in many diverse biological pathways, less is known about the involvement of GNE in muscle development. To address this issue we have studied the role of GNE during in vitro embryogenesis by comparing the developmental profile in culture of embryonic stem cells (ES) from wild type and from GNE KO E3.5 mice embryos, during 45 days. Neuronal cells appeared rarely in GNE KO ES cultures and did not reach an advanced differentiated stage. Although primary cardiac cells appeared at the same time in both normal and GNE KO ES cultures, GNE KO cardiac cells degraded very soon and their beating capacity decayed rapidly. Furthermore very rare skeletal muscle committed cells were detected in the GNE KO ES cultures at any stage of differentiation, as assessed by analysis of the expression of either Pax7, MyoD and MyHC markers. Beyond the supporting evidence that GNE plays an important role in neuronal cell and brain development, these results show that GNE is strongly involved in cardiac tissue and skeletal muscle early survival and organization. These findings could open new avenues in the understanding of muscle function mechanisms in health and in disease

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

AbstractHereditary 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?

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. 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 alpha-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 alpha-actinin 1 localize to an overlapping but not identical region of the myofibrillar apparatus centered on the Z line. The interaction of GNE with alpha-actinin 1 might point to its involvement in alpha-actinin mediated processes. In addition these studies illustrate for the first time the expression of the non-muscle form of alpha-actinin, alpha-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 alpha-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 alpha-actinin 1 in skeletal muscle is relevant to the putative muscle-specific function of alpha-actinin 1, and to the muscle-restricted pathology of HIBM