Rita Gerardy-Schahn

Publications of Rita Gerardy-Schahn

• A high-throughput screen for polysialyltransferase activity.

  Authors: Timothy G Keys, Monika Berger, Rita Gerardy-Schahn

  Analytical biochemistry. 05/2012;

  Polysialic acid is common to humans and a few bacterial pathogens, and holds great potential for the development of new therapeutic reagents. Currently, the bacterial polysialyltransferases (polySTs)Polysialic acid is common to humans and a few bacterial pathogens, and holds great potential for the development of new therapeutic reagents. Currently, the bacterial polysialyltransferases (polySTs) are the only source of polysialic acid for research and biotechnological purposes, either directly, by enzymatic polysialylation of therapeutic proteins, or indirectly, by harvest of polysialic acid from bacterial fermentation. Further engineering and optimisation of these enzymes is hindered by the lack of high-throughput screening methodologies for polysialyltransferase activity. Here we report the development of an efficient in vivo activity screen for bacterial polySTs. The screen exploits complementation of a dormant capsule export complex in the expression strain, E. coli BL21-Gold(DE3). This strain was metabolically engineered to synthesise CMP-Neu5Ac, the donor sugar for the polysialylation reaction. Using the new strain a colony blotting procedure was developed which enables the routine testing of more than 10(4) polyST genes. To test the usefulness of the methodology, we screened a library of N-terminally truncated polySTs derived from the N. meningitidis serogroup B (NmB) polyST. We identified truncations which remove a putative membrane interaction domain resulting in soluble and active enzymes.

• Restoration of Synaptic Plasticity and Learning in Young and Aged NCAM-Deficient Mice by Enhancing Neurotransmission Mediated by GluN2A-Containing NMDA Receptors.

  Authors: Gaga Kochlamazashvili, Olena Bukalo, Oleg Senkov, Benedikt Salmen, Rita Gerardy-Schahn, Andreas K Engel, Melitta Schachner, Alexander Dityatev

  The Journal of neuroscience : the official journal of the Society for Neuroscience. 02/2012; 32(7):2263-2275.

  Neural cell adhesion molecule (NCAM) is the predominant carrier of the unusual glycan polysialic acid (PSA). Deficits in PSA and/or NCAM expression cause impairments in hippocampal long-termNeural cell adhesion molecule (NCAM) is the predominant carrier of the unusual glycan polysialic acid (PSA). Deficits in PSA and/or NCAM expression cause impairments in hippocampal long-term potentiation and depression (LTP and LTD) and are associated with schizophrenia and aging. In this study, we show that impaired LTP in adult NCAM-deficient (NCAM(-/-)) mice is restored by increasing the activity of the NMDA subtype of glutamate receptor (GluN) through either reducing the extracellular Mg(2+) concentration or applying d-cycloserine (DCS), a partial agonist of the GluN glycine binding site. Pharmacological inhibition of the GluN2A subtype reduced LTP to the same level in NCAM(-/-) and wild-type (NCAM(+/+)) littermate mice and abolished the rescue by DCS in NCAM(-/-) mice, suggesting that the effects of DCS are mainly mediated by GluN2A. The insufficient contribution of GluN to LTD in NCAM(-/-) mice was also compensated for by DCS. Furthermore, impaired contextual and cued fear conditioning levels were restored in NCAM(-/-) mice by administration of DCS before conditioning. In 12-month-old NCAM(-/-), but not NCAM(+/+) mice, there was a decline in LTP compared with 3-month-old mice that could be rescued by DCS. In 24-month-old mice of both genotypes, there was a reduction in LTP that could be fully restored by DCS in NCAM(+/+) mice but only partially restored in NCAM(-/-) mice. Thus, several deficiencies of NCAM(-/-) mice can be ameliorated by enhancing GluN2A-mediated neurotransmission with DCS.

• Molecular cloning of a xylosyltransferase that transfers the second xylose to O-glucosylated epidermal growth factor repeats of notch.

  Authors: Maya K Sethi, Falk F R Buettner, Angel Ashikov, Vadim B Krylov, Hideyuki Takeuchi, Nikolay E Nifantiev, Robert S Haltiwanger, Rita Gerardy-Schahn, Hans Bakker

  The Journal of biological chemistry. 11/2011; 287(4):2739-48.

  The extracellular domain of Notch contains epidermal growth factor (EGF) repeats that are extensively modified with different O-linked glycans. O-Fucosylation is essential for receptor function, andThe extracellular domain of Notch contains epidermal growth factor (EGF) repeats that are extensively modified with different O-linked glycans. O-Fucosylation is essential for receptor function, and elongation with N-acetylglucosamine, catalyzed by members of the Fringe family, modulates Notch activity. Only recently, genes encoding enzymes involved in the O-glucosylation pathway have been cloned. In the Drosophila mutant rumi, characterized by a mutation in the protein O-glucosyltransferase, Notch signaling is impaired in a temperature-dependent manner, and a mouse knock-out leads to embryonic lethality. We have previously identified two human genes, GXYLT1 and GXYLT2, encoding glucoside xylosyltransferases responsible for the transfer of xylose to O-linked glucose. The identity of the enzyme further elongating the glycan to generate the final trisaccharide xylose-xylose-glucose, however, remained unknown. Here, we describe that the human gene C3ORF21 encodes a UDP-xylose:α-xyloside α1,3-xylosyltransferase, acting on xylose-α1,3-glucoseβ1-containing acceptor structures. We have, therefore, renamed it XXYLT1 (xyloside xylosyltransferase 1). XXYLT1 cannot act on a synthetic acceptor containing an α-linked xylose alone, but requires the presence of the underlying glucose. Activity on Notch EGF repeats was proven by in vitro xylosylation of a mouse Notch1 fragment recombinantly produced in Sf9 insect cells, a bacterially expressed EGF repeat from mouse Notch2 modified in vitro by Rumi and Gxylt2 and in vivo by co-expression of the enzyme with the Notch1 fragment. The enzyme was shown to be a typical type II membrane-bound glycosyltransferase localized in the endoplasmic reticulum.

• Program and abstracts for the 2011 Meeting of the Society for Glycobiology.

  Authors: M Tony Hollingsworth, Gerald W Hart, James C Paulson, Elizabeth Stansell, Kevin Canis, I-Cheuh Huang, Maria Panico, Howard Morris, Stuart Haslam, Michael Farzan [......] Kazunori Hamamura, Takenosuke Yoshida, Kaoru Akita, Tetsuya Okajima, Keiko Furukawa, Takeshi Urano, Koichi Furukawa, L Renee Ruhaak, Suzanne Miyamoto, Carlito B Lebrilla

  Glycobiology. 11/2011; 21(11):1454-531.

  Cell surface mucins configure the cell surface by presenting extended protein backbones that are heavily O-glycosylated. The glycopeptide structures establish physicochemical properties at the cellCell surface mucins configure the cell surface by presenting extended protein backbones that are heavily O-glycosylated. The glycopeptide structures establish physicochemical properties at the cell surface that enable and block the formation of biologically important molecular complexes. Some mucins, such as MUC1, associate with receptor tyrosine kinases and other cell surface receptors, and engage in signal transduction in order to communicate information regarding conditions at the cell surface to the nucleus. In that context, the MUC1 cytoplasmic tail (MUC1CT) receives phosphorylation signals from receptor tyrosine kinases and serine/threonine kinases, which enables its association with different signaling complexes that conduct these signals to the nucleus and perhaps other subcellular organelles. We have detected the MUC1CT at promoters of over 500 genes, in association with several different transcription factors, and have shown that promoter occupancy can vary under different growth factor conditions. However, the full biochemical nature of the nuclear forms of MUC1 and its function at these promoter regions remain undefined. I will present evidence that nuclear forms of the MUC1CT include extracellular and cytoplasmic tail domains. In addition, I will discuss evidence for a hypothesis that the MUC1CT possesses a novel catalytic function that enables remodeling of the transcription factor occupancy of promoters, and thereby engages in regulation of gene expression.

• ST3GAL3 mutations impair the development of higher cognitive functions.

  Authors: Hao Hu, Katinka Eggers, Wei Chen, Masoud Garshasbi, M Mahdi Motazacker, Klaus Wrogemann, Kimia Kahrizi, Andreas Tzschach, Masoumeh Hosseini, Ideh Bahman, Tim Hucho, Martina Mühlenhoff, Rita Gerardy-Schahn, Hossein Najmabadi, H Hilger Ropers, Andreas W Kuss

  American journal of human genetics. 09/2011; 89(3):407-14.

  The genetic variants leading to impairment of intellectual performance are highly diverse and are still poorly understood. ST3GAL3 encodes the Golgi enzyme β-galactoside-α2,3-sialyltransferase-IIIThe genetic variants leading to impairment of intellectual performance are highly diverse and are still poorly understood. ST3GAL3 encodes the Golgi enzyme β-galactoside-α2,3-sialyltransferase-III that in humans predominantly forms the sialyl Lewis a epitope on proteins. ST3GAL3 resides on chromosome 1 within the MRT4 locus previously identified to associate with nonsyndromic autosomal recessive intellectual disability. We searched for the disease-causing mutations in the MRT4 family and a second independent consanguineous Iranian family by using a combination of chromosome sorting and next-generation sequencing. Two different missense changes in ST3GAL3 cosegregate with the disease but were absent in more than 1000 control chromosomes. In cellular and biochemical test systems, these mutations were shown to cause ER retention of the Golgi enzyme and drastically impair ST3Gal-III functionality. Our data provide conclusive evidence that glycotopes formed by ST3Gal-III are prerequisite for attaining and/or maintaining higher cognitive functions.

• "Add-on" domains of Drosophila β1,4-N-acetylgalactosaminyltransferase B in the stem region and its pilot protein.

  Authors: Benjamin Kraft, Anita Johswich, Gwenda Kauczor, Meike Scharenberg, Rita Gerardy-Schahn, Hans Bakker

  Cellular and molecular life sciences : CMLS. 05/2011; 68(24):4091-100.

  The glycolipid specific Drosophila melanogaster β1,4-N-acetylgalactosaminyltransferase B (β4GalNAcTB) depends on a zinc finger DHHC protein family member named GalNAcTB pilot (GABPI) for activity andThe glycolipid specific Drosophila melanogaster β1,4-N-acetylgalactosaminyltransferase B (β4GalNAcTB) depends on a zinc finger DHHC protein family member named GalNAcTB pilot (GABPI) for activity and translocation to the Golgi. The six-membrane spanning protein actually lacks the cysteine in the cytoplasmic DHHC motif, displaying DHHS instead. Here we show that the whole conserved region around the DHHS sequence, which is essential for palmitoylation in DHHC proteins, is not required for GABPI to interact with β4GalNAcTB. In contrast, the two luminal loops between transmembrane domain 3-4 and 5-6 contain conserved amino acids, which are crucial for activity. Besides the dependence on GABPI, β4GalNAcTB requires its exceptional short stem region for activity. A few hydrophobic amino acids positioned close to the transmembrane domain are essential for the interaction with GABPI. Along with its catalytic domain, β4GalNAcTB, thus, requires an area in its own stem region and two small luminal loops of GABPI as "add-on" domains. Moreover, some inactive GABPI mutants could be rescued by fusion with β4GalNAcTB, indicating their importance in direct GABPI-β4GalNAcTB interaction.

• Comparison of polysialic acid production in Escherichia coli K1 during batch cultivation and fed-batch cultivation applying two different control strategies.

  Authors: Ran Chen, Jinu John, Bastian Rode, Bernd Hitzmann, Rita Gerardy-Schahn, Cornelia Kasper, Thomas Scheper

  Journal of biotechnology. 04/2011; 154(4):222-9.

  The polysialic acid (PSA) production in Escherichia coli (E. coli) K1 was studied using three different cultivation strategies. A batch cultivation, a fed-batch cultivation at a constant specificThe polysialic acid (PSA) production in Escherichia coli (E. coli) K1 was studied using three different cultivation strategies. A batch cultivation, a fed-batch cultivation at a constant specific growth rate of 0.25 h(-1) and a fed-batch cultivation at a constant glucose concentration of 50 mg l(-1) was performed. PSA formation kinetics under different cultivation strategies were analyzed based on the Monod growth model and the Luedeking-Piret equation. The results revealed that PSA formation in E. coli K1 was completely growth associated, the highest specific PSA formation rate (0.0489 g g(-1)h(-1)) was obtained in the batch cultivation. However, comparing biomass and PSA yields on the glucose consumed, both fed-batch cultivations provided higher yields than that of the batch cultivation and acetate formation was prevented. Moreover, PSA yield on glucose was also correlated to the specific growth rate of the cells. The optimal specific growth rate for PSA production was 0.32 h(-1) obtained in the fed-batch cultivation at a constant glucose concentration of 50 mg l(-1), with highest conversion efficiency of 43 mg g(-1).

• Defective polysialylation and sialylation induce opposite effects on gating of the skeletal Na+ channel NaV1.4 in Chinese hamster ovary cells.

  Authors: Jörg Ahrens, Nilufar Foadi, Ania Eberhardt, Gertrud Haeseler, Reinhard Dengler, Andreas Leffler, Martina Mühlenhoff, Rita Gerardy-Schahn, Martin Leuwer

  Pharmacology. 01/2011; 87(5-6):311-7.

  Polysialic acid (polySia) is a large, negatively charged homopolymer of 2,8-linked N-acetylneuraminic acid residues resulting from remodeling and extension of protein-bound sialic acid (Sia) residuesPolysialic acid (polySia) is a large, negatively charged homopolymer of 2,8-linked N-acetylneuraminic acid residues resulting from remodeling and extension of protein-bound sialic acid (Sia) residues and seems to have a key role in regulating neural cell development and function. The aim of this study was to explore and compare the effects of polySia and sialylation on gating of voltage-gated sodium channels. The skeletal muscle α-subunit NaV1.4 was transiently expressed in wild-type Chinese hamster ovary (CHO) cells or in mutant CHO cells with deficits in their capacity to produce sialylated or polysialylated membrane components. Expression in both mutant cell lines resulted in larger peak current amplitudes as compared to wild-type CHO cells. Loss of Sia and polySia also resulted in significant shifts of voltage-dependent activation and steady-state inactivation, however, in opposite directions. Furthermore, only the loss of Sia had a significant effect on recovery from fast inactivation. Our data demonstrate for the first time that gating of voltage-gated sodium channels seems to be differentially regulated by polySia and Sia.

• In vivo evaluation of polysialic acid as part of tissue-engineered nerve transplants.

  Authors: Kirsten Haastert-Talini, Janett Schaper-Rinkel, Ruth Schmitte, Rode Bastian, Martina Mühlenhoff, David Schwarzer, Gerald Draeger, Yi Su, Thomas Scheper, Rita Gerardy-Schahn, Claudia Grothe

  Tissue engineering. Part A. 10/2010; 16(10):3085-98.

  With the aim to develop new biomaterials for peripheral nerve grafts, the current study used bioidentical polysialic acid (polySia) as complement in synthetic conduits. polySia provides an importantWith the aim to develop new biomaterials for peripheral nerve grafts, the current study used bioidentical polysialic acid (polySia) as complement in synthetic conduits. polySia provides an important guidance cue during nervous system development and regeneration. First in vivo results on the use of cell-free and Schwann cell-containing synthetic peripheral nerve grafts complemented with soluble exogenous K1-polySia are presented. Reconstructing 10 mm rat sciatic nerve gaps, K1-polySia complementation significantly improved structural nerve regeneration in comparison to cell-free and K1-polySia-free grafts. Subsequently, long nerve gaps (13 mm) were reconstructed by Schwann cell transplants plus K1-polySia and compared to nerve autotransplantation. Structural but also functional regeneration could be observed using K1-polySia transplants; however, autotransplantation was still significantly more successful. Overall, the current study demonstrates that exogenous K1-polySia has no negative but rather regeneration promoting effects. This is important novel evidence on the applicability of exogenous polySia in vivo. Further studies are required to develop solid three-dimensional polySia-based scaffolds for nerve tissue engineering. Biocompatible and assessable biodegrading materials will ensure long-lasting presence of polySia to allow its applicability and prolonged efficacy in the slow regenerating scenario of human peripheral nerve reconstruction.

• Quantification of nucleotide-activated sialic acids by a combination of reduction and fluorescent labeling.

  Authors: Sebastian P Galuska, Hildegard Geyer, Birgit Weinhold, Maria Kontou, René C Röhrich, Ulrike Bernard, Rita Gerardy-Schahn, Werner Reutter, Anja Münster-Kühnel, Rudolf Geyer

  Analytical chemistry. 06/2010; 82(11):4591-8.

  Sialic acids usually represent the terminal monosaccharide of glycoconjugates and are directly involved in many biological processes. The cellular concentration of their nucleotide-activated form isSialic acids usually represent the terminal monosaccharide of glycoconjugates and are directly involved in many biological processes. The cellular concentration of their nucleotide-activated form is one pacemaker for the highly variable sialylation of glycoconjugates. Hence, the determination of CMP-sialic acid levels is an important factor to understand the complex glycosylation machinery of cells and to standardize the production of glycotherapeutics. We have established a highly sensitive strategy to quantify the concentration of nucleotide-activated sialic acid by a combination of reduction and fluorescent labeling using the fluorophore 1,2-diamino-4,5-methylenedioxybenzene (DMB). The labeling with DMB requires free keto as well as carboxyl groups of the sialic acid molecule. Reduction of the keto group prior to the labeling process precludes the labeling of nonactivated sialic acids. Since the keto group is protected against reduction by the CMP-substitution, labeling of nucleotide-activated sialic acids is still feasible after reduction. Subsequent combination of the DMB-high-performance liquid chromatography (HPLC) application with mass spectrometric approaches, such as matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) and electrospray-ionization (ESI)-MS, allows the unambiguous identification of both natural and modified CMP-sialic acids and localization of potential substituents. Thus, the described strategy offers a sensitive detection, identification, and quantification of nucleotide-activated sialic acid derivatives in the femtomole range without the need for nucleotide-activated standards.

• Structural basis for the recognition and cleavage of polysialic acid by the bacteriophage K1F tailspike protein EndoNF.

  Authors: Eike Christian Schulz, David Schwarzer, Martin Frank, Katharina Stummeyer, Martina Mühlenhoff, Achim Dickmanns, Rita Gerardy-Schahn, Ralf Ficner

  Journal of molecular biology. 03/2010; 397(1):341-51.

  An alpha-2,8-linked polysialic acid (polySia) capsule confers immune tolerance to neuroinvasive, pathogenic prokaryotes such as Escherichia coli K1 and Neisseria meningitidis and supports hostAn alpha-2,8-linked polysialic acid (polySia) capsule confers immune tolerance to neuroinvasive, pathogenic prokaryotes such as Escherichia coli K1 and Neisseria meningitidis and supports host infection by means of molecular mimicry. Bacteriophages of the K1 family, infecting E. coli K1, specifically recognize and degrade this polySia capsule utilizing tailspike endosialidases. While the crystal structure for the catalytic domain of the endosialidase of bacteriophage K1F (endoNF) has been solved, there is yet no structural information on the mode of polySia binding and cleavage available. The crystal structure of activity deficient active-site mutants of the homotrimeric endoNF cocrystallized with oligomeric sialic acid identified three independent polySia binding sites in each endoNF monomer. The bound oligomeric sialic acid displays distinct conformations at each site. In the active site, a Sia(3) molecule is bound in an extended conformation representing the enzyme-product complex. Structural and biochemical data supported by molecular modeling enable to propose a reaction mechanism for polySia cleavage by endoNF.

• Neural cell adhesion molecule-associated polysialic acid regulates synaptic plasticity and learning by restraining the signaling through GluN2B-containing NMDA receptors.

  Authors: Gaga Kochlamazashvili, Oleg Senkov, Sergei Grebenyuk, Catrina Robinson, Mei-Fang Xiao, Katharina Stummeyer, Rita Gerardy-Schahn, Andreas K Engel, Larry Feig, Alexey Semyanov, Vishnu Suppiramaniam, Melitta Schachner, Alexander Dityatev

  The Journal of neuroscience : the official journal of the Society for Neuroscience. 03/2010; 30(11):4171-83.

  The neural cell adhesion molecule (NCAM) is the predominant carrier of alpha2,8 polysialic acid (PSA) in the mammalian brain. Abnormalities in PSA and NCAM expression are associated withThe neural cell adhesion molecule (NCAM) is the predominant carrier of alpha2,8 polysialic acid (PSA) in the mammalian brain. Abnormalities in PSA and NCAM expression are associated with schizophrenia in humans and cause deficits in hippocampal synaptic plasticity and contextual fear conditioning in mice. Here, we show that PSA inhibits opening of recombinant NMDA receptors composed of GluN1/2B (NR1/NR2B) or GluN1/2A/2B (NR1/NR2A/NR2B) but not of GluN1/2A (NR1/NR2A) subunits. Deficits in NCAM/PSA increase GluN2B-mediated transmission and Ca(2+) transients in the CA1 region of the hippocampus. In line with elevation of GluN2B-mediated transmission, defects in long-term potentiation in the CA1 region and contextual fear memory in NCAM/PSA-deficient mice are abrogated by application of a GluN2B-selective antagonist. Furthermore, treatment with the glutamate scavenger glutamic-pyruvic transaminase, ablation of Ras-GRF1 (a mediator of GluN2B signaling to p38 MAPK), or direct inhibition of hyperactive p38 MAPK can restore impaired synaptic plasticity in brain slices lacking PSA/NCAM. Thus, PSA carried by NCAM regulates plasticity and learning by inhibition of the GluN2B-Ras-GRF1-p38 MAPK signaling pathway. These findings implicate carbohydrates carried by adhesion molecules in modulating NMDA receptor signaling in the brain and demonstrate reversibility of cognitive deficits associated with ablation of a schizophrenia-related adhesion molecule.

• Polysialic acid affects pathophysiological consequences of status epilepticus.

  Authors: Anton Pekcec, Birgit Weinhold, Rita Gerardy-Schahn, Heidrun Potschka

  Neuroreport. 03/2010; 21(8):549-53.

  Modulation of the neural cell adhesion molecule by the attachment of polysialic acid residues through the polysialyl-transferase, ST8SiaIV, regulates neuronal plasticity and affects cellularModulation of the neural cell adhesion molecule by the attachment of polysialic acid residues through the polysialyl-transferase, ST8SiaIV, regulates neuronal plasticity and affects cellular alterations in the epileptic brain. Here, we determined the impact of ST8SiaIV deficiency on the pathophysiological consequences of status epilepticus (SE). ST8SiaIV deficiency reduced the latency to SE induction and increased SE-mediated mortality. Analysis of the doublecortin expression showed a reduced number of neuroblasts as a long-term consequence of SE in ST8SiaIV knockouts. Testing in a battery of different behavioral paradigms indicated that loss of ST8SiaIV affects the long-term behavioral consequences. In summary, the data suggest that the polysialic acid-neural cell adhesion molecule system is a putative target for the modulation of pathophysiological events and affects psychiatric comorbidities in epilepsies.

• Crystal structure of an intramolecular chaperone mediating triple-beta-helix folding.

  Authors: Eike C Schulz, Achim Dickmanns, Henning Urlaub, Andreas Schmitt, Martina Mühlenhoff, Katharina Stummeyer, David Schwarzer, Rita Gerardy-Schahn, Ralf Ficner

  Nature structural & molecular biology. 02/2010; 17(2):210-5.

  Protein folding is often mediated by molecular chaperones. Recently, a novel class of intramolecular chaperones has been identified in tailspike proteins of evolutionarily distant viruses, whichProtein folding is often mediated by molecular chaperones. Recently, a novel class of intramolecular chaperones has been identified in tailspike proteins of evolutionarily distant viruses, which require a C-terminal chaperone for correct folding. The highly homologous chaperone domains are interchangeable between pre-proteins and release themselves after protein folding. Here we report the crystal structures of two intramolecular chaperone domains in either the released or the pre-cleaved form, revealing the role of the chaperone domain in the formation of a triple-beta-helix fold. Tentacle-like protrusions enclose the polypeptide chains of the pre-protein during the folding process. After the assembly, a sensory mechanism for correctly folded beta-helices triggers a serine-lysine catalytic dyad to autoproteolytically release the mature protein. Sequence analysis shows a conservation of the intramolecular chaperones in functionally unrelated proteins sharing beta-helices as a common structural motif.

• Structure analysis of endosialidase NF at 0.98 A resolution.

  Authors: Eike C Schulz, Piotr Neumann, Rita Gerardy-Schahn, George M Sheldrick, Ralf Ficner

  Acta crystallographica. Section D, Biological crystallography. 02/2010; 66(Pt 2):176-80.

  Endosialidase NF (endoNF) is a bacteriophage-derived endosialidase that specifically degrades alpha-2,8-linked polysialic acid. The structure of a new crystal form of endoNF in complex with sialicEndosialidase NF (endoNF) is a bacteriophage-derived endosialidase that specifically degrades alpha-2,8-linked polysialic acid. The structure of a new crystal form of endoNF in complex with sialic acid has been refined at 0.98 A resolution. The 210 kDa homotrimeric multi-domain enzyme displays outstanding stability and resistance to SDS. Even at atomic resolution, only a minor fraction of side chains possess alternative conformations. However, multiple conformations of an active-site residue imply that it has an important catalytic function in the cleavage mechanism of polysialic acid.

• Polysialylation of NCAM.

  Authors: Herbert Hildebrandt, Martina Mühlenhoff, Rita Gerardy-Schahn

  Advances in experimental medicine and biology. 01/2010; 663:95-109.

• Neisseria meningitidis Serogroup B Polysialyltransferase: Insights into Substrate Binding.

  Authors: Raphael Böhm, Friedrich Freiberger, Katharina Stummeyer, Rita Gerardy-Schahn, Mark von Itzstein, Thomas Haselhorst

  Chembiochem : a European journal of chemical biology. 12/2009;

• Identification of glycosyltransferase 8 family members as xylosyltransferases acting on O-glucosylated notch EGF repeats.

  Authors: Maya K Sethi, Falk F R Buettner, Vadim B Krylov, Hideyuki Takeuchi, Nikolay E Nifantiev, Robert S Haltiwanger, Rita Gerardy-Schahn, Hans Bakker

  The Journal of biological chemistry. 11/2009;

  The epidermal growth factor repeats of the Notch receptor are extensively glycosylated with three different O-glycans. O-Fucosylation and elongation by the glycosyltransferase Fringe have been wellThe epidermal growth factor repeats of the Notch receptor are extensively glycosylated with three different O-glycans. O-Fucosylation and elongation by the glycosyltransferase Fringe have been well studied and shown to be essential for proper Notch signaling. In contrast, biosynthesis of O-glucose and O-N-acetylglucosamine is less well understood. Recently, the isolation of the Drosophila mutant rumi has shown that absence of O-glucose impairs Notch function. O-Glucose is further extended by two contiguous alpha1,3-linked xylose residues. We have identified two enzymes of the human glycosyltransferase 8 family, now named GXYLT1 and GXYLT2 (glucoside xylosyltransferase), as UDP-D-xylose: beta-D-glucoside alpha1,3-D-xylosyltransferases adding the first xylose. The enzymes are specific for beta-glucose terminating acceptors and UDP-xylose as donor substrate. Generation of the alpha1,3-linkage was confirmed by nuclear magnetic resonance. Activity on a natural acceptor could be shown by in vitro xylosylation of a Notch fragment expressed in a UDP-xylose deficient cell line and in vivo by co-expression of the enzymes and the Notch fragment in insect cells, followed by mass spectrometric analysis of peptide fragments.

• A C-terminal phosphatase module conserved in vertebrate CMP-sialic acid synthetases provides a tetramerization interface for the physiologically active enzyme.

  Authors: Melanie Oschlies, Achim Dickmanns, Thomas Haselhorst, Wiebke Schaper, Katharina Stummeyer, Joe Tiralongo, Birgit Weinhold, Rita Gerardy-Schahn, Mark von Itzstein, Ralf Ficner, Anja K Münster-Kühnel

  Journal of molecular biology. 09/2009;

  The biosynthesis of sialic acid-containing glycoconjugates is crucial for the development of vertebrate life. CMP-sialic acid synthetase (CSS) catalyzes the metabolic activation of sialic acidsThe biosynthesis of sialic acid-containing glycoconjugates is crucial for the development of vertebrate life. CMP-sialic acid synthetase (CSS) catalyzes the metabolic activation of sialic acids (Sia). In vertebrates the enzyme is chimeric, with the N-terminal domain harboring the synthetase activity. The function of the highly conserved C-terminal domain (CSS-CT) is unknown. To shed light on its biological function, we solved the X-ray structure of murine CSS-CT to 1.9 A resolution. CSS-CT is a stable shamrock-like tetramer that superimposes well with phosphatases of the haloacid dehalogenase superfamily. However, a region found exclusively in vertebrate CSS-CT appears to block the active site entrance. Accordingly, no phosphatase activity was observed in vitro which points towards a non-enzymatic function of CSS-CT. A computational 3D-model of full-length CSS, in combination with in vitro oligomerization studies, provides evidence that CSS-CT serves as a platform for the quaternary organization governing the kinetic properties of the physiologically active enzyme as demonstrated in kinetic studies.

• Polysialic acid governs T-cell development by regulating progenitor access to the thymus.

  Authors: Penelope M Drake, Christina M Stock, Jay K Nathan, Phung Gip, Kevin P K Golden, Birgit Weinhold, Rita Gerardy-Schahn, Carolyn R Bertozzi

  Proceedings of the National Academy of Sciences of the United States of America. 08/2009; 106(29):11995-2000.

  Although the polysialyltransferase ST8Sia IV is expressed in both primary and secondary human lymphoid organs, its product, polysialic acid (polySia), has been largely overlooked by immunologists. InAlthough the polysialyltransferase ST8Sia IV is expressed in both primary and secondary human lymphoid organs, its product, polysialic acid (polySia), has been largely overlooked by immunologists. In contrast, polySia expression and function in the nervous system has been well characterized. In this context, polySia modulates cellular adhesion, migration, cytokine response, and contact-dependent differentiation. Provocatively, these same processes are vital components of immune development and function. We previously established that mouse multipotent hematopoietic progenitors use ST8Sia IV to express polySia on their cell surfaces. Here, we demonstrate that, relative to wild-type controls, ST8Sia IV(-/-) mice have a 30% reduction in total thymocytes and a concomitant deficiency in the earliest thymocyte precursors. T-cell progenitors originate in the bone marrow and are mobilized to the blood at regular intervals by unknown signals. We performed in vivo reconstitution experiments in which ST8Sia IV(-/-) progenitors competed with wild-type cells to repopulate depleted or deficient immune subsets. Progenitors lacking polySi exhibited a specific defect in T-cell development because of an inability to access the thymus. This phenotype probably reflects a decreased capacity of the ST8Sia IV(-/-) progenitors to escape from the bone marrow niche. Collectively, these results provide evidence that polySia is involved in hematopoietic development.