Martina Mühlenhoff

Publications of Martina Mühlenhoff

• Homeostatic regulation of NCAM polysialylation is critical for correct synaptic targeting.

  Authors: Johannes Vogt, Robert Glumm, Leslie Schlüter, Dietmar Schmitz, Benjamin R Rost, Nora Streu, Benjamin Rister, B Suman Bharathi, Daniel Gagiannis, Herbert Hildebrandt, Birgit Weinhold, Martina Mühlenhoff, Thomas Naumann, Nic E Savaskan, Anja U Brauer, Werner Reutter, Bernd Heimrich, Robert Nitsch, Rüdiger Horstkorte

  Cellular and molecular life sciences : CMLS. 11/2011;

  During development, axonal projections have a remarkable ability to innervate correct dendritic subcompartments of their target neurons and to form regular neuronal circuits. Altered axonal targetingDuring development, axonal projections have a remarkable ability to innervate correct dendritic subcompartments of their target neurons and to form regular neuronal circuits. Altered axonal targeting with formation of synapses on inappropriate neurons may result in neurodevelopmental sequelae, leading to psychiatric disorders. Here we show that altering the expression level of the polysialic acid moiety, which is a developmentally regulated, posttranslational modification of the neural cell adhesion molecule NCAM, critically affects correct circuit formation. Using a chemically modified sialic acid precursor (N-propyl-D: -mannosamine), we inhibited the polysialyltransferase ST8SiaII, the principal enzyme involved in polysialylation during development, at selected developmental time-points. This treatment altered NCAM polysialylation while NCAM expression was not affected. Altered polysialylation resulted in an aberrant mossy fiber projection that formed glutamatergic terminals on pyramidal neurons of the CA1 region in organotypic slice cultures and in vivo. Electrophysiological recordings revealed that the ectopic terminals on CA1 pyramids were functional and displayed characteristics of mossy fiber synapses. Moreover, ultrastructural examination indicated a "mossy fiber synapse"-like morphology. We thus conclude that homeostatic regulation of the amount of synthesized polysialic acid at specific developmental stages is essential for correct synaptic targeting and circuit formation during hippocampal development.

• 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.

• Polysialic acid controls NCAM signals at cell-cell contacts to regulate focal adhesion independent from FGF receptor activity.

  Authors: Katinka Eggers, Sebastian Werneburg, Andrea Schertzinger, Markus Abeln, Miriam Schiff, Matthias Alexander Scharenberg, Hannelore Burkhardt, Martina Mühlenhoff, Herbert Hildebrandt

  Journal of cell science. 10/2011; 124(Pt 19):3279-91.

  The polysialic acid (polySia) modification of the neural cell adhesion molecule NCAM is a key regulator of cell migration. Yet its role in NCAM-dependent or NCAM-independent modulation of motilityThe polysialic acid (polySia) modification of the neural cell adhesion molecule NCAM is a key regulator of cell migration. Yet its role in NCAM-dependent or NCAM-independent modulation of motility and cell-matrix adhesion is largely unresolved. Here, we demonstrate that loss of polySia attenuates tumour cell migration and augments the number of focal adhesions in a cell-cell contact- and NCAM-dependent manner. In the presence or absence of polySia, NCAM never colocalised with focal adhesions but was enriched at cell-cell contacts. Focal adhesion of polySia- and NCAM-negative cells was enhanced by incubation with soluble NCAM or by removing polySia from heterotypic contacts with polySia-NCAM-positive cells. Focal adhesion was compromised by the src-family kinase inhibitor PP2, whereas loss of polySia or exposure to NCAM promoted the association of p59(Fyn) with the focal adhesion scaffolding protein paxillin. Unlike other NCAM responses, NCAM-induced focal adhesion was not prevented by inhibiting FGF receptor activity and could be evoked by NCAM fragments comprising immunoglobulin domains three and four but not by the NCAM fibronectin domains alone or by an NCAM-derived peptide known to interact with and activate FGF receptors. Together, these data indicate that polySia regulates cell motility through NCAM-induced but FGF-receptor-independent signalling to focal adhesions.

• 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.

• Expression of the neural cell adhesion molecule and polysialic acid in human neuroblastoma cell lines.

  Authors: Ursula Valentiner, Martina Mühlenhoff, Ulrich Lehmann, Herbert Hildebrandt, Udo Schumacher

  International journal of oncology. 08/2011; 39(2):417-24.

  The neural cell adhesion molecule NCAM is a cell surface glycoprotein of the immunoglobulin superfamily and is widely expressed in tumours of neuroectodermal origin such as neuroblastoma. NCAM can beThe neural cell adhesion molecule NCAM is a cell surface glycoprotein of the immunoglobulin superfamily and is widely expressed in tumours of neuroectodermal origin such as neuroblastoma. NCAM can be decorated by the carbohydrate polymer polysialic acid (polySia), which attenuates NCAM-mediated cell adhesion and increases cellular motility. The key enzymes in the biosynthesis of polySia are the two polysialyltransferases ST8SiaII and ST8SiaIV. In the present study, expression of NCAM, polySia-NCAM, ST8SiaII and ST8SiaIV was investigated in five human neuroblastoma cell lines before and after xenografting into SCID mice by immunohistochemistry, Western blot analysis and real-time PCR. Results were correlated with the metastatic potential. In vitro, three cell lines (LAN-1, LAN-5 and SH-SY5Y) were positive for polySia attached to the transmembrane isoforms NCAM-140 and NCAM-180, whereas Kelly and SK-N-SH cells were negative for NCAM and polySia. In the presence of NCAM, the level of polySia correlated with the amount of polysialyltransferase transcripts, which were highest in LAN-1, LAN‑5 and SH-SY5Y cells. In the respective primary tumours grown in SCID mice, the expression patterns of NCAM, polySia and polysialyl-transferases were similar to those observed in vitro. After subcutaneous engraftment, polySia-NCAM-positive neuroblastoma developed disseminated micrometastases, a metastatic pattern that was not observed for tumours derived from NCAM-negative cell lines. Together, this indicates that the presence of polySia reduces the adhesiveness of tumour cells and promotes dissemination.

• Crystal structure analysis of the polysialic acid specific O-acetyltransferase NeuO.

  Authors: Eike C Schulz, Anne K Bergfeld, Ralf Ficner, Martina Mühlenhoff

  PloS one. 01/2011; 6(3):e17403.

  The major virulence factor of the neuroinvasive pathogen Escherichia coli K1 is the K1 capsule composed of α2,8-linked polysialic acid (polySia). K1 strains harboring the CUS-3 prophage modify theirThe major virulence factor of the neuroinvasive pathogen Escherichia coli K1 is the K1 capsule composed of α2,8-linked polysialic acid (polySia). K1 strains harboring the CUS-3 prophage modify their capsular polysaccharide by phase-variable O-acetylation, a step that is associated with increased virulence. Here we present the crystal structure of the prophage-encoded polysialate O-acetyltransferase NeuO. The homotrimeric enzyme belongs to the left-handed β-helix (LβH) family of acyltransferases and is characterized by an unusual funnel-shaped outline. Comparison with other members of the LβH family allowed the identification of active site residues and proposal of a catalytic mechanism and highlighted structural characteristics of polySia specific O-acetyltransferases. As a unique feature of NeuO, the enzymatic activity linearly increases with the length of the N-terminal poly-ψ-domain which is composed of a variable number of tandem copies of an RLKTQDS heptad. Since the poly-ψ-domain was not resolved in the crystal structure it is assumed to be unfolded in the apo-enzyme.

• 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.

• Synaptic cell adhesion molecule SynCAM 1 is a target for polysialylation in postnatal mouse brain.

  Authors: Sebastian P Galuska, Manuela Rollenhagen, Moritz Kaup, Katinka Eggers, Imke Oltmann-Norden, Miriam Schiff, Maike Hartmann, Birgit Weinhold, Herbert Hildebrandt, Rudolf Geyer, Martina Mühlenhoff, Hildegard Geyer

  Proceedings of the National Academy of Sciences of the United States of America. 06/2010; 107(22):10250-5.

  Among the large set of cell surface glycan structures, the carbohydrate polymer polysialic acid (polySia) plays an important role in vertebrate brain development and synaptic plasticity. The mainAmong the large set of cell surface glycan structures, the carbohydrate polymer polysialic acid (polySia) plays an important role in vertebrate brain development and synaptic plasticity. The main carrier of polySia in the nervous system is the neural cell adhesion molecule NCAM. As polySia with chain lengths of more than 40 sialic acid residues was still observed in brain of newborn Ncam(-/-) mice, we performed a glycoproteomics approach to identify the underlying protein scaffolds. Affinity purification of polysialylated molecules from Ncam(-/-) brain followed by peptide mass fingerprinting led to the identification of the synaptic cell adhesion molecule SynCAM 1 as a so far unknown polySia carrier. SynCAM 1 belongs to the Ig superfamily and is a powerful inducer of synapse formation. Importantly, the appearance of polysialylated SynCAM 1 was not restricted to the Ncam(-/-) background but was found to the same extent in perinatal brain of WT mice. PolySia was located on N-glycans of the first Ig domain, which is known to be involved in homo- and heterophilic SynCAM 1 interactions. Both polysialyltransferases, ST8SiaII and ST8SiaIV, were able to polysialylate SynCAM 1 in vitro, and polysialylation of SynCAM 1 completely abolished homophilic binding. Analysis of serial sections of perinatal Ncam(-/-) brain revealed that polySia-SynCAM 1 is expressed exclusively by NG2 cells, a multifunctional glia population that can receive glutamatergic input via unique neuron-NG2 cell synapses. Our findings sug-gest that polySia may act as a dynamic modulator of SynCAM 1 functions during integration of NG2 cells into neural networks.

• 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.

• Mass spectrometric fragmentation analysis of oligosialic and polysialic acids.

  Authors: Sebastian P Galuska, Hildegard Geyer, Christina Bleckmann, René C Röhrich, Kai Maass, Anne K Bergfeld, Martina Mühlenhoff, Rudolf Geyer

  Analytical chemistry. 03/2010; 82(5):2059-66.

  Oligosialic and polysialic acids (oligo/polySia) are characterized by high structural diversity, because of different types of sialic acids and glycosidic linkages. Although several methods have beenOligosialic and polysialic acids (oligo/polySia) are characterized by high structural diversity, because of different types of sialic acids and glycosidic linkages. Although several methods have been described for the analysis of oligo/polySia, only high-performance liquid chromatography (HPLC) analysis in conjunction with 1,2-diamino-4,5-methylenedioxybenzene labeling, fluorometric C7/C9 detection, Western blotting, and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF-MS) of lactonized oligo/polySia species, require submicrogram amounts of analyte. Since these methods do not provide detailed structural information, this study is focused on the characterization of oligo/polySia by tandem mass spectrometry (MS/MS). MALDI-TOF-MS/MS and electrospray ionization tandem mass spectrometry (ESI-MS/MS), employing up to three cycles of ion isolation and fragmentation in an ion trap, have been used for the characterization of nonderivatized glycans, oligoSia species modified at their reducing or nonreducing ends, as well as partially O-acetylated oligoSia derivatives. The obtained spectra were dominated by simultaneous cleavage of glycosidic linkages and the corresponding lactone ring, whereas classical cross-ring fragments were of minor abundance. However, the combined use of the two different types of fragmentation analysis allowed a sensitive and detailed characterization of both short-chained oligoSia and long polySia species. Furthermore, oxidation of the nonreducing end sugar moiety enabled sequence determination and localization of acetylated and nonacetylated sialic acid residues.

• 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.

• Polysialylation of NCAM.

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

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

• O-acetyltransferase gene neuO is segregated according to phylogenetic background and contributes to environmental desiccation resistance in Escherichia coli K1.

  Authors: Ines L Mordhorst, Heike Claus, Christa Ewers, Martin Lappann, Christoph Schoen, Johannes Elias, Julia Batzilla, Ulrich Dobrindt, Lothar H Wieler, Anne K Bergfeld, Martina Mühlenhoff, Ulrich Vogel

  Environmental microbiology. 09/2009;

  Escherichia coli K1 causes disease in humans and birds. Its polysialic acid capsule can be O-acetylated via phase-variable expression of the acetyltransferase NeuO encoded by prophage CUS-3. The roleEscherichia coli K1 causes disease in humans and birds. Its polysialic acid capsule can be O-acetylated via phase-variable expression of the acetyltransferase NeuO encoded by prophage CUS-3. The role of capsule O-acetylation in ecological adaptation or pathogenic invasion of E. coli K1 is largely unclear. A population genetics approach was performed to study the distribution of neuO among E. coli K1 isolates from human and avian sources. Multilocus sequence typing revealed 39 different sequence types (STs) among 183 E. coli K1 strains. The proportion of the ST95 complex (STC95) was 44%. NeuO was found in 98% of the STC95 strains, but only in 24% of other STs. Grouping of STs and prophage genotypes revealed a segregation of prophage types according to STs, suggesting coevolution of CUS-3 and the E. coli K1 host. Within the STC95, which is known to harbour both human and avian pathogenic isolates, CUS-3 genotypes were shared irrespective of the host species. Functional analysis of a variety of strain pairs revealed that NeuO-mediated K1 capsule O-acetylation enhanced desiccation resistance. In contrast, NeuO expression led to a reduced biofilm formation in biofilm positive E. coli K1 isolates. These findings suggest a delicate ecological balance of neuO'on'/'off' switching.

• Imbalance of neural cell adhesion molecule and polysialyltransferase alleles causes defective brain connectivity.

  Authors: Herbert Hildebrandt, Martina Mühlenhoff, Imke Oltmann-Norden, Iris Röckle, Hannelore Burkhardt, Birgit Weinhold, Rita Gerardy-Schahn

  Brain : a journal of neurology. 06/2009;

  The neural cell adhesion molecule (NCAM) and its post-translational modification polysialic acid (polySia) are broadly implicated in neural development. Mice lacking the polysialyltransferasesThe neural cell adhesion molecule (NCAM) and its post-translational modification polysialic acid (polySia) are broadly implicated in neural development. Mice lacking the polysialyltransferases ST8SiaII and ST8SiaIV are devoid of polySia, and show severe malformation of major brain axon tracts. Here, we demonstrate how allelic variation of three interacting gene products (NCAM, ST8SiaII and ST8SiaIV) translates into various degrees of anterior commissure, corpus callosum and internal capsule hypoplasia. Loss of ST8SiaII alone caused mild, but distinct defects and the severity of the pathological phenotype found in mice lacking both polysialyltransferases could be stepwise attenuated by reducing NCAM expression. Analysis of mice with overall nine selected combinations of mutant NCAM and polysialyltransferase alleles revealed that the extent of the fibre tract deficiencies was not linked to the total amount of polySia or NCAM, but correlated strictly with the level of NCAM erroneously devoid of polySia during brain development. The defects implemented by the gain of polySia-free NCAM were reminiscent to abnormalities found in patients with schizophrenia. Since variations in NCAM1 and ST8SIA2 have been implicated in schizophrenia, these findings provide a mechanism how genetic interference with the complex coordination of NCAM polysialylation may lead to a neurodevelopmental predisposition to schizophrenia.

• Brain development needs sugar: the role of polysialic acid in controlling NCAM functions.

  Authors: Martina Mühlenhoff, Imke Oltmann-Norden, Birgit Weinhold, Herbert Hildebrandt, Rita Gerardy-Schahn

  Biological chemistry. 06/2009;

  Abstract Polysialic acid (polySia) is a major regulator of cell-cell-interactions in the developing nervous system and a key factor in maintaining neural plasticity. As a polyanionic molecule withAbstract Polysialic acid (polySia) is a major regulator of cell-cell-interactions in the developing nervous system and a key factor in maintaining neural plasticity. As a polyanionic molecule with high water binding capacity, polySia increases the intercellular space and creates conditions that are permissive for cellular plasticity. While the prevailing model highlights polySia as a non-specific regulator of cell-cell contacts, this review concentrates on recent studies in knock-out mice indicating that a crucial function of polySia resides in controlling interactions mediated by its predominant protein carrier, the neural cell adhesion molecule NCAM.

• Proteolytic release of the intramolecular chaperone domain confers processivity to endosialidase F.

  Authors: David Schwarzer, Katharina Stummeyer, Thomas Haselhorst, Friedrich Freiberger, Bastian Rode, Melanie Grove, Thomas Scheper, Mark von Itzstein, Martina Mühlenhoff, Rita Gerardy-Schahn

  The Journal of biological chemistry. 03/2009;

  Endosialidases (endoN), as identified so far, are tailspike proteins of bacteriophages that specifically bind and degrade the a2,8-linked polysialic acid (polySia) capsules of their hosts. TheEndosialidases (endoN), as identified so far, are tailspike proteins of bacteriophages that specifically bind and degrade the a2,8-linked polysialic acid (polySia) capsules of their hosts. The crystal structure solved for the catalytic domain of endoN from coliphage K1F (endoNF) revealed a functional trimer. Folding of the catalytic trimer is mediated by an intramolecular C terminal chaperone domain. Release of the chaperone from the folded protein confers kinetic stability to endoNF. In mutant c(S), the replacement of serine-911 by alanine prevents proteolysis and generates an enzyme that varies in activity from wild type. Using soluble polySia as substrate a 3-times higher activity was detected whilst evaluation with immobilized polySia revealed a 190-fold reduced activity. Importantly, activity of c(S) did not differ from wild type with tetrameric sialic acid, the minimal endoNF substrate. Furthermore, we show that presence of the chaperone domain in c(S) destabilizes binding to polySia in a similar way as did selective disruption of a polySia binding site in the stalk domain. The improved catalytic efficiency towards soluble polySia observed in these mutants can be explained by higher dissociation and association probabilities, while inversely, an impaired processivity was found. The fact that endoNF is a processive enzyme introduces a new molecular basis to explain capsule degradation by bacteriophages, which until now has been regarded as a result of cooperative interaction of tailspike proteins. Moreover, knowing that release of the chaperone domain confers kinetic stability and processivity, conservation of the proteolytic process can be explained by its importance in phage evolution.

• The polysialic acid specific O-acetyltransferase OatC from Neisseria meningitidis serogroup C evolved apart from other bacterial sialate:O-acetyltransferases.

  Authors: Anne K Bergfeld, Heike Claus, Nina K Lorenzen, Fabian Spielmann, Ulrich Vogel, Martina Mühlenhoff

  The Journal of biological chemistry. 12/2008;

  Neisseria meningitidis serogroup C is a major cause of bacterial meningitis and septicaemia. This human pathogen is protected by a capsule composed of alpha2,9-linked polysialic acid which representsNeisseria meningitidis serogroup C is a major cause of bacterial meningitis and septicaemia. This human pathogen is protected by a capsule composed of alpha2,9-linked polysialic acid which represents an important virulence factor. In the majority of strains, the capsular polysaccharide is modified by O-acetylation at position C-7 or C-8 of the sialic acid residues. The gene encoding the capsule modifying O-acetyltransferase is part of the capsule gene complex and shares no sequence similarities with other proteins. Here we describe the purification and biochemical characterization of recombinant OatC. The enzyme was found as a homodimer, with the first 34 amino acids forming an efficient oligomerization domain that worked even in a different protein context. Using acetyl-CoA as donor substrate, OatC transferred acetyl-groups exclusively onto polysialic acid joined by alpha2,9-linkages and did not act on free or CMP-activated sialic acid. Motif scanning revealed a nucleophile elbow motif (GXS(286)XGG), which is a hallmark of alpha/beta-hydrolase fold enzymes. In a comprehensive site-directed mutagenesis study, we identified a catalytic triad composed of Ser-286, Asp-376, and His 399. Consistent with a double-displacement mechanism common to alpha/beta-hydrolase fold enzymes, a covalent acetyl-enzyme intermediate was found. Together with secondary structure prediction highlighting an alpha/beta-hydrolase fold topology, our data provide strong evidence that OatC belongs to the alpha/beta-hydrolase fold family. This clearly distinguishes OatC from all other bacterial sialate:O-acetyltransferases known so far, since these are members of the hexapeptide repeat family, a class of acyltransferases that adopt a left-handed beta-helix fold and assemble into catalytic trimers.

• Polysialic acid controls NCAM-induced differentiation of neuronal precursors into calretinin-positive olfactory bulb interneurons.

  Authors: Iris Röckle, Ralph Seidenfaden, Birgit Weinhold, Martina Mühlenhoff, Rita Gerardy-Schahn, Herbert Hildebrandt

  Developmental neurobiology. 09/2008; 68(9):1170-84.

  Understanding the mechanisms that regulate neurogenesis is a prerequisite for brain repair approaches based on neuronal precursor cells. One important regulator of postnatal neurogenesis isUnderstanding the mechanisms that regulate neurogenesis is a prerequisite for brain repair approaches based on neuronal precursor cells. One important regulator of postnatal neurogenesis is polysialic acid (polySia), a post-translational modification of the neural cell adhesion molecule NCAM. In the present study, we investigated the role of polySia in differentiation of neuronal precursors isolated from the subventricular zone of early postnatal mice. Removal of polySia promoted neurite induction and selectively enhanced maturation into a calretinin-positive phenotype. Expression of calbindin and Pax6, indicative for other lineages of olfactory bulb interneurons, were not affected. A decrease in the number of TUNEL-positive cells indicated that cell survival was slightly improved by removing polySia. Time lapse imaging revealed the absence of chain migration and low cell motility, in the presence and absence of polySia. The changes in survival and differentiation, therefore, could be dissected from the well-known function of polySia as a promoter of precursor migration. The differentiation response was mimicked by exposure of cells to soluble or substrate-bound NCAM and prevented by the C3d-peptide, a synthetic ligand blocking NCAM interactions. Moreover, a higher degree of differentiation was observed in cultures from polysialyltransferase-depleted mice and after NCAM exposure of precursors from NCAM-knockout mice demonstrating that the NCAM function is mediated via heterophilic binding partners. In conclusion, these data reveal that polySia controls instructive NCAM signals, which direct the differentiation of subventricular zone-derived precursors towards the calretinin-positive phenotype of olfactory bulb interneurons. (c) 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2008.

• Large-scale production and homogenous purification of long chain polysialic acids from E. coli K1.

  Authors: Bastian Rode, Christian Endres, Chen Ran, Frank Stahl, Sascha Beutel, Cornelia Kasper, Sebastian Galuska, Rudolf Geyer, Martina Mühlenhoff, Rita Gerardy-Schahn, Thomas Scheper

  Journal of biotechnology. 06/2008; 135(2):202-9.

  The study of new biomaterials is the objective of many current research projects in biotechnological medicine. A promising scaffold material for the application in tissue engineering or otherThe study of new biomaterials is the objective of many current research projects in biotechnological medicine. A promising scaffold material for the application in tissue engineering or other biomedical applications is polysialic acid (polySia), a homopolymer of alpha2,8-linked sialic acid residues, which represents a posttranslational modification of the neural cell adhesion molecule and occurs in all vertebrate species. Some neuroinvasive bacteria like, e.g. Escherichia coli K1 (E. coli K1) use polySia as capsular polysaccharide. In this latter case long polySia chains with a degree of polymerization of >200 are linked to lipid anchors. Since in vertebrates no polySia degrading enzymes exist, the molecule has a long half-life in the organism, but degradation can be induced by the use of endosialidases, bacteriophage-derived enzymes with pronounced specificity for polySia. In this work a biotechnological process for the production of bacterial polysialic acid is presented. The process includes the development of a multiple fed-batch cultivation of the E. coli K1 strain and a complete downstream strategy of polySia. A controlled feed of substrate at low concentrations resulted in an increase of the carbon yield (C(product)/C(substrate)) from 2.2 to 6.6%. The downstream process was optimized towards purification of long polySia chains. Using a series of adjusted precipitation steps an almost complete depletion of contaminating proteins was achieved. The whole process yielded 1-2g polySia from a 10-l bacterial culture with a purity of 95-99%. Further product analysis demonstrated maximum chain length of >130 for the final product.