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Schulz E.C.,University of Gottingen | Schwarzer D.,Institute For Zellulare Chemie | Frank M.,German Cancer Research Center | Stummeyer K.,Institute For Zellulare Chemie | And 4 more authors.
Journal of Molecular Biology | Year: 2010

An α-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 Sia3 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. © 2010 Elsevier Ltd. All rights reserved.

Wolf S.,University of Hamburg | Warnecke S.,University of Hamburg | Ehrit J.,Institute For Zellulare Chemie | Freiberger F.,Institute For Zellulare Chemie | And 2 more authors.
ChemBioChem | Year: 2012

The cycloSal approach has been used in the past for the synthesis of a range of phosphorylated bioconjugates. In those reports, cycloSal nucleotides were allowed to react with different phosphate nucleophiles. With glycopyranosyl phosphates as nucleophiles, diphosphate-linked sugar nucleotides were formed. Here, cycloSal-nucleotides were used to prepare monophosphate-linked sugar nucleotides successfully in high anomeric purity and high chemical yield. The method was successfully used for the synthesis of three nucleotide glycopyranoses as model compounds. The method was then applied to the syntheses of CMP-N-acetyl-neuraminic acids (CMP-Neu5NAc) and of four derivatives with different modifications at their amino functions (N-propanoyl, N-butanoyl, N-pentanoyl and N-cyclopropylcarbonyl). The compounds were used for initial enzymatic studies with a bacterial polysialyltransferase (polyST). Surprisingly, the enzyme showed marked differences in terms of utilisation of the four derivatives. The N-propanoyl, N-butanoyl, and N-pentanoyl derivatives were efficiently used in a first transfer with a fluorescently labelled trisialo-acceptor. However, elongation of the resulting tetrasialo-acceptors worsened progressively with the size of the N-acyl chain. The N-pentanoyl derivative allowed a single transfer, leading to a capped tetramer. The N-cyclopropylcarbonyl derivative was not transferred. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Schulz E.C.,University of Gottingen | Neumann P.,University of Gottingen | Gerardy-Schahn R.,Institute For Zellulare Chemie | Sheldrick G.M.,University of Gottingen | Ficner R.,University of Gottingen
Acta Crystallographica Section D: Biological Crystallography | Year: 2010

Endosialidase NF (endoNF) is a bacteriophage-derived endosialidase that specifically degrades α-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 Å 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. © 2010 International Union of Crystallography Printed in Singapore - all rights reserved.

Bohm R.,Griffith University | Freiberger F.,Institute For Zellulare Chemie | Stummeyer K.,Institute For Zellulare Chemie | Gerardy-Schahn R.,Institute For Zellulare Chemie | And 2 more authors.
ChemBioChem | Year: 2010

On the loose: We report an STD NMR spectroscopic study of the polysialyltransferase from Neisseria meningitidis serogroup B (NmB-polyST). The spectra reveal that the cytosine and ribose moiety receive more saturation than the sialic acid residue of CMPNeu5Ac. This loose binding enables a fast and efficient sialyl transfer to the acceptor substrate. Our analysis offers a view of the structural determinants necessary for binding to NmB-polyST that provide the basis for the development of novel NmB-polyST inhibitors. (Figure Presented). © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

Schulz E.C.,University of Gottingen | Bergfeld A.K.,Institute For Zellulare Chemie | Bergfeld A.K.,University of California at San Diego | Ficner R.,University of Gottingen | Muhlenhoff M.,Institute For Zellulare Chemie
PLoS ONE | Year: 2011

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 their capsular polysaccharide by phase-variable O-acetlyation, 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-enyzme. © 2011 Schulz et al.

Hadley B.,Griffith University | Maggioni A.,Griffith University | Ashikov A.,Institute For Zellulare Chemie | Ashikov A.,Radboud University Nijmegen | And 3 more authors.
Computational and Structural Biotechnology Journal | Year: 2014

The proteomes of eukaryotes, bacteria and archaea are highly diverse due, in part, to the complex post-translational modification of protein glycosylation. The diversity of glycosylation in eukaryotes is reliant on nucleotide sugar transporters to translocate specific nucleotide sugars that are synthesised in the cytosol and nucleus, into the endoplasmic reticulum and Golgi apparatus where glycosylation reactions occur. Thirty years of research utilising multidisciplinary approaches has contributed to our current understanding of NST function and structure. In this review, the structure and function, with reference to various disease states, of several NSTs including the UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, GDP-fucose, UDP-N-acetylglucosamine/UDP-glucose/GDP-mannose and CMP-sialic acid transporters will be described. Little is known regarding the exact structure of NSTs due to difficulties associated with crystallising membrane proteins. To date, no three-dimensional structure of any NST has been elucidated. What is known is based on computer predictions, mutagenesis experiments, epitope-tagging studies, in-vitro assays and phylogenetic analysis. In this regard the best-characterised NST to date is the CMP-sialic acid transporter (CST). Therefore in this review we will provide the current state-of-play with respect to the structure-function relationship of the (CST). In particular we have summarised work performed by a number groups detailing the affect of various mutations on CST transport activity, efficiency, and substrate specificity. © 2014 Hadley et al.

Maggioni A.,Griffith University | von Itzstein M.,Griffith University | Rodriguez Guzman I.B.,Griffith University | Ashikov A.,Institute For Zellulare Chemie | And 3 more authors.
ChemBioChem | Year: 2013

CMP-sialic acid transporter: We report an in-depth, multidisciplinary, structural study that has identified the amino acid residues intimately involved in CMP-sialic acid transporter (CST) substrate specificity. Our data provide a significant contribution towards a better understanding the structure-function relationship of this important family of transporters and the rational design of CST inhibitors. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Schulz E.C.,University of Gottingen | Dickmanns A.,University of Gottingen | Urlaub H.,Max Planck Institute for Chemistry | Schmitt A.,University of Gottingen | And 5 more authors.
Nature Structural and Molecular Biology | Year: 2010

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, 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-Β-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 Β-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 Β-helices as a common structural motif. © 2010 Nature America, Inc. All rights reserved.

Rey-Gallardo A.,CSIC - Biological Research Center | Delgado-Martin C.,CSIC - Biological Research Center | Gerardy-Schahn R.,Institute For Zellulare Chemie | Rodriguez-Fernandez J.L.,CSIC - Biological Research Center | Vega M.A.,CSIC - Biological Research Center
Glycobiology | Year: 2011

Migration of mature dendritic cells (mDCs) to secondary lymphoid organs is required for the development of immunity. Recently, we reported that polysialic acid (PSA) and the transmembrane glycoprotein neuropilin-2 (NRP2) control mDC chemotaxis to CCL21 and that this process is dependent on the C-terminal basic region of the chemokine. Herein, we provide further insight into the molecular components controlling PSA regulated chemotaxis in mDCs. In the present study, we demonstrate that human mDCs express the NRP2 isoforms NRP2a and NRP2b, that both of them are susceptible to polysialylation and that polysialylation is required to specifically enhance chemotaxis toward CCL21 in mDCs. The results presented suggest that PSA attached to NRP2 isoforms acts as a binding module for the CCL21 chemokine, thereby facilitating its presentation to the chemokine receptor CCR7. To investigate the relevance of polysialylation on mDC migration, a xenograft mouse model was used and the migration of human DCs to mouse lymph nodes analyzed. Here, we demonstrate that the depletion of PSA from mDCs results in a drastic reduction in the migration of the cells to draining popliteal lymph nodes. With this finding, we provide first evidence that PSA is a crucial factor for in vivo migration of mDCs to lymph nodes. © 2010 The Author.

Keys T.G.,Institute For Zellulare Chemie | Gerardy-Schahn R.,Institute For Zellulare Chemie
BioSpektrum | Year: 2015

Polysaccharides are abundant extracellular matrix components and ideal materials for next-generation bioprostheses. Because the chain length critically impacts polymer properties, biotechnological production requires enzymes that allow polymer length control. For the capsule polymerase from Neisseria meningitidis serogroup B, we describe here the switching from processive to distributive polymer elongation. Our methodology delivered a finely tuned enzyme with narrow product distribution even in the absence of advanced high throughput screening platforms. © 2015, Springer-Verlag Berlin Heidelberg.

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