Use of prokaryotic-derived probes to identify poly(sialic acid) in neonatal neuronal membranes.

Vimr, Eric R.; McCoy, Ronald D.; Vollger, Helmuth F.; Wilkison, Nancy C.; Troy, Frederic A.

doi:10.1073/pnas.81.7.1971

Cited by 166 publications

(137 citation statements)

References 21 publications

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“…The identity as PSA of the material that accumulated at the origin of the chromatogram was confirmed in a separate control (not shown) by depolymerization after endo-N pretreatment. sized by K92 neuS by its immunochemical cross-reactivity against ␣GpC antiserum and susceptibility to digestion with endo-N, which is specific for PSA oligomers with at least eight ␣2,8 linkages (18,22). This conclusion was independently supported by proton NMR spectroscopic analysis of the heterologously synthesized K92 PSA capsular polysaccharide expressed in EV240 by complementation with cloned K92 neuS (29).…”

Section: Figsupporting

confidence: 56%

“…Where indicated, linkage assignments were confirmed by endo-N digestion and TLC analysis on silica gel G (Merck Research Laboratories) in an isopropanol, ammonium hydroxide, water (6:1:2) solvent system as previously described (18). Sialyl oligomers of defined length for use as chromatography standards were produced by partial acid hydrolysis of colominic acid as previously described (22). Oligomers were identified colorimetrically with orcinol or by autoradiography using a Kodak BioMax Transcreeen LE intensifying screen for 14 C-labeled sialic acids or Dupont Cronex Quanta-III intensifying screen after autoradiographic enhancement with Fluoro-Hance (rpi Research Products, Mount Prospect, IL).…”

Section: Methodsmentioning

confidence: 99%

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Functional Relationships of the Sialyltransferases Involved in Expression of the Polysialic Acid Capsules ofEscherichia coli K1 and K92 and Neisseria meningitidis Groups B or C

Steenbergen

Vimr

2003

Journal of Biological Chemistry

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Polysialic acid (PSA) capsules are cell-associated homopolymers of ␣2,8-, ␣2,9-, or alternating ␣2,8/2,9-linked sialic acid residues that function as essential virulence factors in neuroinvasive diseases caused by certain strains of Escherichia coli and Neisseria meningitidis. PSA chains structurally identical to the bacterial ␣2,8-linked capsular polysaccharides are also synthesized by the mammalian central nervous system, where they regulate neuronal function in association with the neural cell adhesion molecule (NCAM). Despite the structural identity between bacterial and NCAM PSAs, the respective polysialyltransferases (polySTs) responsible for polymerizing sialyl residues from donor CMP-sialic acid are not homologous glycosyltransferases. To better define the mechanism of capsule biosynthesis, we established the functional interchangeability of bacterial polySTs by complementation of a polymerase-deficient E. coli K1 mutant with the polyST genes from groups B or C N. meningitidis and the control E. coli K92 polymerase gene. The biochemical and immunochemical results demonstrated that linkage specificity is dictated solely by the source of the polymerase structural gene. To determine the molecular basis for linkage specificity, we created chimeras of the K1 and K92 polySTs by overlap extension PCR. Exchanging the first 52 N-terminal amino acids of the K1 NeuS with the C terminus of the K92 homologue did not alter specificity of the resulting chimera, whereas exchanging the first 85 or reciprocally exchanging the first 100 residues did. These results demonstrated that linkage specificity is dependent on residues located between positions 53 and 85 from the N terminus. Site-directed mutagenesis of the K92 polyST N terminus indicated that no single residue alteration was sufficient to affect specificity, consistent with the proposed function of this domain in orienting the acceptor. The combined results provide the first evidence for residues critical to acceptor binding and elongation in polysialyltransferase.

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Section: Figsupporting

confidence: 56%

Section: Methodsmentioning

confidence: 99%

Functional Relationships of the Sialyltransferases Involved in Expression of the Polysialic Acid Capsules ofEscherichia coli K1 and K92 and Neisseria meningitidis Groups B or C

Steenbergen

Vimr

2003

Journal of Biological Chemistry

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“…All previously identified endo-N structural genes encode the tail proteins of K1-specific lytic bacteriophage (phage) that depolymerize polysialic acid as an obligate step in the infectious process (20). K1 phage depolymerases cleave the internal ␣2,8-linkages of the K1 antigen or related polysialic acids composed of at least seven sialyl units (20)(21)(22), and the strain RS218 endo-N was found to be homologous with all known polysialic acid depolymerases (Fig. 4, which is published as supporting information on the PNAS web site).…”

Section: Resultsmentioning

confidence: 99%

Escherichia coliK1 polysialic acidO-acetyltransferase gene,neuO, and the mechanism of capsule form variation involving a mobile contingency locus

Deszo

Steenbergen

Freedberg

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

119

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Potential O-acetylation of the sialic acid residues of Escherichia coli K1, groups W-135, Y, and C meningococci, and group B Streptococcus capsular polysaccharides modifies their immunogenicity and susceptibility to glycosidases. Despite the biological importance of O-acetylation, no sialic or polysialic acid O-acetyltransferase has been identified in any system. Here we show that the E. coli K1 O-acetyltransferase encoded by neuO is genetically linked to the endo-neuraminidase tail protein gene of a chromosomal accretion element, designated CUS-3, with homology to lambdoid bacteriophage. Molecular epidemiological analysis established concordance between O-acetyltransferase and CUS-3 in a set of E. coli K1 strains. Deleting neuO eliminated enzymatic activity, which was restored by complementation in trans, and confirmed by 13 C-NMR analysis of the acetylated product. Analysis of mutants that accumulate intracellular polysialic acid because of export defects (kpsM and kpsS) or an inability to synthesize the sialic acid precursor, N-acetylmannosamine (neuC), indicated that NeuO does not require constant association with its substrate for activity. DNA sequencing and PCR analysis of neuO from strains that had undergone random capsule form variation showed that slip strand DNA mispairing or unequal recombination resulted in gain or loss of (5-AAGACTC-3)n heptanucleotide repeats (where n Ϸ 14 -39) located in the neuO 5 region. These repeats code for a previously undescribed structure designated the poly(⌿) motif. The unexpected discovery of the neuO contingency locus (hypervariable gene controlling expression of a surface epitope) in E. coli, and of a potential phage for redistributing variant neuO alleles, provides a robust system for investigating the functions of localized hypermutability in pathogen evolution.Escherichia coli K1 form variation ͉ polysialic acid capsule acetylation ͉ N-acetylneuraminic acid ͉ lysogenic bacteriophage

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“…This organism can import Neu5Ac thanks to the specific permease NanT (28); hence, with the introduction of the neuS-containing plasmid pWN609 (29) into EV239 cells (EV239 ϩneuS ), addition of labeled Neu5Ac to the culture media produces cells with labeled PSA. Control experiments and ideal growing conditions for these cells expressing the capsule were determined by rocket immunoelectrophoresis (30) and NMR correlation experiments (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Extracellular structure of polysialic acid explored by on cell solution NMR

Azurmendi

Vionnet

Wrightson

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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The capsular polysaccharide of the pathogens Neisseria meningitidis serogroup B and of Escherichia coli K1, ␣(2 3 8) polysialic acid (PSA), is unusual, because when injected into adult humans, it generates little or no antibody. In contrast, people infected with these pathogens generate specific serum antibodies. A structural study on cells is used to address this anomaly by characterizing antigen structures in vivo. We introduce on cell multidimensional solution NMR spectroscopy for direct observation of PSA on E. coli bacteria. Using 13 C, 15 N-labeled PSA, we applied a combination of heteronuclear NMR methods, such as heteronuclear single quantum coherence, HNCA, and HNCO, in vivo. Analysis reveals that free and cell-bound PSA are structurally similar, indicating that the poor immunogenicity of PSA is not due to major structural differences between cells and purified PSA. The 13 C linewidths of PSA on cells are 2 to 3 times larger than the corresponding ones in free PSA. The possible implications of the differences between free and on cell PSA are discussed. In addition, we demonstrate the suitability of the method for in vivo kinetic studies.carbohydrate structure ͉ isotopically labeled carbohydrates ͉ in vivo kinetics ͉ triple resonance NMR

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Use of prokaryotic-derived probes to identify poly(sialic acid) in neonatal neuronal membranes.

Cited by 166 publications

References 21 publications

Functional Relationships of the Sialyltransferases Involved in Expression of the Polysialic Acid Capsules ofEscherichia coli K1 and K92 and Neisseria meningitidis Groups B or C

Functional Relationships of the Sialyltransferases Involved in Expression of the Polysialic Acid Capsules ofEscherichia coli K1 and K92 and Neisseria meningitidis Groups B or C

Escherichia coliK1 polysialic acidO-acetyltransferase gene,neuO, and the mechanism of capsule form variation involving a mobile contingency locus

Extracellular structure of polysialic acid explored by on cell solution NMR

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