Search

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-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 theNMDAsubtype of glutamate receptor (GluN) through either reducing the extracellular Mg2+ 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. [ABSTRACT FROM AUTHOR]

Although the polysialyltransferase ST8Sia IV is expressed in both primary and secondary human lymphoid organs, its product, poly- sialic 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. [ABSTRACT FROM AUTHOR]

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 knockout mice indicating that a crucial function of polySia resides in controlling interactions mediated by its predominant protein carrier, the neural cell adhesion molecule NCAM. [ABSTRACT FROM AUTHOR]

Polysialylation is a post-translational modification of the neural cell adhesion molecule (NCAM), which in the adult brain promotes structural changes in regions of neurogenesis and neuroplasticity. Because a variety of plastic changes including neurogenesis have been suggested to be functionally involved in the pathophysiology of epilepsies, it is of specific interest to define the impact of the polysialic acid (PSA)-NCAM system on development of this disease and associated comorbidities. Therefore, we studied the impact of transient enzymatic depolysialylation of NCAM on the pathophysiology in an electrically induced rat post-status epilepticus (SE) model. Loss of PSA counteracted the SE-induced increase in neurogenesis in a significant manner. This effect of endoneuraminidase (endoN) treatment on hippocampal neurogenesis did not impact the subsequent development of spontaneous seizures. In contrast, transient lack of PSA during SE and in the early phase of epileptogenesis exhibited a cognition sparing effect as revealed in the Morris water maze paradigm. In conclusion, our data do not support a central role of neurogenesis in the development of a hyperexcitable epileptic network. However, in view of the cognition-sparing effect, the transient modulation of the PSA-NCAM system seems to allow beneficial long-term disease modification, which might be mediated by the partial normalization of neurogenesis. [ABSTRACT FROM AUTHOR]

The unique modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) is tightly associated with nervous system development and plasticity. The prevailing view that this large carbohydrate polymer acts as an anti-adhesive factor seems straightforward at first sight. However, during almost 25 years of polySia research it became increasingly clear that the impact of polySia on cell surface interactions can not be explained by one unifying mechanism. Recent progress in the generation of mouse models, which partially or completely lack polySia due to ablation of one or both of the two polySia synthesizing enzymes, provides novel insights into the function of this unique post-translational modification. The present review is focused on a phenotype comparison between the newly established mouse strains which combine polySia-deficiency with normal NCAM expression and the well-characterized NCAM negative mouse model. Analysis of shared and individual phenotypes allows a clear distinction between NCAM and polySia functions and revealed that polySia plays a vital role as a specific control element of NCAM-mediated interactions. [ABSTRACT FROM AUTHOR]

The extracellular polysaccharide capsule is an essential virulence factor of Neisseria meningitidis, a leading cause of severe bacterial meningitis and sepsis. Serogroup B strains, the primary disease causing isolates in Europe and America, are encapsulated in α-2,8 polysialic acid (polySia). The capsular polymer is synthesized from activated sialic acid by action of a membrane-associated polysialyltransferase ( NmB-polyST). Here we present a comprehensive characterization of NmB-polyST. Different from earlier studies, we show that membrane association is not essential for enzyme functionality. Recombinant NmB-polyST was expressed, purified and shown to synthesize long polySia chains in a non-processive manner in vitro. Subsequent structure–function analyses of NmB-polyST based on refined sequence alignments allowed the identification of two functional motifs in bacterial sialyltransferases. Both (D/E-D/E-G and HP motif) are highly conserved among different sialyltransferase families with otherwise little or no sequence identity. Their functional importance for enzyme catalysis and CMP-Neu5Ac binding was demonstrated by mutational analysis of NmB-polyST and is emphasized by structural data available for the Pasteurella multocida sialyltransferase PmST1. Together our data are the first description of conserved functional elements in the highly diverse families of bacterial (poly)sialyltransferases and thus provide an advanced basis for understanding structure–function relations and for phylogenetic sorting of these important enzymes. [ABSTRACT FROM AUTHOR]

Bacterial capsules are not only important virulence factors, but also provide attachment sites for bacteriophages that possess capsule degrading enzymes as tailspike proteins. To gain insight into the evolution of these specialized viruses, we studied a panel of tailed phages specific for Escherichia coli K1, a neuroinvasive pathogen with a polysialic acid capsule. Genome sequencing of two lytic K1-phages and comparative analyses including a K1-prophage revealed that K1-phages did not evolve from a common ancestor. By contrast, each phage is related to a different progenitor type, namely T7-, SP6-, and P22-like phages, and gained new host specificity by horizontal uptake of an endosialidase gene. The new tailspikes emerged by combining endosialidase domains with the capsid binding module of the respective ancestor. For SP6-like phages, we identified a degenerated tailspike protein which now acts as versatile adaptor protein interconnecting tail and newly acquired tailspikes and demonstrate that this adapter utilizes an N-terminal undecapeptide interface to bind otherwise unrelated tailspikes. Combining biochemical and sequence analyses with available structural data, we provide new molecular insight into basic mechanisms that allow changes in host specificity while a conserved head and tail architecture is maintained. Thereby, the present study contributes not only to an improved understanding of phage evolution and host-range extension but may also facilitate the on purpose design of therapeutic phages based on well-characterized template phages. [ABSTRACT FROM AUTHOR]

The neural cell adhesion molecule NCAM and its associated polysialic acid (PSA) play important roles in synaptic plasticity in the CA1 and/or CA3 regions of the hippocampus in vitro. Here, we address the question of whether NCAM and PSA are involved in regulation of synaptic transmission and plasticity also in vivo at synapses formed by entorhinal cortex axons in the dentate gyrus of mice anaesthetized with urethane. We show that basal synaptic transmission, measured as the slope of field excitatory postsynaptic potentials, was reduced strongly in mice lacking ST8SiaII/STX, the enzyme involved in polysialylation of NCAM in stem cell-derived immature granule cells, but not in mice deficient either in the NCAM glycoprotein or the enzyme ST8SiaIV/PST involved in polysialylation of NCAM in mature neurons. Strikingly, only mice deficient in NCAM, but not in PST or STX, were impaired in long-term potentiation (LTP) induced by theta-burst stimulation, suggesting that LTP in the dentate gyrus depends on the NCAM glycoprotein alone rather than on its associated PSA. As also patterns of synaptic activity during and immediately after induction of LTP were impaired in NCAM-deficient mice, it is likely that induction of LTP requires NCAM. These data are the first to describe that NCAM is necessary for induction of synaptic plasticity in identified synapses in vivo and suggest that polysialylation of NCAM expressed by immature granule cells in the dentate gyrus supports development of basal excitatory synaptic transmission in this region. [ABSTRACT FROM AUTHOR]

Galactofuranose metabolism is valued as an important target for the development of new antituberculosis drugs. UDP-galactopyranose mutase, a central enzyme in galactofuranose biosynthesis, is essential for the growth and viability of mycobacteria. This enzyme catalyzes the conversion of UDP-galactopyranose into UDP-galactofuranose, the donor used by various galacto-furanosyltransferases. While D-galactofuranose residues are often found in important surface glycoconjugates of pathogenic bacteria, fungi and protozoan parasites, they are absent in the mammalian host, and thus their biosynthesis is an attractive target for the development of novel therapeutic strategies. In contrast to mycobacteria, the importance of galactofuranose for eukaryotic pathogens has not been ascertained because the enzymes involved in galactofuranose metabolism are unknown. Here, we report the identification and characterization of the first eukaryotic UDP-galactopyranose mutases. The genes encoding the enzymes were cloned from two different human pathogens: the parasite Leishmania major and the opportunistic fungus Aspergillus fumigatus. The newly identified eukaryotic enzymes exhibit 51% sequence identity, but are less than 20% identical to the prokaryotic counterparts. The sequence identity between pro- and eukaryotic enzymes is concentrated at amino acid residues that are involved in substrate and cofactor binding. Therefore, an inhibitor of UDP-galactopyranose mutase might be effective against a wide range of pathogenic organisms. [ABSTRACT FROM AUTHOR]

Phages infecting the polysialic acid (polySia)-encapsulated human pathogen Escherichia coli K1 are equipped with capsule-degrading tailspikes known as endosialidases, which are the only identified enzymes that specifically degrade polySia. As polySia also promotes cellular plasticity and tumor metastasis in vertebrates, endosialidases are widely applied in polySia-related neurosciences and cancer research. Here we report the crystal structures of endosialidase NF and its complex with oligomeric sialic acid. The structure NF, which reveals three distinct domains, indicates that the unique polySia specificity evolved from a combination of structural elements characteristic of exosialidases and bacteriophage tailspike proteins. The endosialidase assembles into a catalytic trimer stabilized by a tripleß-helix. Its active site differs markedly from that of exosialidases, indicating an endosialidase-specific substrate-binding mode and catalytic mechanism. Residues essential for endosialidase activity were identified by structure-based mutational analysis. [ABSTRACT FROM AUTHOR]

The neural cell adhesion molecule (NCAM) plays important rolesduring development, plasticity, and regeneration in the adult nervoussystem. Its function is strongly influenced by attachment of theunusual α2-8-linked polysialic acid (PSA).Here we analyzed the N-glycosylation pattern of polysialylated NCAMfrom brains of newborn calves. Purified PSA-NCAM glycoprotein wasdigested with trypsin, and PSA-glycopeptides were separated by immunoaffinity chromatography.For determining the N-glycosylation sites, PNGase F-treated glycopeptideswere analyzed by Edman degradation and matrix-assisted laser desorption/ionization massspectrometry (MALDI-MS). They were found to be exclusively linkedto the fifth (Asn 439) and sixth (Asn 468) N-glycosylation sitesin the fifth immunoglobulin-like domain of NCAM. The chain lengthof PSA consisted of at least 30 sialic acid residues, as shown byanion exchange chromatography. For analysis of the core structures, endoneuraminidaseN–treated PSA-NCAM was separated by SDS–PAGE anddigested with PNGase F. The core structures of polysialylated glycanswere characterized by MALDI-MS combined with exoglycosidase digestionsand chromatographic fractionation. They include hybrid, di-, tri-, andsmall amounts of tetraantennary carbohydrates, which were all fucosylatedat the innermost N-acetylglucosamine. For the triantennaryglycans, the “2,6” arm was preferred in polysialylatedstructures. High levels of sulfated groups were found on polysialylatedstructures and to a lower extent also on nonpolysialylated glycans.In addition, high-mannose-type glycans could be detected on PSA-NCAMglycoforms ranging from (GlcNAc)2(Man)5 upto (GlcNAc)2(Man)9. In conclusion, we observeda structural variability and high regional selectivity for the PSA-glycansattached to the NCAM molecule that are most likely influencing itsbiological functions. [ABSTRACT FROM PUBLISHER]

2-Keto-3-deoxy-d-glycero-d-galacto-nononic acid (KDN) isa sialic acid (Sia) that is ubiquitously expressed in vertebrates duringnormal development and tumorigenesis. Its expression is thoughtto be regulated by multiple biosynthetic steps catalyzed by severalenzymes, including CMP-Sia synthetase. Using crude enzyme preparations,it was shown that mammalian CMP-Sia synthetases had very low activityto synthesize CMP-KDN from KDN and CTP, and the corresponding enzymefrom rainbow trout testis had high activity to synthesize both CMP-KDNand CMP-N-acetylneuraminic acid (Neu5Ac) (Terada et al. [1993] J.Biol. Chem., 268, 2640–2648). To demonstrate if theunique substrate specificity found in the crude trout enzyme is conveyedby a single enzyme, cDNA cloning of trout CMP-Sia synthetase wascarried out by PCR-based strategy. The trout enzyme was shown toconsist of 432 amino acids with two potential nuclear localizationsignals, and the cDNA sequence displayed 53.8% identityto that of the murine enzyme. Based on the Vmax/Km values, the recombinant trout enzymehad high activity toward both KDN and Neu5Ac (1.1 versus 0.68 min–1).In contrast, the recombinant murine enzyme had 15 times lower activitytoward KDN than Neu5Ac (0.23 versus 3.5 min–1).Northern blot analysis suggested that several sizes of the mRNAare expressed in testis, ovary, and liver in a tissue-specific manner.These results indicate that at least one cloned enzyme has the abilityto utilize both KDN and Neu5Ac as substrates efficiently and isuseful for the production of CMP-KDN. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

Polysialic acid (PSA) is an important regulator of cellular interactions. Two enzymes (ST8SiaII and ST8SiaIV) are capable of synthesizing PSA. In the present study, the gene encoding the murine ST8SiaIV (PST‐1) has been isolated and characterized. In contrast to the ST8SiaII (STX) gene which contains six exons and spans about 80 kb, the ST8SiaIV gene comprises only five exons spanning over at least 55 kb. However, alignment of the two genes revealed that exon–intron boundaries of exons 2–5 of ST8SiaIV and exons 3–6 of ST8SiaII are located at identical sites. Differences are restricted to the 5′‐region encoded by one exon in the case of ST8SiaIV, whereas the corresponding region of ST8SiaII is interrupted by a very long intron. 5′‐RACE analysis of the ST8SiaIV transcript using mRNA from AtT20 cells identified two transcription start sites at positions ‐324 and ‐204 relative to the translation start codon. The promoter region of ST8SiaIV lacks TATA‐ and CAAT‐like sequences and is enriched in G+C (60%). The promoter contains putative Sp1, AP‐1, AP‐2, and PEA3 binding sites, as well as a purine‐ and a pyrimidine‐rich region. Luciferase reporter gene assays demonstrated that the region between nucleotides ‐443 and ‐162 is sufficient to direct gene expression. The induction of luciferase activity was 30‐ and 10‐fold in the PSA‐positive AtT20 and CHO cells, but only 5‐ and 7‐fold in the PSA‐negative NIH‐3T3 cells and in a PSA‐negative subline of AtT20. Thus, although decreased in activity in PSA‐negative cell lines, the basal promoter is not sufficient for the strong cell‐type and tissue specific regulation of the ST8SiaIV gene, suggesting regulatory elements in the more upstream 5′‐region. [ABSTRACT FROM PUBLISHER]

Sialic acid-containing glycoconjugates are generally considered to be unique to the deuterostomes, a lineage of the animal kingdom which includes animals from the echinoderms up to the vertebrates. There are, however, two isolated reports of sialic acid occurring in the insect species Drosophila melanogaster and Galleria mellonella. Since insects are classified as protostomes, these findings call previous assumption on the phylogenetic distribution and thus on the evolution of sialic acids into question. Here, we report the occurrence of N-acetylneuraminic acid (Neu5Ac) in larvae of the cicada Philaenus spumarius. Cytochemical analysis of larval sections with lectins from Sambucus nigra and Limax flavus suggested the presence of sialic acids in the concrement vacuoles of the Malpighian tubules. The monoclonal antibody MAb 735, which is specific for polysialic acid, labelled the same structures. A chemical analysis performed by HPLC of fluorescent derivatives of sialic acids and by GLC-MS provided sound evidence for the presence of Neu5Ac in the Philaenus spumarius larvae. These data suggest that in this cicada Neu5Ac occurs in α2,8-linked polysialic acid structures and in α2,6-linkages. The results provide further evidence for the existence of sialic acids in insects and in linkages known to occur in glycoconjugates of deuterostomate origin. [ABSTRACT FROM AUTHOR]

A mechanism of capsular polysaccharide phase variation in Neisseria meningitidis is described. Meningococcal cells of an encapsulated serogroup B strain were used in invasion assays. Only unencapsulated variants were found to enter epithelial cells. Analysis of one group of capsule-deficient variants indicated that the capsular polysaccharide was re-expressed at a frequency of 10-3, Measurement of enzymatic activities involved in the biosynthesis of the α-2,8 polysialic acid capsule showed that polysialyltransferase (PST) activity was absent in these capsule-negative variants. Nucleotide sequence analysis of siaD revealed an insertion or a deletion of one cytidine residue within a run of (dC)7 residues at position 89, resulting in a frameshift and premature termination of translation. We analysed unencapsulated isolates from carriers and encapsulated case isolates collected during an outbreak of meningococcal disease. Further paired blood-culture isolates and unencapsulated nasopharyngeal isolates from patients with meningococcal meningitis were examined. In all unencapsulated strains analysed we found an insertion or deletion within the oligo-(dC) stretch within siaD, resulting in a frameshift and loss of capsule formation. All encapsulated isolates, however, had seven dC residues at this position, indicating a correlation between capsule phase variation and bacterial invasion and the outbreak of meningococcal disease. [ABSTRACT FROM AUTHOR]

The genes encoding all enzymes necessary for capsular polysaccharide biosynthesis in Neisseria meningitidis B are located on a 5 kb DNA fragment within the chromosomal cps gene cluster. Nucleotide sequence analysis revealed four open reading frames (ORFs), which can encode proteins with molecular masses of 41.4 kDa, 24.9 kDa, 38.3 kDa, and 54.4 kDa, respectively. These ORFs constitute a transcriptional unit as demonstrated by Northern biots. Primer extension analysis revealed that the transcriptional start site is preceded by a nucleotide sequence with homologies to the a70 consensus promoter sequence of Escherichia coli. Functional analysis of the proteins encoded by the ORFs indicated that 0RF2 encodes the CMP-NeuNAc synthetase, 0RF3 encodes the NeuNAc condensing enzyme, and 0RF4 encodes the α-2,8 polysialyltransferase. 0RF1 encodes an enzyme, which provides a precursor molecule for synthesis of monomeric NeuNAc. in E. coli the subcloned ORFs 2-4 were able to synthesize a high-molecular-weight α-2,8 polysialic acid, in contrast, inactivation of 0RF1 in the meningococcal genome resulted in a complete loss of capsule production. A regulatory enzyme, the CMP-NeuNAc hydrolase, which cleaves CMP-NeuNAc to CMP and NeuNAc, was not found as a part of the capsular polysaccharide biosynthesis gene operon or within the cps gene cluster. [ABSTRACT FROM AUTHOR]

Presents a study on the molecular characterization of the murine cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) synthetase. Methodology used in the study; How cloning was generated; Discovery of three potential nuclear localization signals; Discussion on the results of the study.

There is no efficient vaccine against group B meningococcal meningitis because of tolerance induced by host tissue polysialic acid cross-reacting with the capsular polysaccharide. The specificities of polysialic acid-antibody interactions were studied using a ligand binding assay. Antibodies 735, 20-1, 2-1B, 2-2B, 5El, and t5El and antibodies against N-propionylated group B meningococcal polysaccharide-tetanus toxoid conjugate (NP-4, 106-6) bound polysialylated human embryonal brain glycopeptides but not control glycopeptides or disialosyllactose, whereas antibodies 109-3 and 1-627 were more specific for the N-propionylated polysaccharide. Antiganglioside antibodies (KM538, KM641) did not cross-react with polysialic acid. Human classs-witched antibodies 5El (IgM) and t5El (lgG) reacted identically with all compounds tested and no temperature-dependent differences were observed. All anti-polysialosyl antibodies required a polysaccharide chain of 8-10 residues for binding independent of the immunizing antigen, animal species, or immunoglobulin class. The results suggest careful evaluation of polysialic acid cross-reactivity in vaccine development. [ABSTRACT FROM PUBLISHER]