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1.
Transl Psychiatry ; 5: e658, 2015 Oct 13.
Article in English | MEDLINE | ID: mdl-26460482

ABSTRACT

The neural cell adhesion molecule (NCAM) is a glycoprotein implicated in cell-cell adhesion, neurite outgrowth and synaptic plasticity. Polysialic acid (polySia) is mainly attached to NCAM (polySia-NCAM) and has an essential role in regulating NCAM-dependent developmental processes that require plasticity, that is, cell migration, axon guidance and synapse formation. Post-mortem and genetic evidence suggests that dysregulation of polySia-NCAM is involved in schizophrenia (SZ). We enrolled 45 patients diagnosed with SZ and 45 healthy individuals who were submitted to polySia-NCAM peripheral quantification, cognitive and psychopathological assessment and structural neuroimaging (brain volumes and diffusion tensor imaging). PolySia-NCAM serum levels were increased in SZ patients, independently of antipsychotic treatment, and were associated with negative symptoms, blunted affect and declarative memory impairment. The increased polySia-NCAM levels were associated with decreased volume in the left prefrontal cortex, namely Brodmann area 46, in patients and increased volume in the same brain area of healthy individuals. As this brain region is involved in the pathophysiology of SZ and its associated phenomenology, the data indicate that polySia-NCAM deserves further scrutiny because of its possible role in early neurodevelopmental mechanisms of the disorder.


Subject(s)
Brain/pathology , Cognition Disorders/complications , Neural Cell Adhesion Molecules/blood , Schizophrenia/complications , Sialic Acids/blood , Adult , Brain Mapping , Cell Movement/genetics , Cognition Disorders/blood , Cognition Disorders/genetics , Diffusion Tensor Imaging , Female , Humans , Male , Neural Cell Adhesion Molecules/genetics , Neuronal Plasticity/genetics , Organ Size , Schizophrenia/blood , Schizophrenia/genetics , Sialic Acids/genetics
2.
J Biol Chem ; 276(36): 34066-73, 2001 Sep 07.
Article in English | MEDLINE | ID: mdl-11418591

ABSTRACT

Poly-alpha-2,8-sialic acid (polysialic acid) is a post-translational modification of the neural cell adhesion molecule (NCAM) and an important regulator of neuronal cell-cell interactions. The synthesis of polysialic acid depends on the two polysialyltransferases ST8SiaII and ST8SiaIV. Understanding the catalytic mechanisms of the polysialyltransferases is critical toward the aim of influencing physiological and pathophysiological functions mediated by polysialic acid. We recently demonstrated that polysialyltransferases are bifunctional enzymes exhibiting auto- and NCAM polysialylation activity. Autopolysialylation occurs on N-glycans of the enzymes, and glycosylation variants lacking sialic acid and galactose were found to be inactive for both auto- and NCAM polysialylation. In the present study, we have analyzed the number and functional importance of N-linked oligosaccharides present on polysialyltransferases. We demonstrate that autopolysialylation depends on specific N-glycans attached to Asn(74) in ST8SiaIV and Asn(89) and Asn(219) in ST8SiaII. Deletion of polysialic acid acceptor sites by site-directed mutagenesis rendered the polysialyltransferases inactive in vitro and in vivo. The inactivity of autopolysialylation-negative polysialyltransferases in vivo was not caused by the absence or default targeting of the enzymes. The data presented in this study clearly show that active polysialyltransferases are competent to perform autopolysialylation and provide strong evidence for a tight functional link between the two catalytic functions.


Subject(s)
Sialic Acids/metabolism , Sialyltransferases/metabolism , Animals , Asparagine/chemistry , Blotting, Western , CHO Cells , Catalysis , Cricetinae , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique, Indirect , Gene Deletion , Glycosylation , Golgi Apparatus/metabolism , Mice , Mutagenesis, Site-Directed , Mutation , Oligosaccharides/chemistry , Plasmids/metabolism , Polysaccharides/metabolism , Precipitin Tests , Protein Processing, Post-Translational , Transfection
3.
J Biol Chem ; 275(42): 32861-70, 2000 Oct 20.
Article in English | MEDLINE | ID: mdl-10921918

ABSTRACT

Polysialic acid (PSA) is a dynamically regulated posttranslational modification of the neural cell adhesion molecule (NCAM), which modulates NCAM binding functions. PSA biosynthesis is catalyzed by two polysialyltransferases, ST8SiaII and ST8SiaIV. The catalytic mechanisms of these enzymes are unknown. In Chinese hamster ovary cells, ST8SiaIV is responsible for PSA expression. In the complementation group 2A10, the ST8SiaIV gene is disrupted. Investigating the molecular defects in this complementation group, seven clones with missense mutations in ST8SiaIV were found. Mutations cause replacement of amino acids that are highly conserved in alpha2,8-sialyltransferases. To verify the physiological relevance of identified mutations, identical amino acid substitutions were introduced into epitope-tagged variants of hamster ST8SiaIV and murine ST8SiaII and recombinant proteins were tested in vivo and in vitro. None of these constructs reconstituted PSA synthesis in 2A10 cells, although the proteins were expressed and with the exception of the cysteine variants ST8SiaIV-C356F and ST8SiaII-C371F correctly targeted to the Golgi apparatus. Interestingly, two mutations (ST8SiaIV-R277G and -M333V and the corresponding mutants ST8SiaII-R292G and -M348V) could be partially rescued if tested in vitro. Although these mutants were negative for autopolysialylation, partial reconstitution of both auto- and NCAM polysialylation was achieved in the presence of NCAM. The data presented in this study suggest a functional link between auto- and NCAM polysialylation.


Subject(s)
Mutation, Missense , Sialic Acids/biosynthesis , Sialyltransferases/genetics , Amino Acid Sequence , Animals , Base Sequence , CHO Cells , Chickens , Conserved Sequence , Cricetinae , Cysteine , Genetic Complementation Test , Genetic Variation , Golgi Apparatus/enzymology , Humans , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Neural Cell Adhesion Molecules/metabolism , Protein Processing, Post-Translational , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Homology, Amino Acid , Sialic Acids/genetics , Sialyltransferases/chemistry , Transfection
4.
Curr Opin Struct Biol ; 8(5): 558-64, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9818258

ABSTRACT

Polysialic acid is a unique cell surface polysaccharide found in the capsule of neuroinvasive bacteria and as a highly regulated post-translational modification of the neural cell adhesion molecule. Recent progress has been achieved in research on both the physicochemical properties of polysialic acid and the biosynthetic pathways leading to polysialic acid expression in bacteria and mammals.


Subject(s)
Bacteria/metabolism , Sialic Acids/chemistry , Sialic Acids/metabolism , Animals , Gene Expression Regulation , Sialic Acids/genetics
5.
Proc Natl Acad Sci U S A ; 95(16): 9140-5, 1998 Aug 04.
Article in English | MEDLINE | ID: mdl-9689047

ABSTRACT

Sialic acids of cell surface glycoproteins and glycolipids play a pivotal role in the structure and function of animal tissues. The pattern of cell surface sialylation is species- and tissue-specific, is highly regulated during embryonic development, and changes with stages of differentiation. A prerequisite for the synthesis of sialylated glycoconjugates is the activated sugar-nucleotide cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac), which provides a substrate for Golgi sialyltransferases. Although a mammalian enzymatic activity responsible for the synthesis of CMP-Neu5Ac has been described and the enzyme has been purified to near homogeneity, sequence information is restricted to bacterial CMP-Neu5Ac synthetases. In this paper, we describe the molecular characterization, functional expression, and subcellular localization of murine CMP-Neu5Ac synthetase. Cloning was achieved by complementation of the Chinese hamster ovary lec32 mutation that causes a deficiency in CMP-Neu5Ac synthetase activity. A murine cDNA encoding a protein of 432 amino acids rescued the lec32 mutation and also caused polysialic acid to be expressed in the capsule of the CMP-Neu5Ac synthetase negative Escherichia coli mutant EV5. Three potential nuclear localization signals were found in the murine synthetase, and immunofluorescence studies confirmed predominantly nuclear localization of an N-terminally Flag-tagged molecule. Four stretches of amino acids that occur in the N-terminal region are highly conserved in bacterial CMP-Neu5Ac synthetases, providing evidence for an ancestral relationship between the sialylation pathways of bacterial and animal cells.


Subject(s)
Evolution, Molecular , N-Acylneuraminate Cytidylyltransferase/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , CHO Cells , Cloning, Molecular , Cricetinae , DNA, Complementary , Molecular Sequence Data , Mutation , N-Acylneuraminate Cytidylyltransferase/chemistry , N-Acylneuraminate Cytidylyltransferase/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid
6.
Mol Gen Genet ; 257(1): 28-34, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9439566

ABSTRACT

The serogroups B, C, W135 and Y of Neisseria meningitidis express chemically and immunologically distinct capsular polysaccharides containing sialic acid. In the case of serogroup B meningococci sialic acid is synthesized by the gene products of a locus termed sia and forms the homopolymers of the capsule. The organization of the genes required for sialic acid synthesis in serogroups B, C, W135 and Y was elucidated by PCR technology. Cloning, sequencing and the functional expression of the polysialyltransferase (PST) genes of serogroups B and C demonstrated that the difference in capsule composition derives from the presence of related, but distinct siaD genes coding for PSTs. Analysis of meningococci of serogroups W135 and Y expressing sialic acid heteropolymers revealed that the DNA sequences of the corresponding genetic loci in these serogroups were highly homologous, but differed completely from the siaD genes of serogroups B and C. This finding suggests that enzymes unrelated to those of serogroups B and C are required for the formation of sialic acid heteropolymers characteristic of the capsules of serogroups W135 and Y.


Subject(s)
Genetic Variation , Neisseria meningitidis/genetics , Sialic Acids/metabolism , Sialyltransferases/genetics , Biological Evolution , Cloning, Molecular , Genes, Bacterial , Neisseria meningitidis/classification , Neisseria meningitidis/metabolism , Open Reading Frames , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Analysis , Serotyping , Sialyltransferases/metabolism
7.
EMBO J ; 15(24): 6943-50, 1996 Dec 16.
Article in English | MEDLINE | ID: mdl-9003770

ABSTRACT

Polysialic acid (PSA) is a specific and highly regulated post-translational modification of the neural cell adhesion molecule NCAM. Synthesis of PSA depends on the activity of a single enzyme, the polysialyltransferase-1 (PST-1), recently cloned from three mammalian species. The present study was carried out to investigate the catalytic mechanism of PST-1. Using a newly developed in vitro assay system, we demonstrate autopolysialylation for PST-1. The synthesis of PSA chains, which involved N-glycosylation sites, occurred immediately after contact with the activated sugar donor CMP-Neu5Ac. In contrast to the polysialylation of NCAM, where terminal sialylation in either the alpha2,3 or alpha2,6 position is required, the autopolysialylation could be started in the asialo-PST-1 isolated from CHO cells of the Lec2 complementation group. Pre-formed PSA chains were not transferred to NCAM. Nevertheless, the autocatalytic step is likely to be a prerequisite for enzymatic activity, since agalacto-PST-1 isolated from Lec8 cells was functionally inactive. Our data describe a novel route of autocatalytic maturation of a glycosyltransferase and thereby provide a new basis for studies aimed at elucidating and influencing the catalytic functions of PST-1.


Subject(s)
Sialic Acids/metabolism , Sialyltransferases/metabolism , Animals , CHO Cells , Catalysis , Cricetinae , Glycosylation , Kinetics , Neural Cell Adhesion Molecules/metabolism , Recombinant Proteins/metabolism , Staphylococcal Protein A/metabolism
8.
Curr Biol ; 6(9): 1188-91, 1996 Sep 01.
Article in English | MEDLINE | ID: mdl-8805371

ABSTRACT

The addition of poly-alpha2,8-N-acetylneuraminic acid (polysialic acid; PSA) to the neural cell adhesion molecule NCAM plays a crucial role in neural development [1-3], neural regeneration [4], and plastic processes in the vertebrate brain associated with neurite outgrowth [5], axonal pathfinding [6], and learning and memory [7,-9]. PSA levels are decreased in people affected by schizophrenia [10], and PSA has been identified as a specific marker for some neuroendocrine and lymphoblastoid tumours [11-13]; expression of PSA on the surface of these tumour cells modulates their metastatic potential [11-13]. Studies aimed at understanding PSA biosynthesis and the dynamics of its production have largely been promoted by the cloning of polysialyltransferases (PST-1 in hamster; PST in human and mouse) [14-16]. However, the number of enzymes involved in the biosynthesis of PSA has not been identified. Using incompletely glycosylated NCAM variants and soluble recombinant glycosyltransferases, we reconstituted the site at which PST-1 acts to polysialylate NCAM in vitro. The data presented here clearly demonstrate that polysialylation of NCAM is catalyzed by a single enzyme, PST-1, and that terminal sialylation of the N-glycan core is sufficient to generate the PSA acceptor site. Our results also show that PST-1 can act on core structures with the terminal sialic acid connected to galactose via an alpha2,3 or alpha2,6 linkage.


Subject(s)
Neural Cell Adhesion Molecules/metabolism , Sialic Acids/metabolism , Sialyltransferases/metabolism , Animals , Cell Line , Cricetinae , Humans , Mice , Substrate Specificity
9.
Proc Natl Acad Sci U S A ; 93(15): 7572-6, 1996 Jul 23.
Article in English | MEDLINE | ID: mdl-8755516

ABSTRACT

Translocation of nucleotide sugars across the membrane of the Golgi apparatus is a prerequisite for the synthesis of complex carbohydrate structures. While specific transport systems for different nucleotide sugars have been identified biochemically in isolated microsomes and Golgi vesicles, none of these transport proteins has been characterized at the molecular level. Chinese hamster ovary (CHO) mutants of the complementation group Lec2 exhibit a strong reduction in sialylation of glycoproteins and glycolipids due to a defect in the CMP-sialic acid transport system. By complementation cloning in the mutant 6B2, belonging to the Lec2 complementation group, we were able to isolate a cDNA encoding the putative murine Golgi CMP-sialic acid transporter. The cloned cDNA encodes a highly hydrophobic, multiple membrane spanning protein of 36.4 kDa, with structural similarity to the recently cloned ammonium transporters. Transfection of a hemagglutinin-tagged fusion protein into the mutant 6B2 led to Golgi localization of the hemagglutinin epitope. Our results, together with the observation that the cloned gene shares structural similarities to other recently cloned transporter proteins, strongly suggest that the isolated cDNA encodes the CMP-sialic acid transporter.


Subject(s)
Carrier Proteins/biosynthesis , Cytidine Monophosphate N-Acetylneuraminic Acid/metabolism , Glycolipids/biosynthesis , Glycoproteins/biosynthesis , Membrane Proteins/biosynthesis , Nucleotide Transport Proteins , Amino Acid Sequence , Animals , Base Sequence , CHO Cells , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cloning, Molecular , Cricetinae , Genetic Complementation Test , Golgi Apparatus/metabolism , Hemagglutinins/biosynthesis , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Mice , Molecular Sequence Data , Molecular Weight , Recombinant Fusion Proteins/biosynthesis
10.
Mol Microbiol ; 20(6): 1211-20, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8809773

ABSTRACT

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 alpha-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.


Subject(s)
Neisseria meningitidis/genetics , Neisseria meningitidis/pathogenicity , Sialyltransferases/genetics , Amino Acid Sequence , Bacterial Capsules , Base Sequence , Blotting, Northern , DNA, Bacterial , Disease Outbreaks , Genetic Variation , Humans , Meningococcal Infections/epidemiology , Meningococcal Infections/microbiology , Molecular Sequence Data , Neisseria meningitidis/isolation & purification , Sequence Analysis, DNA , Sialyltransferases/metabolism
11.
Mol Microbiol ; 16(3): 441-50, 1995 May.
Article in English | MEDLINE | ID: mdl-7565105

ABSTRACT

Homopolymeric alpha-2,8-linked sialic acid (PSA) has been found as a capsular component of sepsis- and meningitis-causing bacterial pathogens, and on eukaryotic cells as a post-translational modification of the neural cell adhesion molecule (NCAM). The polysaccharide is specifically recognized and degraded by a phage-encoded enzyme, the endo-N-acetylneuraminidase E (Endo NE). Endo NE therefore has become a valuable tool in the study of bacterial pathogenesis and eukaryotic morphogenesis. In this report we describe the molecular cloning of Endo NE and the expression of a functionally active recombinant enzyme. The cloned DNA sequence (2436 bp) encodes a polypeptide of 811 amino acids, which at the 5' end contains a totally conserved neuraminidase motif. Expressed in Escherichia coli, the enzyme migrates as a single band of approximately 74 kDa in SDS-PAGE. A central domain of 669 amino acid residues is about 90% homologous to the recently cloned Endo NF. Both phage-induced lysis of bacteria and the catalysis of PSA degradation by the recombinant enzyme are efficiently inhibited by a polyclonal antiserum raised against the intact phage particle. The C-terminal region seems to be essential to enzymatic functions, as truncation of 32 amino acids outside the homology domain completely abolishes Endo NE activity. Our data also indicate that the 38 kDa protein, previously assumed to be a subunit of the Endo NE holoenzyme, is the product of a separate gene locus and is not necessary for in vitro depolymerase activity.


Subject(s)
Bacteriophages/genetics , Gene Expression Regulation, Viral , Genetic Vectors/genetics , Glycoside Hydrolases/genetics , Neuraminidase/genetics , Viral Proteins/genetics , Amino Acid Sequence , Animals , Bacteriophages/enzymology , Base Sequence , Cloning, Molecular , Enzyme Induction , Escherichia coli , Guinea Pigs , Immune Sera , Molecular Sequence Data , Neuraminidase/biosynthesis , Neutralization Tests , Sequence Alignment , Sialic Acids/metabolism
12.
Nature ; 373(6516): 715-8, 1995 Feb 23.
Article in English | MEDLINE | ID: mdl-7854457

ABSTRACT

Polysialic acid (PSA) is a dynamically regulated product of post-translational modification of the neural cell adhesion molecule, NCAM. Presence of the large anionic carbohydrate modulates NCAM binding properties and, by increasing the intercellular space, influences interactions between other cell surface molecules. PSA expression underlies cell type- and developmental-specific alterations and correlates with stages of cellular motility. In the adult, PSA becomes restricted to regions of permanent neural plasticity and regenerating neural and muscle tissues. Recent data implicate its important function in spatial learning and memory, and in tumour biology. Here we describe the molecular characterization of polysialyltransferase-1, the key enzyme of eukaryotic PSA synthesis. In reconstitution experiments, the newly cloned enzyme induces PSA synthesis in all NCAM-expressing cell lines. Our data therefore represent convincing evidence that the polycondensation of alpha-2,8-linked sialic acids in mammals is the result of a single enzymatic activity and provide a new basis for studying the functional role of PSA in neuro- and tumour biology.


Subject(s)
Sialic Acids/metabolism , Sialyltransferases/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , CHO Cells , Cell Adhesion Molecules, Neuronal/metabolism , Cell Line , Cloning, Molecular , Cricetinae , DNA, Complementary , Mice , Molecular Sequence Data , Mutation , Plasmids , Sialyltransferases/genetics
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