Fluorescent GD2 analog for single-molecule imaging.

Ganglioside GD2 is associated with the proliferation and migration of breast cancer cells. However, the precise role of GD2 is unclear because its tendency to form dynamic and transient domains in cell plasma membranes (PMs), called lipid rafts, makes it difficult to observe. Previously, we developed fluorescent analogs of gangliosides (e.g., GM3 and GM1), which enabled the observation of lipid raft formation for the first time using single-molecule imaging. In this report, we describe the first chemical synthesis of a fluorescent ganglioside, GD2. A biophysical analysis of the synthesized analog revealed its raft-philic character, suggesting its potential to aid single-molecule imaging-based investigations into raft-associated interactions.

The sialyl-O-acetylesterase NanS of Tannerella forsythia encompasses two catalytic modules with different regiospecificity for O7 and O9 of sialic acid.

The periodontal pathogen Tannerella forsythia utilizes host sialic acids as a nutrient source. To also make O-acetylated sialyl residues susceptible to the action of its sialidase and sialic acid uptake system, Tannerella produces NanS, an O-acetylesterase with two putative catalytic domains. Here, we analyzed NanS by homology modeling, predicted a catalytic serine-histidine-aspartate triad for each catalytic domain and performed individual domain inactivation by single alanine exchanges of the triad nucleophiles S32 and S311. Subsequent functional analyses revealed that both domains possess sialyl-O-acetylesterase activity, but differ in their regioselectivity with respect to position O9 and O7 of sialic acid. The 7-O-acetylesterase activity inherent to the C-terminal domain of NanS is unique among sialyl-O-acetylesterases and fills the current gap in tools targeting 7-O-acetylation. Application of the O7-specific variant NanS-S32A allowed us to evidence the presence of cellular 7,9-di-O-acetylated sialoglycans by monitoring the gain in 9-O-acetylation upon selective removal of acetyl groups from O7. Moreover, we established de-7,9-O-acetylation by wild-type NanS as an easy and efficient method to validate the specific binding of three viral lectins commonly used for the recognition of (7),9-O-acetylated sialoglycans. Their binding critically depends on an acetyl group in O9, yet de-7,9-O-acetylation proved advantageous over de-9-O-acetylation as the additional removal of the 7-O-acetyl group eliminated ligand formation by 7,9-ester migration. Together, our data show that NanS gained dual functionality through recruitment of two esterase modules with complementary activities. This enables Tannerella to scavenge 7,9-di-O-acetylated sialyl residues and provides a novel, O7-specific tool for studying sialic acid O-acetylation.

Polysialic Acid Regulates Sympathetic Outflow by Facilitating Information Transfer within the Nucleus of the Solitary Tract.

Expression of the large extracellular glycan, polysialic acid (polySia), is restricted in the adult, to brain regions exhibiting high levels of plasticity or remodeling, including the hippocampus, prefrontal cortex, and the nucleus of the solitary tract (NTS). The NTS, located in the dorsal brainstem, receives constant viscerosensory afferent traffic as well as input from central regions controlling sympathetic nerve activity, respiration, gastrointestinal functions, hormonal release, and behavior. Our aims were to determine the ultrastructural location of polySia in the NTS and the functional effects of enzymatic removal of polySia, both in vitro and in vivo polySia immunoreactivity was found throughout the adult rat NTS. Electron microscopy demonstrated polySia at sites that influence neurotransmission: the extracellular space, fine astrocytic processes, and neuronal terminals. Removing polySia from the NTS had functional consequences. Whole-cell electrophysiological recordings revealed altered intrinsic membrane properties, enhancing voltage-gated K+ currents and increasing intracellular Ca2+ Viscerosensory afferent processing was also disrupted, dampening low-frequency excitatory input and potentiating high-frequency sustained currents at second-order neurons. Removal of polySia in the NTS of anesthetized rats increased sympathetic nerve activity, whereas functionally related enzymes that do not alter polySia expression had little effect. These data indicate that polySia is required for the normal transmission of information through the NTS and that changes in its expression alter sympathetic outflow. polySia is abundant in multiple but discrete brain regions, including sensory nuclei, in both the adult rat and human, where it may regulate neuronal function by mechanisms identified here.SIGNIFICANCE STATEMENT All cells are coated in glycans (sugars) existing predominantly as glycolipids, proteoglycans, or glycoproteins formed by the most complex form of posttranslational modification, glycosylation. How these glycans influence brain function is only now beginning to be elucidated. The adult nucleus of the solitary tract has abundant polysialic acid (polySia) and is a major site of integration, receiving viscerosensory information which controls critical homeostatic functions. Our data reveal that polySia is a determinant of neuronal behavior and excitatory transmission in the nucleus of the solitary tract, regulating sympathetic nerve activity. polySia is abundantly expressed at distinct brain sites in adult, including major sensory nuclei, suggesting that sensory transmission may also be influenced via mechanisms described here. These findings hint at the importance of elucidating how other glycans influence neural function.

Intrabodies against the Polysialyltransferases ST8SiaII and ST8SiaIV inhibit Polysialylation of NCAM in rhabdomyosarcoma tumor cells.

BACKGROUND: Polysialic acid (polySia) is a carbohydrate modification of the neural cell adhesion molecule (NCAM), which is implicated in neural differentiation and plays an important role in tumor development and metastasis. Polysialylation of NCAM is mediated by two Golgi-resident polysialyltransferases (polyST) ST8SiaII and ST8SiaIV. Intracellular antibodies (intrabodies; IB) expressed inside the ER and retaining proteins passing the ER such as cell surface receptors or secretory proteins provide an efficient means of protein knockdown. To inhibit the function of ST8SiaII and ST8SiaIV specific ER IBs were generated starting from two corresponding hybridoma clones. Both IBs αST8SiaII-IB and αST8SiaIV-IB were constructed in the scFv format and their functions characterized in vitro and in vivo. RESULTS: IBs directed against the polySTs prevented the translocation of the enzymes from the ER to the Golgi-apparatus. Co-immunoprecipitation of ST8SiaII and ST8SiaIV with the corresponding IBs confirmed the intracellular interaction with their cognate antigens. In CHO cells overexpressing ST8SiaII and ST8SiaIV, respectively, the transfection with αST8SiaII-IB or αST8SiaIV-IB inhibited significantly the cell surface expression of polysialylated NCAM. Furthermore stable expression of ST8SiaII-IB, ST8SiaIV-IB and luciferase in the rhabdomyosarcoma cell line TE671 reduced cell surface expression of polySia and delayed tumor growth if cells were xenografted into C57BL/6 J RAG-2 mice. CONCLUSION: Data obtained strongly indicate that αST8SiaII-IB and αST8SiaIV-IB are promising experimental tools to analyze the individual role of the two enzymes during brain development and during migration and proliferation of tumor cells.

Polysialylation and lipopolysaccharide-induced shedding of E-selectin ligand-1 and neuropilin-2 by microglia and THP-1 macrophages.

Microglia are tissue macrophages and mediators of innate immune responses in the brain. The protein-modifying glycan polysialic acid (polySia) is implicated in modulating microglia activity. Cultured murine microglia maintain a pool of Golgi-confined polySia, which is depleted in response to lipopolysaccharide (LPS)-induced activation. Polysialylated neuropilin-2 (polySia-NRP2) contributes to this pool but further polySia protein carriers have remained elusive. Here, we use organotypic brain slice cultures to demonstrate that injury-induced activation of microglia initiates Golgi-confined polySia expression in situ. An unbiased glycoproteomic approach with stem cell-derived microglia identifies E-selectin ligand-1 (ESL-1) as a novel polySia acceptor. Together with polySia-NRP2, polySia-ESL-1 is also detected in primary cultured microglia, in brain slice cultures and in phorbol ester-induced THP-1 macrophages. Induction of stem cell-derived microglia, activated microglia in brain slice cultures and THP-1 macrophages by LPS, but not interleukin-4, causes polySia depletion and, as shown for stem cell-derived microglia, a metalloproteinase-dependent release of polySia-ESL-1 and polySia-NRP2. Moreover, soluble polySia attenuates LPS-induced production of nitric oxide and proinflammatory cytokines. Thus, shedding of polySia-ESL-1 and polySia-NRP2 after LPS-induced activation of microglia and THP-1 macrophages may constitute a mechanism for negative feedback regulation. GLIA 2016 GLIA 2016;64:1314-1330.

Polysialic acid on SynCAM 1 in NG2 cells and on neuropilin-2 in microglia is confined to intracellular pools that are rapidly depleted upon stimulation.

NG2 cells comprise a heterogeneous precursor population but molecular markers distinguishing between the assumed NG2 cell subpopulations are lacking. Previously, we described that a subfraction of the synaptic cell adhesion molecule SynCAM 1 is modified with the glycan polysialic acid (polySia) in NG2 cells. As for its major carrier, the neural cell adhesion molecule NCAM, polySia attenuates SynCAM 1 adhesion. Functions, as well as cellular and subcellular distribution of polySia-SynCAM 1 are elusive. Using murine glial cultures we now demonstrate that polySia-SynCAM 1 is confined to the Golgi compartment of a subset of NG2 cells and transiently recruited to the cell surface in response to depolarization. NG2 cells with Golgi-confined polySia were NCAM-negative, but positive for markers of oligodendrocyte precursor cells (OPCs). Consistent with previous data on polySia-SynCAM 1, polySia in Ncam(-/-) NG2 cells was exclusively attached to N-glycans and synthesized by ST8SIA2, one out of two mammalian polysialyltransferases. Unexpectedly, Golgi-confined polySia was also detected in Ncam(-/-) microglia, but this fraction resided on O-glycans and was produced by the second polysialyltransferase, ST8SIA4, indicating the presence of yet another polySia carrier in microglia. Searching for this carrier, we identified polysialylated neuropilin-2, so far only known from dendritic cells and exudate macrophages. Microglia activation by LPS, but not interleukin-4, caused a transient translocation of Golgi-localized polySia to the cell surface, resulting in complete depletion. Finally, NO-production of LPS-stimulated microglia was attenuated by addition of polySia suggesting that the observed loss of polySia-neuropilin-2 is involved in negative feedback regulation of pro-inflammatory microglia polarization.

Biochemical and biophysical characterization of the sialyl-/hexosyltransferase synthesizing the meningococcal serogroup W135 heteropolysaccharide capsule.

Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and sepsis. Crucial virulence determinants of pathogenic Nm strains are the polysaccharide capsules that support invasion by hindering complement attack. In NmW-135 and NmY the capsules are built from the repeating units (→ 6)-α-D-Gal-(1 → 4)-α-Neu5Ac-(2 →)n and (→ 6)-α-D-Glc-(1 → 4)-α-Neu5Ac-(2 →)n, respectively. These unusual heteropolymers represent unique examples of a conjugation between sialic acid and hexosyl-sugars in a polymer chain. Moreover, despite the various catalytic strategies needed for sialic acid and hexose transfer, single enzymes (SiaDW-135/Y) have been identified to form these heteropolymers. Here we used SiaDW-135 as a model system to delineate structure-function relationships. In size exclusion chromatography active SiaDW-135 migrated as a monomer. Fold recognition programs suggested two separate glycosyltransferase domains, both containing a GT-B-fold. Based on conserved motifs predicted folds could be classified as a hexosyl- and sialyltransferase. To analyze enzyme properties and interplay of the two identified glycosyltransferase domains, saturation transfer difference NMR and mutational studies were carried out. Simultaneous and independent binding of UDP-Gal and CMP-Sia was seen in the absence of an acceptor as well as when the catalytic cycle was allowed to proceed. Enzyme variants with only one functionality were generated by site-directed mutagenesis and shown to complement each other in trans when combined in an in vitro test system. Together the data strongly suggests that SiaDW-135 has evolved by fusion of two independent ancestral genes encoding sialyl- and galactosyltransferase activity.

Polysialic acid on neuropilin-2 is exclusively synthesized by the polysialyltransferase ST8SiaIV and attached to mucin-type o-glycans located between the b2 and c domain.

Neuropilin-2 (NRP2) is well known as a co-receptor for class 3 semaphorins and vascular endothelial growth factors, involved in axon guidance and angiogenesis. Moreover, NRP2 was shown to promote chemotactic migration of human monocyte-derived dendritic cells (DCs) toward the chemokine CCL21, a function that relies on the presence of polysialic acid (polySia). In vertebrates, this posttranslational modification is predominantly found on the neural cell adhesion molecule (NCAM), where it is synthesized on N-glycans by either of the two polysialyltransferases, ST8SiaII or ST8SiaIV. In contrast to NCAM, little is known on the biosynthesis of polySia on NRP2. Here we identified the polySia attachment sites and demonstrate that NRP2 is recognized only by ST8SiaIV. Although polySia-NRP2 was found on bone marrow-derived DCs from wild-type and St8sia2(-/-) mice, polySia was completely lost in DCs from St8sia4(-/-) mice despite normal NRP2 expression. In COS-7 cells, co-expression of NRP2 with ST8SiaIV but not ST8SiaII resulted in the formation of polySia-NRP2, highlighting distinct acceptor specificities of the two polysialyltransferases. Notably, ST8SiaIV synthesized polySia selectively on a NRP2 glycoform that was characterized by the presence of sialylated core 1 and core 2 O-glycans. Based on a comprehensive site-directed mutagenesis study, we localized the polySia attachment sites to an O-glycan cluster located in the linker region between b2 and c domain. Combined alanine exchange of Thr-607, -613, -614, -615, -619, and -624 efficiently blocked polysialylation. Restoration of single sites only partially rescued polysialylation, suggesting that within this cluster, polySia is attached to more than one site.

Polysialic acid is present in mammalian semen as a post-translational modification of the neural cell adhesion molecule NCAM and the polysialyltransferase ST8SiaII.

Fertilization in animals is a complex sequence of several biochemical events beginning with the insemination into the female reproductive tract and, finally, leading to embryogenesis. Studies by Kitajima and co-workers (Miyata, S., Sato, C., and Kitajima, K. (2007) Trends Glycosci. Glyc, 19, 85-98) demonstrated the presence of polysialic acid (polySia) on sea urchin sperm. Based on these results, we became interested in the potential involvement of sialic acid polymers in mammalian fertilization. Therefore, we isolated human sperm and performed analyses, including Western blotting and mild 1,2-diamino-4,5-methylenedioxybenzene-HPLC, that revealed the presence α2,8-linked polySia chains. Further analysis by a glyco-proteomics approach led to the identification of two polySia carriers. Interestingly, besides the neural cell adhesion molecule, the polysialyltransferase ST8SiaII has also been found to be a target for polysialylation. Further analysis of testis and epididymis tissue sections demonstrated that only epithelial cells of the caput were polySia-positive. During the epididymal transit, polySia carriers were partially integrated into the sperm membrane of the postacrosomal region. Because polySia is known to counteract histone as well as neutrophil extracellular trap-mediated cytotoxicity against host cells, which plays a role after insemination, we propose that polySia in semen represents a cytoprotective element to increase the number of vital sperm.

ST3GAL3 mutations impair the development of higher cognitive functions.

The genetic variants leading to impairment of intellectual performance are highly diverse and are still poorly understood. ST3GAL3 encodes the Golgi enzyme ß-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.

Soluble polysialylated NCAM: a novel player of the innate immune system in the lung.

Posttranslational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) is well studied in the nervous system and described as a dynamic modulator of plastic processes like precursor cell migration, axon fasciculation, and synaptic plasticity. Here, we describe a novel function of polysialylated NCAM (polySia-NCAM) in innate immunity of the lung. In mature lung tissue of healthy donors, polySia was exclusively attached to the transmembrane isoform NCAM-140 and located to intracellular compartments of epithelial cells. In patients with chronic obstructive pulmonary disease, however, increased polySia levels and processing of the NCAM carrier were observed. Processing of polysialylated NCAM was reproduced in a mouse model by bleomycin administration leading to an activation of the inflammasome and secretion of interleukin (IL)-1ß. As shown in a cell culture model, polySia-NCAM-140 was kept in the late trans-Golgi apparatus of lung epithelial cells and stimulation by IL-1ß or lipopolysaccharide induced metalloprotease-mediated ectodomain shedding, resulting in the secretion of soluble polySia-NCAM. Interestingly, polySia chains of secreted NCAM neutralized the cytotoxic activity of extracellular histones as well as DNA/histone-network-containing "neutrophil extracellular traps", which are formed during invasion of microorganisms. Thus, shedding of polySia-NCAM by lung epithelial cells may provide a host-protective mechanism to reduce tissue damage during inflammatory processes.

The lecticans of mammalian brain perineural net are O-mannosylated.

O-Mannosylation is an important protein modification in brain. During the last years, a few mammalian proteins have been identified as targets of the protein-O-mannosyltransferases 1 and 2. However, these still cannot explain the high content of O-mannosyl glycans in brain and the strong brain involvement of congenital muscular dystrophies caused by POMT mutations (Walker-Warburg syndrome, dystroglycanopathies). By fractionating and analyzing the glycoproteome of mouse and calf brain lysates, we could show that proteins of the perineural net, the lecticans, are O-mannosylated, indicating that major components of neuronal extracellular matrix are O-mannosylated in mammalian brain. This finding corresponds with the high content of O-mannosyl glycans in brain as well as with the brain involvement of dystroglycanopathies. In contrast, the lectican neurocan is not O-mannosylated when recombinantly expressed in EBNA-293 cells, revealing the possibility of different control mechanisms for the initiation of O-mannosylation in different cell types.

Polysialylation of the synaptic cell adhesion molecule 1 (SynCAM 1) depends exclusively on the polysialyltransferase ST8SiaII in vivo.

Polysialic acid is a unique carbohydrate polymer specifically attached to a limited number of glycoproteins. Among them is synaptic cell adhesion molecule 1 (SynCAM 1), a member of the immunoglobulin (Ig) superfamily composed of three extracellular Ig-like domains. Polysialylation of SynCAM 1 is cell type-specific and was exclusively found in NG2 cells, a class of multifunctional progenitor cells that form specialized synapses with neurons. Here, we studied the molecular requirements for SynCAM 1 polysialylation. Analysis of mice lacking one of the two polysialyltransferases, ST8SiaII or ST8SiaIV, revealed that polysialylation of SynCAM 1 is exclusively mediated by ST8SiaII throughout postnatal brain development. Alternative splicing of the three variable exons 8a, 8b, and 8c can theoretically give rise to eight transmembrane isoforms of SynCAM 1. We detected seven transcript variants in the developing mouse brain, including three variants containing exon 8c, which was so far regarded as a cryptic exon in mice. Polysialylation of SynCAM 1 was restricted to four isoforms in perinatal brain. However, cell culture experiments demonstrated that all transmembrane isoforms of SynCAM 1 can be polysialylated by ST8SiaII. Moreover, analysis of domain deletion constructs revealed that Ig1, which harbors the polysialylation site, is not sufficient as an acceptor for ST8SiaII. The minimal polypeptide required for polysialylation contained Ig1 and Ig2, suggesting an important role for Ig2 as a docking site for ST8SiaII.

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

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 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.

A multivalent adsorption apparatus explains the broad host range of phage phi92: a comprehensive genomic and structural analysis.

Bacteriophage phi92 is a large, lytic myovirus isolated in 1983 from pathogenic Escherichia coli strains that carry a polysialic acid capsule. Here we report the genome organization of phi92, the cryoelectron microscopy reconstruction of its virion, and the reinvestigation of its host specificity. The genome consists of a linear, double-stranded 148,612-bp DNA sequence containing 248 potential open reading frames and 11 putative tRNA genes. Orthologs were found for 130 of the predicted proteins. Most of the virion proteins showed significant sequence similarities to proteins of myoviruses rv5 and PVP-SE1, indicating that phi92 is a new member of the novel genus of rv5-like phages. Reinvestigation of phi92 host specificity showed that the host range is not limited to polysialic acid-encapsulated Escherichia coli but includes most laboratory strains of Escherichia coli and many Salmonella strains. Structure analysis of the phi92 virion demonstrated the presence of four different types of tail fibers and/or tailspikes, which enable the phage to use attachment sites on encapsulated and nonencapsulated bacteria. With this report, we provide the first detailed description of a multivalent, multispecies phage armed with a host cell adsorption apparatus resembling a nanosized Swiss army knife. The genome, structure, and, in particular, the organization of the baseplate of phi92 demonstrate how a bacteriophage can evolve into a multi-pathogen-killing agent.

West syndrome caused by ST3Gal-III deficiency.

West syndrome consists of infantile spasms, hypsarrhythmia, and developmental arrest. Most patients remain mentally retarded and many develop Lennox-Gastaut syndrome. Using homozygosity mapping followed by exome sequencing we identified an ST3GAL3 mutation in three infants with West syndrome. ST3GAL3 encodes a sialyltransferase involved in the biosynthesis of sialyl-Lewis epitopes on cell surface-expressed glycoproteins. The mutation affected an essential sialyl-motif and abolished enzymatic activity. Abnormalities in proteins involved in forebrain γ-aminobutyric acid (GABA)ergic synaptic growth and function were recently proposed to account for infantile spasms. Dysfunctional ST3GAL3 may thus result in perturbation of the posttranslational sialylation of proteins in these pathways.

Polysialic acid: versatile modification of NCAM, SynCAM 1 and neuropilin-2.

The glycan polysialic acid is well-known as a unique posttranslational modification of the neural cell adhesion molecule NCAM. Despite remarkable acceptor specificity, however, a few other proteins can be targets of polysialylation. Here, we recapitulate the biosynthesis of polysialic acid by the two polysialyltransferases ST8SIA2 and ST8SIA4 and highlight the increasing evidence that variation in the human ST8SIA2 gene is linked to schizophrenia and possibly other neuropsychiatric disorders. Moreover, we summarize the knowledge on the role of NCAM polysialylation in brain development gained by the analysis of NCAM- and polysialyltransferase-deficient mouse models. The last part of this review is focused on recent advances in identifying SynCAM 1 and neuropilin-2 as novel acceptors of polysialic acid in NG2 cells of the perinatal brain and in dendritic cells of the immune system, respectively.

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

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 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.

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

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 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.

Extending the enzymatic toolbox for heparosan polymerization, depolymerization, and detection.

Heparosan is an acidic polysaccharide expressed as a capsule polymer by pathogenic and commensal bacteria, e.g. by E. coli K5. As a precursor in the biosynthesis of heparan sulfate and heparin, heparosan has a high biocompatibility and is thus of interest for pharmaceutical applications. However, due to its low immunogenicity, developing antibodies against heparosan and detecting the polymer in biological samples has been challenging. In this study, we exploited the enzyme repertoire of E. coli K5 and the E. coli K5-specific bacteriophage ΦK5B for the controlled synthesis and depolymerization of heparosan. A fluorescently labeled heparosan nonamer was used as a priming acceptor to study the elongation mechanism of the E. coli K5 heparosan polymerases KfiA and KfiC. We could demonstrate that the enzymes act in a distributive manner, producing labeled heparosan of low dispersity. The enzymatically synthesized heparosan was a useful tool to identify the tailspike protein KflB of ΦK5B as heparosan lyase and to characterize its endolytic depolymerization mechanism. Most importantly, using site-directed mutagenesis and rational construct design, we generated an inactive version of KflB for the detection of heparosan in ELISA-based assays, on blots, and on bacterial and mammalian cells.