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Sialic acids are found as terminal sugars on glycan structures on cellular surfaces. T cells carry these sialoglycans abundantly, and they are thought to serve multiple functions in cell adhesion, cell migration, and protection from complement attack. We studied the role of sialoglycans on T cells in a mouse model with a T cell-specific deletion of cytidine monophosphate-sialic acid synthase (CMAS), the enzyme that is crucial for the synthesis of sialoglycans. These mice showed a T-cell deficiency in peripheral lymphoid organs. Many T cells with an undeleted Cmas allele were found in the periphery, suggesting that they escaped the Cre-mediated deletion. The remaining peripheral T cells of T cell-specific Cmas KO mice had a memory-like phenotype. Additional depletion of the complement factor C3 could not rescue the phenotype, showing that the T-cell defect was not caused by a host complement activity. Cmas -deficient T cells showed a high level of activated caspase 3, indicating an ongoing apoptosis. In bone marrow chimeric cellular transfer experiments, we observed a strong competitive disadvantage of Cmas -deficient T cells compared to wild-type T cells. These results show that sialoglycans on the surface of T cells are crucial for T-cell survival and maintenance. This function has not been recognized before and is similar to the function of sialoglycans on B cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Schmidt, Linder, Korn, Schellenberg, Meyer, Nimmerjahn, Werner, Abeln, Gerardy-Schahn, Münster-Kühnel and Nitschke.)

Sialic acids (Sias) on the B cell membrane are involved in cell migration, in the control of the complement system and, as sialic acid-binding immunoglobulin-like lectin (Siglec) ligands, in the regulation of cellular signaling. We studied the role of sialoglycans on B cells in a mouse model with B cell-specific deletion of cytidine monophosphate sialic acid synthase (CMAS), the enzyme essential for the synthesis of sialoglycans. Surprisingly, these mice showed a severe B cell deficiency in secondary lymphoid organs. Additional depletion of the complement factor C3 rescued the phenotype only marginally, demonstrating a complement-independent mechanism. The B cell survival receptor BAFF receptor was not up-regulated, and levels of activated caspase 3 and processed caspase 8 were high in B cells of Cmas -deficient mice, indicating ongoing apoptosis. Overexpressed Bcl-2 could not rescue this phenotype, pointing to extrinsic apoptosis. These results show that sialoglycans on the B cell surface are crucial for B cell survival by counteracting several death-inducing pathways.

Genetic replacement of adenoviral fiber knobs by ligands that enable tumor specific targeting of oncolytic adenoviruses is challenging because the fiber knob contributes to virus assembly. Here, we present a novel concept by describing stable recombinant adenoviruses with tumor specific infection mode. The fiber knob was replaced by endosialidaseNF (endoNF), the tailspike protein of bacteriophage K1F. EndoNF recognizes polysialic acid, an oncofetal antigen characteristic for high malignant tumors of neuroendocrine origin. An intramolecular chaperone contained in endoNF warrants folding and compensates for the knob function in virus assembly. Obtained recombinant viruses demonstrated polysialic acid dependent infection modes, strong oncolytic capacity with polysialic acid positive cells in culture and a high potential to inhibit tumor growth in a therapeutic mouse model of subcutaneous neuroblastoma. With a single genetic manipulation we achieved ablation of the fiber knob, introduction of a tumor specific ligand, and folding control over the chimeric fiber construct., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

α2,8-Linked polysialic acid (polySia) is an oncofoetal antigen with high abundance during embryonic development. It reappears in malignant tumours of neuroendocrine origin. Two polysialyltransferases (polySTs) ST8SiaII and IV are responsible for polySia biosynthesis. During development, both enzymes are essential to control polySia expression. However, in tumours ST8SiaII is the prevalent enzyme. Consequently, ST8SiaII is an attractive target for novel cancer therapeutics. A major challenge is the high structural and functional conservation of ST8SiaII and -IV. An assay system that enables differential testing of ST8SiaII and -IV would be of high value to search for specific inhibitors. Here we exploited the different modes of acceptor recognition and elongation for this purpose. With DMB-DP3 and DMB-DP12 (fluorescently labelled sialic acid oligomers with a degree of polymerisation of 3 and 12, respectively) we identified stark differences between the two enzymes. The new acceptors enabled the simple comparative testing of the polyST initial transfer rate for a series of CMP-activated and N-substituted sialic acid derivatives. Of these derivatives, the non-transferable CMP-Neu5Cyclo was found to be a new, competitive ST8SiaII inhibitor., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

The addition of polysialic acid to N- and/or O-linked glycans, referred to as polysialylation, is a rare posttranslational modification that is mainly known to control the developmental plasticity of the nervous system. Here we show that CCR7, the central chemokine receptor controlling immune cell trafficking to secondary lymphatic organs, carries polysialic acid. This modification is essential for the recognition of the CCR7 ligand CCL21. As a consequence, dendritic cell trafficking is abrogated in polysialyltransferase-deficient mice, manifesting as disturbed lymph node homeostasis and unresponsiveness to inflammatory stimuli. Structure-function analysis of chemokine-receptor interactions reveals that CCL21 adopts an autoinhibited conformation, which is released upon interaction with polysialic acid. Thus, we describe a glycosylation-mediated immune cell trafficking disorder and its mechanistic basis., (Copyright © 2016, American Association for the Advancement of Science.)

Polysialic acid (polySia) is expressed on several malignant tumors of neuroendocrine origin, including small cell lung cancer. In this study, we investigated the therapeutic efficacy of tumor-directed T-cell responses, elicited by polySia-retargeted oncolytic adenovirus infection, in an orthotopic murine model of disseminated polySia-positive lung cancer. In several cell lines, we demonstrated highly polySia-selective retargeting of adenoviral infection using a bispecific adapter comprising the ectodomain of the coxsackievirus/adenovirus receptor and a polySia-recognizing single-chain antibody domain. PolySia-dependent systemic infection in vivo facilitated effective uptake of viruses in subcutaneous polySia-expressing human tumors, whereas hepatic viral load and hepatotoxicity were significantly reduced. The impact and nature of antitumoral immune responses triggered by systemic delivery of polySia-retargeted oncolytic adenoviruses were investigated in an orthotopic model of disseminated lung cancer. Interestingly, improved transduction by polySia-retargeted oncolytic adenoviruses led to CD45-positive cell infiltrates in close association with large lytic areas. Consistently, enhanced tumor regression and prolonged survival was only observed in immunocompetent mice, but not in T-cell-deficient mice. To investigate whether improved systemic infection by polySia retargeting would elicit a tumor-specific T-cell response, we screened the used lung cancer cells for mutated oncogenes by complete exon sequencing. In agreement with our other results, only retargeted oncolysis was able to induce a significant response specific for the tumor-associated neoepitope Gsta2-Y9H. In conclusion, we demonstrated that effective retargeting of oncolytic adenovirus against polySia-expressing tumors elicits an effective tumor-directed T-cell response after systemic virus delivery and facilitates therapy of disseminated lung cancer., (©2015 American Association for Cancer Research.)

Unlabelled: In severe liver injury, ductular reactions (DRs) containing bipotential hepatic progenitor cells (HPCs) branch from the portal tract. Neural cell adhesion molecule (NCAM) marks bile ducts and DRs, but not mature hepatocytes. NCAM mediates interactions between cells and surrounding matrix; however, its role in liver development and regeneration is undefined. Polysialic acid (polySia), a unique posttranslational modifier of NCAM, is produced by the enzymes, ST8SiaII and ST8SiaIV, and weakens NCAM interactions. The role of polySia with NCAM synthesizing enzymes ST8SiaII and ST8SiaIV were examined in HPCs in vivo using the choline-deficient ethionine-supplemented and 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet models of liver injury and regeneration, in vitro using models of proliferation, differentiation, and migration, and by use of mouse models with gene defects in the polysialyltransferases (St8sia 2+/-4+/-, and St8sia2-/-4-/-). We show that, during liver development, polySia is required for the correct formation of bile ducts because gene defects in both the polysialyltransferases (St8sia2+/-4+/- and St8sia2-/-4-/- mice) caused abnormal bile duct development. In normal liver, there is minimal polySia production and few ductular NCAM+ cells. Subsequent to injury, NCAM+ cells expand and polySia is produced by DRs/HPCs through ST8SiaIV. PolySia weakens cell-cell and cell-matrix interactions, facilitating HGF-induced migration. Differentiation of HPCs to hepatocytes in vitro results in both transcriptional down-regulation of polySia and cleavage of polySia-NCAM. Cleavage of polySia by endosialidase (endoN) during liver regeneration reduces migration of DRs into parenchyma., Conclusion: PolySia modification of NCAM+ ductules weakens cell-cell and cell-matrix interactions, allowing DRs/HPCs to migrate for normal development and regeneration. Modulation of polySia levels may provide a therapeutic option in liver regeneration., (© 2014 by the American Association for the Study of Liver Diseases.)

Polysialic acid (polySia) is a unique glycan modification of the neural cell adhesion molecule NCAM and a major determinant of brain development. Polysialylation of NCAM is implemented by the two polysialyltransferases (polySTs) ST8SIA2 and ST8SIA4. Dysregulation of the polySia-NCAM system and variation in ST8SIA2 has been linked to schizophrenia and other psychiatric disorders. Here, we show reduced interneuron densities in the medial prefrontal cortex (mPFC) of mice with either partial or complete loss of polySia synthesizing capacity by ablation of St8sia2, St8sia4, or both. Cells positive for parvalbumin and perineuronal nets as well as somatostatin-positive cells were reduced in the mPFC of all polyST-deficient lines, whereas calretinin-positive cells and the parvalbumin-negative fraction of calbindin-positive cells were unaffected. Reduced interneuron numbers were corroborated by analyzing polyST-deficient GAD67-GFP knock-in mice. The accumulation of precursors in the ganglionic eminences and reduced numbers of tangentially migrating interneurons in the pallium were observed in polyST-deficient embryos. Removal of polySia by endosialidase treatment of organotypic slice cultures led to decreased entry of GAD67-GFP-positive interneurons from the ganglionic eminences into the pallium. Moreover, the acute loss of polySia caused significant reductions in interneuron velocity and leading process length. Thus, attenuation of polySia interferes with the developmental migration of cortical interneurons and causes pathological changes in specific interneuron subtypes. This provides a possible link between genetic variation in polyST genes, neurodevelopmental alterations and interneuron dysfunction in neuropsychiatric disease., (© 2014. Published by The Company of Biologists Ltd.)

Every cell in nature carries a rich surface coat of glycans, its glycocalyx, which constitutes the cell's interface with its environment. In eukaryotes, the glycocalyx is composed of glycolipids, glycoproteins, and proteoglycans, the compositions of which vary among different tissues and cell types. Many of the linear and branched glycans on cell surface glycoproteins and glycolipids of vertebrates are terminated with sialic acids, nine-carbon sugars with a carboxylic acid, a glycerol side-chain, and an N-acyl group that, along with their display at the outmost end of cell surface glycans, provide for varied molecular interactions. Among their functions, sialic acids regulate cell-cell interactions, modulate the activities of their glycoprotein and glycolipid scaffolds as well as other cell surface molecules, and are receptors for pathogens and toxins. In the brain, two families of sialoglycans are of particular interest: gangliosides and polysialic acid. Gangliosides, sialylated glycosphingolipids, are the most abundant sialoglycans of nerve cells. Mouse genetic studies and human disorders of ganglioside metabolism implicate gangliosides in axon-myelin interactions, axon stability, axon regeneration, and the modulation of nerve cell excitability. Polysialic acid is a unique homopolymer that reaches >90 sialic acid residues attached to select glycoproteins, especially the neural cell adhesion molecule in the brain. Molecular, cellular, and genetic studies implicate polysialic acid in the control of cell-cell and cell-matrix interactions, intermolecular interactions at cell surfaces, and interactions with other molecules in the cellular environment. Polysialic acid is essential for appropriate brain development, and polymorphisms in the human genes responsible for polysialic acid biosynthesis are associated with psychiatric disorders including schizophrenia, autism, and bipolar disorder. Polysialic acid also appears to play a role in adult brain plasticity, including regeneration. Together, vertebrate brain sialoglycans are key regulatory components that contribute to proper development, maintenance, and health of the nervous system.

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)-1β. 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-1β 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.

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.

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.

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.

Sialic acid polymers of glycoproteins and glycolipids are characterized by a high diversity in nature and are involved in distinct biological processes depending inter alia on the glycosidic linkages between the present sialic acid residues. Though suitable protocols are available for chain length and sialic acid determination, sensitive methods for linkage analysis of di-, oligo-, and polysialic acids (di/oligo/polySia) are still pending. In this study, we have established a highly sensitive glycomic strategy for this purpose which is based on permethylation of di/oligo/polySia after tagging their reducing ends with the fluorescent dye 1,2-diamino-4,5-methylenedioxybenzene (DMB). Using DMB-labeled sialic acid di/oligo/polymers glycosidic linkages could be efficiently determined and, optionally, the established working procedure can be combined with HPLC for in depth characterization of distinct di/oligo/polySia chains. Moreover, the outlined approach can be directly applied to mammalian tissue samples and linkage analysis of sialic acid polymers present in biopsy samples of neuroblastoma tissue demonstrating the usefulness of the outlined work flow to screen, for example, cancer tissue for the presence of distinct variants of di/oligo/polySia as potentially novel biomarkers. Hence, the described strategy offers a highly sensitive and efficient strategy for identification of glycosidic linkages in sialic acid di/oligo/polymers of glycoproteins and glycolipids., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Hormones such as leptin and ghrelin can rapidly rewire hypothalamic feeding circuits when injected into rodent brains. These experimental manipulations suggest that the hypothalamus might reorganize continually in adulthood to integrate the metabolic status of the whole body. In this study, we examined whether hypothalamic plasticity occurs in naive animals according to their nutritional conditions. For this purpose, we fed mice with a short-term high-fat diet (HFD) and assessed brain remodeling through its molecular and functional signature. We found that HFD for 3 d rewired the hypothalamic arcuate nucleus, increasing the anorexigenic tone due to activated pro-opiomelanocortin (POMC) neurons. We identified the polysialic acid molecule (PSA) as a mediator of the diet-induced rewiring of arcuate POMC. Moreover, local pharmacological inhibition and genetic disruption of the PSA signaling limits the behavioral and metabolic adaptation to HFD, as treated mice failed to normalize energy intake and showed increased body weight gain after the HFD challenge. Altogether, these findings reveal the existence of physiological hypothalamic rewiring involved in the homeostatic response to dietary fat. Furthermore, defects in the hypothalamic plasticity-driven adaptive response to HFD are obesogenic and could be involved in the development of metabolic diseases.

Polysialic acid is common to humans and a few bacterial pathogens and it holds great potential for the development of new therapeutic reagents. Currently, the bacterial polysialyltransferases (polySTs) are the only source of polysialic acid for research and biotechnological purposes either directly, by enzymatic polysialylation of therapeutic proteins, or indirectly, by harvest of polysialic acid from bacterial fermentation. Further engineering and optimization of these enzymes is hindered by the lack of high-throughput screening methodologies for polysialyltransferase activity. Here we report the development of an efficient in vivo activity screen for bacterial polySTs. The screen exploits complementation of a dormant capsule export complex in the expression strain, Escherichia coli BL21-Gold(DE3). This strain was metabolically engineered to synthesize CMP-Neu5Ac, the donor sugar for the polysialylation reaction. Using the new strain, a colony blotting procedure that enables the routine testing of more than 10(4) polyST genes was developed. To test the usefulness of the methodology, we screened a library of N-terminally truncated polySTs derived from the Neisseria meningitidis serogroup B (NmB)-polyST. We identified truncations that remove a putative membrane interaction domain, resulting in soluble and active enzymes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The polysialic acid (PSA) production in Escherichia coli (E. coli) K1 was studied using three different cultivation strategies. A batch cultivation, a fed-batch cultivation at a constant specific growth rate of 0.25 h(-1) and a fed-batch cultivation at a constant glucose concentration of 50 mg l(-1) was performed. PSA formation kinetics under different cultivation strategies were analyzed based on the Monod growth model and the Luedeking-Piret equation. The results revealed that PSA formation in E. coli K1 was completely growth associated, the highest specific PSA formation rate (0.0489 g g(-1)h(-1)) was obtained in the batch cultivation. However, comparing biomass and PSA yields on the glucose consumed, both fed-batch cultivations provided higher yields than that of the batch cultivation and acetate formation was prevented. Moreover, PSA yield on glucose was also correlated to the specific growth rate of the cells. The optimal specific growth rate for PSA production was 0.32 h(-1) obtained in the fed-batch cultivation at a constant glucose concentration of 50 mg l(-1), with highest conversion efficiency of 43 mg g(-1)., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

Polysialic acid (polySia) is a large, negatively charged homopolymer of 2,8-linked N-acetylneuraminic acid residues resulting from remodeling and extension of protein-bound sialic acid (Sia) residues and seems to have a key role in regulating neural cell development and function. The aim of this study was to explore and compare the effects of polySia and sialylation on gating of voltage-gated sodium channels. The skeletal muscle α-subunit NaV1.4 was transiently expressed in wild-type Chinese hamster ovary (CHO) cells or in mutant CHO cells with deficits in their capacity to produce sialylated or polysialylated membrane components. Expression in both mutant cell lines resulted in larger peak current amplitudes as compared to wild-type CHO cells. Loss of Sia and polySia also resulted in significant shifts of voltage-dependent activation and steady-state inactivation, however, in opposite directions. Furthermore, only the loss of Sia had a significant effect on recovery from fast inactivation. Our data demonstrate for the first time that gating of voltage-gated sodium channels seems to be differentially regulated by polySia and Sia., (Copyright © 2011 S. Karger AG, Basel.)

With the aim to develop new biomaterials for peripheral nerve grafts, the current study used bioidentical polysialic acid (polySia) as complement in synthetic conduits. polySia provides an important guidance cue during nervous system development and regeneration. First in vivo results on the use of cell-free and Schwann cell-containing synthetic peripheral nerve grafts complemented with soluble exogenous K1-polySia are presented. Reconstructing 10 mm rat sciatic nerve gaps, K1-polySia complementation significantly improved structural nerve regeneration in comparison to cell-free and K1-polySia-free grafts. Subsequently, long nerve gaps (13 mm) were reconstructed by Schwann cell transplants plus K1-polySia and compared to nerve autotransplantation. Structural but also functional regeneration could be observed using K1-polySia transplants; however, autotransplantation was still significantly more successful. Overall, the current study demonstrates that exogenous K1-polySia has no negative but rather regeneration promoting effects. This is important novel evidence on the applicability of exogenous polySia in vivo. Further studies are required to develop solid three-dimensional polySia-based scaffolds for nerve tissue engineering. Biocompatible and assessable biodegrading materials will ensure long-lasting presence of polySia to allow its applicability and prolonged efficacy in the slow regenerating scenario of human peripheral nerve reconstruction.

An alpha-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 Sia(3) 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., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

The neural cell adhesion molecule (NCAM) is the predominant carrier of alpha2,8 polysialic acid (PSA) in the mammalian brain. Abnormalities in PSA and NCAM expression are associated with schizophrenia in humans and cause deficits in hippocampal synaptic plasticity and contextual fear conditioning in mice. Here, we show that PSA inhibits opening of recombinant NMDA receptors composed of GluN1/2B (NR1/NR2B) or GluN1/2A/2B (NR1/NR2A/NR2B) but not of GluN1/2A (NR1/NR2A) subunits. Deficits in NCAM/PSA increase GluN2B-mediated transmission and Ca(2+) transients in the CA1 region of the hippocampus. In line with elevation of GluN2B-mediated transmission, defects in long-term potentiation in the CA1 region and contextual fear memory in NCAM/PSA-deficient mice are abrogated by application of a GluN2B-selective antagonist. Furthermore, treatment with the glutamate scavenger glutamic-pyruvic transaminase, ablation of Ras-GRF1 (a mediator of GluN2B signaling to p38 MAPK), or direct inhibition of hyperactive p38 MAPK can restore impaired synaptic plasticity in brain slices lacking PSA/NCAM. Thus, PSA carried by NCAM regulates plasticity and learning by inhibition of the GluN2B-Ras-GRF1-p38 MAPK signaling pathway. These findings implicate carbohydrates carried by adhesion molecules in modulating NMDA receptor signaling in the brain and demonstrate reversibility of cognitive deficits associated with ablation of a schizophrenia-related adhesion molecule.

Although the polysialyltransferase ST8Sia IV is expressed in both primary and secondary human lymphoid organs, its product, polysialic 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.

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.

PolySia, the most striking post-translational modification of the neural cell adhesion molecule, is down-regulated during postnatal development. After peripheral nerve lesion, polySia is located on neuronal and glial cells normally not synthesizing polySia. However, structural consequences of reduced polySia content for peripheral nerve regeneration have not yet been clear. Furthermore, the contribution of sialyltransferases ST8SiaII and ST8SiaIV for the up-regulation of polySia has not been studied so far. In order to investigate the impact of polySia on regeneration processes of myelinated axons, we examined mouse mutants retaining only one functional sialyltransferase allele. In the absence of ST8SiaII, quantification of myelinated axons revealed a significant decrease in number and size of regenerated fibers without impairment of remyelination. In contrast, St8SiaIV deficiency resulted in increased fiber outgrowth and axonal maturation. Western blot analysis demonstrated that both ST8SiaII and St8SiaIV direct up-regulation of polySia. Cell-specific induction of polySia in myelinating Schwann cells and on regenerated axons in the presence of ST8SiaIV, but not ST8SiaII, indicates that not only the amount of polySia but also its cellular localization has a high impact on the regeneration progress of peripheral nerves.

Polysialic acid (polySia) is a large glycan with restricted expression, typically found attached to the protein scaffold neural cell adhesion molecule (NCAM). PolySia is best known for its proposed role in modulating neuronal development. Its presence and potential functions outside the nervous systems are essentially unexplored. Herein we show the expression of polySia on hematopoietic progenitor cells, and demonstrate a role for this glycan in immune response using both acute inflammatory and tumor models. Specifically, we found that human NK cells modulate expression of NCAM and the degree of polymerization of its polySia glycans according to activation state. This contrasts with the mouse, where polySia and NCAM expression are restricted to multipotent hematopoietic progenitors and cells developing along a myeloid lineage. Sialyltransferase 8Sia IV(-/-) mice, which lacked polySia expression in the immune compartment, demonstrated an increased contact hypersensitivity response and decreased control of tumor growth as compared with wild-type animals. This is the first demonstration of polySia expression and regulation on myeloid cells, and the results in animal models suggest a role for polySia in immune regulation.

Restorative medicine has a constant need for improved scaffold materials. Degradable biopolymers often suffer from uncontrolled chemical or enzymatic hydrolysis by the host. The need for a second surgery on the other hand is a major drawback for nondegradable scaffold materials. In this paper we report the design and synthesis of a novel polysialic acid-based hydrogel with promising properties. Hydrogel synthesis was optimized and enzymatic degradation was studied using a phage-born endosialidase. After addition of endosialidase, hydrogels readily degraded depending on the amount of initially used cross-linker within 2 to 11 days. This polysialic acid hydrogel is not cytotoxic, completely stable under physiological conditions, and could be evaluated as growth support for PC12 cells. Here, additional coating with collagen I, poly-L-lysine or matrigel is mandatory to improve the properties of the material.

Understanding the mechanisms that regulate neurogenesis is a prerequisite for brain repair approaches based on neuronal precursor cells. One important regulator of postnatal neurogenesis is polysialic acid (polySia), a post-translational modification of the neural cell adhesion molecule NCAM. In the present study, we investigated the role of polySia in differentiation of neuronal precursors isolated from the subventricular zone of early postnatal mice. Removal of polySia promoted neurite induction and selectively enhanced maturation into a calretinin-positive phenotype. Expression of calbindin and Pax6, indicative for other lineages of olfactory bulb interneurons, were not affected. A decrease in the number of TUNEL-positive cells indicated that cell survival was slightly improved by removing polySia. Time lapse imaging revealed the absence of chain migration and low cell motility, in the presence and absence of polySia. The changes in survival and differentiation, therefore, could be dissected from the well-known function of polySia as a promoter of precursor migration. The differentiation response was mimicked by exposure of cells to soluble or substrate-bound NCAM and prevented by the C3d-peptide, a synthetic ligand blocking NCAM interactions. Moreover, a higher degree of differentiation was observed in cultures from polysialyltransferase-depleted mice and after NCAM exposure of precursors from NCAM-knockout mice demonstrating that the NCAM function is mediated via heterophilic binding partners. In conclusion, these data reveal that polySia controls instructive NCAM signals, which direct the differentiation of subventricular zone-derived precursors towards the calretinin-positive phenotype of olfactory bulb interneurons., ((c) 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2008.)

The study of new biomaterials is the objective of many current research projects in biotechnological medicine. A promising scaffold material for the application in tissue engineering or other biomedical applications is polysialic acid (polySia), a homopolymer of alpha2,8-linked sialic acid residues, which represents a posttranslational modification of the neural cell adhesion molecule and occurs in all vertebrate species. Some neuroinvasive bacteria like, e.g. Escherichia coli K1 (E. coli K1) use polySia as capsular polysaccharide. In this latter case long polySia chains with a degree of polymerization of >200 are linked to lipid anchors. Since in vertebrates no polySia degrading enzymes exist, the molecule has a long half-life in the organism, but degradation can be induced by the use of endosialidases, bacteriophage-derived enzymes with pronounced specificity for polySia. In this work a biotechnological process for the production of bacterial polysialic acid is presented. The process includes the development of a multiple fed-batch cultivation of the E. coli K1 strain and a complete downstream strategy of polySia. A controlled feed of substrate at low concentrations resulted in an increase of the carbon yield (C(product)/C(substrate)) from 2.2 to 6.6%. The downstream process was optimized towards purification of long polySia chains. Using a series of adjusted precipitation steps an almost complete depletion of contaminating proteins was achieved. The whole process yielded 1-2g polySia from a 10-l bacterial culture with a purity of 95-99%. Further product analysis demonstrated maximum chain length of >130 for the final product.

In this study we present the enzymatic and biological analysis of polysialic acid (polySia) based hydrogel in terms of its degradation and cytocompatibility. PolySia based hydrogel is completely degradable by endosialidase enzyme which may avoid second surgery after tissue recovery. Viability assay showed that soluble components of polySia hydrogel did not cause any toxic effect on cultured Schwann cells. Moreover, green fluorescence protein transfected neonatal and adult Schwann cells, neural stem cells and dorsal root ganglionic cells (unlabelled) were seeded on polySia hydrogel modified with poly-L-lysine (Pll), poly-L-ornithine-laminin (porn-laminin) or collagen. Water soluble tetrazolium salt assay revealed that modification of the hydrogel significantly improved cell adhesion and viability. These results infer that polySia based scaffolds in combination with cell adhesion molecules and cells genetically modified to express growth factors would potentially be promising alternative in reconstructive therapeutic strategies.

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.

Polysialic acid (polySia), a post-translational modification of the neural cell adhesion molecule (NCAM), is the key regulator of NCAM-mediated functions and crucial for normal brain development, postnatal growth, and survival. Two polysialyltransferases, ST8SiaII and ST8SiaIV, mediate polySia biosynthesis. To dissect the impact of each enzyme during postnatal brain development, we monitored the developmental changes in NCAM polysialylation in wild-type, ST8SiaII-, and ST8SiaIV-deficient mice using whole brain lysates obtained at 10 time points from postnatal days 1 to 21 and from adult mice. In wild-type and ST8SiaIV-null brain, polySia biosynthesis kept pace with the rapid increase in brain weight until day 9, and nearly all NCAM was polysialylated. Thereafter, polySia dropped by approximately 70% within 1 week, accompanied by the first occurrence of polySia-free NCAM-140 and NCAM-180. In ST8SiaII-null brain, polySia declined immediately after birth, leading to 60% less polySia at day 9 combined with the untimely appearance of polySia-free NCAM. Polysialyltransferase deficiency did not alter NCAM expression level or isoform pattern. In all three genotypes, NCAM-140 and NCAM-180 were expressed at constant levels from days 1 to 21 and provided the major polySia acceptors. By contrast, NCAM-120 first appeared at day 5, followed by a strong up-regulation inverse to the decrease in polySia. Together, we provide a comprehensive quantitative analysis of the developmental changes in polySia level, NCAM polysialylation status, and polysialyltransferase transcript levels and show that the predominant role of ST8SiaII during postnatal brain development is restricted to the first 15 days.

Polysialic acid (polySia), an alpha2,8-linked polymer of N-acetylneuraminic acid, represents an essential regulator of neural cell adhesion molecule (NCAM) functions. Two polysialyltransferases, ST8SiaII and ST8SiaIV, account for polySia synthesis, but their individual roles in vivo are still not fully understood. Previous in vitro studies defined differences between the two enzymes in their usage of the two NCAM N-glycosylation sites affected and suggested a synergistic effect. Using mutant mice, lacking either enzyme, we now assessed in vivo the contribution of ST8SiaII and ST8SiaIV to polysialylation of NCAM. PolySia-NCAM was isolated from mouse brains and trypsinized, and polysialylated glycopeptides as well as glycans were analyzed in detail. Our results revealed an identical glycosylation and almost complete polysialylation of N-glycosylation sites 5 and 6 in polySia-NCAM irrespective of the enzyme present. The same sets of glycans were substituted by identical numbers of polySia chains in vivo, the length distribution of which, however, differed with the enzyme setting. Expression of ST8SiaIV alone led to higher amounts of short polySia chains and gradual decrease with length, whereas exclusive action of ST8SiaII evoked a slight reduction in long polySia chains only. These variations were most pronounced at N-glycosylation site 5, whereas the polysialylation pattern at N-glycosylation site 6 did not differ between NCAM from wild-type and ST8SiaII- or ST8SiaIV-deficient mice. Thus, our fine structure analyses suggest a comparable quality of polysialylation by ST8SiaII and ST8SiaIV and a distinct synergistic action of the two enzymes in the synthesis of long polySia chains at N-glycosylation site 5 in vivo.

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.

Polysialic acid (PSA) is a unique linear homopolymer of alpha2,8-linked sialic acid that has been identified as a posttranslational modification on only five mammalian proteins. Studied predominantly on neural cell adhesion molecule (NCAM) during development of the vertebrate nervous system, PSA modulates cell interactions mediated by NCAM and other adhesion molecules. An isoform of NCAM (CD56) on natural killer (NK) cells is the only protein known to be polysialylated in cells of the immune system, yet the function of PSA in NK cells remains unclear. We show here that neuropilin-2 (NRP-2), a receptor for the semaphorin and vascular endothelial growth factor families in neurons and endothelial cells, respectively, is expressed on the surface of human dendritic cells and is polysialylated. Expression of NRP-2 is up-regulated during dendritic cell maturation, coincident with increased expression of ST8Sia IV, one of the key enzymes of PSA biosynthesis, and with the appearance of PSA on the cell surface. PSA on NRP-2 is resistant to digestion with peptide N-glycosidase F but is sensitive to release under alkaline conditions, suggesting that PSA chains are added to O-linked glycans of NRP-2. Removal of polysialic acid from the surface of dendritic cells or binding of NRP-2 with specific IgG promoted dendritic cell-induced activation and proliferation of T lymphocytes. Thus, this newly recognized polysialylated protein on the surface of dendritic cells influences dendritic cell-T lymphocyte interactions through one or more of its distinct extracellular domains.

Polysialylation is a posttranslational modification of the neural cell adhesion molecule (NCAM). In the adult brain, polysialylated NCAM (PSA-NCAM) is restricted to regions of neurogenesis and neuroplasticity, where PSA promotes plastic changes. 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 PSA-NCAM system on development and progression of this disease and associated comorbidities. Here, we studied the impact of transient enzymatic depolysialylation of NCAM on the pathophysiology in the amygdala kindling model, a chronic rodent model of temporal lobe epilepsy. The investigations focused on seizure-induced neurogenesis, seizure progression, and on the development of kindling-associated changes in behavior and cognition. Loss of PSA decreased the number of hippocampal newborn cells that incorporated BrdU during the kindling process and the number of new neurons that were ectopically located in the hilus. The persistence of basal dendrites has been suggested to be a hallmark of newborn granule cells in the epileptic brain. Loss of PSA increased the number of cells with persistent basal dendrites. The modification of the hippocampal cell proliferation rate and the fate of newborn neurons which occurred as a consequence of PSA removal did not affect the generation of a hyperexcitable kindled network or associated behavioral changes. Kindling progression was comparable in rats with and without removal of PSA. In contrast, loss of PSA increased acute seizure susceptibility as indicated by reduced seizure thresholds before kindling. The data indicate that hippocampal proliferation rates and ectoptic hilar newborn neurons are less critical for epileptic network generation. The PSA-NCAM system was not substantiated as a target for antiepileptogenic strategies. However, its impact on ectopic newborn neurons gives evidence that modulation of PSA-NCAM function may be a strategy to promote neuroregeneration in different central nervous system insults.

The post-translational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) represents a remarkable example of dynamic modulation of homo- and heterophilic cell interactions by glycosylation. The synthesis of this unique carbohydrate polymer depends on the polysialyltransferases ST8SiaII and ST8SiaIV. Aiming to understand in more detail the contributions of ST8SiaII and ST8SiaIV to polySia biosynthesis in vivo, we used mutant mouse lines that differ in the number of functional polysialyltransferase alleles. The 1,2-diamino-4,5-methylenedioxybenzene method was used to qualitatively and quantitatively assess the polySia patterns. Similar to the wild-type genotype, long polySia chains (>50 residues) were detected in all genotypes expressing at least one functional polysialyltransferase allele. However, variant allelic combinations resulted in distinct alterations in the total amount of poly-Sia; the relative abundance of long, medium, and short polymers; and the ratio of polysialylated to non-polysialylated NCAM. In ST8SiaII-null mice, 45% of the brain NCAM was non-polysialylated, whereas a single functional allele of ST8SiaII was sufficient to polysialylate approximately 90% of the NCAM pool. Our data reveal a complex polysialylation pattern and show that, under in vivo conditions, the coordinated action of ST8SiaII and ST8SiaIV is crucial to fine-tune the amount and structure of polySia on NCAM.

Polysialic acid (PSA) regulates functions of the neural cell adhesion molecule (NCAM) during development and in neuroplasticity in the adult; the underlying mechanisms at different phases of learning and memory consolidation are, however, unknown. To investigate the contributions of PSA versus the extracellular domain of the NCAM glycoprotein backbone to synaptic plasticity, we applied NCAM, PSA-NCAM, and PSA to acute slices of the hippocampal CA1 region of NCAM-deficient mice and measured their effects on long-term potentiation (LTP). Remarkably, only PSA and PSA-NCAM, but not NCAM restored normal LTP. Application of these molecules to the dorsal hippocampus of wild-type mice showed that PSA-NCAM and PSA, but not NCAM, injected before fear conditioning, impaired formation of hippocampus-dependent contextual memory. Consolidation of contextual memory was affected by PSA-NCAM only when injected during its late, but not early phases. None of the tested compounds disturbed extrahippocampal-cued memory. Mice lacking the polysialyltransferase (ST8SialV/PST) responsible for attachment of PSA to NCAM in adulthood showed a mild deficit only in hippocampal contextual learning, when compared with NCAM-deficient mice that were disturbed in both contextual and cued memories. Contextual and tone memory in NCAM-deficient mice could be partially restored by injection of PSA-NCAM, but not of NCAM, into the hippocampus, suggesting that the impact of PSA-NCAM in synaptic plasticity and learning is not mediated by modulation of NCAM-NCAM homophilic interactions. In conclusion, our data support the view that polysialylated NCAM is involved in both formation and late consolidation of contextual memory.

The architectural conservation of nucleotide sugar transport proteins (NSTs) enabled the theoretical prediction of putative NSTs in diverse gene databases. In the human genome, 17 NST sequences have been identified but only six have been unequivocally characterized with respect to their transport specificities. Defining transport characteristics of recombinant NSTs has become a major challenge because true zero background systems are widely absent. Production of recombinant NSTs in heterologous systems has developed multifunctionality for some NSTs leading to a novel level of complexity in the field. Assuming that (1) the specificity of NSTs is determined at the primary sequence level and (2) the proteins are autonomously functional units, final definition of the substrate specificity will depend on the use of isolated transport proteins. Herein, we describe the first report of the functional expression of mouse CMP-sialic acid transporter (CST) in Escherichia coli and thus provide significant progress towards the production of transporter proteins in quantities suitable for functional and structural analyses. Recovery of the active NST from inclusion bodies was achieved after solubilization with 8 M urea and stepwise renaturation. After reconstitution into phospholipid vesicles, the recombinant protein demonstrated specific transport for CMP-N-acetylneuraminic acid (CMP-Neu5Ac) with no transport of UDP-sugars. Kinetic studies carried out with CMP-Neu5Ac and established CMP-Neu5Ac antagonist's evaluated natural conformation of the reconstituted protein and clearly demonstrate that the transporter acts as a simple mobile carrier.

Poly-alpha2,8-sialic acid (polySia) is a unique modification of the neural cell adhesion molecule, NCAM, tightly associated with neural development and plasticity. However, the vital role attributed to this carbohydrate polymer has been challenged by the mild phenotype of mice lacking polySia due to NCAM-deficiency. To dissect polySia and NCAM functions, we generated polySia-negative but NCAM-positive mice by simultaneous deletion of the two polysialyltransferase genes, St8sia-II and St8sia-IV. Beyond features shared with NCAM-null animals, a severe phenotype with specific brain wiring defects, progressive hydrocephalus, postnatal growth retardation, and precocious death was observed. These drastic defects were selectively rescued by additional deletion of NCAM, demonstrating that they originate from a gain of NCAM functions because of polySia deficiency. The data presented in this study reveal that the essential role of polySia resides in the control and coordination of NCAM interactions during mouse brain development. Moreover, this first demonstration in vivo that a highly specific glycan structure is more important than the glycoconjugate as a whole provides a novel view on the relevance of protein glycosylation for the complex process of building the vertebrate brain.

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 triple beta-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.

Polysialic acid (PSA), a carbohydrate polymer attached to the neural cell adhesion molecule (NCAM), promotes neural plasticity and tumor malignancy, but its mode of action is controversial. Here we establish that PSA controls tumor cell growth and differentiation by interfering with NCAM signaling at cell-cell contacts. Interactions between cells with different PSA and NCAM expression profiles were initiated by enzymatic removal of PSA and by ectopic expression of NCAM or PSA-NCAM. Removal of PSA from the cell surface led to reduced proliferation and activated extracellular signal-regulated kinase (ERK), inducing enhanced survival and neuronal differentiation of neuroblastoma cells. Blocking with an NCAM-specific peptide prevented these effects. Combinatorial transinteraction studies with cells and membranes with different PSA and NCAM phenotypes revealed that heterophilic NCAM binding mimics the cellular responses to PSA removal. In conclusion, our data demonstrate that PSA masks heterophilic NCAM signals, having a direct impact on tumor cell growth. This provides a mechanism for how PSA may promote the genesis and progression of highly aggressive PSA-NCAM-positive tumors.

Polysialylated neural cell adhesion molecule (NCAM) was immunoaffinity-purified from the brains of newborn calves. A degree of polymerization of up to 40 was chromatographically determined for released polysialic acid (PSA) chains. For characterization of N-glycan structures and attachment sites, PSA-NCAM was digested with trypsin, and the generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specific for PSA or the HNK1 epitope, i.e., HSO(3)-3GlcA(beta 1-3)Gal(beta 1-4)GlcNAc(beta 1-, yielding PSA-glycopeptides, HNK-glycopeptides and non-PSA/HNK1-(glyco) peptides. Using a combination of enzymatic deglycosylation, peptide fractionation, mass spectrometry and Edman degradation, HNK1-N-glycans could be assigned to glycosylation sites 2, 4, 5 and 6. Non-PSA/HNK1-glycans were assigned to glycosylation site 2, whereas PSA-N-glycans of bovine NCAM had been already previously shown to be restricted to glycosylation sites 5 and 6 (Glycobiology 12 (2002) 47). Respective oligosaccharides were enzymatically released, labeled with 2-aminopyridine and characterized by linkage analysis and mass spectrometry. Carbohydrate chains bearing PSA or the HNK1 epitope comprised mainly fucosylated, partially sulfated diantennary, triantennary or tetraantennary glycans without bisecting GlcNAc or fucosylated diantennary and triantennary species carrying, in part, bisecting GlcNAc residues, respectively. Some N-glycans simultaneously contained both the HNK1-epitope and PSA. Non-PSA/HNK1-glycans exhibited a heterogeneous pattern of partially truncated, mostly diantennary structures with one to three fucose residues, bisecting GlcNAc and/or sulfate residues. In addition, they were demonstrated to carry, to some extent, the Lewis X epitope. When compared with previous data on murine NCAM glycosylation, our results indicate a conservation of structural features and attachment sites for the different types of NCAM N-glycans.

The neural cell adhesion molecule (NCAM) plays important roles during development, plasticity, and regeneration in the adult nervous system. Its function is strongly influenced by attachment of the unusual alpha 2-8-linked polysialic acid (PSA). Here we analyzed the N-glycosylation pattern of polysialylated NCAM from brains of newborn calves. Purified PSA-NCAM glycoprotein was digested with trypsin, and PSA-glycopeptides were separated by immunoaffinity chromatography. For determining the N-glycosylation sites, PNGase F-treated glycopeptides were analyzed by Edman degradation and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). They were found to be exclusively linked to the fifth (Asn 439) and sixth (Asn 468) N-glycosylation sites in the fifth immunoglobulin-like domain of NCAM. The chain length of PSA consisted of at least 30 sialic acid residues, as shown by anion exchange chromatography. For analysis of the core structures, endoneuraminidase N-treated PSA-NCAM was separated by SDS-PAGE and digested with PNGase F. The core structures of polysialylated glycans were characterized by MALDI-MS combined with exoglycosidase digestions and chromatographic fractionation. They include hybrid, di-, tri-, and small amounts of tetraantennary carbohydrates, which were all fucosylated at the innermost N-acetylglucosamine. For the triantennary glycans, the "2,6" arm was preferred in polysialylated structures. High levels of sulfated groups were found on polysialylated structures and to a lower extent also on nonpolysialylated glycans. In addition, high-mannose-type glycans could be detected on PSA-NCAM glycoforms ranging from (GlcNAc)(2)(Man)(5) up to (GlcNAc)(2)(Man)(9). In conclusion, we observed a structural variability and high regional selectivity for the PSA-glycans attached to the NCAM molecule that are most likely influencing its biological functions.