Characterization of N-glycans from mouse brain neural cell adhesion molecule.

The N-glycosylation pattern of the neural cell adhesion molecule (NCAM), isolated from brains of newborn mice, has been analyzed. Following digestion with trypsin, generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specifically recognizing polysialic acid (PSA) units or the HNK1-carbohydrate epitope. Subsequent analyses of the resulting (glyco)peptides by Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed polysialylated glycans to be exclusively linked to glycosylation sites 5 (Asn(431)) and 6 (Asn(460)), whereas glycans carrying the HNK1-epitope could be assigned to sites 2 (Asn(297)), 5, 6, and, to a lesser extent, site 3 (Asn(329)). PSA-, HNK1-, and non-PSA/HNK1-glycan fractions were characterized by carbohydrate constituent and methylation analyses as well as MALDI-TOF-MS in conjunction with chromatographic fractionation techniques. The results revealed that the core structures of PSA-glycans represented predominantly fucosylated, partially sulfated 2,6-branched isomers of triantennary as well as tetraantennary complex-type glycans, whereas carbohydrate chains bearing the HNK1-epitope were dominated by diantennary species carrying in part bisecting GlcNAc residues. Non-PSA/HNK1-glycans exhibited a highly heterogeneous pattern of partially truncated, mostly diantennary structures being characterized by the presence of additional fucose, bisecting GlcNAc and/or sulfate residues. In conclusion, our results revealed that the glycosylation pattern of murine NCAM displays high structural and regional selectivity, which might play an important role in controlling the biological activities of this molecule.     

Degree of polymerization (DP) of polysialic acid (polySia) on neural cell adhesion molecules (N-CAMS): development and application of a new strategy to accurately determine the DP of polySia chains on N-CAMS

Alpha2,8-linked polysialic acid (polySia) is a structurally unique antiadhesive glycotope that covalently modifies N-linked glycans on neural cell adhesion molecules (N-CAMs). These sugar chains play a key role in modulating cell-cell interactions, principally during embryonic development, neural plasticity, and tumor metastasis. The degree of polymerization (DP) of polySia chains on N-CAM is postulated to be of critical importance in regulating N-CAM function. There are limitations, however, in the conventional methods to accurately determine the DP of polySia on N-CAM, the most serious being partial acid hydrolysis of internal alpha2,8-ketosidic linkages that occur during fluorescent derivatization, a step necessary to enhance chromatographic detection. To circumvent this problem, we have developed a facile method that combines the use of Endo-beta-galactosidase to first release linear polySia chains from N-CAM, with high resolution high pressure liquid chromatography profiling. This strategy avoids acid hydrolysis prior to chromatographic profiling and thus provides an accurate determination of the DP and distribution of polySia on N-CAM. The potential of this new method was evaluated using a nonpolysialylated construct of N-CAM that was polysialylated in vitro using a soluble construct of ST8Sia II or ST8Sia IV. Whereas most of the oligosialic acid/polySia chains consisted of DPs approximately 50-60 or less, a subpopulation of chains with DPs approximately 150 to approximately 180 and extending to DP approximately 400 were detected. The DP of this subpopulation is considerably greater than reported previously for N-CAM. Endo-beta-galactosidase can also release polySia chains from polysialylated membranes expressed in the neuroblastoma cell line, Neuro2A, and native N-CAM from embryonic chick brains.     

The relationship of polysialic acid and the neural cell adhesion molecule N-CAM in Wilms tumor and their subcellular distributions

In previous studies we have reported that polysialic acid is an oncodevelopmental antigen in human kidney but its relationship to the neural cell adhesion molecule (N-CAM) remained undefined. In the present study, we showed by the combination of immunoprecipitation and immunoblotting that renal polysialic acid is a structural component of N-CAM polypeptide and that two highly sialylated N-CAM isoforms of approximately 120 kDa and 140 kDa existed in Wilms tumor. The presence of a cell surface coat composed of polysialic acid and N-CAM was revealed by immunoelectron microscopy, and morphological evidence for its involvement in modulating cell-cell adhesion has been provided. Furthermore, highly sialylated N-CAM was detectable extracellularly. N-CAM immunolabeling was present in compartments from the nuclear envelope to the plasma membrane. However, polysialic acid was only detectable at the cell surface suggesting that in Wilms tumor cells sialyl polymer synthesis may occur partially or exclusively at this site.     

Localization of defined carbohydrate epitopes in bovine polysialylated NCAM

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.     

Localization and characterization of polysialic acid-containing N-linked glycans from bovine NCAM

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.     

The minimal structural domains required for neural cell adhesion molecule polysialylation by PST/ST8Sia IV and STX/ST8Sia II

A limited number of mammalian proteins are modified by polysialic acid, with the neural cell adhesion molecule (NCAM) being the most abundant of these. We hypothesize that polysialylation is a protein-specific glycosylation event and that an initial protein-protein interaction between polysialyltransferases and glycoprotein substrates mediates this specificity. To evaluate the regions of NCAM required for recognition and polysialylation by PST/ST8Sia IV and STX/ST8Sia II, a series of domain deletion proteins were generated, co-expressed with each enzyme, and their polysialylation analyzed. A protein consisting of the fifth immunoglobulin-like domain (Ig5), which contains the reported sites of polysialylation, and the first fibronectin type III repeat (FN1) was polysialylated by both enzymes, whereas a protein consisting of Ig5 alone was not polysialylated by either enzyme. This demonstrates that the Ig5 domain of NCAM and FN1 are sufficient for polysialylation, and suggests that the FN1 may constitute an enzyme recognition and docking site. Two other NCAM mutants, NCAM-6 (Ig1-5) and NCAM-7 (FN1-FN2), were weakly polysialylated by PST/ST8Sia IV, suggesting that a weaker enzyme recognition site may exist within the Ig domains, and that glycans in the FN region are polysialylated. Further analysis indicated that O-linked oligosaccharides in NCAM-7, and O-linked and N-linked glycans in full-length NCAM, are polysialylated when these proteins are co-expressed with the polysialyltransferases in COS-1 cells. Our data support a model in which the polysialyltransferases bind to the FN1 of NCAM to polymerize polysialic acid chains on appropriately presented glycans in adjacent regions.     

Polysialyltransferase-1 autopolysialylation is not requisite for polysialylation of neural cell adhesion molecule

Polysialyltransferase-1 (PST; ST8Sia IV) is one of the alpha2, 8-polysialyltransferases responsible for the polysialylation of the neural cell adhesion molecule (NCAM). The presence of polysialic acid on NCAM has been shown to modulate cell-cell and cell-matrix interactions. We previously reported that the PST enzyme itself is modified by alpha2,8-linked polysialic acid chains in vivo. To understand the role of autopolysialylation in PST enzymatic activity, we employed a mutagenesis approach. We found that PST is modified by five Asn-linked oligosaccharides and that the vast majority of the polysialic acid is found on the oligosaccharide modifying Asn-74. In addition, the presence of the oligosaccharide on Asn-119 appeared to be required for folding of PST into an active enzyme. Co-expression of the PST Asn mutants with NCAM demonstrated that autopolysialylation is not required for PST polysialyltransferase activity. Notably, catalytically active, non-autopolysialylated PST does not polysialylate any endogenous COS-1 cell proteins, highlighting the protein specificity of polysialylation. Immunoblot analyses of NCAM polysialylation by polysialylated and non-autopolysialylated PST suggests that the NCAM is polysialylated to a higher degree by autopolysialylated PST. We conclude that autopolysialylation of PST is not required for, but does enhance, NCAM polysialylation.     

Polysialylated neuropilin-2 is expressed on the surface of human dendritic cells and modulates dendritic cell-T lymphocyte 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.     

Polysialic acid as a regulator of intramuscular nerve branching during embryonic development

The role of polysialic acid (PSA) during initial innervation of chick muscle was examined. Previously, the adhesion molecules L1 and N-CAM were shown to be important in balancing axon-axon and axon-muscle adhesion during this process. Here we demonstrate developmental changes in the pattern of innervation that are not correlated with levels of L1 or N-CAM expression, but rather with the amount of PSA at the axon surface. Removal of PSA by a specific endoneuraminidase (Endo-N) increased axon fasciculation and reduced nerve branching. In contrast, the nerve trunk defasciculation and increased branching produced by neuromuscular activity blockade were associated with an increase in axonal PSA levels. Furthermore, Endo-N prevented these inactivity-induced effects on branching. Together these results illustrate the potential of PSA as a regulator of cell-cell interactions and provide a direct example of a molecular link between the morphogenic effects of adhesion-mediated and synaptic activity-dependent processes.     

An IgG monoclonal antibody to group B meningococci cross-reacts with developmentally regulated polysialic acid units of glycoproteins in neural and extraneural tissues

The structurally similar polysialic acid capsules of group B meningococci and Escherichia coli K1 are poor immunogens, and attempts are currently being made to improve their immunogenicity by chemical modifications. An IgG monoclonal antibody to these polysialic acid capsules was used for the study of the presence of structurally similar components in tissue glycoproteins to investigate the reasons for the poor immunogenicity and to evaluate potential dangers in active or passive immunization. By immunoblotting polysialic acid was detected outside the brain in newborn rat kidney, heart, and muscle. It appeared in immunoblots as one component and with similar mobility to the neural cell adhesion molecule N-CAM. Specificity studies of the antibody and endosialidase treatment showed that the polysialic acid glycans detected were composed of chains as long as eight sialic acid residues or more. The polysialic acid was not detected in the corresponding tissues of the adult animal. These results indicate that polysialic acid units are developmentally regulated components of both neural and extraneural tissues, and are bound to components with properties similar to a known cell-adhesion molecule. This together with the presence of low amounts of polysialic acid even in the adult brain, suggests potential hazards in vaccination trials and suggested immunotherapy of meningitis caused by group B meningococci or E. coli K1, which should be carefully assessed.     

The neural cell adhesion molecule NCAM regulates neuritogenesis by multiple mechanisms of interaction

The neural cell adhesion molecule NCAM and its glycosylation with polysialic acid (polySia) are crucially involved in proliferation, migration and differentiation of neural progenitors. Modification with polySia, homophilic and heterophilic interactions set the function of NCAM, but little is known on their interplay. We have shown recently that removal of polySia induces neuronal differentiation via heterophilic NCAM interactions at cell contacts between SH-SY5Y neuroblastoma cells. Here we analyze the additional impact of NCAM-positive fibroblasts as a ligand-presenting cellular environment, a model often used to demonstrate the neuritogenic effect of homophilic NCAM interactions. Native SH-SY5Y cells did not respond to interactions with fibroblast NCAM. However, after induction of neuronal differentiation by retinoic acid the previously ineffective NCAM signals activated extracellular signal-regulated kinase (ERK) and promoted neuritogenesis. Removal of polySia increased neuritogenesis in retinoic acid-treated cells additive to the NCAM substrate effect. The change in responsiveness to substrate NCAM was associated with a rearrangement of polysialylated NCAM away from its enrichment at homotypic cell-cell contacts and with the appearance of non-polysialylated NCAM, i.e. changes facilitating NCAM interactions with the substrate. Thus, heterophilic and homophilic NCAM interactions are integrated into the cell's response yet they have the capacity to independently trigger neuritogenesis. The actual occurrence of each of these interactions, however, depends on the cellular context, targeted cell surface presentation of NCAM and the dynamic regulation of its modification by polysialic acid. In summary, this study reveals how the complex interplay of NCAM interactions and polysialylation provides an elaborate system to regulate neuritogenesis.     

Up-regulation of embryonic NCAM in an EC cell line by retinoic acid

The impact of retinoic acid (RA) on the expression of the neural cell adhesion molecules (NCAMs) and their developmentally regulated polysialic acid (PSA) moiety was studied in embryonal carcinoma (EC) cell lines. These cell lines are known to be capable of RA-induced differentiation into neurons (murine P19 cells) or parietal endoderm (murine F9 cells), respectively. Monoclonal antibodies were employed to monitor expression of NCAM and PSA. F9 and P19 cells were both found to express NCAM but only P19 cells carried the highly polysialylated "embryonic form" of NCAM (E-NCAM). The amount of NCAM in aggregated P19 cells but not in F9 cells was dramatically increased upon treatment with RA. Since NCAMs play an important role in cell interactions during embryogenesis it is tempting to speculate that the regulative impact of RA on NCAMs is related to its morphogenic property.     

The intracellular concentration of sialic acid regulates the polysialylation of the neural cell adhesion molecule

Sialic acids are expressed as terminal sugars in many glycoconjugates and play an important role during development and regeneration, as they are involved as polysialic acid in a variety of cell-cell interactions mediated by the neural cell adhesion molecule NCAM. The key enzyme for the biosynthesis of sialic acid is the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE). Mutations in the binding site of the feedback inhibitor CMP-sialic acid of the GNE leads to sialuria, a disease in which patients produce sialic acid in gram scale. Here, we report on the consequences after expression of a sialuria-mutated GNE. Expression of the sialuria-mutated GNE leads to a dramatic increase of both cellular sialic acid and polysialic acid on NCAM. This could also be achieved by application of the sialic acid precursor N-acetylmannosamine. Our data suggest that biosynthesis of sialic acid regulates and limits the synthesis of polysialic acid.     

NCAM and PSA-NCAM dependent membrane spreading and F-actin reorganization in suspended adhering neural cells

Mediation of synchronous cell-cell interactions by NCAM and PSA-NCAM is examined here in aggregates (monolayers) of C6 polysialylated embryonic neural cells, formed rapidly (within 30 s) in suspension in an ultrasound trap. These cells express all three main isoforms of neural cell adhesion molecule (NCAM). The rate of extension of perimeter contact (i.e., membrane spreading) between closely adjacent cells and the temporal reinforcement of the Filamentous (F)-actin cytoskeleton at those regions were measured. Enzymatic removal of the cell-cell repelling polysialic acid (PSA) increases the rate of NCAM-induced membrane spreading, while removal of NCAM-120 had no detectable effect. Competitive peptide inhibition of the third immunoglobulin domain of NCAM significantly reduced the rate of membrane spreading, while NCAM siRNA transfected cells lost their ability to spread. It is argued that NCAM induced contact is the initial requirement for membrane spreading and facilitates conditions for subsequent cytoskeletal reorganization in these neural cells.     

NCAM and PSA-NCAM dependent membrane spreading and F-actin reorganization in suspended adhering neural cells

Mediation of synchronous cell-cell interactions by NCAM and PSA-NCAM is examined here in aggregates (monolayers) of C6 polysialylated embryonic neural cells, formed rapidly (within 30 s) in suspension in an ultrasound trap. These cells express all three main isoforms of neural cell adhesion molecule (NCAM). The rate of extension of perimeter contact (i.e., membrane spreading) between closely adjacent cells and the temporal reinforcement of the Filamentous (F)-actin cytoskeleton at those regions were measured. Enzymatic removal of the cell-cell repelling polysialic acid (PSA) increases the rate of NCAM-induced membrane spreading, while removal of NCAM-120 had no detectable effect. Competitive peptide inhibition of the third immunoglobulin domain of NCAM significantly reduced the rate of membrane spreading, while NCAM siRNA transfected cells lost their ability to spread. It is argued that NCAM induced contact is the initial requirement for membrane spreading and facilitates conditions for subsequent cytoskeletal reorganization in these neural cells.     

The impact of N-glycosylation on the functions of polysialyltransferases.

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

ST8Sia II and ST8Sia IV polysialyltransferases exhibit marked differences in utilizing various acceptors containing oligosialic acid and short polysialic acid. The basis for cooperative polysialylation by two enzymes

Polysialylation of the neural cell adhesion molecule (NCAM) is thought to play a critical role in neural development. Two polysialyltransferases, ST8Sia II and ST8Sia IV, play dominant roles in polysialic acid synthesis on NCAM. However, the individual roles and mechanisms by which these two enzymes form large amounts of polysialic acid on NCAM were heretofore unknown. Previous studies indicate that ST8Sia IV forms more highly polysialylated N-glycans on NCAM than ST8Sia II in vitro. In the present study, we first demonstrated that a combination of ST8Sia II and ST8Sia IV cooperatively polysialylated NCAM, resulting in NCAM N-glycans containing more, and thus longer, polysialic acid than when the enzymes were used individually. There was also an increase in polysialylated NCAM when we used ST8Sia II and ST8Sia IV sequentially, whereas there appeared to be a subtle increase when the enzymes were used in the reverse order. Furthermore, ST8Sia IV was able to add polysialic acid to oligosialylated oligosaccharides and unpolysialylated antennas in N-glycans attached to NCAM, even when polysialic acid was attached to at least one of the other antennas. By contrast, ST8Sia II added little polysialic acid to the same acceptors. On the other hand, neither ST8Sia II nor ST8Sia IV could add polysialic acid to a polysialylated antenna of NCAM N-glycans. These combined results indicate that the synergistic effect of ST8Sia II and ST8Sia IV is caused by: 1) the ability of ST8Sia IV to add polysialic acid to oligosialic acid formed by ST8Sia II, 2) the potential of ST8Sia IV to act on more antennas of N-glycans than ST8Sia II, and 3) the ability of ST8Sia II and ST8Sia IV in combination to act on the fifth and sixth N-glycosylation sites of NCAM.