Consolidation of passive avoidance learning is associated with transient increases of polysialylated neurons in layer II of the rat medial temporal cortex

Within the rat medial temporal lobe, transient modulations of neural cell adhesion molecule (NCAM) polysialylation have been observed to follow spatial learning. These have been attributed to neuroplastic events associated with the processing of information destined for long term memory consolidation. To determine if similar events are associated with avoidance learning, we investigated change in polysialylated cell number in the entorhinal, perirhinal, and piriform cortex, following acquisition of a passive avoidance task in the rat. Direct quantification of polysialylated neurons in layer II of these cortical regions revealed a significant increase in polysialylated cell frequency at 12 h following passive avoidance training. Unlike spatial learning, the increased expression of polysialylated neurons persisted for up to 24-48 h following training. In the more dorsal aspect of the perirhinal/entorhinal cortex, this increase was found to be specific to learning, as it was not observed in animals rendered amnesic with scopolamine. By contrast, change in polysialylated cell frequency in the ventral aspect of the medial temporal lobe was only partially reduced by amnesic doses of scopolamine. The persisting activation of NCAM polysialylation in the more dorsal aspects of the perirhinal and entorhinal cortex is suggested to reflect the need for more extensive synaptic alterations, as compared to those required for the consolidation of spatial learning. Moreover, the neuroplastic modulations observed in the more ventral regions of the entorhinal and perirhinal cortex appear to be a unique aspect of avoidance conditioning that reflects the activation of alternative learning strategies associated with motivational and/or contextual parameters of the task.     

Spatial Learning Activates Neural Cell Adhesion Molecule Polysialylation in a Corticohippocampal Pathway Within the Medial Temporal Lobe

Abstract: Transient and time-dependent modulations of neural cell adhesion molecule (NCAM) polysialylation in the dentate gyrus of the rodent hippocampus are a feature of spatial and nonspatial forms of learning. In the hippocampal formation, polysialic acid immunoreactivity was localized to granule-like cells and their mossy fibre axons. We now demonstrate the latter to extend to the CA3 region where apparent recurrent and Schaffer collaterals were labelled. The axons of the CA1 pyramidal cell layer were immunopositive, as was the subiculum that they innervate. Layers I and III of the entorhinal cortex stained intensely for polysialic acid; however, these were not visible in the more lateral aspect of this region and were replaced by a single band of immunopositive neurons that extended to include the perirhinal and piriform cortices. After Morris water maze training, the number of polysialylated neurons within the entorhinal cortex exhibited a two- to threefold increase at the 10–12-h posttraining time with respect to that observed immediately after training. This increase was task specific, as no change was observed in freely swimming animals or those required to locate a visible platform. These results suggest the presence of a corticohippocampal pathway involved in the eventual consolidation of memory.     

Memory Consolidation Induces a Transient and Time-Dependent Increase in the Frequency of Neural Cell Adhesion Molecule Polysialylated Cells in the Adult Rat Hippocampus

Abstract: Animals trained in a passive avoidance task exhibit a transient time-dependent increase in hippocampal neural cell adhesion molecule (NCAM) polysialylation at 12–24 h following the initial learning trial. Using immunocytochemical techniques with a monoclonal antibody that specifically recognises NCAM-polysialic acid homopolymers, a distinct population of granule-like cells, at the border of the granule cell layer and the hilus in the dentate gyrus of the adult rat hippocampus, has been demonstrated to exhibit time-dependent change in frequency at 10–12 h following the initial learning of a one-trial, step-through, passive avoidance response. These changes were paradigm specific as they failed to occur in those animals rendered amnesic with scopolamine. These polysialylated dentate neurons are not de novo granule cell precursors as administration of 5-bromo-2'-deoxyuridine every 2 h from the point of learning to the 12-h posttraining time showed no significant difference between trained and passive animals in the small number of heterogeneously distributed, labelled cells. These findings directly identify a morphological substrate of memory, implied by previous correlative and interventive studies on NCAM function.     

Pentyl-4-yn-valproic acid enhances both spatial and avoidance learning, and attenuates age-related NCAM-mediated neuroplastic decline within the rat medial temporal lobe

2-N-Pentyl-4-pentynoic acid [pentyl-4-yn-valproic acid (VPA)] is an analogue of valproic acid that induces neuritogenesis and increases neural cell adhesion molecule (NCAM) prevalence in cultured neural cells. As memory consolidation involves synapse growth, aided by cell adhesion molecule function, we determined whether or not pentyl-4-yn-VPA had cognition-enhancing properties. Pentyl-4-yn-VPA (16-85 mg/kg) significantly improved water maze learning and task retention when given prior to each training session. Acute administration of pentyl-4-yn-VPA also influenced memory consolidation processes as, when given at 3 h post-passive avoidance training, the amnesia induced by scopolamine given 6 h post-training was prevented in a dose-dependent manner. Chronic administration of pentyl-4-yn-VPA (16.8 or 50.4 mg/kg) also significantly reduced escape latencies in the water maze task, 24 h following the last drug administration. This improved spatial learning was accompanied by enhanced neuroplasticity as the expression of NCAM polysialylated neurons in the infragranular zone of the dentate gyrus and in layer II of the perirhinal and piriform cortex was increased significantly following chronic drug treatment. The cognition-enhancing qualities of pentyl-4-yn-VPA, combined with its ability to attenuate the age-related loss of the NCAM polysialylation state, suggest that it may effectively slow the onset of cognitive decline.     

The Polysialyltransferases Interact with Sequences in Two Domains of the Neural Cell Adhesion Molecule to Allow Its Polysialylation

The neural cell adhesion molecule (NCAM) is the major substrate for the polysialyltransferases (polySTs), ST8SiaII/STX and ST8SiaIV/PST. The polysialylation of NCAM N-glycans decreases cell adhesion and alters signaling. Previous work demonstrated that the first fibronectin type III repeat (FN1) of NCAM is required for polyST recognition and the polysialylation of the N-glycans on the adjacent Ig5 domain. In this work, we highlight the importance of an FN1 acidic patch in polyST recognition and also reveal that the polySTs are required to interact with sequences in the Ig5 domain for polysialylation to occur. We find that features of the Ig5 domain of the olfactory cell adhesion molecule (OCAM) are responsible for its lack of polysialylation. Specifically, two basic OCAM Ig5 residues (Lys and Arg) found near asparagines equivalent to those carrying the polysialylated N-glycans in NCAM substantially decrease or eliminate polysialylation when used to replace the smaller and more neutral residues (Ser and Asn) in analogous positions in NCAM Ig5. This decrease in polysialylation does not reflect altered glycosylation but instead is correlated with a decrease in polyST-NCAM binding. In addition, inserting non-conserved OCAM sequences into NCAM Ig5, including an “extra” N-glycosylation site, decreases or completely blocks NCAM polysialylation. Taken together, these results indicate that the polySTs not only recognize an acidic patch in the FN1 domain of NCAM but also must contact sequences in the Ig5 domain for polysialylation of Ig5 N-glycans to occur.     

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.     

Structure and Mutagenesis of Neural Cell Adhesion Molecule Domains: EVIDENCE FOR FLEXIBILITY IN THE PLACEMENT OF POLYSIALIC ACID ATTACHMENT SITES*

The addition of α2,8-polysialic acid to the N-glycans of the neural cell adhesion molecule, NCAM, is critical for brain development and plays roles in synaptic plasticity, learning and memory, neuronal regeneration, and the growth and invasiveness of cancer cells. Our previous work indicates that the polysialylation of two N-glycans located on the fifth immunoglobulin domain (Ig5) of NCAM requires the presence of specific sequences in the adjacent fibronectin type III repeat (FN1). To understand the relationship of these two domains, we have solved the crystal structure of the NCAM Ig5-FN1 tandem. Unexpectedly, the structure reveals that the sites of Ig5 polysialylation are on the opposite face from the FN1 residues previously found to be critical for N-glycan polysialylation, suggesting that the Ig5-FN1 domain relationship may be flexible and/or that there is flexibility in the placement of Ig5 glycosylation sites for polysialylation. To test the latter possibility, new Ig5 glycosylation sites were engineered and their polysialylation tested. We observed some flexibility in glycosylation site location for polysialylation and demonstrate that the lack of polysialylation of a glycan attached to Asn-423 may be in part related to a lack of terminal processing. The data also suggest that, although the polysialyltransferases do not require the Ig5 domain for NCAM recognition, their ability to engage with this domain is necessary for polysialylation to occur on Ig5 N-glycans.     

The polysialyltransferase ST8Sia II/STX: posttranslational processing and role of autopolysialylation in the polysialylation of neural cell adhesion molecule.

The presence of alpha2,8-linked polysialic acid on the neural cell adhesion molecule (NCAM) is known to modulate cell interactions during development and oncogenesis. Two enzymes, the alpha2,8-polysialyltransferases ST8Sia IV()/PST and ST8Sia II()/STX are responsible for the polysialylation of NCAM. We previously reported that both ST8Sia IV/PST and ST8Sia II/STX enzymes are themselves modified by alpha2,8-linked polysialic acid chains, a process called autopolysialylation. In the case of ST8Sia IV/PST, autopolysialylation is not required for enzymatic activity. However, whether the autopolysialylation of ST8Sia II/STX is required for its ability to polysialylate NCAM is unknown. To understand how autopolysialylation impacts ST8Sia II/STX enzymatic activity, we employed a mutagenesis approach. We found that ST8Sia II/STX is modified by six Asn-linked oligosaccharides and that polysialic acid is distributed among the oligosaccharides modifying Asn 89, 219, and 234. Coexpression of a nonautopolysialylated ST8Sia II/STX mutant with NCAM demonstrated that autopolysialylation is not required for ST8Sia II/STX polysialyltransferase activity. In addition, catalytically active, nonautopolysialylated ST8Sia II/STX does not polysialylate any endogenous COS-1 cell proteins, highlighting the protein specificity of polysialylation. Furthermore, immunoblot analysis of NCAM polysialylation by autopolysialylated and nonautopolysialylated ST8Sia II/STX suggests that the NCAM is polysialylated to a higher degree by autopolysialylated ST8Sia II/STX. Therefore, we conclude that autopolysialylation of ST8Sia II/STX, like that of ST8Sia IV/PST, is not required for, but does enhance, NCAM polysialylation.     

Altered expression of NCAM in hippocampus and cortex may underlie memory and learning deficits in rats with streptozotocin-induced diabetes mellitus

Neurological and structural changes are paralleled by cognitive deficits in diabetes mellitus. The present study was designed to evaluate the expression of neural cell adhesion molecules (NCAM) in the hippocampus, cortex and cerebellum and to examine cognitive functions in diabetic rats. Diabetes was induced in male albino rats via intraperitoneal streptozotocin injection. Learning and memory behaviors were investigated using a passive avoidance test and a spatial version of the Morris water maze test. NCAM expression was detected in the hippocampus, cortex and cerebellum by an immunoblotting method. The diabetic rats developed significant impairment in learning and memory behaviours as indicated by deficits in passive avoidance and water maze tests as compared to control rats. Expression of NCAM 180 and 120 kDa were found to be higher in hippocampus and cortex of diabetic rat brains compared to those of control, whereas expression of NCAM 140 kDa decreased in these brain regions. Our findings suggest that streptozotocin-induced diabetes impairs cognitive functions and causes an imbalance in expression of NCAM in those brain regions involved in learning and memory. Altered expression of NCAM in hippocampus may be an important cause of learning and memory deficits that occur in diabetes mellitus.     

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.     

Direct evidence that neural cell adhesion molecule (NCAM) polysialylation increases intermembrane repulsion and abrogates adhesion

Molecular force measurements quantified the impact of polysialylation on the adhesive properties both of membrane-bound neural cell adhesion molecule (NCAM) and of other proteins on the same membrane. These results show quantitatively that NCAM polysialylation increases the range and magnitude of intermembrane repulsion. The repulsion is sufficient to overwhelm both homophilic NCAM and cadherin attraction at physiological ionic strength, and it abrogates the protein-mediated intermembrane adhesion. The steric repulsion is ionic strength dependent and decreases substantially at high monovalent salt concentrations with a concomitant increase in the intermembrane attraction. The magnitude of the repulsion also depends on the amount of polysialic acid (PSA) on the membranes, and the PSA-dependent attenuation of cadherin adhesion increases with increasing PSA-NCAM:cadherin ratios. These findings agree qualitatively with independent reports based on cell adhesion studies and reveal the likely molecular mechanism by which NCAM polysialylation regulates cell adhesion and intermembrane space.     

Polysialytation as a regulator of neural plasticity in rodent learning and aging

Although generally accepted to play an important role in development, the precise functional significance of NCAM remains to be elucidated. Correlative and interventive studies suggest a role for polysialylated NCAM in neurite elaboration. In the adult NCAM polysialylation continues to be expressed in regions of the central nervous system which retain neuroplastic potential. During memory formation modulation of polysialylation on the synapse-enriched isoform of NCAM occurs in the hippocampus. The polysialylated neurons of this structure have been located at the border of the granule cell layer and hilar region of the dentate and their number increases dramatically during memory consolidation. The converse is also true for a profound decline in the basal number of polysialylated neurons occurs with ageing when neural plasticity becomes attenuated. In conclusion, it is suggested that NCAM polysialylation regulates ultrastructural plasticity associated with synaptic elaboration.Abbreviations PSA polysialic acid - NCAM neural cell adhesion molecule - SGL sub-granular cell layer - MF mossy fibers Special issue dedicated to Dr. Robert Balazs.     

A novel alpha-helix in the first fibronectin type III repeat of the neural cell adhesion molecule is critical for N-glycan polysialylation

Polysialic acid is a developmentally regulated, anti-adhesive glycan that is added to the neural cell adhesion molecule, NCAM. Polysialylated NCAM is critical for brain development and plays roles in synaptic plasticity, axon guidance, and cell migration. The first fibronectin type III repeat of NCAM, FN1, is necessary for the polysialylation of N-glycans on the adjacent immunoglobulin domain. This repeat cannot be replaced by other fibronectin type III repeats. We solved the crystal structure of human NCAM FN1 and found that, in addition to a unique acidic surface patch, it possesses a novel alpha-helix that links strands 4 and 5 of its beta-sandwich structure. Replacement of the alpha-helix did not eliminate polysialyltransferase recognition, but shifted the addition of polysialic acid from the N-glycans modifying the adjacent immunoglobulin domain to O-glycans modifying FN1. Other experiments demonstrated that replacement of residues in the acidic surface patch alter the polysialylation of both N- and O-glycans in the same way, while the alpha-helix is only required for the polysialylation of N-glycans. Our data are consistent with a model in which the FN1 alpha-helix is involved in an Ig5-FN1 interaction that is critical for the correct positioning of Ig5 N-glycans for polysialylation.     

Molecular dissection of the ST8Sia IV polysialyltransferase. Distinct domains are required for neural cell adhesion molecule recognition and polysialylation

Polysialic acid, a homopolymer of alpha2,8-linked sialic acid expressed on the neural cell adhesion molecule (NCAM), is thought to play critical roles in neural development. Two highly homologous polysialyltransferases, ST8Sia II and ST8Sia IV, which belong to the sialyltransferase gene family, synthesize polysialic acid on NCAM. By contrast, ST8Sia III, which is moderately homologous to ST8Sia II and ST8Sia IV, adds oligosialic acid to itself but very inefficiently to NCAM. Here, we report domains of polysialyltransferases required for NCAM recognition and polysialylation by generating chimeric enzymes between ST8Sia IV and ST8Sia III or ST8Sia II. We first determined the catalytic domain of ST8Sia IV by deletion mutants. To identify domains responsible for NCAM polysialylation, different segments of the ST8Sia IV catalytic domain, identified by the deletion experiments, were replaced with corresponding segments of ST8Sia II and ST8Sia III. We found that larger polysialic acid was formed on the enzymes themselves (autopolysialylation) when chimeric enzymes contained the carboxyl-terminal region of ST8Sia IV. However, chimeric enzymes that contain only the carboxyl-terminal segment of ST8Sia IV and the amino-terminal segment of ST8Sia III showed very weak activity toward NCAM, even though they had strong activity in polysialylating themselves. In fact, chimeric enzymes containing the amino-terminal portion of ST8Sia IV fused to downstream sequences of ST8Sia III inhibited NCAM polysialylation in vitro, although they did not polysialylate NCAM. These results suggest that in polysialyltransferases the NCAM recognition domain is distinct from the polysialylation domain and that some chimeric enzymes may act as a dominant negative enzyme for NCAM polysialylation.     

Regulation of cell adhesion by polysialic acid. Effects on cadherin, immunoglobulin cell adhesion molecule, and integrin function and independence from neural cell adhesion molecule binding or signaling activity.

The polysialylation of neural cell adhesion molecule (NCAM) evolved in vertebrates to carry out biological functions related to changes in cell position and morphology. Many of these effects involve the attenuation of cell interactions that are not mediated through NCAM's own adhesion properties. A proposed mechanism for this global effect on cell interaction is the steric inhibition of membrane-membrane apposition based solely on polysialic acid (PSA) biophysical properties. However, it remains possible that the intrinsic binding or signaling properties of the NCAM polypeptide are also involved. To help resolve this issue, this study uses a quantitative cell detachment assay together with cells engineered to display different adhesion receptors together with a variety of polysialylated NCAM polypeptide isoforms and functional domain deletion mutations. The results obtained indicate that regulation by PSA occurs with adhesion receptors as diverse as an IgCAM, a cadherin and an integrin, and does not require NCAM functional domains other than those minimally required for polysialylation. These findings are most consistent with the cell apposition mechanism for PSA action, as this model predicts that the inhibitory effects of PSA-NCAM on cell adhesion should be independent of the nature of the adhesion system and of any intrinsic binding or signaling properties of the NCAM polypeptide itself.     

Sequences prior to conserved catalytic motifs of polysialyltransferase ST8Sia IV are required for substrate recognition

Polysialic acid on the neural cell adhesion molecule (NCAM) modulates cell-cell adhesion and signaling, is required for proper brain development, and plays roles in neuronal regeneration and the growth and invasiveness of tumor cells. Evidence indicates that NCAM polysialylation is highly protein-specific, requiring an initial polysialyltransferase-NCAM protein-protein interaction. Previous work suggested that a polybasic region located prior to the conserved polysialyltransferase catalytic motifs may be involved in NCAM recognition, but not overall enzyme activity (Foley, D. A., Swartzentruber, K. G., and Colley, K. J. (2009) J. Biol. Chem. 284, 15505-15516). Here, we employ a competition assay to evaluate the role of this region in substrate recognition. We find that truncated, catalytically inactive ST8SiaIV/PST proteins that include the polybasic region, but not those that lack this region, compete with endogenous ST8SiaIV/PST and reduce NCAM polysialylation in SW2 small cell lung carcinoma cells. Replacing two polybasic region residues, Arg(82) and Arg(93), eliminates the ability of a full-length, catalytically inactive enzyme (PST H331K) to compete with SW2 cell ST8SiaIV/PST and block NCAM polysialylation. Replacing these residues singly or together in ST8SiaIV/PST substantially reduces or eliminates NCAM polysialylation, respectively. In contrast, replacing Arg(82), but not Arg(93), substantially reduces the ability of ST8SiaIV/PST to polysialylate neuropilin-2 and SynCAM 1, suggesting that Arg(82) plays a general role in substrate recognition, whereas Arg(93) specifically functions in NCAM recognition. Taken together, our results indicate that the ST8SiaIV/PST polybasic region plays a critical role in substrate recognition and suggest that different combinations of basic residues may mediate the recognition of distinct substrates.     

Enzyme-dependent variations in the polysialylation of the neural cell adhesion molecule (NCAM) in vivo

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 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.     

Sequences from the First Fibronectin Type III Repeat of the Neural Cell Adhesion Molecule Allow O-Glycan Polysialylation of an Adhesion Molecule Chimera

Polysialic acid is a developmentally regulated, anti-adhesive polymer that is added to N-glycans on the fifth immunoglobulin domain (Ig5) of the neural cell adhesion molecule (NCAM). We found that the first fibronectin type III repeat (FN1) of NCAM is required for the polysialylation of N-glycans on the adjacent Ig5 domain, and we proposed that the polysialyltransferases recognize specific sequences in FN1 to position themselves for Ig5 N-glycan polysialylation. Other studies identified a novel FN1 acidic surface patch and α-helix that play roles in NCAM polysialylation. Here, we characterize the contribution of two additional FN1 sequences, Pro⁵¹⁰-Tyr⁵¹¹-Ser⁵¹² (PYS) and Gln⁵¹⁶-Val⁵¹⁷-Gln⁵¹⁸ (QVQ). Replacing PYS or the acidic patch dramatically decreases the O-glycan polysialylation of a truncated NCAM protein, and replacing the α-helix or QVQ shifts polysialic acid to FN1 O-glycans in full-length NCAM. We also found that the FN1 domain of the olfactory cell adhesion molecule, a homologous but unpolysialylated protein, could partially replace NCAM FN1. Inserting Pro⁵¹⁰-Tyr⁵¹¹ eliminated N-glycan polysialylation and enhanced O-glycosylation of an NCAM- olfactory cell adhesion molecule chimera, and inserting other FN1 sequences unique to NCAM, predominantly the acidic patch, created a new polysialyltransferase recognition site. Taken together, our results highlight the role of the FN1 α-helix and QVQ sequences in N-glycan polysialylation and demonstrate that the acidic patch primarily functions in O-glycan polysialylation.     

Specific amino acids in the first fibronectin type III repeat of the neural cell adhesion molecule play a role in its recognition and polysialylation by the polysialyltransferase ST8Sia IV/PST

Polysialic acid is an anti-adhesive protein modification that promotes cell migration and the plasticity of cell interactions. Because so few proteins carry polysialic acid, we hypothesized that polysialylation is a protein-specific event and that a specific polysialyltransferase-substrate interaction is the basis of this specificity. The major substrate for the polysialyltransferases is the neural cell adhesion molecule, NCAM. Previous work demonstrates that the first fibronectin type III repeat of NCAM (FN1) was necessary for the polysialylation of the N-glycans on the adjacent immunoglobulin domain (Ig5) (Close, B. E., Mendiratta, S. S., Geiger, K. M., Broom, L. J., Ho, L. L., and Colley, K. J. (2003) J. Biol. Chem. 278, 30796-30805). This suggested that FN1 may be a recognition site for the polysialyltransferases. In this study, we showed that the second fibronectin type III repeat (FN2) of NCAM cannot replace FN1. Arg substitution of three unique acidic amino acids on the surface of FN1 eliminated polysialylation not only of a minimal Ig5-FN1 substrate but also of full-length NCAM. Ala substitution of these residues eliminated Ig5-FN1 polysialylation but not that of full-length NCAM, suggesting that the two proteins are interacting differently with the enzymes and that multiple residues are involved in the enzyme-NCAM interaction. By using another truncated protein, Ig5-FN1-FN2, we confirmed the importance of enzyme-substrate positioning for optimal recognition and polysialylation. In sum, we have found that acidic residues on the surface of FN1 are part of a larger protein interaction region that is critical for NCAM recognition and polysialylation by the polysialyltransferases.