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.     

Metalloprotease-induced ectodomain shedding of neural cell adhesion molecule (NCAM)

Transmembrane forms of neural cell adhesion molecule (NCAM140, NCAM180(1)) are key regulators of neuronal development. The extracellular domain of NCAM can occur as a soluble protein in normal brain, and its levels are elevated in neuropsychiatric disorders, such as schizophrenia; however the mechanism of ectodomain release is obscure. Ectodomain shedding of NCAM140, releasing a fragment of 115 kD, was found to be induced in NCAM-transfected L-fibroblasts by the tyrosine phosphatase inhibitor pervanadate, but not phorbol esters. Pervanadate-induced shedding was mediated by a disintegrin metalloprotease (ADAM), regulated by ERK1/2 MAP kinase. In primary cortical neurons, NCAM was shed at high levels, and the metalloprotease inhibitor GM6001 significantly increased NCAM-dependent neurite branching and outgrowth. Moreover, NCAM-dependent neurite outgrowth and branching were inhibited in neurons isolated from a transgenic mouse model of NCAM shedding. These results suggest that regulated metalloprotease-induced ectodomain shedding of NCAM down-regulates neurite branching and neurite outgrowth. Thus, increased levels of soluble NCAM in schizophrenic brain have the potential to impair neuronal connectivity.     

Induction of neural cell adhesion molecule (NCAM) in Xenopus embryos

Using a classical neural induction protocol (H. Spemann and H. Mangold (1924). Roux' Arch. Entwicklungsmech. Org. 123, 389-517), it has been demonstrated that the sustained presence of NCAM in Xenopus embryos, as detected by immunohistochemistry, was confined to the experimentally induced nervous system and the primary host nervous system. Furthermore, in vitro NCAM expression by dorsal blastopore lip and animal pole tissue was detected only when the two tissues were cultured in contact. These and other results show that readily detected and sustained levels of NCAM expression in Xenopus can be used as a marker for neural tissue and an early positive indicator that neural induction has occurred. They suggest that the observed levels of NCAM are a consequence of and not a prerequisite for induction. Using NCAM expression in vitro to determine the minimum time necessary for this induction to occur in vivo, it was found that NCAM was first detected in cultured animal pole that had been removed at stage 10.75 or later. Thus, an inductive step necessary and sufficient for stimulation of NCAM expression in animal pole tissues had not occurred or was reversible prior to the first 2 to 2.5 hr of gastrulation.     

Hamster pulmonary endocrine cells with neural cell adhesion molecule (NCAM) immunostaining

Pulmonary endocrine cells of Syrian golden hamster were stained for neural cell adhesion molecule (NCAM) with indirect fluorescent immunostaining and observed with a confocal laser scanning microscope equipped with an argon laser. Sections 100 m thick of hamster lung fixed with phosphate-buffered 4% paraformaldehyde were prepared. The sections were incubated with rat monoclonal antibody against NCAM, followed by fluorescence-labeled antibody against rat immunoglobulin. Some were doubly immunostained for NCAM and one of the following endocrine markers: neuron-specific enolase, calcitonin gene-related peptide and serotonin. Expression of NCAM in the hamster airway epithelium was seen in cell nests resembling neuroepithelial bodies (NEBs). NCAM immunostaining was positive at the lateral cell borders between the cells composing the nest, but negative at the border with the adjacent, presumably non-endocrine cells. Double immunostaining confirmed that the grouped cells with NCAM immunoreactivity were of an endocrine nature, but that single endocrine cells did not show NCAM immunoreactivity. An electron microscopic study with NCAM immunostaining confirmed the light microscopic study. These suggest that NCAM expression could be important for the morphogenesis of NEBs. A confocal laser microscope was used to make theee-dimensional images of NEBs after NCAM immunostaining and the spatial interaction between NEBs and the surrounding microenvironment was studied.     

Expression of the neural cell adhesion molecule NCAM in endocrine cells

We examined the expression of the neural cell adhesion molecule NCAM in a number of endocrine tissues of adult rat and in an endocrine tumor cell line. NCAM was found by immunoelectron microscopy to be present on the surface of all endocrine cells in the three lobes of the hypophysis, although staining was relatively less intense in the intermediate lobe, and in pancreatic islets. Pituicytes, hypophyseal glial cells, were also labeled for NCAM. A rat insulinoma cell line (RIN A2) also expressed NCAM as judged by immunocytochemistry. Analysis of NCAM antigenic determinants (Mr 180, 140, and 120 KD) revealed large variations in the relative proportions of NCAM polypeptides present in the different tissues. Although all tissues and cell lines expressed NCAM-140, NCAM-180 was not detected in the adenohypophysis, pancreas, or adrenal medulla, and NCAM-120 was found in none of the endocrine tissues or cell lines except at low levels in the neurohypophysis. The tumor cell line expressed significant levels of NCAM-180, which was most abundant in the neurohypophysis. These results show that NCAM expression appears to be a general property of endocrine cells, although the antigenic composition differs markedly from that in brain tissue. These data are discussed with regard to the embryological origins of the different endocrine tissues, and possible functional implications are suggested.     

Experimental approaches to interfere with the polysialylation of the neural cell adhesion molecule in vitro and in vivo

Sialic acid (Sia) is expressed as terminal sugar in many glycoconjugates and plays an important role during development and regeneration. Addition of homopolymers of Sia (polysialic acid; polySia/PSA) is a unique and highly regulated post-translational modification of the neural cell adhesion molecule (NCAM). The presence of polySia affects NCAM-dependent cell adhesion and plays an important role during brain development, neural regeneration, and plastic processes including learning and memory. PolySia-NCAM is expressed on several neuroendocrine tumors of high malignancy and correlates with poor prognosis. Two closely related enzymes, the polysialyltransferases ST8SiaII and ST8SiaIV, catalyze the biosynthesis of polySia. This review summarizes recent knowledge on Sia biosynthesis and the correlation between Sia biosynthesis and polysialylation of NCAM and report on approaches to modify the degree of polySia on NCAM in vitro and in vivo. First, we describe the inhibition of polysialylation of NCAM in ST8SiaII-expressing cells using synthetic Sia precursors. Second, we demonstrate that the key enzyme of the Sia biosynthesis (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase) regulates and limits the synthesis of polySia by controlling the cellular Sia concentration.     

Metalloprotease‐induced ectodomain shedding of neural cell adhesion molecule (NCAM)

Transmembrane forms of neural cell adhesion molecule (NCAM140, NCAM180¹) are key regulators of neuronal development. The extracellular domain of NCAM can occur as a soluble protein in normal brain, and its levels are elevated in neuropsychiatric disorders, such as schizophrenia; however the mechanism of ectodomain release is obscure. Ectodomain shedding of NCAM140, releasing a fragment of 115 kD, was found to be induced in NCAM‐transfected L‐fibroblasts by the tyrosine phosphatase inhibitor pervanadate, but not phorbol esters. Pervanadate‐induced shedding was mediated by a disintegrin metalloprotease (ADAM), regulated by ERK1/2 MAP kinase. In primary cortical neurons, NCAM was shed at high levels, and the metalloprotease inhibitor GM6001 significantly increased NCAM‐dependent neurite branching and outgrowth. Moreover, NCAM‐dependent neurite outgrowth and branching were inhibited in neurons isolated from a transgenic mouse model of NCAM shedding. These results suggest that regulated metalloprotease‐induced ectodomain shedding of NCAM down‐regulates neurite branching and neurite outgrowth. Thus, increased levels of soluble NCAM in schizophrenic brain have the potential to impair neuronal connectivity. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Proteolytic modification of neural cell adhesion molecule (NCAM) by the intracellular proteinase calpain

The neural cell adhesion molecule, NCAM, is concentrated in synaptic regions and thus may contribute to the formation and maintenance of connections between brain cells. We present evidence that the cytoplasmic domain of NCAM can be experimentally modified by the intracellular calcium-dependent proteinase, calpain. This degradation could provide a mechanism for rapidly uncoupling and reorganizing synaptic contacts.     

Neural cell adhesion molecule (NCAM) and myoblast fusion

Considerable evidence points to an involvement of neural cell adhesion molecule (NCAM) in myoblast fusion. Changes in the level of NCAM expression, isoform specificity, and localization in muscle cells and tissues correspond to key morphogenetic events during muscle differentiation and repair. Furthermore, anti-NCAM antibodies have been shown by others to reduce the rate of myoblast fusion, whereas overexpression of NCAM cDNAs increases the rate of myoblast fusion compared to controls. In this study we have used a novel fusion assay based on intracistronic complementation of lacZ, in combination with fluorescent X-gal histochemistry and immunocytochemistry to assess levels of NCAM expression in individual muscle cells. Our results indicate that a substantial proportion of newly fused myoblasts have NCAM expression levels unchanged from the levels of the surrounding unfused population suggesting that increased expression of NCAM is not required for wild-type myoblasts to fuse. Moreover, pure populations of primary myoblasts isolated from mice homozygous null for NCAM and therefore lacking the molecule, when placed in differentiation medium, consistently fused to form contractile myotubes with kinetics equivalent to wild-type primary myoblasts. We conclude that the increase in expression of NCAM, although typically observed during myogenesis, is not essential to myoblast fusion to form myotubes.     

Differences in aggregation properties and levels of the neural cell adhesion molecule (NCAM) between islet cell types

Cells within rat islets of Langerhans are typically organized as a core of B-cells, surrounded by the other cell types. When mixed in culture, primary islet cells and insulinoma (RIN2A) cells form aggregates where B-cells are centrally located, surrounded by non-B-cells, while RIN-cells segregate as the outermost layer. To gain insight into the molecular basis underlying this nonrandom cellular organization, the aggregation properties of the three cell populations were studied. Isolated islet cells were separated into B-cells and non-B-cells by autofluorescence-activated cell sorting (FACS). In a short-term aggregation assay, primary B-cell aggregation in the absence of calcium was only 19 +/- 3.7%, compared to the 67 +/- 2.9% seen in the presence of calcium (mean +/- SEM; P less than 0.001; n = 7). By contrast, non-B-cell aggregation and RIN cell aggregation in the absence of calcium (62 +/- 2 and 66 +/- 2%, respectively) were only slightly less than with calcium (70 +/- 3 and 76 +/- 3%). The surface density of the Ca2(+)-independent neural CAM (NCAM) was therefore measured by flow cytometry and found to be 2.64 +/- 0.82-fold higher in non-B-cells, compared to that in B-cells (P less than 0.01; n = 3). Even higher levels were found on RIN cells. In the three cell types, NCAM-140 was the only molecular form detected by immunoblotting. In conclusion, differences in the calcium dependency of aggregation and in the levels of NCAM are demonstrated among islet B-cells, non-B-cells, and RIN cells. Because cell-cell adhesion is crucial for the maintenance of adult tissue, these aggregation specificities might contribute to the concentric segregation of islet cell types in culture and to the nonrandom distribution of cells within rat islets.     

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.     

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.     

Differential expression of the neural cell adhesion molecule NCAM 140 in human pituitary tumors

We have analyzed the expression of the intracellular marker protein neuron specific enolase (NSE), synaptophysin (SYN) and of the cell surface marker NCAM (neural cell adhesion molecule) in both normal hypophysis and in pituitary adenomas in order to explore their potential use as diagnostic tools. All adenomas (4 prolactinomas, 3 growth hormone (GH) producing adenomas and 4 inactive adenomas) showed SYN and NSE immunoreactivity on tissue sections and this was confirmed by immunoblots. NCAM 140 (an isoform of NCAM with molecular mass 140 kDa) was detected by immunoblotting in normal human adenohypophysis, in all GH adenomas, and in three out of four inactive adenomas, but not in prolactinomas. Using highly sensitive techniques, NCAM immunoreactivity was observed by electron microscopy in all adenomas. These data indicate that NCAM 140 is a constituent of the cell surface of endocrine cells in both normal human adenohypophysis and its tumors. Since prolactinomas express very low levels of NCAM 140 compared to other hypophyseal tumors its virtual absence could be used for differential diagnosis. A combined analysis of NCAM, SYN and NSE could be useful to characterize inactive adenomas which are not immunoreactive for pituitary hormones and which may contain no or only low levels of the alpha chain of the glycoprotein hormones.     

SNPs in the neural cell adhesion molecule 1 gene (NCAM1) may be associated with human neural tube defects

Neural tube defects (NTDs) are common birth defects, occurring in approximately 1/1,000 births; both genetic and environmental factors are implicated. To date, no major genetic risk factors have been identified. Throughout development, cell adhesion molecules are strongly implicated in cell–cell interactions, and may play a role in the formation and closure of the neural tube. To evaluate the role of neural cell adhesion molecule 1 (NCAM1) in risk of human NTDs, we screened for novel single-nucleotide polymorphisms (SNPs) within the gene. Eleven SNPs across NCAM1 were genotyped using TaqMan. We utilized a family-based approach to evaluate evidence for association and/or linkage disequilibrium. We evaluated American Caucasian simplex lumbosacral myelomeningocele families (n=132 families) using the family based association test (FBAT) and the pedigree disequilibrium test (PDT). Association analysis revealed a significant association between risk for NTDs and intronic SNP rs2298526 using both the FBAT test (P=0.0018) and the PDT (P=0.0025). Using the HBAT version of the FBAT to look for haplotype association, all pairwise comparisons with SNP rs2298526 were also significant. A replication study set, consisting of 72 additional families showed no significant association; however, the overall trend for overtransmission of the less common allele of SNP rs2298526 remained significant in the combined sample set. In addition, we analyzed the expression pattern of the NCAM1 protein in human embryos, and while NCAM1 is not expressed within the neural tube at the time of closure, it is expressed in the surrounding and later in differentiated neurons of the CNS. These results suggest variations in NCAM1 may influence risk for human NTDs.Other members of NTD Collaborative Group involved in this study are listed in the appendix     

Polysialylation of the neural cell adhesion molecule: interfering with polysialylation and migration in neuroblastoma cells

Polysialic acid represents a unique posttranslational modification of the neural cell adhesion molecule (NCAM). It is built as a homopolymer of up to 150 molecules of alpha 2-8-linked sialic acids on N-glycans of the fifth immunoglobulin-like domain of NCAM. Besides its role in cell migration and axonal growth during development, polysialic acids are closely related to tumor malignancy as they are linked to the malignant potential of several tumors, such as undifferentiated neuroblastoma. Polysialic acid expression is significantly more frequent in high-grade tumors than in low-grade tumors. It is synthesized in the Golgi apparatus by the activity of two closely related enzymes, the polysialyltransferases ST8SiaII and ST8SiaIV. Interestingly, polysialylation of tumors is not equally synthesized by both polysialyltransferases. It has been shown that especially the ST8SiaII gene is not expressed in some normal tissue, but is strongly expressed in tumor tissue. Here we summarize some knowledge on the role of polysialic acid in cell migration and tumor progression and present novel evidence that interfering with polysialylation using unnatural sialic acid precursors decreases the migration of neuroblastoma cells.     

Ammonia inhibits neural cell adhesion molecule polysialylation in Chinese hamster ovary and small cell lung cancer cells

Ammonia is a major concern in biotechnology because it often limits recombinant protein production by animal cells. Conditions, such as ammonia accumulation, in large-scale production systems can parallel those that develop within fast-growing solid tumors such as small cell lung cancer (SCLC). Ammonia's specific inhibition of the sialylation of secreted glycoproteins is well documented, but it is not known how ammonia affects membrane-bound proteins, nor what role it may have on important glycosylation determinants in cancer. We therefore examined the effects of NH₄Cl on polysialic acid (PolySia) in the neural cell adhesion molecule (NCAM). By using flow cytometry combined with two NCAM antibodies, one specific for the peptide backbone and another that recognizes PolySia chains, we show that ammonia causes rapid, dose-dependent, and reversible inhibition of NCAM polysialylation in Chinese hamster ovary (CHO) and SCLC NCI-N417 cells. The decrease in PolySia was accompanied by a small increase in NCAM, suggesting that the changes were specific to the oligosaccharide. Inhibition by ammonia was greater for CHO cells, with PolySia cell surface content decreasing to 10% of control after a 4-day culture with 10 mM NH₄Cl, while N417 cell PolySia was reduced by only 35%. Ammonia caused a 60% decrease in the CHO cell yield from glucose, while N417 cells were barely affected, suggesting that increased resistance to ammonia by N417 cells is a global rather than glycosylation-specific phenomenon. The data presented show that the tumor microenvironment may be an important factor in the regulation of PolySia expression. J. Cell. Physiol. 177:248–263, 1998. © 1998 Wiley-Liss, Inc.     

Genetic linkage of the human apolipoprotein AI-CIII-AIV gene cluster and the neural cell adhesion molecule (NCAM) gene

The genes encoding apolipoproteins AI, CIII, and AIV, three plasma proteins involved in lipid metabolism, are clustered within a 15-kb DNA segment (apoAI-CIII-AIV gene cluster) located on human chromosome 11 at band q23. The gene encoding the neural cell adhesion molecule (NCAM), a cell surface glycoprotein involved in cell-cell recognition during morphogenesis, is also located on chromosome 11, band q23. In this report, 12 previously described restriction fragment length polymorphisms (RFLPs) in the apoAI-CIII-AIV gene cluster were tested for cosegregation with a newly identified BamHI RFLP in the NCAM gene using 13 families. The results show that the apoAI-CIII-AIV gene cluster and the NCAM gene loci are linked with a maximum lod score of 15.9 at a recombination fraction of 0.028. In addition, an approach for the most efficient use of the apoAI-CIII-AIV gene cluster polymorphisms, based on the evaluation of their individual and cumulative heterozygosities, is presented.     

A synthetic peptide ligand of neural cell adhesion molecule (NCAM) IgI domain prevents NCAM internalization and disrupts passive avoidance learning

The neural cell adhesion molecule (NCAM) mediates cell adhesion and signal transduction through trans-homophilic- and/or cis-heterophilic-binding mechanisms. Intraventricular infusions of anti-NCAM have revealed a functional requirement of NCAM for the consolidation of memory in rats and chicks in a specific interval 6-8 h after training. We have now extended these studies to a synthetic peptide ligand of NCAM (C3) with an affinity for the IgI domain and the capability of inhibiting NCAM-mediated neurite outgrowth in vitro. Intraventricular administration of a single 5 microg bolus of C3 strongly inhibited recall of a passive avoidance response in adult rats, when given during training or in the 6-8-h posttraining period. The effect of C3 on memory consolidation was similar to that obtained with anti-NCAM as the amnesia was not observed until the 48-h recall time. The unique amnesic action of C3 during training could be related to disrupted NCAM internalization following training. In the 3-4-h posttraining period NCAM 180, the synapse-associated isoform, was down-regulated in the hippocampal dentate gyrus. This effect was mediated by ubiquitination and was prevented by C3 administration during training. These findings indicate NCAM to be involved in both the acquisition and consolidation of a passive avoidance response in the rat. Moreover, the study provides the first in vivo evidence for NCAM internalization in learning and identifies a synthetic NCAM ligand capable of modulating memory processes 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.