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.     

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.     

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.     

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.     

The neural cell adhesion molecule NCAM is an alternative signaling receptor for GDNF family ligands

Intercellular communication involves either direct cell-cell contact or release and uptake of diffusible signals, two strategies mediated by distinct and largely nonoverlapping sets of molecules. Here, we show that the neural cell adhesion molecule NCAM can function as a signaling receptor for members of the GDNF ligand family. Association of NCAM with GFRalpha1, a GPI-anchored receptor for GDNF, downregulates NCAM-mediated cell adhesion and promotes high-affinity binding of GDNF to p140(NCAM), resulting in rapid activation of cytoplasmic protein tyrosine kinases Fyn and FAK in cells lacking RET, a known GDNF signaling receptor. GDNF stimulates Schwann cell migration and axonal growth in hippocampal and cortical neurons via binding to NCAM and activation of Fyn, but independently of RET. These results uncover an unexpected intersection between short- and long-range mechanisms of intercellular communication and reveal a pathway for GDNF signaling that does not require the RET receptor.     

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.     

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     

Multiple forms of mouse vascular endothelial growth factor-D are generated by RNA splicing and proteolysis

The secreted glycoprotein vascular endothelial growth factor-D (VEGF-D) is angiogenic, lymphangiogenic, and promotes metastatic spread of tumor cells via lymphatic vessels. VEGF-D consists of a receptor-binding domain (VEGF homology domain) and N- and C-terminal propeptides. Proteolytic processing produces numerous forms of human VEGF-D, including fully processed derivatives (containing only the VEGF homology domain), partially processed, and unprocessed derivatives. Proteolysis is essential to generate human VEGF-D that binds the angiogenic receptor VEGF receptor-2 (VEGFR-2) and the lymphangiogenic receptor VEGFR-3 with high affinity. Here, we report that alternative use of an RNA splice donor site in exon 6 of the mouse VEGF-D gene produces two different protein isoforms, VEGF-D(358) and VEGF-D(326), with distinct C termini. The two isoforms were both expressed in all adult mouse tissues and embryonic stages of development analyzed. Both isoforms are proteolytically processed in a similar fashion to human VEGF-D to generate a range of secreted derivatives and bind and cross-link VEGFR-3 with similar potency. The isoforms are differently glycosylated when expressed in vitro. This study demonstrates that RNA splicing, protein glycosylation, and proteolysis are mechanisms for generating structural diversity of mouse VEGF-D.     

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.     

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.     

Opioid-binding cell adhesion molecule (OBCAM)-related clones from a rat brain cDNA library.

The AKin10 gene from Arabidopsis thaliana encoding a putative Ser/Thr protein kinase (PK) has been isolated and characterized. The AKin10-encoding gene is located on a genomic 5.4-kb BamHI fragment and contains ten introns, one being located in the 5' untranslated region. The deduced amino acid sequence of AKin10 is 65% identical over the catalytic domain to the yeast PK (SNF1). SNF1 is essential for the derepression of many glucose-repressible genes, including Suc2 which encodes invertase. Southern blot hybridization experiments suggested the presence of one copy of the gene per haploid genome of A. thaliana. Northern hybridization experiments indicated that this gene is expressed in roots, shoots and leaves. AKin10 may play an important role in a signal transduction cascade regulating gene expression and carbohydrate metabolism in higher plants.     

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.     

Molecular cloning of cDNA encoding the rat neural cell adhesion molecule L1. Two L1 isoforms in the cytoplasmic region are produced by differential splicing

We have isolated and sequenced a full-length cDNA encoding the rat neural cell adhesion molecule L1. The deduced amino acid sequence as a whole shows high homology to mouse L1 sequence. In addition to this complete form of L1, we found an isoform, L1cs, which lacks four amino acid residues (RSLE) in the cytoplasmic domain and probably is derived from the same single L1 gene by tissue-specific alternative splicing. While L1 mRNA was predominantly expressed in the brain, L1cs mRNA was found exclusively in peripheral nervous tissue. Differential splicing in the highly conserved cytoplasmic domain may play an important role in modulating the function of L1 in different cells.     

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.     

Mitochondrial and nuclear forms of Wnt13 are generated via alternative promoters, alternative RNA splicing, and alternative translation start sites

Wnt proteins play a key role in cell survival, cell proliferation, and cell fate during development. In endothelial cells, we identified the expression of Wnt13A, Wnt13B, and Wnt13C mRNAs, which are generated by alternative promoters and alternative RNA splicing. Wnt13A and Wnt13B proteins differ only in their N-terminal sequences. Wnt13A, a typical Wnt, is N-glycosylated and localized in the endoplasmic reticulum, with only a small fraction being secreted. Wnt13B proteins appear as a protein doublet, L-Wnt13B and S-Wnt13B, which are neither N-glycosylated nor secreted. Wnt13B proteins localized mainly to mitochondria, as demonstrated using detection in mitochondria enriched fractions and colocalization with Mitotracker and HSP60. A nuclear localization was also observed in 20% of Wnt13B-expressing cells. Both the N-terminal hydrophobic stretch (residues 1-17) and alpha-helix (residues 26-50) were the main determinants for Wnt13B mitochondrial targeting. Serial deletions of Wnt13B N-terminal sequences abolished its association with mitochondria and favored instead a nuclear localization. The production of S-Wnt13B was independent of the mitochondrial targeting but dependent on an alternative translation start corresponding to Met(74) in L-Wnt13B. The same translation start is used in Wnt13C mRNA to encode a protein undistinguishable from S-Wnt13B. S-Wnt13B when expressed alone localized to the nucleus like Wnt13C, whereas L-Wnt13B localized to mitochondria. Wnt13 nuclear forms increased the beta-catenin/T-cell factor activity in HEK293 cells and increased apoptosis in bovine aortic endothelial cells. Altogether our results demonstrate that, in addition to alternative promoters and RNA splicing, an alternative translation start in Wnt13B and Wnt13C mRNAs increases the complexity of both human wnt13 expression and functions.     

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.