Molecule Neural Cell Adhesion Molecule Research Articles

Ekspresija NCAM i FGFR1 molekula i njihov uticaj na biološko ponašanje karcinoma bubrežnih ćelija

Introduction: The incidence of renal cell tumors (RCT) and the deaths caused by them has been increasing in recent decades. Although renal cell carcinomas (RCCs) represent only 2% of all cancers, these tumors are among the top ten causes of death in Europe, when cancers are concerned. Aim: As it is known that the neural cell adhesion molecule (NCAM) and fibroblast growth factor receptor 1 (FGFR1) interact on the surface of the cell membrane and can also be expressed in other cellular localizations, we decided to examine the potential influence of different patterns of their co-expression on the clinical and pathological characteristics of renal tumors. Material and methods: A total of 100 renal tumors, diagnosed at the Institute of Pathology, Faculty of Medicine, University of Belgrade, were analyzed. Immunohistochemical analysis was performed on tissue microarray slides, using NCAM (1:50, clone123C3.D5) and FGFR1 (1:100, clone M19B2) antibodies. Clinical and pathohistological characteristics of renal tumors were examined in relation to the presence and localization of the co-expression of NCAM and FGFR1 molecules. Results: Co-expression of NCAM and FGFR1 molecules in renal tumors was observed in the cytoplasm and on the membrane, however, these patterns did not depend on the pathohistological type of tumor. Each tumor in which FGFR1 immunopositivity was observed in the nucleus also showed membranous positivity for both tested molecules. It was observed that the frequency of co-expression of NCAM and FGFR1 molecules increased with increasing T stage, but the finding was not statistically significant. Conclusion: Membranous co-expression was not observed in any benign tumor, despite the presence of cytoplasmic co-expression. There is also a possibility that the presence of FGFR in the nucleus induces the occurrence of membranous co-expression.

Ekspresija neuralnog ćelijskog adhezionog molekula u intersticijumu bubrega sa različitim stepenom fibroze

Introduction: In healthy adult kidneys, differentiated tubular epithelial cells do not express the neural cell adhesion molecule (NCAM), while a small number of NCAM-expressing cells can be detected in the renal interstitium. The role and the significance of these cells have not yet been clarified, but it has been observed that the number of NCAM-expressing cells increases in the initial stage of interstitial fibrosis. Aim: The aim of the study is to examine the significance of the expression of NCAM molecules in the renal interstitium, in etiologically different diseases, with varying degrees of interstitial fibrosis, as well as to define the pathohistological and clinical indicators (predictors) of impaired kidney function. Materials and methods: The study included 69 patients who underwent needle biopsies of the kidneys in 2011 and 2012. Clinical and laboratory data were collected at the time of the biopsy and at the time of the latest follow-up examination. Pathohistological characteristics were defined optically-microscopically, while NCAM-expressing interstitial cells were detected with immunohistochemical staining, using the primary NCAM antibody (1:50, clone 123C3.D5). Results: NCAM-expressing interstitial cells were detected in 59.4% of kidney biopsies, the presence of these cells was significantly more frequent in the initial stages of interstitial fibrosis than in the remaining stages (p < 0.001), and it did not depend on the pathohistological diagnosis (p = 0.995). Patients in whom NCAM cells were detected had significantly lower proteinuria levels at the time of biopsy, as compared to patients without NCAM interstitial cells (p = 0.024). The levels of serum creatinine (p < 0.001) and urea (p = 0.007) significantly influenced the probability of the deterioration of renal function. Conclusion: The presence of NCAM cells in the kidney interstitium is a characteristic of the early stages of chronic kidney disease with incipient interstitial fibrosis and a lesser degree of proteinuria

Polysialic acid chains exhibit enhanced affinity for ordered regions of membranes

Polysialic acid (polySia) forms linear chains which are usually attached to the external surface of the plasma membrane mainly through the Neural Cell Adhesion Molecule (NCAM) protein. It is exposed on neural cells, several types of cancer cells, dendritic cells, and egg and sperm cells. There are several lipid raft-related phenomena in which polySia is involved; however the mechanisms of polySia action as well as determinants of its localization in lipid raft microdomains are still unknown, although the majority of NCAM molecules in the liquid-ordered raft membrane fractions of neural cells appear to be polysialylated. Here we investigate the affinity of polySia (both soluble and NCAM-dependent plasma membrane-bound) for liquid-ordered- and liquid-disordered regions of lipid vesicle and neuroblastoma cell membranes. Our studies indicate that polySia chains have a higher affinity for ordered regions of membranes as determined by the dissociation constant values for polySia-lipid bilayer complex, the fluorescence intensity of polySia bound to giant vesicles, the polySia-to-membrane FRET signal at the plasma membrane of live cells, and the decrease of the FRET signals after Endo-N treatment of the cells. These results suggest that polysialylation may be one of the determinants of protein association with liquid-ordered membrane lipid raft domains.

Autopolysialylation of polysialyltransferases is required for polysialylation and polysialic acid chain elongation on select glycoprotein substrates

Polysialic acid (polySia) is a large glycan polymer that is added to some glycoproteins by two polysialyltransferases (polySTs), ST8Sia-II and ST8Sia-IV. As polySia modulates cell adhesion and signaling, immune cell function, and tumor metastasis, it is of interest to determine how the polySTs recognize their select substrates. We have recently identified residues within the ST8Sia-IV polybasic region (PBR) that are required for neural cell adhesion molecule (NCAM) recognition and subsequent polysialylation. Here, we compared the PBR sequence requirements for NCAM, neuropilin-2 (NRP-2), and synaptic cell adhesion molecule 1 (SynCAM 1) for polysialylation by their respective polySTs. We found that the polySTs use unique but overlapping sets of PBR residues for substrate recognition, that the NCAM-recognizing PBR sites in ST8Sia-II and ST8Sia-IV include homologous residues, but that the ST8Sia-II site is larger, and that fewer PBR residues are involved in NRP-2 and SynCAM 1 recognition than in NCAM recognition. Noting that the two sites for ST8Sia-IV autopolysialylation flank the PBR, we evaluated the role of PBR residues in autopolysialylation and found that the requirements for polyST autopolysialylation and substrate polysialylation overlap. These data together with the evaluation of the polyST autopolysialylation mechanism enabled us to further identify PBR residues potentially playing dual roles in substrate recognition and in polySia chain polymerization. Finally, we found that ST8Sia-IV autopolysialylation is required for NRP-2 polysialylation and that ST8Sia-II autopolysialylation promotes the polymerization of longer polySia chains on SynCAM 1, suggesting a critical role for polyST autopolysialylation in substrate selection and polySia chain elongation.

Immobilized Pool of NCAM180 in the Postsynaptic Membrane Is Homeostatically Replenished by the Flux of NCAM180 from Extrasynaptic Regions

Homeostatic mechanisms maintaining high levels of adhesion molecules in synapses over prolonged periods of time remain incompletely understood. We used fluorescence recovery after photobleaching experiments to analyze the steady state turnover of the immobile pool of green fluorescent protein-labeled NCAM180, the largest postsynaptically accumulating isoform of the neural cell adhesion molecule (NCAM). We show that there is a continuous flux of NCAM180 to the postsynaptic membrane from nonsynaptic regions of dendrites by diffusion. In the postsynaptic membrane, the newly delivered NCAM180 slowly intermixes with the immobilized pool of NCAM180. Preferential immobilization and accumulation of NCAM180 in the postsynaptic membrane is reduced after disruption of the association of NCAM180 with the spectrin cytoskeleton and in the absence of the homophilic interactions of NCAM180 in synapses. Our observations indicate that the homophilic interactions and binding to the cytoskeleton promote immobilization of NCAM180 and its accumulation in the postsynaptic membrane. Flux of NCAM180 from extrasynaptic regions and its slow intermixture with the immobile pool of NCAM180 in the postsynaptic membrane may be important for the continuous homeostatic replenishment of NCAM180 protein at synaptic contacts without compromising the long term synaptic contact stability.

A peptide derived from a trans‐homophilic binding site in neural cell adhesion molecule induces neurite outgrowth and neuronal survival

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and synaptic plasticity. The crystal structure of a fragment of NCAM comprising the three N-terminal immunoglobulin (Ig)-like modules indicates that the first and second Ig modules bind to each other, thereby presumably mediating dimerization of NCAM molecules expressed on the same cell surface (cis-interactions), whereas the third Ig module, through interactions with the first or second Ig module, mediates interactions between NCAM molecules expressed on the surface of opposing cells (trans-interactions). We have designed a new potent peptide ligand of NCAM, termed plannexin, based on a discontinuous sequence in the second NCAM Ig module that represents a homophilic binding site for an opposing third Ig module. The peptide was found by surface plasmon resonance analysis to bind the third NCAM Ig module. It promoted survival of cultured cerebellar granule neurons (CGNs) and also induced neurite extension in cultures of dopaminergic neurons and CGNs; the latter effect was shown to be dependent on NCAM expression, indicating that plannexin mimics the neuritogenic effect of homophilic NCAM binding.

Specific Detection of CD56 (NCAM) Isoforms for the Identification of Aggressive Malignant Neoplasms with Progressive Development

Alternative splicing of transcripts from many cancer-associated genes is believed to play a major role in carcinogenesis as well as in tumor progression. Alternative splicing of one such gene, the neural cell adhesion molecule CD56 (NCAM), impacts the progression, inadequate therapeutic response, and reduced total survival of patients who suffer from numerous malignant neoplasms. Although previous investigations have determined that CD56 exists in three major isoforms (CD56(120kD), CD56(140kD), and CD56(180kD)) with individual structural and functional properties, neither the expression profiles nor the functional relevance of these isoforms in malignant tumors have been consistently investigated. Using new quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) strategies and novel CD56 isoform-specific antibodies, CD56(140kD) was shown to be exclusively expressed in a number of highly malignant CD56(+) neoplasms and was associated with the progression of CD56(+) precursor lesions of unclear malignant potential. Moreover, only CD56(140kD) induced antiapoptotic/proliferative pathways and specifically phosphorylated calcium-dependent kinases that are relevant for tumorigenesis. We conclude, therefore, that the specific detection of CD56 isoforms will help to elucidate their individual functions in the pathogenesis and progression of malignant neoplasms and may have a positive impact on the development of CD56-based immunotherapeutic strategies.

S68-04 Depression-like behaviour in neural cell adhesion molecule (NCAM) deficient mice and its reversal by an NCAM-derived peptide, FGL

Neural cell adhesion (NCAM) plays important roles in the structural and functional plasticity in the CNS. Recent years much attention was put on the role brain plasticity in relation to depressive state. Mice deficient in NCAM molecule demonstrated signs of depression-like behavior in the tail suspension- and sucrose consumption tests. Analysis of hippocampal neurogenesis in NCAM deficient mice revealed a reduced survival of the newborn cells and their differentiation into calbindin-positive granule neurons. NCAM-deficient mice have also demonstrated reduction of CREB phosphorylation in the basolateral nucleus of amygdala, pre- and frontal cortex and CA3 regions of hippocampus. Since BDNF signaling system plays a significant role in the mechanisms of depression and in the effects of antidepressants, we measured the levels of BDNF and the level of phosphorylation of TrkB receptor in the brain of NCAM-/- mice. Experiments demonstrated a reduced BDNF signaling in the brains of NCAM-/- ice as a reduced level of phosphorylated Trk B receptor evidenced it. The depression-like signs in NCAM-/- mice were reversed by the NCAM-mimetic peptide FGL. In addition, repeated FGL administration enhanced survival of the newly generated cells in the dentate gyrus of NCAM-/- mice. Our data suggest that reduced brain plasticity due to deficiency in NCAM plays a role in the development of depressive state and NCAM mimetic peptide FGL might represent a new class of drugs with antidepressant activity.

Extracellular Protein Interactions Mediated by the Neural Cell Adhesion Molecule, NCAM: Heterophilic Interactions Between NCAM and Cell Adhesion Molecules, Extracellular Matrix Proteins, and Viruses

Cell adhesion molecules (CAMs) mediate cell-to-cell interactions and interactions between cells and the extracellular matrix (ECM). The neural cell adhesion molecule (NCAM), a prototypic member of the immunoglobulin (Ig) superfamily of CAMs, mediates adhesion through homophilic and heterophilic interactions, thereby modulating a range of biological processes. This review summarizes interactions between NCAM and other CAMs and ECM proteins. Additionally, the role of NCAM as a receptor for rabies virus, and its implications in rabies infections is briefly described. Interactions between NCAM and its heterophilic partners involve most of the NCAM extracellular modules and are mediated via amino acids and carbohydrates. The interactions promote cell adhesion and trigger signal transduction and alterations in cytoskeletal dynamics and organization. Heterophilic NCAM interactions may modulate, or be modulated by, homophilic NCAM interactions. Furthermore, some of the interactions are mutually exclusive, whereas others might lead to the formation of multimeric protein complexes. Consequently, biological processes affected by NCAM interactions are regulated in a complex manner involving many extracellular protein interactions.

WITHDRAWN: NCAM and the FGF-Receptor.

In this review, the structural biology of interaction between the neural cell adhesion molecule (NCAM) and the fibroblast growth factor (FGF) receptor is described and a possible mechanism of the FGF-receptor activation by NCAM is discussed. Most of the FGF-receptor molecules are thought to be constantly involved in a transient interaction with NCAM. However, the FGF-receptor becomes activated only when NCAM is involved the trans-homophilic binding (mediating cell-cell adhesion). The trans-homophilic binding between the NCAM molecules is believed to result in formation of either one- or two-dimensional 'zipper'-like arrays of the NCAM molecules, which leads to NCAM clustering and as a result to clustering of the FGF-receptor, which in turn may lead to its activation through a direct receptor-receptor dimerization (and thus activation) due to an increase in the local concentration of the receptor.

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.

Effects of P2, a peptide derived from a homophilic binding site in the neural cell adhesion molecule on learning and memory in rats

The neural cell adhesion molecule (NCAM) plays a pivotal role in neural development, regeneration, synaptic plasticity, and memory processes. P2 is a 12-amino-acid peptide derived from the second immunoglobulin-like (Ig) module of NCAM mediating cis-homophilic interactions between NCAM molecules present on the same cell. P2 is a potent NCAM agonist, capable of promoting neuronal differentiation and survival in vitro. The aim of this study was to assess the effect of P2 on learning and memory. Rats treated with P2 intracerebroventricularly (1 h prior to test) performed significantly better than controls in the reinforced T-maze, a test of spatial working memory. Further, rats treated with P2 exhibited decreased anxiety-like behavior while learning the T-maze task. In the social recognition test, both intracerebroventricular (1 h prior to test) and systemic (1 and 24 h prior to test) P2 treatment enhanced short-term social memory and counteracted (administration 24 h prior test) scopolamine-induced social memory impairment. In contrast, P2 (1 h prior to test) did not significantly improve long-term (24 h) retention of social memory, nor did it have any significant effects on long-term memory evaluated by the Morris water maze (administration between 2 days before training and 5.5 h posttraining). In the open field test, P2 (1 h prior to test) decreased general locomotion and rearing, but did not influence any other anxiety-related behaviors, indicating only a minimal influence on baseline anxiety levels. Taken together, these data indicate that in vivo P2 enhances short-term memory and protects against the amnestic effects of scopolamine, while modulating emotional behavior in a learning or novelty-related task.

Blockade of natural killer cell‐mediated lysis by NCAM140 expressed on tumor cells

Expression of the neural cell adhesion molecule (NCAM) on malignant cells of neuroendocrine, epithelial and hematopoeitic origin has been reported, but its role for tumor cell recognition by the immune system remained uncertain so far. We have studied the cytotoxicity of the natural killer (NK) cell line NK92 and polyclonal NK cells from different donors, against NCAM-deficient and NCAM-transfected tumors. While the pancreatic carcinoma PANC-1 and the glioblastoma T98G showed no enhanced susceptibility to NK lysis after NCAM transfection, de novo NCAM expression in HeLa cervical carcinoma, SHEP neuroblastoma and the multiple myeloma lines RPMI-8226 and LP-1 was associated with significantly decreased lysis by NK cells. Binding of an NCAM-specific monoclonal antibody to NCAM-positive target cells was able to reverse the reduced lysis susceptibility. Conjugate formation of NCAM-expressing tumor cells with NK cells was blocked and could be restored by anti-NCAM. NK cell-expressed NCAM molecules which might engage in homotypic cis- or trans-interactions had no apparent inhibitory function. The known cis-ligands of NCAM, heparan sulfate proteoglycan and L1-CAM, were also not directly involved in NK inhibition. ICAM-1 mRNA and cell surface expression was downmodulated in NCAM-transfected HeLa cells. ICAM-1 is involved in killer cell immune synapse formation. Its downmodulation may therefore contribute to the reduced lysis of NCAM-expressing target cells. We conclude that aberrant expression of NCAM on tumor cells of different histogenetic origin can lead to inhibition of target cell recognition and lysis by NK cells.

Disruption of the GDNF Binding Site in NCAM Dissociates Ligand Binding and Homophilic Cell Adhesion

Most plasma membrane proteins are capable of sensing multiple cell-cell and cell-ligand interactions, but the extent to which this functional versatility is founded on their modular design is less clear. We have identified the third immunoglobulin domain of the Neural Cell Adhesion Molecule (NCAM) as the necessary and sufficient determinant for its interaction with Glial Cell Line-derived Neurotrophic Factor (GDNF). Four charged contacts were identified by molecular modeling as the main contributors to binding energy. Their mutation abolished GDNF binding to NCAM but left intact the ability of NCAM to mediate cell adhesion, indicating that the two functions are genetically separable. The GDNF-NCAM interface allows complex formation with the GDNF family receptor alpha1, shedding light on the molecular architecture of a multicomponent GDNF receptor.

Structural biology of NCAM homophilic binding and activation of FGFR

In this review, we analyse the structural basis of the homophilic interactions of the neural cell adhesion molecule (NCAM) and the NCAM-mediated activation of the fibroblast growth factor receptor (FGFR). Recent structural evidence suggests that NCAM molecules form cis-dimers in the cell membrane through a high affinity interaction. These cis-dimers, in turn, mediate low affinity trans-interactions between cells via formation of either one- or two-dimensional 'zippers'. We provide evidence that FGFR is probably activated by NCAM very differently from the way by which it is activated by FGFs, reflecting the different conditions for NCAM-FGFR and FGF-FGFR interactions. The affinity of FGF for FGFR is approximately 10(6) times higher than that of NCAM for FGFR. Moreover, in the brain NCAM is constantly present on the cell surface in a concentration of about 50 microm, whereas FGFs only appear transiently in the extracellular environment and in concentrations in the nanomolar range. We discuss the structural basis for the regulation of NCAM-FGFR interactions by two molecular 'switches', polysialic acid (PSA) and adenosine triphosphate (ATP), which determine whether NCAM acts as a signalling or an adhesion molecule.

Recovery of evoked potentials, metabolic activity and behavior in a mouse model of somatosensory cortex lesion: role of the neural cell adhesion molecule (NCAM).

Understanding the processes that underlie functional recovery after cortical injury is a major challenge for neurobiology and clinical neurology. The aim of the present study was to establish a mouse model of functional recovery that would facilitate the investigation of the molecular and cellular events involved in cortical dynamics. We show that a focal injury of approximately 0.5 mm of diameter and 1 mm depth made in the barrel cortex of adult mice induced a transitory deficit that could be characterized using somatosensory evoked potential (SEP), metabolic mapping and a behavioral test. SEP recordings of short latency responses using an epicranial multi-array system showed a decreased cortical activity in the peri-lesion regions 2 weeks after the injury and a partial recovery to normal pattern 6 weeks after the lesion. Delayed SEP signals over the motor cortex were not altered by the injury. Metabolic mapping with [14C]deoxyglucose uptake in the surround of the injury reproduced the time course of deficit and recovery. Finally, a deficit in vibrissae related performance in a gap-crossing test 1 week after injury was followed by a functional recovery in the following 2 weeks. We show in addition that the recovery process is deficient and significantly delayed in NCAM knockout mice lacking all isoforms of NCAM (neural cell adhesion molecule)and PSA-NCAM. These results support the hypothesis that impairment and recovery of functions after focal cortical lesion involves remodeling of intact circuits surrounding the lesion and that the NCAM molecule participate in this process. The model opens new possibilities for investigating the role of candidate molecules in functional recovery using genetically modified mice.

Localization of neural cell adhesion molecule and pan-cadherin immunoreactivity in intrauterine growth-retarded newborn rat kidneys.

To examine the expression and distribution pattern of neural cell adhesion molecule (N-CAM) and pan-cadherin immunoreactivity in intrauterine growth retardation (IUGR) newborn kidneys, we used a rat model of maternal protein restriction throughout pregnancy. Weak or moderate immunoreactivity for N-CAM and pan-cadherin was seen in some proximal tubules, in the thick segments of Henle, and in the collecting tubules of the control sections. However, the number of tubules expressing N-CAM and pan-cadherin was increased in the IUGR group compared with the control group. Increased density of N-CAM and pan-cadherin immunoreactivity was observed mostly in the proximal tubules, in the thick segments of Henle, and in the collecting tubules of IUGR newborn rat kidneys. Furthermore, N-CAM and pan-cadherin immunoreactivity was present in the thin limb of Henle in the IUGR group, whereas it was absent in the control group. Glomeruli were negative in both groups except for some glomeruli that showed very weak N-CAM staining in the IUGR group. Thus it was demonstrated for the first time that IUGR newborn rat kidneys express N-CAM and cadherin adhesion molecules at specific sites of the nephron.

A Synthetic Peptide Ligand of Neural Cell Adhesion Molecule (NCAM), C3d, Promotes Neuritogenesis and Synaptogenesis and Modulates Presynaptic Function in Primary Cultures of Rat Hippocampal Neurons

The neural cell adhesion molecule (NCAM) plays a key role in morphogenesis of the nervous system and in remodeling of neuronal connections accompanying regenerative and cognitive processes. Recently, a new synthetic ligand of NCAM, the C3-peptide, which binds to the NCAM IgI module, has been identified by means of combinatorial chemistry (Rønn, L. C. B, Olsen, M., Ostergaard, S., Kiselyov, V., Berezin, V., Mortensen, M. T., Lerche, M. H., Jensen, P. H., Soroka, V., Saffell, J. L., Doherty, P., Poulsen, F. M., Bock, E., Holm, A., and Saffells, J. L. (1999) Nat. Biotechnol. 17, 1000-1005). In vitro, the dendrimeric form of C3, termed C3d, disrupts NCAM-mediated cell adhesion, induces neurite outgrowth, and triggers intracellular signaling cascades similar to those activated by homophilic NCAM binding. The peptide may therefore be expected to regulate regeneration and synaptic plasticity. Here we demonstrate that in primary cultures of hippocampal neurons: 1) C3d induces a sustained neuritogenic response, the neuritogenic activity of the compound being dependent on the dose, starting time, and duration of peptide application; 2) the peptide triggers the neuritogenic response by forming an adhesive substratum necessary for NCAM-mediated neurite formation and elongation; 3) C3d promotes synapse formation; and 4) C3d modulates the presynaptic function, causing a transient increase of the function at low (2 and 5 microm) doses and a reduction when applied at a higher concentration (10 microm). The effect of the peptide is dependent on the activation of the fibroblast growth factor receptor. We suggest that C3d may constitute a useful lead for the development of compounds for treatment of various neurodegenerative disorders.

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.

Soluble forms of NCAM and F3 neuronal cell adhesion molecules promote Schwann cell migration: identification of protein tyrosine phosphatases zeta/beta as the putative F3 receptors on Schwann cells.

Neural cell adhesion molecule (NCAM) and F3 are both axonal adhesion molecules which display homophilic (NCAM) or heterophilic (NCAM, F3) binding activities and participate in bidirectional exchange of information between neurones and glial cells. Engineered Fc chimeric molecules are fusion proteins that contain the extracellular part of NCAM or F3 and the Fc region of human IgG1. Here, we investigated the effect of NCAM-Fc and F3-Fc chimeras on Schwann cell (SC) migration. Binding sites were identified at the surface of cultured SCs by chimera coated fluorospheres. The functional effect of NCAM-Fc and F3-Fc binding was studied in two different SC migration models. In the first, migration is monitored at specific time intervals inside a 1-mm gap produced in a monolayer culture of SCs. In the second, SCs from a dorsal root ganglion explant migrate on a sciatic nerve cryosection. In both systems addition of the chimeras significantly increased the extent of SC migration and this effect could be prevented by the corresponding anti-NCAM or anti-F3 blocking antibodies. Furthermore, antiproteoglycan-type protein tyrosine phosphatase zeta/beta (RPTPzeta/beta) antibodies identified the presence of RPTPzeta/beta on SCs and prevented the enhancing effect of soluble F3 on SC motility by 95%. The F3-Fc coated Sepharose beads precipitated RPTPzeta/beta from SC lysates. Altogether these data point to RPTPzeta/beta is the putative F3 receptor on SCs. These results identify F3 and NCAM receptors on SC as potential mediators of signalling occurring between axons and glial cells during peripheral nerve development and regeneration.