Comparison of two cell surface molecules involved in neural cell adhesion

Two cell surface molecules found in mouse brain, N-CAM and the L1 antigen, were compared in terms of their cell adhesion function, polypeptide structures, antigenic determinants and distribution in cerebellar tissue. Fab fragments of polyclonal antibodies to either N-CAM or L1 antigen only partially inhibited the rate of calcium-independent aggregation of neuroblastoma N2A cells, whereas complete and more efficient inhibition was obtained when they were used in combination. Despite the functional similarity, comparison of the electrophoretic behaviour of the purified molecules and of their proteolytic fragments shows that the L1 antigen polypeptide is distinct from that of N-CAM. In addition, no antigenic cross-reactivity was detected between the two molecules. In cryostat sections of cerebellum from young post-natal mice, N-CAM was found to be present in all cell and neurite layers, whereas L1 antigen was expressed only in regions containing post-mitotic cells. These results indicate that two chemically and histochemically distinct cell surface polypeptides can contribute to the calcium-independent adhesiveness of neural cells, and suggest that their differential expression might cause adhesive specificity among cells of developing neural tissues.     

The neural cell adhesion molecule N-CAM enhances L1-dependent cell-cell interactions

On neural cells, the cell adhesion molecule L1 is generally found coexpressed with N-CAM. The two molecules have been suggested, but not directly shown, to affect each other's function. To investigate the possible functional relationship between the two molecules, we have characterized the adhesive interactions between the purified molecules and between cultured cells expressing them. Latex beads were coated with purified L1 and found to aggregate slowly. N-CAM-coated beads did not aggregate, but did so after addition of heparin. Beads coated with both L1 and N-CAM aggregated better than L1-coated beads. Strongest aggregation was achieved when L1-coated beads were incubated together with beads carrying both L1 and N-CAM. In a binding assay, the complex of L1 and N-CAM bound strongly to immobilized L1, but not to the cell adhesion molecules J1 or myelin-associated glycoprotein. N-CAM alone did not bind to these glycoproteins. Cerebellar neurones adhered to and sent out processes on L1 immobilized on nitrocellulose. N-CAM was less effective as substrate. Neurones interacted most efficiently with the immobilized complex of L1 and N-CAM. They adhered to this complex even when its concentration was at least 10 times lower than the lowest concentration of L1 found to promote adhesion. The complex became adhesive for cells only when the two glycoproteins were preincubated together for approximately 30 min before their immobilization on nitrocellulose. The adhesive properties between cells that express L1 only or both L1 and N-CAM were also studied. ESb-MP cells, which are L1-positive, but N-CAM negative, aggregated slowly under low Ca2+. Their aggregation could be completely inhibited by antibodies to L1 and enhanced by addition of soluble N-CAM to the cells before aggregation. N2A cells, which are L1 and N-CAM positive aggregated well under low Ca2+. Their aggregation was partially inhibited by either L1 or N-CAM antibodies and almost completely by the combination of both antibodies. N2A and ESb-MP cells coaggregated rapidly and their interaction was similarly inhibited by L1 and N-CAM antibodies. These results indicate that L1 is involved in two types of binding mechanisms. In one type, L1 serves as its own receptor with slow binding kinetics. In the other, L1 is modulated in the presence of N-CAM on one cell (cis-binding) to form a more potent receptor complex for L1 on another cell (trans-binding).     

Characterization of the cell adhesion molecules L1, N-CAM and J1 in the mouse intestine.

To gain insight into the cellular and molecular mechanisms underlying epithelial cell surface interactions in the adult mouse intestine, we have characterized the cell adhesion molecules L1, N-CAM and J1 by immunocytological, biochemical and cell biological methods. Whereas N-CAM and J1 expression was found to be confined to the mesenchymal and neuroectodermally-derived parts of the intestine, L1 was localized in the proliferating epithelial progenitor cells of crypts, but not in the more differentiated epithelial cells of villi. L1 was detected in crypt cells by Western blot analysis in the molecular forms characteristic of peripheral neural cells, with apparent mol. wts of 230, 180 and 150 kd. Aggregation of single, enriched crypt, but not villus cells, was strongly inhibited in the presence of Fab fragments of polyclonal L1 antibodies. These observations show that L1 is not confined to the nervous system and that it may play a functional role in the histogenesis of the intestine in the adult animal.     

Studies of adhesion molecules mediating interactions between cells of peripheral nervous system indicate a major role for L1 in mediating sensory neuron growth on Schwann cells in culture

The involvement of the adhesion molecules L1, N-CAM, and J1 in adhesion and neurite outgrowth in the peripheral nervous system was investigated. We prepared Schwann cells and fibroblasts (from sciatic nerves) and neurons (from dorsal root ganglia) from 1-d mice. These cells were allowed to interact with each other in a short-term adhesion assay. We also measured outgrowth of dorsal root ganglion neurons on Schwann cell and fibroblast monolayers. Schwann cells (which express L1, N-CAM, and J1) adhered most strongly to dorsal root ganglion neurons by an L1-dependent mechanism and less by N-CAM and J1. Schwann cell-Schwann cell adhesion was mediated by L1 and N-CAM, but not J1. Adhesion of fibroblasts (which express N-CAM, but not L1 or J1) to neurons or Schwann cells was mediated by L1 and N-CAM and not J1. However, inhibition by L1 and N-CAM antibodies was found to be less pronounced with fibroblasts than with Schwann cells. N-CAM was also strongly involved in fibroblast-fibroblast adhesion. Neurite outgrowth was most extensive on Schwann cells and less on fibroblasts. A difference in extent of neurite elongation was seen between small- (10-20 microns) and large- (20-35 microns) diameter neurons, with the larger neurons tending to exhibit longer neurites. Fab fragments of polyclonal L1, N-CAM, and J1 antibodies exerted slightly different inhibitory effects on neurite outgrowth, depending on whether the neurites were derived from small or large neurons. L1 antibodies interfered most strikingly with neurite outgrowth on Schwann cells (inhibition of 88% for small and 76% for large neurons), while no inhibition was detectable on fibroblasts. Similarly, although to a smaller extent than L1, N-CAM appeared to be involved in neurite outgrowth on Schwann cells and not on fibroblasts. Antibodies to J1 only showed a very small effect on neurite outgrowth of large neurons on Schwann cells. These observations show for the first time that identified adhesion molecules are potent mediators of glia-dependent neurite formation and attribute to L1 a predominant role in neurite outgrowth on Schwann cells which may be instrumental in regeneration.     

Molecular and functional analysis of human natural killer cell-associated neural cell adhesion molecule (N-CAM/CD56).

The neural cell adhesion molecule (N-CAM/CD56) is a member of the Ig supergene family that has been shown to mediate homophilic binding. Several isoforms of N-CAM have been identified that are expressed preferentially in different tissues and stages of embryonic development. To examine the primary structure of N-CAM expressed in leukocytes, N-CAM cDNA were generated by polymerase chain reaction from RNA isolated from normal human NK cells and the KG1a hematopoietic leukemia cell line. The sequence of leukocyte-derived N-CAM cDNA was essentially identical with N-CAM cDNA from human neuroblastoma cells that encode the 140-kDa isoform of N-CAM. Inasmuch as N-CAM is preferentially expressed on human NK cells and a subset of T lymphocytes that mediate MHC-unrestricted cell-mediated cytotoxicity, we examined the potential role of N-CAM in cell-mediated cytotoxicity and heterotypic lymphocyte-tumor cell adhesion. N-CAM loss mutants were established from the human N-CAM+ KG1a leukemia cell line, and N-CAM cDNA was transfected into a human colon carcinoma cell line and murine L cells. Using this panel of mutants and transfectants, it was determined that expression of N-CAM on these target cells does not affect susceptibility to resting or IL-2-activated NK cell-mediated cytotoxicity. Moreover, expression of N-CAM in these transfectants failed to induce homotypic or heterotypic cellular adhesion. Collectively, these studies indicate that homophilic N-CAM interactions probably do not mediate a major role in the cytolytic interaction between NK cells and N-CAM+ tumor cell targets.     

Antibodies that recognize astrotactin block granule neuron binding to astroglia

To provide a rapid, specific assay for receptor systems involved in the binding of cerebellar granule neurons to astroglia, granule cells, purified from early postnatal mice, or from E15-E16 chicks, were radiolabeled with [35S]methionine and plasma membranes were prepared. The kinetics of binding of radiolabeled material to primary mouse or chick glia or to the mouse G26-24 astrocytoma cell line was measured in the presence or absence of antibodies against astrotactin, neural cell adhesion molecules, cadherins, or integrins. Addition of Fab fragments of astrotactin antibodies reduced the amount of granule cell membrane binding to astroglia by 70%. In contrast, Fab fragments of antibodies against the neural adhesion molecules N-CAM, L1, and N-cadherin and against integrin did not reduce the level of granule cell membrane binding to astroglia. Combinations of antibodies against N-CAM, L1, N-cadherin, and integrin also did not impair neuron binding to glia.     

Topography of cell ahesion molecules CD9, CD24, L1 and N-CAM on the surface of neuroblastoma cells studied using chemical cross linking

Cell-adhesion molecules are thought to play crucial roles in development and plasticity in the nervous system. Four neural cell adhesion molecules CD9, CD24, L1 and N-CAM are associated in the surface membrane of cultured neuroblastoma cells as studied by chemical cross-linking with bifunctional reagent 3,3'-dithiobis (sulphosuccinimidyl-propionate) followed by a subsequent immunodetection using antibodies directed against the above molecules. We obtained direct evidence of CD9 and L1, but not CD9 and N-CAM clasterisation, also interactions of CD24 with L1, CD24 with N-CAM and some others. These observations illustrate topography of neural cell adhesion molecules located in the vicinity to each other and imply the basis for their functional cooperativity.     

The neural cell adhesion molecule L1 is distinct from the N-CAM related group of surface antigens BSP-2 and D2.

The neural cell adhesion molecule L1 and the group of N-CAM related molecules, BSP-2 and D2 antigen, are immunochemically distinct molecular species. The two groups of surface molecules are also functionally distinct entities, since inhibition of Ca2+-independent adhesion among early post-natal mouse cerebellar cells by Fab fragments of both antibodies are at least additive, when compared with equal concentrations of the individual antibodies.     

Molecular recognition between glyconectins as an adhesion self-assembly pathway to multicellularity

The appearance of multicellular forms of life has been tightly coupled to the ability of an organism to retain its own anatomical integrity and to distinguish self from non-self. Large glycoconjugates, which make up the outermost cell surface layer of all Metazoans, are the primary candidates for the primordial adhesion and recognition functions in biological self-assembly systems. Atomic force microscopy experiments demonstrated that the binding strength between a single pair of Porifera cell surface glyconectin 1 glycoconjugates from Microciona prolifera can hold the weight of 1600 cells, proving their adhesion functions. Here, measurement of molecular self-recognition of glyconectins (GNs) purified from three Porifera species was used as an experimental model for primordial xenogeneic self/non-self discrimination. Physicochemical and biochemical characterization of the three glyconectins, their glycans, and peptides using gel electrophoresis, ultracentrifugation, NMR, mass spectrometry, glycosaminoglycan-degrading enzyme treatment, amino acid and carbohydrate analyses, and peptide mapping showed that GNs define a new family of proteoglycan-like molecules exhibiting species-specific structures with complex and repetitive acidic carbohydrate motives different from the classical proteoglycans and mucins. In functional self-assembly color-coded bead, cell, and blotting assays, glyconectins displayed species-specific recognition and adhesion. Affinity-purified monospecific polyclonal antibodies prepared against GN1, -2, and -3 glycans selectively inhibited cell adhesion of the respective sponge species. These results together with species-specific coaggregation of GN carbohydrate-coated beads with cells showed that GN glycans are functional in cell recognition and adhesion. The specificity of carbohydrate-mediated homophilic GN interactions in Porifera approaches the binding selectivity of the evolutionarily advanced immunoglobulin superfamily. Xenoselectivity of primordial glyconectin to glyconectin recognition may be a new paradigm in the self-assembly and non-self discrimination pathway of cellular adhesion leading to multicellularity.     

Differential contributions of Ng-CAM and N-CAM to cell adhesion in different neural regions

Individual neurons can express both the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) at their cell surfaces. To determine how the functions of the two molecules may be differentially controlled, we have used specific antibodies to each cell adhesion molecule (CAM) to perturb its function, first in brain membrane vesicle aggregation and then in tissue culture assays testing the fasciculation of neurite outgrowths from cultured dorsal root ganglia, the migration of granule cells in cerebellar explants, and the formation of histological layers in the developing retina. Our strategy was initially to delineate further the binding mechanisms for each CAM. Antibodies to Ng-CAM and N-CAM each inhibited brain membrane vesicle aggregation but the binding mechanisms of the two CAMs differed. As expected from the known homophilic binding mechanism of N-CAM, anti-N- CAM-coated vesicles did not co-aggregate with uncoated vesicles. Anti- Ng-CAM-coated vesicles readily co-aggregated with uncoated vesicles in accord with a postulated heterophilic binding mechanism. It was also shown that N-CAM was not a ligand for Ng-CAM. In contrast to assays with brain membrane vesicles, cellular systems can reveal functional differences for each CAM reflecting its relative amount (prevalence modulation) and location (polarity modulation). Consistent with this, each of the three cellular processes examined in vitro was preferentially inhibited only by anti-N-CAM or by anti-Ng-CAM antibodies. Both neurite fasciculation and the migration of cerebellar granule cells were preferentially inhibited by anti-Ng-CAM antibodies. Anti-N-CAM antibodies inhibited the formation of histological layers in the retina. The data on perturbation by antibodies were correlated with the relative levels of expression of Ng-CAM and N-CAM in each of these different neural regions. Quantitative immunoblotting experiments indicated that the relative Ng-CAM/N-CAM ratios in comparable extracts of brain, dorsal root ganglia, and retina were respectively 0.32, 0.81, and 0.04. During culture of dorsal root ganglia in the presence of nerve growth factor, the Ng-CAM/N-CAM ratio rose to 4.95 in neurite outgrowths and 1.99 in the ganglion proper, reflecting both polarity and prevalence modulation. These results suggest that the relative ability of anti-Ng-CAM and anti-N-CAM antibodies to inhibit cell-cell interactions in different neural tissues is strongly correlated with the local Ng-CAM/N-CAM ratio.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cell adhesion molecules in the early developing mouse retina: Retinal neurons show preferential outgrowth in vitro on L1 but not N-CAM

Both L1 and N-CAM are present on optic axons early in the developing mouse retina and optic nerve. In in vitro assays on substrates of purified cell adhesion molecules cells derived from E13 mouse retinae showed vigorous neurite extension on L1 but not on N-CAM. Although retinal neurons on N-CAM showed only limited attachment to the substrate, they were able to form lamellipodia immediately around the cell perimeter. In contrast, similarly derived cortical cells showed extensive neurite outgrowth on both substrates. Under these culture conditions, nearly all of the L1 and N-CAM present in the cell membrane appeared to be sequestered on the lower surface of the growth cones and neurites, indicating that most of these cell adhesion molecules were involved in homophilic interactions. Our results suggest differential roles for L1 and N-CAM in intitiation and establishment of the optic pathway. © 1994 John Wiley & Sons, Inc.     

Homophilic and heterophilic binding activities of Nr-CAM, a nervous system cell adhesion molecule

Nr-CAM is a membrane glycoprotein that is expressed on neurons. It is structurally related to members of the N-CAM superfamily of neural cell adhesion molecules having six immunoglobulin-like domains and five fibronectin type III repeats in the extracellular region. We have found that the aggregation of chick brain cells was inhibited by anti-Nr-CAM Fab' fragments, indicating that Nr-CAM can act as a cell adhesion molecule. To clarify the mode of action of Nr-CAM, a mouse fibroblast cell line L-M(TK-) (or L cells) was transfected with a DNA expression construct encoding an entire chicken Nr-CAM cDNA sequence. After transfection, L cells expressed Nr-CAM on their surface and aggregated. Aggregation was specifically inhibited by anti-Nr-CAM Fab' fragments. To check the specificity of this aggregation, a fusion protein (FGTNr) consisting of glutathione S-transferase linked to the six immunoglobulin domains and the first fibronectin type III repeat of Nr-CAM was expressed in Escherichia coli. Addition of FGTNr to the transfected cells blocked their aggregation. Further analysis using a combination of cell aggregation assays, binding of cells to FGTNr-coated substrates, aggregation of FGTNr-coated Covaspheres and binding of FGTNr-coated Covaspheres to FGTNr-coated substrates revealed that Nr-CAM mediates two types of cell interactions: a homophilic, divalent cation-independent binding, and a heterophilic, divalent cation-dependent binding. Homophilic binding was demonstrated between transfected L cells, between chick embryo brain cells and FGTNr, and between Covaspheres to which FGTNr was covalently attached. Heterophilic binding was shown to occur between transfected and untransfected L cells, and between FGTNr and primary chick embryo fibroblasts; in all cases, it was dependent on the presence of either calcium or magnesium. Primary chick embryo glia or a human glial cell line did not bind to FGTNr-coated substrates. The results indicate that Nr-CAM is a cell adhesion molecule of the nervous system that can bind by two distinct mechanisms, a homophilic mechanism that can mediate interactions between neurons and a heterophilic mechanism that can mediate binding between neurons and other cells such as fibroblasts.     

Biochemical and functional characterization of a novel neuron-glia adhesion molecule that is involved in neuronal migration

Adhesion molecule on glia (AMOG) is a novel neural cell adhesion molecule that mediates neuron-astrocyte interaction in vitro. In situ AMOG is expressed in the cerebellum by glial cells at the critical developmental stages of granule neuron migration. Granule neuron migration that is guided by surface contacts between migrating neurons and astroglial processes is inhibited by monoclonal AMOG antibody, probably by disturbing neuron-glia adhesion. AMOG is an integral cell surface glycoprotein of 45-50-kD molecular weight with a carbohydrate content of at least 30%. It does not belong to the L2/HNK-1 family of neural cell adhesion molecules but expresses another carbohydrate epitope that is shared with the adhesion molecules L1 and myelin-associated glycoprotein, but is not present on N-CAM or J1.     

Neural cell adhesion molecule (N-CAM) homophilic binding mediated by the two N-terminal Ig domains is influenced by intramolecular domain-domain interactions

The mechanism by which the neural cell adhesion molecule, N-CAM, mediates homophilic interactions between cells has been variously attributed to an isologous interaction of the third immunoglobulin (Ig) domain, to reciprocal binding of the two N-terminal Ig domains, or to reciprocal interactions of all five Ig domains. Here, we have used a panel of recombinant proteins in a bead binding assay, as well as transfected and primary cells, to clarify the molecular mechanism of N-CAM homophilic binding. The entire extracellular region of N-CAM mediated bead aggregation in a concentration- and temperature-dependent manner. Interactions of the N-terminal Ig domains, Ig1 and Ig2, were essential for bead binding, based on deletion and mutation experiments and on antibody inhibition studies. These findings were largely in accord with aggregation experiments using transfected L cells or primary chick brain cells. Additionally, maximal binding was dependent on the integrity of the intramolecular domain-domain interactions throughout the extracellular region. We propose that these interactions maintain the relative orientation of each domain in an optimal configuration for binding. Our results suggest that the role of Ig3 in homophilic binding is largely structural. Several Ig3-specific reagents failed to affect N-CAM binding on beads or on cells, while an inhibitory effect of an Ig3-specific monoclonal antibody is probably due to perturbations at the Ig2-Ig3 boundary. Thus, it appears that reciprocal interactions between Ig1 and Ig2 are necessary and sufficient for N-CAM homophilic binding, but that maximal binding requires the quaternary structure of the extracellular region defined by intramolecular domain-domain interactions.     

Intercellular adhesion mediated by human muscle neural cell adhesion molecule: effects of alternative exon use

Mouse 3T3 fibroblasts were permanently transfected with cDNAs encoding isoforms of the neural cell adhesion molecule (N-CAM) present in human skeletal muscle and brain. Parental and transfected cells were then used in a range of adhesion assays. In the absence of external shear forces, transfection with cDNAs encoding either transmembrane or glycosylphosphatidylinositol (GPI)-linked N-CAM species significantly increased the intercellular adhesiveness of 3T3 cells in suspension. Transfection of a cDNA encoding a secreted N-CAM isoform was without effect on adhesion. Cells transfected with cDNAs containing or lacking the muscle-specific domain 1 sequence, a four-exon group spliced into the muscle but not the brain GPI-linked N-CAM species, were equally adhesive in the assays used. We also demonstrate that N-CAM-mediated intercellular adhesiveness is inhibited by 0.2 mg/ml heparin; but, at higher concentrations, reduced adhesion of parental cells was also seen. Coaggregation of fluorescently labeled and unlabeled cell populations was performed and measured by comparing their distribution within aggregates with distributions that assume nonspecific (random) aggregation. These studies demonstrate that random aggregation occurs between transfected cells expressing the transmembrane and GPI-linked N- CAM species and between parental cells and those expressing the secreted N-CAM isoform. Other combinations of these populations tested exhibited partial adhesive specificity, indicating homophilic binding between surface-bound N-CAM. Thus, the approach exploited here allows for a full analysis of the requirements, characteristics, and specificities of the adhesive behavior of individual N-CAM isoforms.     

L1 and N-CAM Antibodies Trigger Protein Phosphatase Activity in Growth Cone-Enriched Membranes

Abstract: Triggering of the cell adhesion molecules L1 or N-CAM in a nerve growth cone membrane fraction from fetal rat brain with purified L1 or N-CAM or specific antibodies decreases the steady-state levels of protein tyrosine phosphorylation in the membranes. Here we report that triggering of L1 and N-CAM in the growth cone-enriched membrane fraction with a subset of antibodies directed against the extracellular region of L1 and N-CAM elicited dephosphorylation of endogenous protein substrates, indicating the presence of a cell adhesion molecule-activated phosphatase. The most prominent substrates were a membrane-associated 200-kDa protein and tubulin, both of which were dephosphorylated on tyrosine and serine/threonine residues in response to L1 or N-CAM triggering. The antibody-induced phosphatase was inhibited by agents that blocked tyrosine and serine/threonine phosphatases, including sodium orthovanadate, vanadyl sulfate, zinc cations, heparin, and sodium pyrophosphate. Purified L1 and N-CAM fragments and other antibodies reacting with the extracellular region of these adhesion molecules did not activate the phosphatase but did inhibit tyrosine phosphorylation. These properties suggested that triggering of L1 and N-CAM can lead to either phosphatase activation or tyrosine kinase inhibition in growth cone membranes. These findings implicate protein phosphatases in addition to tyrosine kinases as components of L1 and N-CAM intracellular signaling pathways in growth cones.     

Expression of the neural cell adhesion molecules L1 and N-CAM and their common carbohydrate epitope L2/HNK-1 during development and after transection of the mouse sciatic nerve

The expression of the neural cell adhesion molecules L1 and N-CAM and of their shared carbohydrate epitope L2/HNK-1 was studied during the development and after the transection of mouse sciatic nerves. During development, L1 and N-CAM were detectable on most, if not all, Schwann cells at embryonic day 17, the earliest stage tested. With increasing age, the immunoreactivity was reduced being confined to non-myelinating Schwann cells by post-natal day 10, at which stage the staining pattern resembled that seen in adult sciatic nerves. Double-immunolabelling experiments revealed a complete overlap between L1 and N-CAM antibodies. The L2/HNK-1 epitope was not detectable in developing sciatic nerves until the end of the 2nd post-natal week, when it appeared to be associated with the outer profiles of thick myelin sheets, as also seen in adult sciatic nerves. Three days after the transection of adult sciatic nerves, L1 antigen and N-CAM was detectable in more Schwann cells in the distal nerve end than in untreated control nerves. The peak level of the reappearance of L1 antigen and N-CAM in Schwann cells occurred between 2 and 4 weeks after transection. The reduction of L1-antigen expression to its normal adult level took more than a year, thus recapitulating normal development, but on a more protracted time scale. Similarly, the L2/HNK-1 epitope remained undetectable until the transected nerve had returned to its normal state of myelination, i.e. approximately 1 year after transection.     

The L2/HNK-1 carbohydrate of neural cell adhesion molecules is involved in cell interactions

We investigated whether the L2/HNK-1 carbohydrate epitope, expressed by two unusual glycolipids and several neural adhesion molecules, including L1, neural cell adhesion molecule, J1, and the myelin-associated glycoprotein, is involved in adhesion. Monoclonal L2 antibodies, the L2/HNK-1-reactive, sulfate-3-glucuronyl residue carrying glycolipids (L2 glycolipid) and a tetrasaccharide derived from the L2 glycolipid (L2 tetrasaccharide) were added to microexplant cultures of early postnatal mouse cerebellum, and cell migration and process extension were monitored. On the substrate poly-D-lysine, Fab fragments of L2 antibodies, L2 glycolipid, and L2 tetrasaccharide inhibited outgrowth of astrocytic processes and migration of cell bodies, but only L2 glycolipid and L2 tetrasaccharide reduced neurite outgrowth. On laminin, L2 antibodies, L2 glycolipid, and L2 tetrasaccharide inhibited outgrowth of astrocytic processes. Additionally, L2 glycolipid and L2 tetrasaccharide inhibited cell migration and neurite outgrowth. Several negatively charged glycolipids, lipids, and saccharides were tested for control and found to have no effect on outgrowth patterns, except for sulfatide and heparin, which modified outgrowth patterns in a similar fashion as L2 glycolipid and L2 tetrasaccharide. On astrocytes none of the tested compounds interfered with explant outgrowth. In short-term adhesion assays L2 glycolipid, sulfatide, and heparin inhibited adhesion of neural cells to laminin. L2 glycolipid and sulfatide interfered with neuron to astrocyte and astrocyte to astrocyte adhesion, but not with neuron-neuron adhesion. The most straightforward interpretation of these observations is that the L2/HNK-1 carbohydrate and the sulfated carbohydrates, sulfatide and heparin, act as ligands in cell adhesion.     

Cell adhesion mechanisms in gangliogenesis studied in avian embryo and in a model system

Neural crest cells undergo rapid changes in their cell-to-cell and cell-to-extracellular matrix adhesion during the ontogeny of the peripheral nervous system. The mechanisms of adhesion have been analyzed to assess the respective roles played by the cell adhesion molecules (CAMs) and the differentiated junctions. Crest cells which lose their terminal bar junctions after emigration from the neural tube contain only very few gap junctions during gangliogenesis. The calcium-dependent cell adhesion molecules, L-CAM, disappear from the neural crest and never reappear in crest cell derivatives. In contrast, the number of calcium-independent cell adhesion molecules, N-CAM, diminishes transiently during the migratory phase. In vitro, N-CAM is expressed de novo either just before or at the onset of aggregation into autonomic ganglion rudiments, whereas it is delayed in the dorsal root ganglion cells. In vitro, N-CAM mediates the calcium-independent aggregation mechanism; the rate of aggregation is, however, similar whether crest cells are derived from well-spread cultures or from two- and three-dimensional clusters. Crest cells also exhibit a calcium-dependent mechanism of adhesion controlled by molecules differing from N-CAM but which may codistribute on many different cell types during embryogenesis. These two classes of cell adhesion molecules are present on the surface of neural precursors prior to their differentiation into neurons and glial cells.     

Myelin-Associated Glycoprotein Shares an Antigenic Determinant with a Glycoprotein of Human Melanoma Cells

A sulfated 100K-dalton glycoprotein has been shown to be released into the culture medium of melanoma cells. Monoclonal antibodies 10C5 and 11B5, which were raised to human melanoma cells, as well as HNK-1 bind to this glycoprotein. It is shown here that mouse anti-myelin-associated glycoprotein (MAG) carbohydrate antibodies raised to human MAG and a human IgM paraprotein associated with neuropathy also bind to the same 100K molecule. However, anti-MAG antibodies recognizing peptide epitopes do not appear to react with this glycoprotein of melanoma cells, a result suggesting that its similarity to MAG is restricted to shared carbohydrate moieties. The anti-melanoma antibodies (10C5 and 11B5) resemble HNK-1 in binding to MAG and to some 19-28K-dalton glycoproteins and sulfated, glucuronic acid-containing sphingoglycolipids of the peripheral nervous system (PNS). In addition, the anti-melanoma antibodies cross-react with neural cell adhesion molecule (N-CAM), an observation emphasizing the shared antigenicity between MAG and other adhesion molecules. The results demonstrate that the anti-melanoma antibodies fall into a class of monoclonal antibodies (including HNK-1, human IgM paraproteins associated with neuropathy, anti-human MAG antibodies, and L2 antibodies) that are characterized by reactivity against related carbohydrate determinants shared by human MAG, N-CAM, and several protein and lipid glycoconjugates of the PNS.