The neuronal chondroitin sulfate proteoglycan neurocan binds to the neural cell adhesion molecules Ng-CAM/L1/NILE and N-CAM, and inhibits neuronal adhesion and neurite outgrowth

We have previously shown that aggregation of microbeads coated with N- CAM and Ng-CAM is inhibited by incubation with soluble neurocan, a chondroitin sulfate proteoglycan of brain, suggesting that neurocan binds to these cell adhesion molecules (Grumet, M., A. Flaccus, and R. U. Margolis. 1993. J. Cell Biol. 120:815). To investigate these interactions more directly, we have tested binding of soluble 125I- neurocan to microwells coated with different glycoproteins. Neurocan bound at high levels to Ng-CAM and N-CAM, but little or no binding was detected to myelin-associated glycoprotein, EGF receptor, fibronectin, laminin, and collagen IV. The binding to Ng-CAM and N-CAM was saturable and in each case Scatchard plots indicated a high affinity binding site with a dissociation constant of approximately 1 nM. Binding was significantly reduced after treatment of neurocan with chondroitinase, and free chondroitin sulfate inhibited binding of neurocan to Ng-CAM and N-CAM. These results indicate a role for chondroitin sulfate in this process, although the core glycoprotein also has binding activity. The COOH-terminal half of neurocan was shown to have binding properties essentially identical to those of the full-length proteoglycan. To study the potential biological functions of neurocan, its effects on neuronal adhesion and neurite growth were analyzed. When neurons were incubated on dishes coated with different combinations of neurocan and Ng-CAM, neuronal adhesion and neurite extension were inhibited. Experiments using anti-Ng-CAM antibodies as a substrate also indicate that neurocan has a direct inhibitory effect on neuronal adhesion and neurite growth. Immunoperoxidase staining of tissue sections showed that neurocan, Ng-CAM, and N-CAM are all present at highest concentration in the molecular layer and fiber tracts of developing cerebellum. The overlapping localization in vivo, the molecular binding studies, and the striking effects on neuronal adhesion and neurite growth support the view that neurocan may modulate neuronal adhesion and neurite growth during development by binding to neural cell adhesion molecules.

[1]  P. Maurel,et al.  Phosphacan, a chondroitin sulfate proteoglycan of brain that interacts with neurons and neural cell-adhesion molecules, is an extracellular variant of a receptor-type protein tyrosine phosphatase. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[2]  A. Oohira,et al.  Core proteins of soluble chondroitin sulfate proteoglycans purified from the rat brain block the cell cycle of PC12D cells , 1993, Journal of cellular physiology.

[3]  J. Silver,et al.  Putative inhibitory extracellular matrix molecules at the dorsal root entry zone of the spinal cord during development and after root and sciatic nerve lesions. , 1993, Developmental biology.

[4]  R. U. Margolis,et al.  Functional characterization of chondroitin sulfate proteoglycans of brain: interactions with neurons and neural cell adhesion molecules , 1993, The Journal of cell biology.

[5]  P. Sonderegger,et al.  Regulation of axonal growth in the vertebrate nervous system by interactions between glycoproteins belonging to two subgroups of the immunoglobulin superfamily , 1992, The Journal of cell biology.

[6]  M. Schachner,et al.  Cell Type‐specific Effects of the Neural Adhesion Molecules L1 and N‐CAM on Diverse Second Messenger Systems , 1992, The European journal of neuroscience.

[7]  F. Walsh,et al.  Cell adhesion molecules, second messengers and axonal growth , 1992, Current Opinion in Neurobiology.

[8]  E. Qwarnstrom,et al.  The role of proteoglycans in cell adhesion, migration and proliferation. , 1992 .

[9]  R. Falchetto,et al.  Neuronal cell adhesion molecule contactin/F11 binds to tenascin via its immunoglobulin-like domains , 1992, The Journal of cell biology.

[10]  P. Maurel,et al.  Cloning and primary structure of neurocan, a developmentally regulated, aggregating chondroitin sulfate proteoglycan of brain. , 1992, The Journal of biological chemistry.

[11]  A. Hennig,et al.  Two immunologically and developmentally distinct chondroitin sulfate proteolglycans in embryonic chick brain. , 1992, The Journal of biological chemistry.

[12]  D. Snow,et al.  Neurite outgrowth on a step gradient of chondroitin sulfate proteoglycan (CS-PG). , 1992, Journal of neurobiology.

[13]  V. Lemmon,et al.  Neurite growth on different substrates: permissive versus instructive influences and the role of adhesive strength , 1992, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[14]  J. Silver,et al.  Chondroitin sulfate as a regulator of neuronal patterning in the retina. , 1992, Science.

[15]  R. Hynes,et al.  Contact and adhesive specificities in the associations, migrations, and targeting of cells and axons , 1992, Cell.

[16]  M. Grumet.  Structure, expression, and function of, Ng‐CAM a member of the immunoglobulin superfamily involved in neuron‐neuron and neuron‐glia adhesion , 1992, Journal of neuroscience research.

[17]  J. Silver,et al.  A chondroitin sulfate proteoglycan may influence the direction of retinal ganglion cell outgrowth. , 1991, Development.

[18]  A. Lander,et al.  Relationship between neuronal migration and cell-substratum adhesion: laminin and merosin promote olfactory neuronal migration but are anti- adhesive , 1991, The Journal of cell biology.

[19]  J. Prince,et al.  Molecular cloning of NILE glycoprotein and evidence for its continued expression in mature rat CNS , 1991, Journal of neuroscience research.

[20]  Kathryn W. Tosney,et al.  Peanut agglutinin and chondroitin-6-sulfate are molecular markers for tissues that act as barriers to axon advance in the avian embryo. , 1991, Developmental biology.

[21]  R. U. Margolis,et al.  Isolation and characterization of developmentally regulated chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of brain identified with monoclonal antibodies. , 1991, The Journal of biological chemistry.

[22]  A. Ullrich,et al.  Epidermal growth factor (EGF) induces oligomerization of soluble, extracellular, ligand-binding domain of EGF receptor. A low resolution projection structure of the ligand-binding domain. , 1991, The Journal of biological chemistry.

[23]  A. Prochiantz,et al.  J1/tenascin in substrate-bound and soluble form displays contrary effects on neurite outgrowth , 1991, The Journal of cell biology.

[24]  R. U. Margolis,et al.  Effects of beta-xylosides on proteoglycan biosynthesis and morphology of PC12 pheochromocytoma cells and primary cultures of rat cerebellum. , 1991, Journal of cell science.

[25]  G. Edelman,et al.  Structure of the chicken neuron-glia cell adhesion molecule, Ng-CAM: origin of the polypeptides and relation to the Ig superfamily , 1991, The Journal of cell biology.

[26]  M. Bronner‐Fraser,et al.  Spatial and temporal changes in the distribution of proteoglycans during avian neural crest development. , 1991, Development.

[27]  J. Salzer,et al.  The myelin-associated glycoproteins: membrane disposition, evidence of a novel disulfide linkage between immunoglobulin-like domains, and posttranslational palmitylation , 1990, The Journal of cell biology.

[28]  K. Hattori,et al.  O-linked sugar chain of human granulocyte colony-stimulating factor protects it against polymerization and denaturation allowing it to retain its biological activity. , 1990, The Journal of biological chemistry.

[29]  J. Silver,et al.  Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro , 1990, Experimental Neurology.

[30]  D. Friedlander,et al.  Expression of adhesion molecules and the establishment of boundaries during embryonic and neural development , 1990, Experimental Neurology.

[31]  D. Steindler,et al.  Molecular and cellular characterization of the glial roof plate of the spinal cord and optic tectum: a possible role for a proteoglycan in the development of an axon barrier. , 1990, Developmental biology.

[32]  E. Ruoslahti.  Proteoglycans in cell regulation. , 1989, Journal of Biological Chemistry.

[33]  M. Lohse,et al.  Neural cell adhesion molecules influence second messenger systems , 1989, Neuron.

[34]  G. Edelman,et al.  Topology of cell adhesion molecules. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[35]  G. Edelman,et al.  Functional mapping of cytotactin: proteolytic fragments active in cell- substrate adhesion , 1988, The Journal of cell biology.

[36]  D. Teplow,et al.  Neural adhesion molecule L1 as a member of the immunoglobulin superfamily with binding domains similar to fibronectin , 1988, Nature.

[37]  J. Schlessinger.  The epidermal growth factor receptor as a multifunctional allosteric protein. , 1988, Biochemistry.

[38]  G. Edelman,et al.  Neuron-glia cell adhesion molecule interacts with neurons and astroglia via different binding mechanisms , 1988, The Journal of cell biology.

[39]  Melitta Schachner,et al.  Selective expression of the 180-kD component of the neural cell adhesion molecule N-CAM during development , 1985, The Journal of cell biology.

[40]  W. Stallcup,et al.  Involvement of the nerve growth factor-inducible large external glycoprotein (NILE) in neurite fasciculation in primary cultures of rat brain. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[41]  G. Edelman,et al.  Polypeptide components and binding functions of neuron-glia cell adhesion molecules. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[42]  M. Schachner,et al.  Neural cell adhesion molecules and myelin-associated glycoprotein share a common carbohydrate moiety recognized by monoclonal antibodies L2 and HNK-1 , 1984, Nature.

[43]  G. Edelman,et al.  Heterotypic binding between neuronal membrane vesicles and glial cells is mediated by a specific cell adhesion molecule , 1984, The Journal of cell biology.

[44]  U. Rutishauser,et al.  Comparison of two cell surface molecules involved in neural cell adhesion. , 1984, The EMBO journal.

[45]  G. Edelman,et al.  Kinetics of homophilic binding by embryonic and adult forms of the neural cell adhesion molecule. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[46]  R. U. Margolis,et al.  Fractionation and properties of a chondroitin sulfate proteoglycan and the soluble glycoproteins of brain. , 1981, The Journal of biological chemistry.

[47]  D Rodbard,et al.  Ligand: a versatile computerized approach for characterization of ligand-binding systems. , 1980, Analytical biochemistry.

[48]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[49]  A. Reddi,et al.  Characteristics of proteoglycans extracted from the Swarm rat chondrosarcoma with associative solvents. , 1979, The Journal of biological chemistry.

[50]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[51]  G. Scatchard,et al.  THE ATTRACTIONS OF PROTEINS FOR SMALL MOLECULES AND IONS , 1949 .

[52]  J. Kapfhammer,et al.  Inhibitors of neurite growth. , 1993, Annual review of neuroscience.

[53]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. 1979. , 1992, Biotechnology.

[54]  L. Reichardt,et al.  Extracellular matrix molecules and their receptors: functions in neural development. , 1991, Annual review of neuroscience.

[55]  G. Edelman,et al.  Cell adhesion molecules: implications for a molecular histology. , 1991, Annual review of biochemistry.

[56]  G. Edelman,et al.  Two antigenically related neuronal cell adhesion molecules of different specificities mediate neuron-neuron and neuron-glia adhesion. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[57]  G. Edelman,et al.  Distinct calcium-independent and calcium-dependent adhesion systems of chicken embryo cells. , 1981, Proceedings of the National Academy of Sciences of the United States of America.