Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 May 1;125(3):669–680. doi: 10.1083/jcb.125.3.669

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

PMCID: PMC2119998  PMID: 7513709

Abstract

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.

Full Text

The Full Text of this article is available as a PDF (2.7 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Becker J. W., Erickson H. P., Hoffman S., Cunningham B. A., Edelman G. M. Topology of cell adhesion molecules. Proc Natl Acad Sci U S A. 1989 Feb;86(3):1088–1092. doi: 10.1073/pnas.86.3.1088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brackenbury R., Rutishauser U., Edelman G. M. Distinct calcium-independent and calcium-dependent adhesion systems of chicken embryo cells. Proc Natl Acad Sci U S A. 1981 Jan;78(1):387–391. doi: 10.1073/pnas.78.1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brittis P. A., Canning D. R., Silver J. Chondroitin sulfate as a regulator of neuronal patterning in the retina. Science. 1992 Feb 7;255(5045):733–736. doi: 10.1126/science.1738848. [DOI] [PubMed] [Google Scholar]
  4. Burgoon M. P., Grumet M., Mauro V., Edelman G. M., Cunningham B. A. Structure of the chicken neuron-glia cell adhesion molecule, Ng-CAM: origin of the polypeptides and relation to the Ig superfamily. J Cell Biol. 1991 Mar;112(5):1017–1029. doi: 10.1083/jcb.112.5.1017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Calof A. L., Lander A. D. Relationship between neuronal migration and cell-substratum adhesion: laminin and merosin promote olfactory neuronal migration but are anti-adhesive. J Cell Biol. 1991 Nov;115(3):779–794. doi: 10.1083/jcb.115.3.779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Crossin K. L., Prieto A. L., Hoffman S., Jones F. S., Friedlander D. R. Expression of adhesion molecules and the establishment of boundaries during embryonic and neural development. Exp Neurol. 1990 Jul;109(1):6–18. doi: 10.1016/s0014-4886(05)80004-4. [DOI] [PubMed] [Google Scholar]
  7. Doherty P., Walsh F. S. Cell adhesion molecules, second messengers and axonal growth. Curr Opin Neurobiol. 1992 Oct;2(5):595–601. doi: 10.1016/0959-4388(92)90024-f. [DOI] [PubMed] [Google Scholar]
  8. Edelman G. M., Crossin K. L. Cell adhesion molecules: implications for a molecular histology. Annu Rev Biochem. 1991;60:155–190. doi: 10.1146/annurev.bi.60.070191.001103. [DOI] [PubMed] [Google Scholar]
  9. Faltz L. L., Reddi A. H., Hascall G. K., Martin D., Pita J. C., Hascall V. C. Characteristics of proteoglycans extracted from the Swarm rat chondrosarcoma with associative solvents. J Biol Chem. 1979 Feb 25;254(4):1375–1380. [PubMed] [Google Scholar]
  10. Friedlander D. R., Hoffman S., Edelman G. M. Functional mapping of cytotactin: proteolytic fragments active in cell-substrate adhesion. J Cell Biol. 1988 Dec;107(6 Pt 1):2329–2340. doi: 10.1083/jcb.107.6.2329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grumet M., Edelman G. M. Heterotypic binding between neuronal membrane vesicles and glial cells is mediated by a specific cell adhesion molecule. J Cell Biol. 1984 May;98(5):1746–1756. doi: 10.1083/jcb.98.5.1746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Grumet M., Edelman G. M. Neuron-glia cell adhesion molecule interacts with neurons and astroglia via different binding mechanisms. J Cell Biol. 1988 Feb;106(2):487–503. doi: 10.1083/jcb.106.2.487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grumet M., Flaccus A., Margolis R. U. Functional characterization of chondroitin sulfate proteoglycans of brain: interactions with neurons and neural cell adhesion molecules. J Cell Biol. 1993 Feb;120(3):815–824. doi: 10.1083/jcb.120.3.815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grumet M., Hoffman S., Chuong C. M., Edelman G. M. Polypeptide components and binding functions of neuron-glia cell adhesion molecules. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7989–7993. doi: 10.1073/pnas.81.24.7989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grumet M., Hoffman S., Edelman G. M. Two antigenically related neuronal cell adhesion molecules of different specificities mediate neuron-neuron and neuron-glia adhesion. Proc Natl Acad Sci U S A. 1984 Jan;81(1):267–271. doi: 10.1073/pnas.81.1.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grumet M. Structure, expression, and function of Ng-CAM, a member of the immunoglobulin superfamily involved in neuron-neuron and neuron-glia adhesion. J Neurosci Res. 1992 Jan;31(1):1–13. doi: 10.1002/jnr.490310102. [DOI] [PubMed] [Google Scholar]
  17. Hoffman S., Edelman G. M. Kinetics of homophilic binding by embryonic and adult forms of the neural cell adhesion molecule. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5762–5766. doi: 10.1073/pnas.80.18.5762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hynes R. O., Lander A. D. Contact and adhesive specificities in the associations, migrations, and targeting of cells and axons. Cell. 1992 Jan 24;68(2):303–322. doi: 10.1016/0092-8674(92)90472-o. [DOI] [PubMed] [Google Scholar]
  19. Katoh-Semba R., Oohira A. Core proteins of soluble chondroitin sulfate proteoglycans purified from the rat brain block the cell cycle of PC12D cells. J Cell Physiol. 1993 Jul;156(1):17–23. doi: 10.1002/jcp.1041560104. [DOI] [PubMed] [Google Scholar]
  20. Kiang W. L., Margolis R. U., Margolis R. K. Fractionation and properties of a chondroitin sulfate proteoglycan and the soluble glycoproteins of brain. J Biol Chem. 1981 Oct 25;256(20):10529–10537. [PubMed] [Google Scholar]
  21. Krueger R. C., Jr, Hennig A. K., Schwartz N. B. Two immunologically and developmentally distinct chondroitin sulfate proteolglycans in embryonic chick brain. J Biol Chem. 1992 Jun 15;267(17):12149–12161. [PubMed] [Google Scholar]
  22. Kruse J., Mailhammer R., Wernecke H., Faissner A., Sommer I., Goridis C., Schachner M. Neural cell adhesion molecules and myelin-associated glycoprotein share a common carbohydrate moiety recognized by monoclonal antibodies L2 and HNK-1. Nature. 1984 Sep 13;311(5982):153–155. doi: 10.1038/311153a0. [DOI] [PubMed] [Google Scholar]
  23. Lax I., Mitra A. K., Ravera C., Hurwitz D. R., Rubinstein M., Ullrich A., Stroud R. M., Schlessinger J. 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. J Biol Chem. 1991 Jul 25;266(21):13828–13833. [PubMed] [Google Scholar]
  24. Lemmon V., Burden S. M., Payne H. R., Elmslie G. J., Hlavin M. L. Neurite growth on different substrates: permissive versus instructive influences and the role of adhesive strength. J Neurosci. 1992 Mar;12(3):818–826. doi: 10.1523/JNEUROSCI.12-03-00818.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lochter A., Vaughan L., Kaplony A., Prochiantz A., Schachner M., Faissner A. J1/tenascin in substrate-bound and soluble form displays contrary effects on neurite outgrowth. J Cell Biol. 1991 Jun;113(5):1159–1171. doi: 10.1083/jcb.113.5.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Margolis R. K., Goossen B., Tekotte H., Hilgenberg L., Margolis R. U. Effects of beta-xylosides on proteoglycan biosynthesis and morphology of PC12 pheochromocytoma cells and primary cultures of rat cerebellum. J Cell Sci. 1991 Jun;99(Pt 2):237–246. doi: 10.1242/jcs.99.2.237. [DOI] [PubMed] [Google Scholar]
  27. Margolis R. K., Margolis R. U. Nervous tissue proteoglycans. Experientia. 1993 May 15;49(5):429–446. doi: 10.1007/BF01923587. [DOI] [PubMed] [Google Scholar]
  28. Maurel P., Rauch U., Flad M., Margolis R. K., Margolis R. U. 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. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2512–2516. doi: 10.1073/pnas.91.7.2512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Moos M., Tacke R., Scherer H., Teplow D., Früh K., Schachner M. Neural adhesion molecule L1 as a member of the immunoglobulin superfamily with binding domains similar to fibronectin. Nature. 1988 Aug 25;334(6184):701–703. doi: 10.1038/334701a0. [DOI] [PubMed] [Google Scholar]
  30. Munson P. J., Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep 1;107(1):220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  31. Oakley R. A., Tosney K. W. Peanut agglutinin and chondroitin-6-sulfate are molecular markers for tissues that act as barriers to axon advance in the avian embryo. Dev Biol. 1991 Sep;147(1):187–206. doi: 10.1016/s0012-1606(05)80017-x. [DOI] [PubMed] [Google Scholar]
  32. Oh-eda M., Hasegawa M., Hattori K., Kuboniwa H., Kojima T., Orita T., Tomonou K., Yamazaki T., Ochi N. O-linked sugar chain of human granulocyte colony-stimulating factor protects it against polymerization and denaturation allowing it to retain its biological activity. J Biol Chem. 1990 Jul 15;265(20):11432–11435. [PubMed] [Google Scholar]
  33. Pedraza L., Owens G. C., Green L. A., Salzer J. L. The myelin-associated glycoproteins: membrane disposition, evidence of a novel disulfide linkage between immunoglobulin-like domains, and posttranslational palmitylation. J Cell Biol. 1990 Dec;111(6 Pt 1):2651–2661. doi: 10.1083/jcb.111.6.2651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Perris R., Krotoski D., Lallier T., Domingo C., Sorrell J. M., Bronner-Fraser M. Spatial and temporal changes in the distribution of proteoglycans during avian neural crest development. Development. 1991 Feb;111(2):583–599. doi: 10.1242/dev.111.2.583. [DOI] [PubMed] [Google Scholar]
  35. Pindzola R. R., Doller C., Silver J. Putative inhibitory extracellular matrix molecules at the dorsal root entry zone of the spinal cord during development and after root and sciatic nerve lesions. Dev Biol. 1993 Mar;156(1):34–48. doi: 10.1006/dbio.1993.1057. [DOI] [PubMed] [Google Scholar]
  36. Pollerberg E. G., Sadoul R., Goridis C., Schachner M. Selective expression of the 180-kD component of the neural cell adhesion molecule N-CAM during development. J Cell Biol. 1985 Nov;101(5 Pt 1):1921–1929. doi: 10.1083/jcb.101.5.1921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Prince J. T., Alberti L., Healy P. A., Nauman S. J., Stallcup W. B. Molecular cloning of NILE glycoprotein and evidence for its continued expression in mature rat CNS. J Neurosci Res. 1991 Nov;30(3):567–581. doi: 10.1002/jnr.490300315. [DOI] [PubMed] [Google Scholar]
  38. Rathjen F. G., Rutishauser U. Comparison of two cell surface molecules involved in neural cell adhesion. EMBO J. 1984 Feb;3(2):461–465. doi: 10.1002/j.1460-2075.1984.tb01828.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rauch U., Gao P., Janetzko A., Flaccus A., Hilgenberg L., Tekotte H., Margolis R. K., Margolis R. U. Isolation and characterization of developmentally regulated chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of brain identified with monoclonal antibodies. J Biol Chem. 1991 Aug 5;266(22):14785–14801. [PubMed] [Google Scholar]
  40. Rauch U., Karthikeyan L., Maurel P., Margolis R. U., Margolis R. K. Cloning and primary structure of neurocan, a developmentally regulated, aggregating chondroitin sulfate proteoglycan of brain. J Biol Chem. 1992 Sep 25;267(27):19536–19547. [PubMed] [Google Scholar]
  41. Reichardt L. F., Tomaselli K. J. Extracellular matrix molecules and their receptors: functions in neural development. Annu Rev Neurosci. 1991;14:531–570. doi: 10.1146/annurev.ne.14.030191.002531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ruoslahti E. Proteoglycans in cell regulation. J Biol Chem. 1989 Aug 15;264(23):13369–13372. [PubMed] [Google Scholar]
  43. Schlessinger J. The epidermal growth factor receptor as a multifunctional allosteric protein. Biochemistry. 1988 May 3;27(9):3119–3123. doi: 10.1021/bi00409a002. [DOI] [PubMed] [Google Scholar]
  44. Schuch U., Lohse M. J., Schachner M. Neural cell adhesion molecules influence second messenger systems. Neuron. 1989 Jul;3(1):13–20. doi: 10.1016/0896-6273(89)90111-6. [DOI] [PubMed] [Google Scholar]
  45. Schwab M. E., Kapfhammer J. P., Bandtlow C. E. Inhibitors of neurite growth. Annu Rev Neurosci. 1993;16:565–595. doi: 10.1146/annurev.ne.16.030193.003025. [DOI] [PubMed] [Google Scholar]
  46. Snow D. M., Lemmon V., Carrino D. A., Caplan A. I., Silver J. Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro. Exp Neurol. 1990 Jul;109(1):111–130. doi: 10.1016/s0014-4886(05)80013-5. [DOI] [PubMed] [Google Scholar]
  47. Snow D. M., Letourneau P. C. Neurite outgrowth on a step gradient of chondroitin sulfate proteoglycan (CS-PG). J Neurobiol. 1992 Apr;23(3):322–336. doi: 10.1002/neu.480230311. [DOI] [PubMed] [Google Scholar]
  48. Snow D. M., Steindler D. A., Silver J. 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. Dev Biol. 1990 Apr;138(2):359–376. doi: 10.1016/0012-1606(90)90203-u. [DOI] [PubMed] [Google Scholar]
  49. Snow D. M., Watanabe M., Letourneau P. C., Silver J. A chondroitin sulfate proteoglycan may influence the direction of retinal ganglion cell outgrowth. Development. 1991 Dec;113(4):1473–1485. doi: 10.1242/dev.113.4.1473. [DOI] [PubMed] [Google Scholar]
  50. Sonderegger P., Rathjen F. G. Regulation of axonal growth in the vertebrate nervous system by interactions between glycoproteins belonging to two subgroups of the immunoglobulin superfamily. J Cell Biol. 1992 Dec;119(6):1387–1394. doi: 10.1083/jcb.119.6.1387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Von Bohlen Und Halbach Friedrich, Taylor Joanne, Schachner Melitta. Cell Type-specific Effects of the Neural Adhesion Molecules L1 and N-CAM on Diverse Second Messenger Systems. Eur J Neurosci. 1992;4(10):896–909. doi: 10.1111/j.1460-9568.1992.tb00116.x. [DOI] [PubMed] [Google Scholar]
  53. Wight T. N., Kinsella M. G., Qwarnström E. E. The role of proteoglycans in cell adhesion, migration and proliferation. Curr Opin Cell Biol. 1992 Oct;4(5):793–801. doi: 10.1016/0955-0674(92)90102-i. [DOI] [PubMed] [Google Scholar]
  54. Zisch A. H., D'Alessandri L., Ranscht B., Falchetto R., Winterhalter K. H., Vaughan L. Neuronal cell adhesion molecule contactin/F11 binds to tenascin via its immunoglobulin-like domains. J Cell Biol. 1992 Oct;119(1):203–213. doi: 10.1083/jcb.119.1.203. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES