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. 1986 Oct 1;103(4):1383–1398. doi: 10.1083/jcb.103.4.1383

A monoclonal antibody that blocks the activity of a neurite regeneration-promoting factor: studies on the binding site and its localization in vivo

PMCID: PMC2114357  PMID: 2945826

Abstract

Work from several laboratories has identified a proteoglycan complex secreted by a variety of non-neuronal cells that can promote neurite regeneration when applied to the surface of culture dishes. Using a novel immunization protocol, a monoclonal antibody (INO) was produced that blocks the activity of this outgrowth-promoting factor (Matthew, W. D., and P. H. Patterson, 1983, Cold Spring Harbor Symp. Quant. Biol. 48:625-631). We have used the antibody to analyze the components of the active site and to localize the complex in vivo. INO binding is lost when the complex is dissociated; if its components are selectively reassociated, INO binds only to a complex containing two different molecular weight species. These are likely to be laminin and heparan sulfate proteoglycan, respectively. On frozen sections of adult rat tissues, INO binding is present on the surfaces of glial cells of the peripheral, but not the central, nervous system. INO also binds to the basement membrane surrounding cardiac and skeletal muscle cells, and binding to the latter greatly increases after denervation. In the adrenal gland and kidney, INO selectively reacts with areas rich in basement membranes, staining a subset of structures that are immunoreactive for both laminin and heparan sulfate proteoglycan. In general, the outgrowth-blocking antibody binds to areas known to promote axonal regeneration and is absent from areas known to lack this ability. This suggests that this complex, which is active in culture, may be the physiological substrate supporting nerve regeneration in vivo.

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Selected References

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  1. Adler R., Manthorpe M., Skaper S. D., Varon S. Polyornithine-attached neurite-promoting factors (PNPFs). Culture sources and responsive neurons. Brain Res. 1981 Feb 9;206(1):129–144. doi: 10.1016/0006-8993(81)90105-0. [DOI] [PubMed] [Google Scholar]
  2. Akers R. M., Mosher D. F., Lilien J. E. Promotion of retinal neurite outgrowth by substratum-bound fibronectin. Dev Biol. 1981 Aug;86(1):179–188. doi: 10.1016/0012-1606(81)90328-6. [DOI] [PubMed] [Google Scholar]
  3. Barde Y. A., Edgar D., Thoenen H. New neurotrophic factors. Annu Rev Physiol. 1983;45:601–612. doi: 10.1146/annurev.ph.45.030183.003125. [DOI] [PubMed] [Google Scholar]
  4. Baron-Van Evercooren A., Kleinman H. K., Ohno S., Marangos P., Schwartz J. P., Dubois-Dalcq M. E. Nerve growth factor, laminin, and fibronectin promote neurite growth in human fetal sensory ganglia cultures. J Neurosci Res. 1982;8(2-3):179–193. doi: 10.1002/jnr.490080208. [DOI] [PubMed] [Google Scholar]
  5. Benfey M., Aguayo A. J. Extensive elongation of axons from rat brain into peripheral nerve grafts. Nature. 1982 Mar 11;296(5853):150–152. doi: 10.1038/296150a0. [DOI] [PubMed] [Google Scholar]
  6. Brown M. C., Holland R. L., Hopkins W. G. Motor nerve sprouting. Annu Rev Neurosci. 1981;4:17–42. doi: 10.1146/annurev.ne.04.030181.000313. [DOI] [PubMed] [Google Scholar]
  7. Calof A. L., Reichardt L. F. Motoneurons purified by cell sorting respond to two distinct activities in myotube-conditioned medium. Dev Biol. 1984 Nov;106(1):194–210. doi: 10.1016/0012-1606(84)90075-7. [DOI] [PubMed] [Google Scholar]
  8. Carbonetto S., Gruver M. M., Turner D. C. Nerve fiber growth in culture on fibronectin, collagen, and glycosaminoglycan substrates. J Neurosci. 1983 Nov;3(11):2324–2335. doi: 10.1523/JNEUROSCI.03-11-02324.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cole G. J., Glaser L. A heparin-binding domain from N-CAM is involved in neural cell-substratum adhesion. J Cell Biol. 1986 Feb;102(2):403–412. doi: 10.1083/jcb.102.2.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cole G. J., Loewy A., Glaser L. Neuronal cell-cell adhesion depends on interactions of N-CAM with heparin-like molecules. Nature. 1986 Apr 3;320(6061):445–447. doi: 10.1038/320445a0. [DOI] [PubMed] [Google Scholar]
  11. Collins F. Induction of neurite outgrowth by a conditioned-medium factor bound to the culture substratum. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5210–5213. doi: 10.1073/pnas.75.10.5210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Coughlin M. D., Bloom E. M., Black I. B. Characterization of a neuronal growth factor from mouse heart-cell-conditioned medium. Dev Biol. 1981 Feb;82(1):56–68. doi: 10.1016/0012-1606(81)90428-0. [DOI] [PubMed] [Google Scholar]
  13. Courtney K., Roper S. Sprouting of synapses after partial denervation of frag cardic ganglion. Nature. 1976 Jan 29;259(5541):317–319. doi: 10.1038/259317a0. [DOI] [PubMed] [Google Scholar]
  14. Covault J., Sanes J. R. Neural cell adhesion molecule (N-CAM) accumulates in denervated and paralyzed skeletal muscles. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4544–4548. doi: 10.1073/pnas.82.13.4544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Davis G. E., Manthorpe M., Engvall E., Varon S. Isolation and characterization of rat schwannoma neurite-promoting factor: evidence that the factor contains laminin. J Neurosci. 1985 Oct;5(10):2662–2671. doi: 10.1523/JNEUROSCI.05-10-02662.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Edgar D., Timpl R., Thoenen H. The heparin-binding domain of laminin is responsible for its effects on neurite outgrowth and neuronal survival. EMBO J. 1984 Jul;3(7):1463–1468. doi: 10.1002/j.1460-2075.1984.tb01997.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Eldridge C. F., Sanes J. R., Chiu A. Y., Bunge R. P., Cornbrooks C. J. Basal lamina-associated heparan sulphate proteoglycan in the rat PNS: characterization and localization using monoclonal antibodies. J Neurocytol. 1986 Feb;15(1):37–51. doi: 10.1007/BF02057903. [DOI] [PubMed] [Google Scholar]
  18. Fedarko N. S., Conrad H. E. A unique heparan sulfate in the nuclei of hepatocytes: structural changes with the growth state of the cells. J Cell Biol. 1986 Feb;102(2):587–599. doi: 10.1083/jcb.102.2.587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gurney M. E. Suppression of sprouting at the neuromuscular junction by immune sera. Nature. 1984 Feb 9;307(5951):546–548. doi: 10.1038/307546a0. [DOI] [PubMed] [Google Scholar]
  20. Hassell J. R., Robey P. G., Barrach H. J., Wilczek J., Rennard S. I., Martin G. R. Isolation of a heparan sulfate-containing proteoglycan from basement membrane. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4494–4498. doi: 10.1073/pnas.77.8.4494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hawrot E., Patterson P. H. Long-term culture of dissociated sympathetic neurons. Methods Enzymol. 1979;58:574–584. doi: 10.1016/s0076-6879(79)58174-9. [DOI] [PubMed] [Google Scholar]
  22. Jousimaa J., Merenmies J., Rauvala H. Neurite outgrowth of neuroblastoma cells induced by proteins covalently coupled to glass coverslips. Eur J Cell Biol. 1984 Sep;35(1):55–61. [PubMed] [Google Scholar]
  23. Klinman N. R. The mechanism of antigenic stimulation of primary and secondary clonal precursor cells. J Exp Med. 1972 Aug 1;136(2):241–260. doi: 10.1084/jem.136.2.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lander A. D., Fujii D. K., Gospodarowicz D., Reichardt L. F. Characterization of a factor that promotes neurite outgrowth: evidence linking activity to a heparan sulfate proteoglycan. J Cell Biol. 1982 Sep;94(3):574–585. doi: 10.1083/jcb.94.3.574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lander A. D., Fujii D. K., Reichardt L. F. Laminin is associated with the "neurite outgrowth-promoting factors" found in conditioned media. Proc Natl Acad Sci U S A. 1985 Apr;82(7):2183–2187. doi: 10.1073/pnas.82.7.2183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lander A. D., Tomaselli K., Calof A. L., Reichardt L. F. Studies on extracellular matrix components that promote neurite outgrowth. Cold Spring Harb Symp Quant Biol. 1983;48(Pt 2):611–623. doi: 10.1101/sqb.1983.048.01.065. [DOI] [PubMed] [Google Scholar]
  27. Liesi P., Dahl D., Vaheri A. Neurons cultured from developing rat brain attach and spread preferentially to laminin. J Neurosci Res. 1984;11(3):241–251. doi: 10.1002/jnr.490110304. [DOI] [PubMed] [Google Scholar]
  28. Manthorpe M., Engvall E., Ruoslahti E., Longo F. M., Davis G. E., Varon S. Laminin promotes neuritic regeneration from cultured peripheral and central neurons. J Cell Biol. 1983 Dec;97(6):1882–1890. doi: 10.1083/jcb.97.6.1882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Matthew W. D., Greenspan R. J., Lander A. D., Reichardt L. F. Immunopurification and characterization of a neuronal heparan sulfate proteoglycan. J Neurosci. 1985 Jul;5(7):1842–1850. doi: 10.1523/JNEUROSCI.05-07-01842.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Matthew W. D., Patterson P. H. The production of a monoclonal antibody that blocks the action of a neurite outgrowth-promoting factor. Cold Spring Harb Symp Quant Biol. 1983;48(Pt 2):625–631. doi: 10.1101/sqb.1983.048.01.066. [DOI] [PubMed] [Google Scholar]
  31. Okayama M., Pacifici M., Holtzer H. Differences among sulfated proteoglycans synthesized in nonchondrogenic cells, presumptive chondroblasts, and chondroblasts. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3224–3228. doi: 10.1073/pnas.73.9.3224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Osterlund E., Eronen I., Osterlund K., Vuento M. Secondary structure of human plasma fibronectin: conformational change induced by calf alveolar heparan sulfates. Biochemistry. 1985 May 21;24(11):2661–2667. doi: 10.1021/bi00332a011. [DOI] [PubMed] [Google Scholar]
  33. Raisman G., Field P. M. A quantitative investigation of the development of collateral reinnervation after partial deafferentation of the septal nuclei. Brain Res. 1973 Feb 28;50(2):241–264. doi: 10.1016/0006-8993(73)90729-4. [DOI] [PubMed] [Google Scholar]
  34. Ravdin P., Axelrod D. Fluorescent tetramethyl rhodamine derivatives of alpha-bungarotoxin: preparation, separation, and characterization. Anal Biochem. 1977 Jun;80(2):585–592. doi: 10.1016/0003-2697(77)90682-0. [DOI] [PubMed] [Google Scholar]
  35. Rogers S. L., Letourneau P. C., Palm S. L., McCarthy J., Furcht L. T. Neurite extension by peripheral and central nervous system neurons in response to substratum-bound fibronectin and laminin. Dev Biol. 1983 Jul;98(1):212–220. doi: 10.1016/0012-1606(83)90350-0. [DOI] [PubMed] [Google Scholar]
  36. Sanes J. R., Chiu A. Y. The basal lamina of the neuromuscular junction. Cold Spring Harb Symp Quant Biol. 1983;48(Pt 2):667–678. doi: 10.1101/sqb.1983.048.01.070. [DOI] [PubMed] [Google Scholar]
  37. Sanes J. R., Hall Z. W. Antibodies that bind specifically to synaptic sites on muscle fiber basal lamina. J Cell Biol. 1979 Nov;83(2 Pt 1):357–370. doi: 10.1083/jcb.83.2.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sanes J. R., Schachner M., Covault J. Expression of several adhesive macromolecules (N-CAM, L1, J1, NILE, uvomorulin, laminin, fibronectin, and a heparan sulfate proteoglycan) in embryonic, adult, and denervated adult skeletal muscle. J Cell Biol. 1986 Feb;102(2):420–431. doi: 10.1083/jcb.102.2.420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Smalheiser N. R., Crain S. M., Reid L. M. Laminin as a substrate for retinal axons in vitro. Brain Res. 1984 Jan;314(1):136–140. doi: 10.1016/0165-3806(84)90184-6. [DOI] [PubMed] [Google Scholar]
  40. Valnes K., Brandtzaeg P. Retardation of immunofluorescence fading during microscopy. J Histochem Cytochem. 1985 Aug;33(8):755–761. doi: 10.1177/33.8.3926864. [DOI] [PubMed] [Google Scholar]
  41. Wan Y. J., Wu T. C., Chung A. E., Damjanov I. Monoclonal antibodies to laminin reveal the heterogeneity of basement membranes in the developing and adult mouse tissues. J Cell Biol. 1984 Mar;98(3):971–979. doi: 10.1083/jcb.98.3.971. [DOI] [PMC free article] [PubMed] [Google Scholar]

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