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. 1985 Sep 1;101(3):898–913. doi: 10.1083/jcb.101.3.898

Purification of a factor that promotes neurite outgrowth: isolation of laminin and associated molecules

PMCID: PMC2113739  PMID: 4030898

Abstract

When culture medium, conditioned by any of several cell types, is applied to a polycationic substratum, a substance is adsorbed that causes neurons cultured on that substratum to extend processes (neurites) rapidly and profusely. We have purified the factor responsible for this effect from medium conditioned by bovine corneal endothelial cells, and have shown that it is composed of the glycoprotein laminin and two associated laminin-binding molecules: a sulfated protein known as entactin, and a large heparan sulfate proteoglycan. Of these molecules, only laminin was found to be present throughout the purification in all fractions possessing neurite outgrowth-promoting activity and absent from all fractions lacking activity. Laminin, purified from other sources, has been shown previously to promote extensive outgrowth by cultured neurons. These and other data presented here support the conclusion that laminin is responsible for the neurite outgrowth-promoting activity of the conditioned medium factor. Evidence is also presented that the association of a proteoglycan with laminin promotes efficient attachment of laminin to polycationic substrata, particularly in the presence of competing molecules.

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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. 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]
  4. Batteiger B., Newhall W. J., 5th, Jones R. B. The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J Immunol Methods. 1982 Dec 30;55(3):297–307. doi: 10.1016/0022-1759(82)90089-8. [DOI] [PubMed] [Google Scholar]
  5. Berg D. K. New neuronal growth factors. Annu Rev Neurosci. 1984;7:149–170. doi: 10.1146/annurev.ne.07.030184.001053. [DOI] [PubMed] [Google Scholar]
  6. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  7. CRAVEN G. R., STEERS E., Jr, ANFINSEN C. B. PURIFICATION, COMPOSITION, AND MOLECULAR WEIGHT OF THE BETA-GALACTOSIDASE OF ESCHERICHIA COLI K12. J Biol Chem. 1965 Jun;240:2468–2477. [PubMed] [Google Scholar]
  8. 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]
  9. Carlin B. E., Durkin M. E., Bender B., Jaffe R., Chung A. E. Synthesis of laminin and entactin by F9 cells induced with retinoic acid and dibutyryl cyclic AMP. J Biol Chem. 1983 Jun 25;258(12):7729–7737. [PubMed] [Google Scholar]
  10. Carlin B., Jaffe R., Bender B., Chung A. E. Entactin, a novel basal lamina-associated sulfated glycoprotein. J Biol Chem. 1981 May 25;256(10):5209–5214. [PubMed] [Google Scholar]
  11. Clarke S. The size and detergent binding of membrane proteins. J Biol Chem. 1975 Jul 25;250(14):5459–5469. [PubMed] [Google Scholar]
  12. 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]
  13. Collins F., Lee M. R. The spatial control of ganglionic neurite growth by the substrate-associated material from conditioned medium: an experimental model of haptotaxis. J Neurosci. 1984 Nov;4(11):2823–2829. doi: 10.1523/JNEUROSCI.04-11-02823.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Collins F. Neurite outgrowth induced by the substrate associated material from nonneuronal cells. Dev Biol. 1980 Sep;79(1):247–252. doi: 10.1016/0012-1606(80)90089-5. [DOI] [PubMed] [Google Scholar]
  15. Cooper A. R., Kurkinen M., Taylor A., Hogan B. L. Studies on the biosynthesis of laminin by murine parietal endoderm cells. Eur J Biochem. 1981 Sep;119(1):189–197. doi: 10.1111/j.1432-1033.1981.tb05593.x. [DOI] [PubMed] [Google Scholar]
  16. Cornbrooks C. J., Carey D. J., McDonald J. A., Timpl R., Bunge R. P. In vivo and in vitro observations on laminin production by Schwann cells. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3850–3854. doi: 10.1073/pnas.80.12.3850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Coughlin M. D., Kessler J. A. Antiserum to a new neuronal growth factor: effects on neurite outgrowth. J Neurosci Res. 1982;8(2-3):289–302. doi: 10.1002/jnr.490080219. [DOI] [PubMed] [Google Scholar]
  19. Del Rosso M., Cappelletti R., Viti M., Vannucchi S., Chiarugi V. Binding of the basement-membrane glycoprotein laminin to glycosaminoglycans. An affinity-chromatography study. Biochem J. 1981 Dec 1;199(3):699–704. doi: 10.1042/bj1990699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Edgar D., Thoenen H. Modulation of NGF-induced survival of chick sympathetic neurons by contact with a conditioned medium factor bound to the culture substrate. Brain Res. 1982 Sep;281(1):89–92. doi: 10.1016/0165-3806(82)90115-8. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Engel J., Odermatt E., Engel A., Madri J. A., Furthmayr H., Rohde H., Timpl R. Shapes, domain organizations and flexibility of laminin and fibronectin, two multifunctional proteins of the extracellular matrix. J Mol Biol. 1981 Jul 25;150(1):97–120. doi: 10.1016/0022-2836(81)90326-0. [DOI] [PubMed] [Google Scholar]
  23. Fujii D. K., Massoglia S. L., Savion N., Gospodarowicz D. Neurite outgrowth and protein synthesis by PC12 cells as a function of substratum and nerve growth factor. J Neurosci. 1982 Aug;2(8):1157–1175. doi: 10.1523/JNEUROSCI.02-08-01157.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gospodarowicz D., Greenburg G., Foidart J. M., Savion N. The production and localization of laminin in cultured vascular and corneal endothelial cells. J Cell Physiol. 1981 May;107(2):171–183. doi: 10.1002/jcp.1041070203. [DOI] [PubMed] [Google Scholar]
  25. Gospodarowicz D., Greenburg G. The coating of bovine and rabbit corneas denuded of their endothelium with bovine corneal endothelial cells. Exp Eye Res. 1979 Mar;28(3):249–265. doi: 10.1016/0014-4835(79)90087-3. [DOI] [PubMed] [Google Scholar]
  26. Gospodarowicz D., Mescher A. L., Birdwell C. R. Stimulation of corneal endothelial cell proliferations in vitro by fibroblast and epidermal growth factors. Exp Eye Res. 1977 Jul;25(1):75–89. doi: 10.1016/0014-4835(77)90248-2. [DOI] [PubMed] [Google Scholar]
  27. Hedman K., Johansson S., Vartio T., Kjellén L., Vaheri A., Hök M. Structure of the pericellular matrix: association of heparan and chondroitin sulfates with fibronectin-procollagen fibers. Cell. 1982 Mar;28(3):663–671. doi: 10.1016/0092-8674(82)90221-5. [DOI] [PubMed] [Google Scholar]
  28. Helfand S. L., Riopelle R. J., Wessells N. K. Non-equivalence of conditioned medium and nerve growth factor for sympathetic, parasympathetic, and sensory neurons. Exp Cell Res. 1978 Apr;113(1):39–45. doi: 10.1016/0014-4827(78)90085-x. [DOI] [PubMed] [Google Scholar]
  29. Hogan B. L., Taylor A., Kurkinen M., Couchman J. R. Synthesis and localization of two sulphated glycoproteins associated with basement membranes and the extracellular matrix. J Cell Biol. 1982 Oct;95(1):197–204. doi: 10.1083/jcb.95.1.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Johnston R. N., Wessells N. K. Regulation of the elongating nerve fiber. Curr Top Dev Biol. 1980;16:165–206. doi: 10.1016/s0070-2153(08)60156-8. [DOI] [PubMed] [Google Scholar]
  31. Kleinman H. K., McGarvey M. L., Liotta L. A., Robey P. G., Tryggvason K., Martin G. R. Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry. 1982 Nov 23;21(24):6188–6193. doi: 10.1021/bi00267a025. [DOI] [PubMed] [Google Scholar]
  32. Kühl U., Timpl R., von der Mark K. Synthesis of type IV collagen and laminin in cultures of skeletal muscle cells and their assembly on the surface of myotubes. Dev Biol. 1982 Oct;93(2):344–354. doi: 10.1016/0012-1606(82)90122-1. [DOI] [PubMed] [Google Scholar]
  33. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. 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]
  37. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  38. Letourneau P. C. Cell-to-substratum adhesion and guidance of axonal elongation. Dev Biol. 1975 May;44(1):92–101. doi: 10.1016/0012-1606(75)90379-6. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Manthorpe M., Varon S., Adler R. Neurite-promoting factor in conditioned medium from RN22 Schwannoma cultures: bioassay, fractionation, and properties. J Neurochem. 1981 Sep;37(3):759–767. doi: 10.1111/j.1471-4159.1982.tb12552.x. [DOI] [PubMed] [Google Scholar]
  41. March S. C., Parikh I., Cuatrecasas P. A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal Biochem. 1974 Jul;60(1):149–152. doi: 10.1016/0003-2697(74)90139-0. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Oohira A., Wight T. N., Bornstein P. Sulfated proteoglycans synthesized by vascular endothelial cells in culture. J Biol Chem. 1983 Feb 10;258(3):2014–2021. [PubMed] [Google Scholar]
  45. Ott U., Odermatt E., Engel J., Furthmayr H., Timpl R. Protease resistance and conformation of laminin. Eur J Biochem. 1982 Mar;123(1):63–72. doi: 10.1111/j.1432-1033.1982.tb06499.x. [DOI] [PubMed] [Google Scholar]
  46. Palm S. L., Furcht L. T. Production of laminin and fibronectin by Schwannoma cells: cell-protein interactions in vitro and protein localization in peripheral nerve in vivo. J Cell Biol. 1983 May;96(5):1218–1226. doi: 10.1083/jcb.96.5.1218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Reading C. L. Theory and methods for immunization in culture and monoclonal antibody production. J Immunol Methods. 1982 Sep 30;53(3):261–291. doi: 10.1016/0022-1759(82)90175-2. [DOI] [PubMed] [Google Scholar]
  48. Robinson J., Gospodarowicz D. Glycosaminoglycans synthesized by cultured bovine corneal endothelial cells. J Cell Physiol. 1983 Dec;117(3):368–376. doi: 10.1002/jcp.1041170312. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Rohde H., Bächinger H. P., Timpl R. Characterization of pepsin fragments of laminin in a tumor basement membrane. Evidence for the existence of related proteins. Hoppe Seylers Z Physiol Chem. 1980 Nov;361(11):1651–1660. doi: 10.1515/bchm2.1980.361.2.1651. [DOI] [PubMed] [Google Scholar]
  51. Rohrer H., Thoenen H., Edgar D. Presence of nerve growth factor receptors and catecholamine uptake in subpopulations of chick sympathetic neurons: correlation with survival factor requirements in culture. Dev Biol. 1983 Sep;99(1):34–40. doi: 10.1016/0012-1606(83)90251-8. [DOI] [PubMed] [Google Scholar]
  52. Ruoslahti E., Hayman E. G., Pierschbacher M., Engvall E. Fibronectin: purification, immunochemical properties, and biological activities. Methods Enzymol. 1982;82(Pt A):803–831. doi: 10.1016/0076-6879(82)82103-4. [DOI] [PubMed] [Google Scholar]
  53. Sakashita S., Engvall E., Ruoslahti E. Basement membrane glycoprotein laminin binds to heparin. FEBS Lett. 1980 Jul 28;116(2):243–246. doi: 10.1016/0014-5793(80)80654-5. [DOI] [PubMed] [Google Scholar]
  54. Sanes J. R. Laminin, fibronectin, and collagen in synaptic and extrasynaptic portions of muscle fiber basement membrane. J Cell Biol. 1982 May;93(2):442–451. doi: 10.1083/jcb.93.2.442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Schlesinger M. J., Barrett K. The reversible dissociation of the alkaline phosphatase of Escherichia coli. I. Formation and reactivation of subunits. J Biol Chem. 1965 Nov;240(11):4284–4292. [PubMed] [Google Scholar]
  56. Suissa M. Spectrophotometric quantitation of silver grains eluted from autoradiograms. Anal Biochem. 1983 Sep;133(2):511–514. doi: 10.1016/0003-2697(83)90117-3. [DOI] [PubMed] [Google Scholar]
  57. Timpl R., Johansson S., van Delden V., Oberbäumer I., Hök M. Characterization of protease-resistant fragments of laminin mediating attachment and spreading of rat hepatocytes. J Biol Chem. 1983 Jul 25;258(14):8922–8927. [PubMed] [Google Scholar]
  58. Timpl R., Rohde H., Risteli L., Ott U., Robey P. G., Martin G. R. Laminin. Methods Enzymol. 1982;82(Pt A):831–838. doi: 10.1016/0076-6879(82)82104-6. [DOI] [PubMed] [Google Scholar]
  59. Timpl R., Rohde H., Robey P. G., Rennard S. I., Foidart J. M., Martin G. R. Laminin--a glycoprotein from basement membranes. J Biol Chem. 1979 Oct 10;254(19):9933–9937. [PubMed] [Google Scholar]
  60. Vlodavsky I., Levi A., Lax I., Fuks Z., Schlessinger J. Induction of cell attachment and morphological differentiation in a pheochromocytoma cell line and embryonal sensory cells by the extracellular matrix. Dev Biol. 1982 Oct;93(2):285–300. doi: 10.1016/0012-1606(82)90118-x. [DOI] [PubMed] [Google Scholar]
  61. Warburton M. J., Monaghan P., Ferns S. A., Rudland P. S., Perusinghe N., Chung A. E. Distribution of entactin in the basement membrane of the rat mammary gland. Evidence for a non-epithelial origin. Exp Cell Res. 1984 May;152(1):240–254. doi: 10.1016/0014-4827(84)90249-0. [DOI] [PubMed] [Google Scholar]

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