Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 May 1;113(3):637–644. doi: 10.1083/jcb.113.3.637

Laminin fragment E8 mediates PC12 cell neurite outgrowth by binding to cell surface beta 1,4 galactosyltransferase

PMCID: PMC2288965  PMID: 1901868

Abstract

A number of cell surface receptors bind to distinct laminin domains, thereby mediating laminin's diverse biological activities. Cell surface beta 1,4-galactosyltransferase (GalTase) functions as one of these laminin receptors, facilitating mesenchymal cell migration and PC12 cell neurite outgrowth on laminin. In this study, the GalTase binding site within laminin was identified as the E8 fragment by assaying purified fragments and by immunoprecipitating and immunoblotting galactosylated laminin using E8-reactive antibodies. Compared with intact laminin and other laminin fragments, E8 possessed the highest GalTase binding activity, using both membrane-bound and solubilized GalTase. More significantly, the neurite-promoting activity of fragment E8 was shown to be dependent upon its interaction with GalTase. Pregalactosylating purified E8 eliminated subsequent GalTase binding and consequently inhibited neurite initiation; parallel studies on laminin fragments E1-4 or E1 failed to affect neurite outgrowth. Furthermore, anti-GalTase IgG inhibited neurite initiation on purified E8 substrates; control IgG had no effect. These results localize the predominant GalTase binding domain in laminin to fragment E8 and demonstrate that the neurite-promoting activity of E8 is dependent upon its interaction with GalTase.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

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

  1. Arumugham R. G., Hsieh T. C., Tanzer M. L., Laine R. A. Structures of the asparagine-linked sugar chains of laminin. Biochim Biophys Acta. 1986 Aug 6;883(1):112–126. doi: 10.1016/0304-4165(86)90142-x. [DOI] [PubMed] [Google Scholar]
  2. Aumailley M., Gerl M., Sonnenberg A., Deutzmann R., Timpl R. Identification of the Arg-Gly-Asp sequence in laminin A chain as a latent cell-binding site being exposed in fragment P1. FEBS Lett. 1990 Mar 12;262(1):82–86. doi: 10.1016/0014-5793(90)80159-g. [DOI] [PubMed] [Google Scholar]
  3. Aumailley M., Nurcombe V., Edgar D., Paulsson M., Timpl R. The cellular interactions of laminin fragments. Cell adhesion correlates with two fragment-specific high affinity binding sites. J Biol Chem. 1987 Aug 25;262(24):11532–11538. [PubMed] [Google Scholar]
  4. Bayna E. M., Shaper J. H., Shur B. D. Temporally specific involvement of cell surface beta-1,4 galactosyltransferase during mouse embryo morula compaction. Cell. 1988 Apr 8;53(1):145–157. doi: 10.1016/0092-8674(88)90496-5. [DOI] [PubMed] [Google Scholar]
  5. Begovac P. C., Shur B. D. Cell surface galactosyltransferase mediates the initiation of neurite outgrowth from PC12 cells on laminin. J Cell Biol. 1990 Feb;110(2):461–470. doi: 10.1083/jcb.110.2.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bozyczko D., Horwitz A. F. The participation of a putative cell surface receptor for laminin and fibronectin in peripheral neurite extension. J Neurosci. 1986 May;6(5):1241–1251. doi: 10.1523/JNEUROSCI.06-05-01241.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Charonis A. S., Skubitz A. P., Koliakos G. G., Reger L. A., Dege J., Vogel A. M., Wohlhueter R., Furcht L. T. A novel synthetic peptide from the B1 chain of laminin with heparin-binding and cell adhesion-promoting activities. J Cell Biol. 1988 Sep;107(3):1253–1260. doi: 10.1083/jcb.107.3.1253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Charonis A. S., Tsilibary E. C., Saku T., Furthmayr H. Inhibition of laminin self-assembly and interaction with type IV collagen by antibodies to the terminal domain of the long arm. J Cell Biol. 1986 Nov;103(5):1689–1697. doi: 10.1083/jcb.103.5.1689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Charonis A. S., Tsilibary E. C., Yurchenco P. D., Furthmayr H. Binding of laminin to type IV collagen: a morphological study. J Cell Biol. 1985 Jun;100(6):1848–1853. doi: 10.1083/jcb.100.6.1848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chung A. E., Jaffe R., Freeman I. L., Vergnes J. P., Braginski J. E., Carlin B. Properties of a basement membrane-related glycoprotein synthesized in culture by a mouse embryonal carcinoma-derived cell line. Cell. 1979 Feb;16(2):277–287. doi: 10.1016/0092-8674(79)90005-9. [DOI] [PubMed] [Google Scholar]
  11. Dannenberg A., Rotenberg M., Zakim D. Regulation of UDP-glucuronosyltransferase by lipid-protein interactions. Comparison of the thermotropic properties of pure reconstituted enzyme with microsomal enzyme. J Biol Chem. 1989 Jan 5;264(1):238–242. [PubMed] [Google Scholar]
  12. Dean J. W., 3rd, Chandrasekaran S., Tanzer M. L. A biological role of the carbohydrate moieties of laminin. J Biol Chem. 1990 Jul 25;265(21):12553–12562. [PubMed] [Google Scholar]
  13. Dean J. W., 3rd, Chandrasekaran S., Tanzer M. L. Lectins inhibit cell binding and spreading on a laminin substrate. Biochem Biophys Res Commun. 1988 Oct 14;156(1):411–416. doi: 10.1016/s0006-291x(88)80856-8. [DOI] [PubMed] [Google Scholar]
  14. Deutzmann R., Huber J., Schmetz K. A., Oberbäumer I., Hartl L. Structural study of long arm fragments of laminin. Evidence for repetitive C-terminal sequences in the A-chain, not present in the B-chains. Eur J Biochem. 1988 Oct 15;177(1):35–45. doi: 10.1111/j.1432-1033.1988.tb14342.x. [DOI] [PubMed] [Google Scholar]
  15. Douville P. J., Harvey W. J., Carbonetto S. Isolation and partial characterization of high affinity laminin receptors in neural cells. J Biol Chem. 1988 Oct 15;263(29):14964–14969. [PubMed] [Google Scholar]
  16. Eckstein D. J., Shur B. D. Laminin induces the stable expression of surface galactosyltransferase on lamellipodia of migrating cells. J Cell Biol. 1989 Jun;108(6):2507–2517. doi: 10.1083/jcb.108.6.2507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Edgar D., Timpl R., Thoenen H. Structural requirements for the stimulation of neurite outgrowth by two variants of laminin and their inhibition by antibodies. J Cell Biol. 1988 Apr;106(4):1299–1306. doi: 10.1083/jcb.106.4.1299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Engvall E., Davis G. E., Dickerson K., Ruoslahti E., Varon S., Manthorpe M. Mapping of domains in human laminin using monoclonal antibodies: localization of the neurite-promoting site. J Cell Biol. 1986 Dec;103(6 Pt 1):2457–2465. doi: 10.1083/jcb.103.6.2457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Engvall E., Krusius T., Wewer U., Ruoslahti E. Laminin from rat yolk sac tumor: isolation, partial characterization, and comparison with mouse laminin. Arch Biochem Biophys. 1983 Apr 15;222(2):649–656. doi: 10.1016/0003-9861(83)90562-3. [DOI] [PubMed] [Google Scholar]
  21. Fujiwara S., Shinkai H., Deutzmann R., Paulsson M., Timpl R. Structure and distribution of N-linked oligosaccharide chains on various domains of mouse tumour laminin. Biochem J. 1988 Jun 1;252(2):453–461. doi: 10.1042/bj2520453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Goodman S. L., Deutzmann R., von der Mark K. Two distinct cell-binding domains in laminin can independently promote nonneuronal cell adhesion and spreading. J Cell Biol. 1987 Jul;105(1):589–598. doi: 10.1083/jcb.105.1.589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Graf J., Iwamoto Y., Sasaki M., Martin G. R., Kleinman H. K., Robey F. A., Yamada Y. Identification of an amino acid sequence in laminin mediating cell attachment, chemotaxis, and receptor binding. Cell. 1987 Mar 27;48(6):989–996. doi: 10.1016/0092-8674(87)90707-0. [DOI] [PubMed] [Google Scholar]
  24. Greene L. A., Tischler A. S. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2424–2428. doi: 10.1073/pnas.73.7.2424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hall D. E., Reichardt L. F., Crowley E., Holley B., Moezzi H., Sonnenberg A., Damsky C. H. The alpha 1/beta 1 and alpha 6/beta 1 integrin heterodimers mediate cell attachment to distinct sites on laminin. J Cell Biol. 1990 Jun;110(6):2175–2184. doi: 10.1083/jcb.110.6.2175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hathaway H. J., Romagnano L. C., Babiarz B. S. Analysis of cell surface galactosyltransferase activity during mouse trophectodermal differentiation. Dev Biol. 1989 Aug;134(2):351–361. doi: 10.1016/0012-1606(89)90107-3. [DOI] [PubMed] [Google Scholar]
  27. Horwitz A., Duggan K., Greggs R., Decker C., Buck C. The cell substrate attachment (CSAT) antigen has properties of a receptor for laminin and fibronectin. J Cell Biol. 1985 Dec;101(6):2134–2144. doi: 10.1083/jcb.101.6.2134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kleinman H. K., Cannon F. B., Laurie G. W., Hassell J. R., Aumailley M., Terranova V. P., Martin G. R., DuBois-Dalcq M. Biological activities of laminin. J Cell Biochem. 1985;27(4):317–325. doi: 10.1002/jcb.240270402. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Kleinman H. K., Ogle R. C., Cannon F. B., Little C. D., Sweeney T. M., Luckenbill-Edds L. Laminin receptors for neurite formation. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1282–1286. doi: 10.1073/pnas.85.4.1282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Knibbs R. N., Perini F., Goldstein I. J. Structure of the major concanavalin A reactive oligosaccharides of the extracellular matrix component laminin. Biochemistry. 1989 Jul 25;28(15):6379–6392. doi: 10.1021/bi00441a034. [DOI] [PubMed] [Google Scholar]
  32. Kouzi-Koliakos K., Koliakos G. G., Tsilibary E. C., Furcht L. T., Charonis A. S. Mapping of three major heparin-binding sites on laminin and identification of a novel heparin-binding site on the B1 chain. J Biol Chem. 1989 Oct 25;264(30):17971–17978. [PubMed] [Google Scholar]
  33. Liesi P., Närvänen A., Soos J., Sariola H., Snounou G. Identification of a neurite outgrowth-promoting domain of laminin using synthetic peptides. FEBS Lett. 1989 Feb 13;244(1):141–148. doi: 10.1016/0014-5793(89)81180-9. [DOI] [PubMed] [Google Scholar]
  34. Lopez L. C., Bayna E. M., Litoff D., Shaper N. L., Shaper J. H., Shur B. D. Receptor function of mouse sperm surface galactosyltransferase during fertilization. J Cell Biol. 1985 Oct;101(4):1501–1510. doi: 10.1083/jcb.101.4.1501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lopez L. C., Maillet C. M., Oleszkowicz K., Shur B. D. Cell surface and Golgi pools of beta-1,4-galactosyltransferase are differentially regulated during embryonal carcinoma cell differentiation. Mol Cell Biol. 1989 Jun;9(6):2370–2377. doi: 10.1128/mcb.9.6.2370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Malinoff H. L., Wicha M. S. Isolation of a cell surface receptor protein for laminin from murine fibrosarcoma cells. J Cell Biol. 1983 May;96(5):1475–1479. doi: 10.1083/jcb.96.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Martin G. R., Timpl R. Laminin and other basement membrane components. Annu Rev Cell Biol. 1987;3:57–85. doi: 10.1146/annurev.cb.03.110187.000421. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Paulsson M., Aumailley M., Deutzmann R., Timpl R., Beck K., Engel J. Laminin-nidogen complex. Extraction with chelating agents and structural characterization. Eur J Biochem. 1987 Jul 1;166(1):11–19. doi: 10.1111/j.1432-1033.1987.tb13476.x. [DOI] [PubMed] [Google Scholar]
  40. Paulsson M., Deutzmann R., Timpl R., Dalzoppo D., Odermatt E., Engel J. Evidence for coiled-coil alpha-helical regions in the long arm of laminin. EMBO J. 1985 Feb;4(2):309–316. doi: 10.1002/j.1460-2075.1985.tb03630.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rao C. N., Margulies I. M., Tralka T. S., Terranova V. P., Madri J. A., Liotta L. A. Isolation of a subunit of laminin and its role in molecular structure and tumor cell attachment. J Biol Chem. 1982 Aug 25;257(16):9740–9744. [PubMed] [Google Scholar]
  42. Runyan R. B., Versalovic J., Shur B. D. Functionally distinct laminin receptors mediate cell adhesion and spreading: the requirement for surface galactosyltransferase in cell spreading. J Cell Biol. 1988 Nov;107(5):1863–1871. doi: 10.1083/jcb.107.5.1863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Sasaki M., Kato S., Kohno K., Martin G. R., Yamada Y. Sequence of the cDNA encoding the laminin B1 chain reveals a multidomain protein containing cysteine-rich repeats. Proc Natl Acad Sci U S A. 1987 Feb;84(4):935–939. doi: 10.1073/pnas.84.4.935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sasaki M., Kleinman H. K., Huber H., Deutzmann R., Yamada Y. Laminin, a multidomain protein. The A chain has a unique globular domain and homology with the basement membrane proteoglycan and the laminin B chains. J Biol Chem. 1988 Nov 15;263(32):16536–16544. [PubMed] [Google Scholar]
  46. Sasaki M., Yamada Y. The laminin B2 chain has a multidomain structure homologous to the B1 chain. J Biol Chem. 1987 Dec 15;262(35):17111–17117. [PubMed] [Google Scholar]
  47. Sephel G. C., Burrous B. A., Kleinman H. K. Laminin neural activity and binding proteins. Dev Neurosci. 1989;11(4-5):313–331. doi: 10.1159/000111909. [DOI] [PubMed] [Google Scholar]
  48. Sephel G. C., Tashiro K. I., Sasaki M., Greatorex D., Martin G. R., Yamada Y., Kleinman H. K. Laminin A chain synthetic peptide which supports neurite outgrowth. Biochem Biophys Res Commun. 1989 Jul 31;162(2):821–829. doi: 10.1016/0006-291x(89)92384-x. [DOI] [PubMed] [Google Scholar]
  49. Sephel G. C., Tashiro K., Sasaki M., Kandel S., Yamada Y., Kleinman H. K. A laminin-pepsin fragment with cell attachment and neurite outgrowth activity at distinct sites. Dev Biol. 1989 Sep;135(1):172–181. doi: 10.1016/0012-1606(89)90167-x. [DOI] [PubMed] [Google Scholar]
  50. Shur B. D. Cell surface glycosyltransferase activities during normal and mutant (T/T) mesenchyme migration. Dev Biol. 1982 May;91(1):149–162. doi: 10.1016/0012-1606(82)90018-5. [DOI] [PubMed] [Google Scholar]
  51. Shur B. D., Hall N. G. A role for mouse sperm surface galactosyltransferase in sperm binding to the egg zona pellucida. J Cell Biol. 1982 Nov;95(2 Pt 1):574–579. doi: 10.1083/jcb.95.2.574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tashiro K., Sephel G. C., Weeks B., Sasaki M., Martin G. R., Kleinman H. K., Yamada Y. A synthetic peptide containing the IKVAV sequence from the A chain of laminin mediates cell attachment, migration, and neurite outgrowth. J Biol Chem. 1989 Sep 25;264(27):16174–16182. [PubMed] [Google Scholar]
  53. Terranova V. P., Rao C. N., Kalebic T., Margulies I. M., Liotta L. A. Laminin receptor on human breast carcinoma cells. Proc Natl Acad Sci U S A. 1983 Jan;80(2):444–448. doi: 10.1073/pnas.80.2.444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. 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]
  56. Tomaselli K. J., Damsky C. H., Reichardt L. F. Interactions of a neuronal cell line (PC12) with laminin, collagen IV, and fibronectin: identification of integrin-related glycoproteins involved in attachment and process outgrowth. J Cell Biol. 1987 Nov;105(5):2347–2358. doi: 10.1083/jcb.105.5.2347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Tomaselli K. J., Damsky C. H., Reichardt L. F. Purification and characterization of mammalian integrins expressed by a rat neuronal cell line (PC12): evidence that they function as alpha/beta heterodimeric receptors for laminin and type IV collagen. J Cell Biol. 1988 Sep;107(3):1241–1252. doi: 10.1083/jcb.107.3.1241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Turley E. A., Roth S. Spontaneous glycosylation of glycosaminoglycan substrates by adherent fibroblasts. Cell. 1979 May;17(1):109–115. doi: 10.1016/0092-8674(79)90299-x. [DOI] [PubMed] [Google Scholar]
  59. Wallace R. A., Selman K. Major protein changes during vitellogenesis and maturation of Fundulus oocytes. Dev Biol. 1985 Aug;110(2):492–498. doi: 10.1016/0012-1606(85)90106-x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES