Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1993 May;12(5):1879–1885. doi: 10.1002/j.1460-2075.1993.tb05836.x

A single EGF-like motif of laminin is responsible for high affinity nidogen binding.

U Mayer 1, R Nischt 1, E Pöschl 1, K Mann 1, K Fukuda 1, M Gerl 1, Y Yamada 1, R Timpl 1
PMCID: PMC413408  PMID: 8491180

Abstract

A major nidogen binding site of mouse laminin was previously localized to about three EGF-like repeats (Nos 3-5) of its B2 chain domain III [M. Gerl et al. (1991) Eur. J. Biochem., 202, 167]. The corresponding cDNA was amplified by polymerase chain reaction and inserted into a eukaryotic expression vector tagged with a signal peptide. Stably transfected human kidney cell clones were shown to process and secrete the resulting fragment B2III3-5 in substantial quantities. It possessed high binding activity for recombinant nidogen in ligand assays, with an affinity comparable with that of authentic laminin fragments. In addition, complexes of B2III3-5 and nidogen could be efficiently converted into a covalent complex by cross-linking reagents. Proteolytic degradation of the covalent complex demonstrated the association of B2III3-5 with a approximately 80 residue segment of nidogen domain G3 to which laminin binding has previously been attributed. The correct formation of most of the 12 disulfide bridges in B2III3-5 was indicated from its protease resistance and the complete loss of cross-reacting epitopes as well as of nidogen-binding activity after reduction and alkylation. Smaller fragments were prepared by the same recombinant procedure and showed that combinations of EGF-like repeats 3-4 and 4-5 and the single repeat 4 but not repeats 3 or 5 possess full nidogen-binding activity. This identifies repeat 4 as the only binding structure. The sequence of repeat 4 is well conserved in the human and in part in the Drosophila laminin B2 chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Full text

PDF
1884

Images in this article

1881Image
on p.1881
1883Image
on p.1883

Selected References

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

  1. Appella E., Weber I. T., Blasi F. Structure and function of epidermal growth factor-like regions in proteins. FEBS Lett. 1988 Apr 11;231(1):1–4. doi: 10.1016/0014-5793(88)80690-2. [DOI] [PubMed] [Google Scholar]
  2. Aumailley M., Battaglia C., Mayer U., Reinhardt D., Nischt R., Timpl R., Fox J. W. Nidogen mediates the formation of ternary complexes of basement membrane components. Kidney Int. 1993 Jan;43(1):7–12. doi: 10.1038/ki.1993.3. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Aumailley M., Wiedemann H., Mann K., Timpl R. Binding of nidogen and the laminin-nidogen complex to basement membrane collagen type IV. Eur J Biochem. 1989 Sep 1;184(1):241–248. doi: 10.1111/j.1432-1033.1989.tb15013.x. [DOI] [PubMed] [Google Scholar]
  5. Battaglia C., Mayer U., Aumailley M., Timpl R. Basement-membrane heparan sulfate proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein core. Eur J Biochem. 1992 Sep 1;208(2):359–366. doi: 10.1111/j.1432-1033.1992.tb17195.x. [DOI] [PubMed] [Google Scholar]
  6. Beck K., Hunter I., Engel J. Structure and function of laminin: anatomy of a multidomain glycoprotein. FASEB J. 1990 Feb 1;4(2):148–160. doi: 10.1096/fasebj.4.2.2404817. [DOI] [PubMed] [Google Scholar]
  7. Chi H. C., Hui C. F. Primary structure of the Drosophila laminin B2 chain and comparison with human, mouse, and Drosophila laminin B1 and B2 chains. J Biol Chem. 1989 Jan 25;264(3):1543–1550. [PubMed] [Google Scholar]
  8. Engel J. EGF-like domains in extracellular matrix proteins: localized signals for growth and differentiation? FEBS Lett. 1989 Jul 17;251(1-2):1–7. doi: 10.1016/0014-5793(89)81417-6. [DOI] [PubMed] [Google Scholar]
  9. Engel J., Schalch W. Antibody binding constants from Farr test and other radioimmunoassays. A theoretical and experimental analysis. Mol Immunol. 1980 May;17(5):675–680. doi: 10.1016/0161-5890(80)90166-2. [DOI] [PubMed] [Google Scholar]
  10. Fessler L. I., Campbell A. G., Duncan K. G., Fessler J. H. Drosophila laminin: characterization and localization. J Cell Biol. 1987 Nov;105(5):2383–2391. doi: 10.1083/jcb.105.5.2383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fox J. W., Mayer U., Nischt R., Aumailley M., Reinhardt D., Wiedemann H., Mann K., Timpl R., Krieg T., Engel J. Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV. EMBO J. 1991 Nov;10(11):3137–3146. doi: 10.1002/j.1460-2075.1991.tb04875.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Geisler N., Schünemann J., Weber K. Chemical cross-linking indicates a staggered and antiparallel protofilament of desmin intermediate filaments and characterizes one higher-level complex between protofilaments. Eur J Biochem. 1992 Jun 15;206(3):841–852. doi: 10.1111/j.1432-1033.1992.tb16992.x. [DOI] [PubMed] [Google Scholar]
  13. Gerl M., Mann K., Aumailley M., Timpl R. Localization of a major nidogen-binding site to domain III of laminin B2 chain. Eur J Biochem. 1991 Nov 15;202(1):167–174. doi: 10.1111/j.1432-1033.1991.tb16358.x. [DOI] [PubMed] [Google Scholar]
  14. Kallunki P., Sainio K., Eddy R., Byers M., Kallunki T., Sariola H., Beck K., Hirvonen H., Shows T. B., Tryggvason K. A truncated laminin chain homologous to the B2 chain: structure, spatial expression, and chromosomal assignment. J Cell Biol. 1992 Nov;119(3):679–693. doi: 10.1083/jcb.119.3.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kallunki P., Tryggvason K. Human basement membrane heparan sulfate proteoglycan core protein: a 467-kD protein containing multiple domains resembling elements of the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor. J Cell Biol. 1992 Jan;116(2):559–571. doi: 10.1083/jcb.116.2.559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lankat-Buttgereit B., Mann K., Deutzmann R., Timpl R., Krieg T. Cloning and complete amino acid sequences of human and murine basement membrane protein BM-40 (SPARC, osteonectin). FEBS Lett. 1988 Aug 29;236(2):352–356. doi: 10.1016/0014-5793(88)80054-1. [DOI] [PubMed] [Google Scholar]
  17. Laurie G. W., Bing J. T., Kleinman H. K., Hassell J. R., Aumailley M., Martin G. R., Feldmann R. J. Localization of binding sites for laminin, heparan sulfate proteoglycan and fibronectin on basement membrane (type IV) collagen. J Mol Biol. 1986 May 5;189(1):205–216. doi: 10.1016/0022-2836(86)90391-8. [DOI] [PubMed] [Google Scholar]
  18. Mann K., Deutzmann R., Aumailley M., Timpl R., Raimondi L., Yamada Y., Pan T. C., Conway D., Chu M. L. Amino acid sequence of mouse nidogen, a multidomain basement membrane protein with binding activity for laminin, collagen IV and cells. EMBO J. 1989 Jan;8(1):65–72. doi: 10.1002/j.1460-2075.1989.tb03349.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mann K., Deutzmann R., Timpl R. Characterization of proteolytic fragments of the laminin-nidogen complex and their activity in ligand-binding assays. Eur J Biochem. 1988 Dec 1;178(1):71–80. doi: 10.1111/j.1432-1033.1988.tb14430.x. [DOI] [PubMed] [Google Scholar]
  20. Mayer U., Aumailley M., Mann K., Timpl R., Engel J. Calcium-dependent binding of basement membrane protein BM-40 (osteonectin, SPARC) to basement membrane collagen type IV. Eur J Biochem. 1991 May 23;198(1):141–150. doi: 10.1111/j.1432-1033.1991.tb15996.x. [DOI] [PubMed] [Google Scholar]
  21. Murdoch A. D., Dodge G. R., Cohen I., Tuan R. S., Iozzo R. V. Primary structure of the human heparan sulfate proteoglycan from basement membrane (HSPG2/perlecan). A chimeric molecule with multiple domains homologous to the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor. J Biol Chem. 1992 Apr 25;267(12):8544–8557. [PubMed] [Google Scholar]
  22. Nischt R., Pottgiesser J., Krieg T., Mayer U., Aumailley M., Timpl R. Recombinant expression and properties of the human calcium-binding extracellular matrix protein BM-40. Eur J Biochem. 1991 Sep 1;200(2):529–536. doi: 10.1111/j.1432-1033.1991.tb16214.x. [DOI] [PubMed] [Google Scholar]
  23. Noonan D. M., Fulle A., Valente P., Cai S., Horigan E., Sasaki M., Yamada Y., Hassell J. R. The complete sequence of perlecan, a basement membrane heparan sulfate proteoglycan, reveals extensive similarity with laminin A chain, low density lipoprotein-receptor, and the neural cell adhesion molecule. J Biol Chem. 1991 Dec 5;266(34):22939–22947. [PubMed] [Google Scholar]
  24. 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]
  25. Panayotou G., End P., Aumailley M., Timpl R., Engel J. Domains of laminin with growth-factor activity. Cell. 1989 Jan 13;56(1):93–101. doi: 10.1016/0092-8674(89)90987-2. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Paulsson M. Basement membrane proteins: structure, assembly, and cellular interactions. Crit Rev Biochem Mol Biol. 1992;27(1-2):93–127. doi: 10.3109/10409239209082560. [DOI] [PubMed] [Google Scholar]
  28. Pikkarainen T., Kallunki T., Tryggvason K. Human laminin B2 chain. Comparison of the complete amino acid sequence with the B1 chain reveals variability in sequence homology between different structural domains. J Biol Chem. 1988 May 15;263(14):6751–6758. [PubMed] [Google Scholar]
  29. Rupp F., Payan D. G., Magill-Solc C., Cowan D. M., Scheller R. H. Structure and expression of a rat agrin. Neuron. 1991 May;6(5):811–823. doi: 10.1016/0896-6273(91)90177-2. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Specks U., Mayer U., Nischt R., Spissinger T., Mann K., Timpl R., Engel J., Chu M. L. Structure of recombinant N-terminal globule of type VI collagen alpha 3 chain and its binding to heparin and hyaluronan. EMBO J. 1992 Dec;11(12):4281–4290. doi: 10.1002/j.1460-2075.1992.tb05527.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Timpl R. Antibodies to collagens and procollagens. Methods Enzymol. 1982;82(Pt A):472–498. doi: 10.1016/0076-6879(82)82079-x. [DOI] [PubMed] [Google Scholar]
  34. Tsim K. W., Ruegg M. A., Escher G., Kröger S., McMahan U. J. cDNA that encodes active agrin. Neuron. 1992 Apr;8(4):677–689. doi: 10.1016/0896-6273(92)90089-v. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES