Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1986 Nov;5(11):2825–2830. doi: 10.1002/j.1460-2075.1986.tb04575.x

Mapping the collagen-binding site of human fibronectin by expression in Escherichia coli.

R J Owens, F E Baralle
PMCID: PMC1167230  PMID: 3024962

Abstract

The collagen-binding domain of human fibronectin has been expressed as a cro/beta-galactosidase fusion protein in Escherichia coli. The hybrid polypeptide was recognized by an anti-(human plasma fibronectin) serum and bound specifically to gelatin-Sepharose. The collagen-binding region was subdivided by constructing a series of overlapping bacterial expression plasmids. The fusion proteins produced by these constructs were analysed for gelatin-binding activity. The results indicate that the binding site lies within an approximately 12.5 kd fragment of fibronectin, and show that the following 14 amino acid sequence is critical for gelatin-binding activity: Ala-Ala-His-Glu-Glu-Ile-Cys-Thr-Thr-Asn-Glu-Gly-Val-Met. This sequence links the second type II homology unit with the adjacent type I repeat in the amino-terminal third of the fibronectin molecule.

Full text

PDF
2825

Images in this article

2827Fig. 2.
on p.2827
2828Fig. 3.
on p.2828

Selected References

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

  1. Balian G., Click E. M., Crouch E., Davidson J. M., Bornstein P. Isolation of a collagen-binding fragment from fibronectin and cold-insoluble globulin. J Biol Chem. 1979 Mar 10;254(5):1429–1432. [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Cheng Y. S. Increased cell buoyant densities of protein overproducing Escherichia coli cells. Biochem Biophys Res Commun. 1983 Feb 28;111(1):104–111. doi: 10.1016/s0006-291x(83)80123-5. [DOI] [PubMed] [Google Scholar]
  4. Dessau W., Adelmann B. C., Timpl R. Identification of the sites in collagen alpha-chains that bind serum anti-gelatin factor (cold-insoluble globulin). Biochem J. 1978 Jan 1;169(1):55–59. doi: 10.1042/bj1690055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Engvall E., Ruoslahti E. Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. Int J Cancer. 1977 Jul 15;20(1):1–5. doi: 10.1002/ijc.2910200102. [DOI] [PubMed] [Google Scholar]
  6. Engvall E., Ruoslahti E., Miller E. J. Affinity of fibronectin to collagens of different genetic types and to fibrinogen. J Exp Med. 1978 Jun 1;147(6):1584–1595. doi: 10.1084/jem.147.6.1584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Forastieri H., Ingham K. C. Interaction of gelatin with a fluorescein-labeled 42-kDa chymotryptic fragment of fibronectin. J Biol Chem. 1985 Sep 5;260(19):10546–10550. [PubMed] [Google Scholar]
  8. Gergen J. P., Stern R. H., Wensink P. C. Filter replicas and permanent collections of recombinant DNA plasmids. Nucleic Acids Res. 1979 Dec 20;7(8):2115–2136. doi: 10.1093/nar/7.8.2115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gold L. I., Garcia-Pardo A., Frangione B., Franklin E. C., Pearlstein E. Subtilisin and cyanogen bromide cleavage products of fibronectin that retain gelatin-binding activity. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4803–4807. doi: 10.1073/pnas.76.10.4803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hynes R. O., Yamada K. M. Fibronectins: multifunctional modular glycoproteins. J Cell Biol. 1982 Nov;95(2 Pt 1):369–377. doi: 10.1083/jcb.95.2.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hynes R. Molecular biology of fibronectin. Annu Rev Cell Biol. 1985;1:67–90. doi: 10.1146/annurev.cb.01.110185.000435. [DOI] [PubMed] [Google Scholar]
  12. Kleinman H. K., McGoodwin E. B. Localization of the cell attachment region in types I and II collagens. Biochem Biophys Res Commun. 1976 Sep 20;72(2):426–432. doi: 10.1016/s0006-291x(76)80060-5. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  16. McDonald J. A., Kelley D. G., Broekelmann T. J. Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix. J Cell Biol. 1982 Feb;92(2):485–492. doi: 10.1083/jcb.92.2.485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nagata K., Humphries M. J., Olden K., Yamada K. M. Collagen can modulate cell interactions with fibronectin. J Cell Biol. 1985 Aug;101(2):386–394. doi: 10.1083/jcb.101.2.386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Owen M. J., Kissonerghis A. M., Lodish H. F. Biosynthesis of HLA-A and HLA-B antigens in vivo. J Biol Chem. 1980 Oct 25;255(20):9678–9684. [PubMed] [Google Scholar]
  19. Petersen T. E., Thøgersen H. C., Skorstengaard K., Vibe-Pedersen K., Sahl P., Sottrup-Jensen L., Magnusson S. Partial primary structure of bovine plasma fibronectin: three types of internal homology. Proc Natl Acad Sci U S A. 1983 Jan;80(1):137–141. doi: 10.1073/pnas.80.1.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Petersen T. E., Thøgersen H. C., Skorstengaard K., Vibe-Pedersen K., Sahl P., Sottrup-Jensen L., Magnusson S. Partial primary structure of bovine plasma fibronectin: three types of internal homology. Proc Natl Acad Sci U S A. 1983 Jan;80(1):137–141. doi: 10.1073/pnas.80.1.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pierschbacher M. D., Ruoslahti E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature. 1984 May 3;309(5963):30–33. doi: 10.1038/309030a0. [DOI] [PubMed] [Google Scholar]
  22. Remaut E., Tsao H., Fiers W. Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication. Gene. 1983 Apr;22(1):103–113. doi: 10.1016/0378-1119(83)90069-0. [DOI] [PubMed] [Google Scholar]
  23. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  24. Ruoslahti E., Engvall E. Immunochemical and collagen-binding properties of fibronectin. Ann N Y Acad Sci. 1978 Jun 20;312:178–191. doi: 10.1111/j.1749-6632.1978.tb16802.x. [DOI] [PubMed] [Google Scholar]
  25. Ruoslahti E., Hayman E. G., Kuusela P., Shively J. E., Engvall E. Isolation of a tryptic fragment containing the collagen-binding site of plasma fibronectin. J Biol Chem. 1979 Jul 10;254(13):6054–6059. [PubMed] [Google Scholar]
  26. 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]
  27. Rüther U., Wagner E. F., Müller R. Analysis of the differentiation-promoting potential of inducible c-fos genes introduced into embryonal carcinoma cells. EMBO J. 1985 Jul;4(7):1775–1781. doi: 10.1002/j.1460-2075.1985.tb03850.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Skorstengaard K., Thøgersen H. C., Petersen T. E. Complete primary structure of the collagen-binding domain of bovine fibronectin. Eur J Biochem. 1984 Apr 16;140(2):235–243. doi: 10.1111/j.1432-1033.1984.tb08092.x. [DOI] [PubMed] [Google Scholar]
  29. Skorstengaard K., Thøgersen H. C., Vibe-Pedersen K., Petersen T. E., Magnusson S. Purification of twelve cyanogen bromide fragments from bovine plasma fibronectin and the amino acid sequence of eight of them. Overlap evidence aligning two plasmic fragments, internal homology in gelatin-binding region and phosphorylation site near C terminus. Eur J Biochem. 1982 Nov 15;128(2-3):605–623. doi: 10.1111/j.1432-1033.1982.tb07007.x. [DOI] [PubMed] [Google Scholar]
  30. Stanley K. K., Luzio J. P. Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins. EMBO J. 1984 Jun;3(6):1429–1434. doi: 10.1002/j.1460-2075.1984.tb01988.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stanley K. K. Solubilization and immune-detection of beta-galactosidase hybrid proteins carrying foreign antigenic determinants. Nucleic Acids Res. 1983 Jun 25;11(12):4077–4092. doi: 10.1093/nar/11.12.4077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Vaheri A., Kurkinen M., Lehto V. P., Linder E., Timpl R. Codistribution of pericellular matrix proteins in cultured fibroblasts and loss in transformation: fibronectin and procollagen. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4944–4948. doi: 10.1073/pnas.75.10.4944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vuento M., Salonen E., Osterlund K., Stenman U. H. Essential charged amino acids in the binding of fibronectin to gelatin. Biochem J. 1982 Jan 1;201(1):1–8. doi: 10.1042/bj2010001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Williams D. C., Van Frank R. M., Muth W. L., Burnett J. P. Cytoplasmic inclusion bodies in Escherichia coli producing biosynthetic human insulin proteins. Science. 1982 Feb 5;215(4533):687–689. doi: 10.1126/science.7036343. [DOI] [PubMed] [Google Scholar]
  36. Williams E. C., Janmey P. A., Ferry J. D., Mosher D. F. Conformational states of fibronectin. Effects of pH, ionic strength, and collagen binding. J Biol Chem. 1982 Dec 25;257(24):14973–14978. [PubMed] [Google Scholar]
  37. Zabeau M., Stanley K. K. Enhanced expression of cro-beta-galactosidase fusion proteins under the control of the PR promoter of bacteriophage lambda. EMBO J. 1982;1(10):1217–1224. doi: 10.1002/j.1460-2075.1982.tb00016.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES