Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Jul;86(13):4872–4876. doi: 10.1073/pnas.86.13.4872

Identification of critical regions in mouse granulocyte-macrophage colony-stimulating factor by scanning-deletion analysis.

A B Shanafelt 1, R A Kastelein 1
PMCID: PMC297517  PMID: 2662186

Abstract

Structure-function relationships for mouse granulocyte-macrophage colony-stimulating factor were examined by generating a series of small deletions scanning the entire length of the molecule. Deletions of three amino acids were introduced at intervals of five amino acids by site-directed mutagenesis of the mature mouse granulocyte-macrophage colony-stimulating factor gene. The mutant proteins were expressed in Escherichia coli and assayed for biological activity. This procedure identified four regions critical to activity. These critical regions were further delineated by additional three-amino acid deletion mutants. Larger deletions at each terminus were also made, as well as changes of specific amino acid residues. The four critical regions span amino acid residues 18-22, 34-41, 52-61, and 94-115. The disulfide bridge between Cys-51 and Cys-93 was also shown to be essential for activity, whereas that between Cys-85 and Cys-118 could be removed without loss of activity. The possible structural and/or functional roles of the critical regions are discussed.

Full text

PDF
4872

Images in this article

4873Image
on p.4873

Selected References

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

  1. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  2. Clark-Lewis I., Lopez A. F., To L. B., Vadas M. A., Schrader J. W., Hood L. E., Kent S. B. Structure-function studies of human granulocyte-macrophage colony-stimulating factor. Identification of residues required for activity. J Immunol. 1988 Aug 1;141(3):881–889. [PubMed] [Google Scholar]
  3. Ghrayeb J., Kimura H., Takahara M., Hsiung H., Masui Y., Inouye M. Secretion cloning vectors in Escherichia coli. EMBO J. 1984 Oct;3(10):2437–2442. doi: 10.1002/j.1460-2075.1984.tb02151.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gough N. M., Grail D., Gearing D. P., Metcalf D. Mutagenesis of murine granulocyte/macrophage-colony-stimulating factor reveals critical residues near the N terminus. Eur J Biochem. 1987 Dec 1;169(2):353–358. doi: 10.1111/j.1432-1033.1987.tb13619.x. [DOI] [PubMed] [Google Scholar]
  5. Gough N. M., Metcalf D., Gough J., Grail D., Dunn A. R. Structure and expression of the mRNA for murine granulocyte-macrophage colony stimulating factor. EMBO J. 1985 Mar;4(3):645–653. doi: 10.1002/j.1460-2075.1985.tb03678.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. HOWARD-FLANDERS P., SIMSON E., THERIOT L. A LOCUS THAT CONTROLS FILAMENT FORMATION AND SENSITIVITY TO RADIATION IN ESCHERICHIA COLI K-12. Genetics. 1964 Feb;49:237–246. doi: 10.1093/genetics/49.2.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Koshland D., Botstein D. Secretion of beta-lactamase requires the carboxy end of the protein. Cell. 1980 Jul;20(3):749–760. doi: 10.1016/0092-8674(80)90321-9. [DOI] [PubMed] [Google Scholar]
  8. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Lee F., Yokota T., Otsuka T., Gemmell L., Larson N., Luh J., Arai K., Rennick D. Isolation of cDNA for a human granulocyte-macrophage colony-stimulating factor by functional expression in mammalian cells. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4360–4364. doi: 10.1073/pnas.82.13.4360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lopez A. F., Williamson D. J., Gamble J. R., Begley C. G., Harlan J. M., Klebanoff S. J., Waltersdorph A., Wong G., Clark S. C., Vadas M. A. Recombinant human granulocyte-macrophage colony-stimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression, and survival. J Clin Invest. 1986 Nov;78(5):1220–1228. doi: 10.1172/JCI112705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  13. Metcalf D. The granulocyte-macrophage colony-stimulating factors. Science. 1985 Jul 5;229(4708):16–22. doi: 10.1126/science.2990035. [DOI] [PubMed] [Google Scholar]
  14. Metcalf D. The molecular biology and functions of the granulocyte-macrophage colony-stimulating factors. Blood. 1986 Feb;67(2):257–267. [PubMed] [Google Scholar]
  15. Miyajima A., Otsu K., Schreurs J., Bond M. W., Abrams J. S., Arai K. Expression of murine and human granulocyte-macrophage colony-stimulating factors in S. cerevisiae: mutagenesis of the potential glycosylation sites. EMBO J. 1986 Jun;5(6):1193–1197. doi: 10.1002/j.1460-2075.1986.tb04346.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983 Dec 16;65(1-2):55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]
  17. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Schrimsher J. L., Rose K., Simona M. G., Wingfield P. Characterization of human and mouse granulocyte-macrophage-colony-stimulating factors derived from Escherichia coli. Biochem J. 1987 Oct 1;247(1):195–199. doi: 10.1042/bj2470195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Wingfield P., Graber P., Moonen P., Craig S., Pain R. H. The conformation and stability of recombinant-derived granulocyte-macrophage colony stimulating factors. Eur J Biochem. 1988 Apr 5;173(1):65–72. doi: 10.1111/j.1432-1033.1988.tb13967.x. [DOI] [PubMed] [Google Scholar]
  21. Zurawski S. M., Zurawski G. Identification of three critical regions within mouse interleukin 2 by fine structural deletion analysis. EMBO J. 1988 Apr;7(4):1061–1069. doi: 10.1002/j.1460-2075.1988.tb02914.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES