Skip to main content
NCBI 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
. 1993 Nov 1;90(21):10056–10060. doi: 10.1073/pnas.90.21.10056

Molecular cloning and functional expression of a cDNA encoding glycosylation-inhibiting factor.

T Mikayama 1, T Nakano 1, H Gomi 1, Y Nakagawa 1, Y C Liu 1, M Sato 1, A Iwamatsu 1, Y Ishii 1, W Y Weiser 1, K Ishizaka 1
PMCID: PMC47712  PMID: 8234256

Abstract

By using probes based on partial amino acid sequence of glycosylation-inhibiting factor (GIF) from a mouse T-cell hybridoma, a full-length cDNA encoding mouse GIF was isolated. A cDNA clone encoding human GIF was isolated from cDNA libraries of a GIF-producing human T-cell hybridoma by using mouse GIF cDNA as a probe. The cDNAs encode a putative 12.5-kDa peptide of 115 amino acids. Northern blot analysis demonstrated a single, 0.6-kb transcript. Polyclonal rabbit antibodies against the Escherichia coli-derived recombinant 13-kDa peptide bound hybridoma-derived GIF. Although the peptide did not contain a signal peptide sequence, transfection of the cDNA into COS-1 cells resulted in secretion of 13-kDa peptide, but the peptide had substantially less bioactivity than the hybridoma-derived GIF. However, expression of a chimeric cDNA encoding a fusion protein consisting of the N-terminal pro region of calcitonin precursor and human GIF and cotransfection with furin cDNA to allow intracellular cleavage of the fusion protein resulted in secretion of 13-kDa peptide that was comparable to hybridoma-derived GIF in its bioactivity. Both the 13-kDa peptide and GIF bioactivity in the transfected COS-1 supernatant bound to a monoclonal antibody against hybridoma-derived human GIF. These results indicate that the 13-kDa peptide represents recombinant GIF, but posttranslational modification of the peptide is important for generation of the bioactivity. The GIF cDNA had high homology with the cDNA encoding macrophage migration inhibitory factor. However, the recombinant GIF failed to inhibit migration of human monocytes, and recombinant human macrophage migration inhibitory factor did not have GIF bioactivity.

Full text

PDF
10056

Images in this article

10058Fig. 2
on p.10058
10059Fig. 3
on p.10059

Selected References

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

  1. Akasaki M., Jardieu P., Ishizaka K. Immunosuppressive effects of glycosylation inhibiting factor on the IgE and IgG antibody response. J Immunol. 1986 May 1;136(9):3172–3179. [PubMed] [Google Scholar]
  2. Hosaka M., Nagahama M., Kim W. S., Watanabe T., Hatsuzawa K., Ikemizu J., Murakami K., Nakayama K. Arg-X-Lys/Arg-Arg motif as a signal for precursor cleavage catalyzed by furin within the constitutive secretory pathway. J Biol Chem. 1991 Jul 5;266(19):12127–12130. [PubMed] [Google Scholar]
  3. Ishizaka K. Regulation of IgE synthesis. Annu Rev Immunol. 1984;2:159–182. doi: 10.1146/annurev.iy.02.040184.001111. [DOI] [PubMed] [Google Scholar]
  4. Iwamatsu A., Aoyama H., Dibó G., Tsunasawa S., Sakiyama F. Amino acid sequence of nuclease S1 from Aspergillus oryzae. J Biochem. 1991 Jul;110(1):151–158. doi: 10.1093/oxfordjournals.jbchem.a123534. [DOI] [PubMed] [Google Scholar]
  5. Iwamatsu A. S-carboxymethylation of proteins transferred onto polyvinylidene difluoride membranes followed by in situ protease digestion and amino acid microsequencing. Electrophoresis. 1992 Mar;13(3):142–147. doi: 10.1002/elps.1150130129. [DOI] [PubMed] [Google Scholar]
  6. Iwata M., Ishizaka K. Construction of antigen-specific suppressor T cell hybridomas from spleen cells of mice primed for the persistent IgE antibody formation. J Immunol. 1988 Nov 15;141(10):3270–3277. [PubMed] [Google Scholar]
  7. Jardieu P., Akasaki M., Ishizaka K. Carrier-specific suppression of antibody responses by antigen-specific glycosylation-inhibiting factors. J Immunol. 1987 Mar 1;138(5):1494–1501. [PubMed] [Google Scholar]
  8. Kozak M. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev. 1983 Mar;47(1):1–45. doi: 10.1128/mr.47.1.1-45.1983. [DOI] [PMC free article] [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. Lanahan A., Williams J. B., Sanders L. K., Nathans D. Growth factor-induced delayed early response genes. Mol Cell Biol. 1992 Sep;12(9):3919–3929. doi: 10.1128/mcb.12.9.3919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Liu Y. C., Kawagishi M., Mikayama T., Inagaki Y., Takeuchi T., Ohashi H. Processing of a fusion protein by endoprotease in COS-1 cells for secretion of mature peptide by using a chimeric expression vector. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):8957–8961. doi: 10.1073/pnas.90.19.8957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Matsuki S., Ozawa T., Nagao S., Hirata H., Kanoh H., Nozawa Y. High-level expression of complementary DNA encoding rat calmodulin in Escherichia coli. Biotechnol Appl Biochem. 1990 Jun;12(3):284–291. [PubMed] [Google Scholar]
  13. Nichols B. P., Yanofsky C. Plasmids containing the trp promoters of Escherichia coli and Serratia marcescens and their use in expressing cloned genes. Methods Enzymol. 1983;101:155–164. doi: 10.1016/0076-6879(83)01011-3. [DOI] [PubMed] [Google Scholar]
  14. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  15. Remold H. G., Mednis A. D. Migration inhibitory factor. Methods Enzymol. 1985;116:379–394. doi: 10.1016/s0076-6879(85)16030-1. [DOI] [PubMed] [Google Scholar]
  16. Rubartelli A., Cozzolino F., Talio M., Sitia R. A novel secretory pathway for interleukin-1 beta, a protein lacking a signal sequence. EMBO J. 1990 May;9(5):1503–1510. doi: 10.1002/j.1460-2075.1990.tb08268.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Steele J. K., Kuchroo V. K., Kawasaki H., Jayaraman S., Iwata M., Ishizaka K., Dorf M. E. A monoclonal antibody raised to lipomodulin recognizes T suppressor factors in two independent hapten-specific suppressor networks. J Immunol. 1989 Apr 1;142(7):2213–2220. [PubMed] [Google Scholar]
  18. Tagaya Y., Mori A., Ishizaka K. Biochemical characterization of murine glycosylation-inhibiting factor. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9117–9121. doi: 10.1073/pnas.88.20.9117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Takebe Y., Seiki M., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol. 1988 Jan;8(1):466–472. doi: 10.1128/mcb.8.1.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Thomas P., Gomi H., Takeuchi T., Carini C., Tagaya Y., Ishizaka K. Glycosylation-inhibiting factor from human T cell hybridomas constructed from peripheral blood lymphocytes of a bee venom-sensitive allergic patient. J Immunol. 1992 Feb 1;148(3):729–737. [PubMed] [Google Scholar]
  21. Weiser W. Y., Greineder D. K., Remold H. G., David J. R. Studies on human migration inhibitory factor: characterization of three molecular species. J Immunol. 1981 May;126(5):1958–1962. [PubMed] [Google Scholar]
  22. Weiser W. Y., Temple P. A., Witek-Giannotti J. S., Remold H. G., Clark S. C., David J. R. Molecular cloning of a cDNA encoding a human macrophage migration inhibitory factor. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7522–7526. doi: 10.1073/pnas.86.19.7522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Weiser W. Y., Temple P. A., Witek-Giannotti J. S., Remold H. G., Clark S. C., David J. R. Molecular cloning of a cDNA encoding a human macrophage migration inhibitory factor. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7522–7526. doi: 10.1073/pnas.86.19.7522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wise R. J., Barr P. J., Wong P. A., Kiefer M. C., Brake A. J., Kaufman R. J. Expression of a human proprotein processing enzyme: correct cleavage of the von Willebrand factor precursor at a paired basic amino acid site. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9378–9382. doi: 10.1073/pnas.87.23.9378. [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