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A soybean cDNA encoding a chromatin-binding peptide inhibits mitosis of mammalian cells

Nature Biotechnology volume 17, pages 495–500 (1999)Cite this article

Abstract

A soybean cDNA encoding the small subunit peptide of a cotyledon-specific 2S albumin (Gm2S-1) is thought to play a role in arresting mitosis during the DNA endoreduplication and cell expansion phase of seed development. The peptide (termed lunasin) contains the cell adhesion motif Arg-Gly-Asp (RGD) followed by eight aspartic acid residues at its C-terminal end. A chimeric gene encoding the lunasin peptide tagged with green fluorescent protein (GFP) arrested cell division, caused abnormal spindle fiber elongation, chromosomal fragmentation, and cell lysis when transiently transfected into murine embryo fibroblast, murine hepatoma, and human breast cancer cells. Deletion of the polyaspartyl end abolished the antimitotic effect. Subcellular localization of lunasin and immunobinding assay using synthetic peptides revealed the preferential adherence of lunasin to chromatin. Immunofluorescence showed that kinetochore proteins were displaced from the centromere in lunasin-transfected cells. These observations suggest that lunasin binds to the chromatin, leading to disruption of kinetochore formation and inhibition of mitosis.

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Figure 1: Induced expression of (A) lunasin and (B) lunasin-del in transformed DH5α E. coli.
Figure 2: Transient expression of lunasin-del and lunasin in mammalian cells: (A) Murine hepatoma cells (Hepa 1c1c7).
Figure 3
Figure 4: Indirect immunofluorescence (green) of microtubules in (A) lunasin-del and (B–D) lunasin-transfected murine hepatoma cells.
Figure 5: Solid-phase immunobinding assay for synthetic lunasin and lunasin-del peptides to (A) histones and (B) MAP2.
Figure 6: Double indirect immunofluorescence of kinetochore proteins and lunasin using CREST human antisera and lunasin polyclonal antisera, respectively.

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Acknowledgements

We thank L. Bjeldanes, J. Riby, and G.H. Chang (Division of Nutritional Sciences and Toxicology, University of California at Berkeley [UCB]) for the cancer cell lines and the technical support in the transfection experiments; S. Ruzin and D. Schichnes (Center for Biological Imaging, UCB) for the microscopy and imaging assistance; P. Schow (Cancer Research Lab, UCB) for the technical help in flow cytometry and cell cycle analyses; H. Stotz (Department of Biological Sciences, Stanford University) for the technical advice on tubulin indirect immunofluorescence; B.R. Brinkley and R-H. Chen for generously providing the CREST and XMAD2 antibodies, respectively; W. Lei, R. Cho, V. Gurtu, Y. Lam, N. Duldulao, and C. Malacaman for their technical help; D.C. Krenz, I. Atkinson, and M.J. Revilleza for helpful discussions; and S. Nandi (Cancer Research Lab, UCB), Bob Buchanan (Department of Plant Biology, UCB), and L. Bjeldanes for critical review of the manuscript.

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  1. Division of Nutritional Sciences and Toxicology, University of California, Berkeley, 94720-3104, CA

    Alfredo F. Galvez & Benito O. de Lumen

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Correspondence to Benito O. de Lumen.

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Galvez, A., de Lumen, B. A soybean cDNA encoding a chromatin-binding peptide inhibits mitosis of mammalian cells. Nat Biotechnol 17, 495–500 (1999). https://doi.org/10.1038/8676

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