Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Feb 1;369(Pt 3):697–703. doi: 10.1042/BJ20021401

Cytotoxic activity of Bacillus thuringiensis Cry proteins on mammalian cells transfected with cadherin-like Cry receptor gene of Bombyx mori (silkworm).

Yoko Tsuda 1, Fumiki Nakatani 1, Keiko Hashimoto 1, Satoshi Ikawa 1, Chikako Matsuura 1, Takashi Fukada 1, Kenji Sugimoto 1, Michio Himeno 1
PMCID: PMC1223121  PMID: 12403648

Abstract

Cry1Aa, an insecticidal protein produced by Bacillus thuringiensis, has been shown to bind to cadherin-like protein, BtR175, in Bombyx mori (silkworm) midgut. We previously reported three variant alleles of BtR175 (BtR175a, b and c). When transiently expressed in COS7 cells, all the three BtR175 variants bound to Cry1Aa. We stably expressed BtR175b in HEK293 cells. These BtR175b-expressing cells swelled and died in the presence of activated Cry1Aa in a dose- and time-dependent manner, showing that BtR175b itself can impart Cry1Aa-susceptibility to mammalian cells. These cells were more susceptible to Cry1Aa than to Cry1Ab and Cry1Ac. Since dispersed B. mori midgut cells were reported to be highly susceptible to Cry1Ac, this result suggested that other Cry1Ac-specific receptor(s) were simultaneously working with BtR175 in the midgut cells. Advantages are also discussed of applying these transfected mammalian cells to toxicity assays of mutant Cry proteins.

Full Text

The Full Text of this article is available as a PDF (254.0 KB).

Selected References

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

  1. Gahan L. J., Gould F., Heckel D. G. Identification of a gene associated with Bt resistance in Heliothis virescens. Science. 2001 Aug 3;293(5531):857–860. doi: 10.1126/science.1060949. [DOI] [PubMed] [Google Scholar]
  2. Ge A. Z., Shivarova N. I., Dean D. H. Location of the Bombyx mori specificity domain on a Bacillus thuringiensis delta-endotoxin protein. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4037–4041. doi: 10.1073/pnas.86.11.4037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Gringorten J. L., Witt D. P., Milne R. E., Fast P. G., Sohi S. S., van Frankenhuyzen K. An in vitro system for testing Bacillus thuringiensis toxins: the lawn assay. J Invertebr Pathol. 1990 Sep;56(2):237–242. doi: 10.1016/0022-2011(90)90106-g. [DOI] [PubMed] [Google Scholar]
  4. HUNTER W. M., GREENWOOD F. C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature. 1962 May 5;194:495–496. doi: 10.1038/194495a0. [DOI] [PubMed] [Google Scholar]
  5. Ihara H., Uemura T., Masuhara M., Ikawa S., Sugimoto K., Wadano A., Himeno M. Purification and partial amino acid sequences of the binding protein from Bombyx mori for CryIAa delta-endotoxin of Bacillus thuringiensis. Comp Biochem Physiol B Biochem Mol Biol. 1998 May;120(1):197–204. doi: 10.1016/s0305-0491(98)10009-3. [DOI] [PubMed] [Google Scholar]
  6. Ikawa S., Tsuda Y., Fukada T., Sugimoto K., Himeno M. cDNA cloning of the Cry1Aa receptor variants from Bombyx mori and their expression in mammalian cells. Biosci Biotechnol Biochem. 2000 Dec;64(12):2682–2685. doi: 10.1271/bbb.64.2682. [DOI] [PubMed] [Google Scholar]
  7. Jenkins J. L., Dean D. H. Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors. BMC Biochem. 2001 Oct 16;2:12–12. doi: 10.1186/1471-2091-2-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Keeton T. P., Bulla L. A., Jr Ligand specificity and affinity of BT-R1, the Bacillus thuringiensis Cry1A toxin receptor from Manduca sexta, expressed in mammalian and insect cell cultures. Appl Environ Microbiol. 1997 Sep;63(9):3419–3425. doi: 10.1128/aem.63.9.3419-3425.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Knight P. J., Crickmore N., Ellar D. J. The receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N. Mol Microbiol. 1994 Feb;11(3):429–436. doi: 10.1111/j.1365-2958.1994.tb00324.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Luo K., Sangadala S., Masson L., Mazza A., Brousseau R., Adang M. J. The heliothis virescens 170 kDa aminopeptidase functions as "receptor A" by mediating specific Bacillus thuringiensis Cry1A delta-endotoxin binding and pore formation. Insect Biochem Mol Biol. 1997 Aug-Sep;27(8-9):735–743. doi: 10.1016/s0965-1748(97)00052-0. [DOI] [PubMed] [Google Scholar]
  11. Masson L., Mazza A., Gringorten L., Baines D., Aneliunas V., Brousseau R. Specificity domain localization of Bacillus thuringiensis insecticidal toxins is highly dependent on the bioassay system. Mol Microbiol. 1994 Dec;14(5):851–860. doi: 10.1111/j.1365-2958.1994.tb01321.x. [DOI] [PubMed] [Google Scholar]
  12. Nagamatsu Y., Koike T., Sasaki K., Yoshimoto A., Furukawa Y. The cadherin-like protein is essential to specificity determination and cytotoxic action of the Bacillus thuringiensis insecticidal CryIAa toxin. FEBS Lett. 1999 Oct 29;460(2):385–390. doi: 10.1016/s0014-5793(99)01327-7. [DOI] [PubMed] [Google Scholar]
  13. Nagamatsu Y., Toda S., Koike T., Miyoshi Y., Shigematsu S., Kogure M. Cloning, sequencing, and expression of the Bombyx mori receptor for Bacillus thuringiensis insecticidal CryIA(a) toxin. Biosci Biotechnol Biochem. 1998 Apr;62(4):727–734. doi: 10.1271/bbb.62.727. [DOI] [PubMed] [Google Scholar]
  14. Potvin L, Laprade R, Schwartz JL. Cry1Ac, a bacillus thuringiensis toxin, triggers extracellular Ca2+ influx and Ca2+ release from intracellular stores in Cf1 cells. J Exp Biol. 1998 May 21;201(Pt 12):1851–1858. doi: 10.1242/jeb.201.12.1851. [DOI] [PubMed] [Google Scholar]
  15. Schnepf E., Crickmore N., Van Rie J., Lereclus D., Baum J., Feitelson J., Zeigler D. R., Dean D. H. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev. 1998 Sep;62(3):775–806. doi: 10.1128/mmbr.62.3.775-806.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Smith G. P., Ellar D. J. Mutagenesis of two surface-exposed loops of the Bacillus thuringiensis CryIC delta-endotoxin affects insecticidal specificity. Biochem J. 1994 Sep 1;302(Pt 2):611–616. doi: 10.1042/bj3020611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sugimoto K., Tasaka H., Dotsu M. Molecular behavior in living mitotic cells of human centromere heterochromatin protein HPLalpha ectopically expressed as a fusion to red fluorescent protein. Cell Struct Funct. 2001 Dec;26(6):705–718. doi: 10.1247/csf.26.705. [DOI] [PubMed] [Google Scholar]
  18. Tabashnik B. E., Liu Y. B., Malvar T., Heckel D. G., Masson L., Ballester V., Granero F., Ménsua J. L., Ferré J. Global variation in the genetic and biochemical basis of diamondback moth resistance to Bacillus thuringiensis. Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):12780–12785. doi: 10.1073/pnas.94.24.12780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Vadlamudi R. K., Ji T. H., Bulla L. A., Jr A specific binding protein from Manduca sexta for the insecticidal toxin of Bacillus thuringiensis subsp. berliner. J Biol Chem. 1993 Jun 15;268(17):12334–12340. [PubMed] [Google Scholar]
  20. Vadlamudi R. K., Weber E., Ji I., Ji T. H., Bulla L. A., Jr Cloning and expression of a receptor for an insecticidal toxin of Bacillus thuringiensis. J Biol Chem. 1995 Mar 10;270(10):5490–5494. doi: 10.1074/jbc.270.10.5490. [DOI] [PubMed] [Google Scholar]
  21. Villalon M., Vachon V., Brousseau R., Schwartz J. L., Laprade R. Video imaging analysis of the plasma membrane permeabilizing effects of Bacillus thuringiensis insecticidal toxins in Sf9 cells. Biochim Biophys Acta. 1998 Jan 5;1368(1):27–34. doi: 10.1016/s0005-2736(97)00184-3. [DOI] [PubMed] [Google Scholar]
  22. Wright D. J., Iqbal M., Granero F., Ferre J. A Change in a Single Midgut Receptor in the Diamondback Moth (Plutella xylostella) Is Only in Part Responsible for Field Resistance to Bacillus thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai. Appl Environ Microbiol. 1997 May;63(5):1814–1819. doi: 10.1128/aem.63.5.1814-1819.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Yaoi K., Kadotani T., Kuwana H., Shinkawa A., Takahashi T., Iwahana H., Sato R. Aminopeptidase N from Bombyx mori as a candidate for the receptor of Bacillus thuringiensis Cry1Aa toxin. Eur J Biochem. 1997 Jun 15;246(3):652–657. doi: 10.1111/j.1432-1033.1997.t01-1-00652.x. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Multimedia adjunct for figure 5 (small movie)
Download video file (5.6MB, mov)
Multimedia adjunct for figure 5(large movie)
Download video file (9.5MB, mov)

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES