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. 1997 Sep;63(9):3419–3425. doi: 10.1128/aem.63.9.3419-3425.1997

Ligand specificity and affinity of BT-R1, the Bacillus thuringiensis Cry1A toxin receptor from Manduca sexta, expressed in mammalian and insect cell cultures.

T P Keeton 1, L A Bulla Jr 1
PMCID: PMC168650  PMID: 9292994

Abstract

The Manduca sexta receptor for the Bacillus thuringiensis Cry1Aa, Cry1Ab, and Cry1Ac toxins, BT-R1, has been expressed in heterologous cell culture, and its ligand binding characteristics have been determined. When transfected with the BT-R1 cDNA, insect and mammalian cell cultures produce a binding protein of approximately 195 kDa, in contrast to natural BT-R1 from M. sexia, which has an apparent molecular weight of 210 kDa. Transfection of cultured Spodoptera frugiperda cells with the BT-R1 cDNA imparts Cry1A-specific high-affinity binding activity typical of membranes prepared from larval M. sexta midguts. Competition assays with BT-R1 prepared from larval M. sexta midguts and transiently expressed in cell culture reveal virtually identical affinities for the Cry1Aa, Cry1Ab, and Cry1Ac toxins, clearly demonstrating the absolute specificity of the receptor for toxins of the lepidopteran-specific Cry1A family. BT-R1 therefore remains the only M. sexta Cry1A binding protein to be purified, cloned, and functionally expressed in heterologous cell culture, and for the first time, we are able to correlate the Cry1Aa, Cry1Ab, and Cry1Ac toxin sensitivities of M. sexta to the identity and ligand binding characteristics of a single midgut receptor molecule.

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Selected References

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  1. Adamo H. P., Caride A. J., Penniston J. T. Use of expression mutants and monoclonal antibodies to map the erythrocyte Ca2+ pump. J Biol Chem. 1992 Jul 15;267(20):14244–14249. [PubMed] [Google Scholar]
  2. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chilcott C. N., Ellar D. J. Comparative toxicity of Bacillus thuringiensis var. israelensis crystal proteins in vivo and in vitro. J Gen Microbiol. 1988 Sep;134(9):2551–2558. doi: 10.1099/00221287-134-9-2551. [DOI] [PubMed] [Google Scholar]
  4. Denolf P., Jansens S., Peferoen M., Degheele D., Van Rie J. Two Different Bacillus thuringiensis Delta-Endotoxin Receptors in the Midgut Brush Border Membrane of the European Corn Borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae). Appl Environ Microbiol. 1993 Jun;59(6):1828–1837. doi: 10.1128/aem.59.6.1828-1837.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Du C., Nickerson K. W. The Bacillus thuringiensis insecticidal toxin binds biotin-containing proteins. Appl Environ Microbiol. 1996 Aug;62(8):2932–2939. doi: 10.1128/aem.62.8.2932-2939.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Estada U., Ferre J. Binding of Insecticidal Crystal Proteins of Bacillus thuringiensis to the Midgut Brush Border of the Cabbage Looper, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), and Selection for Resistance to One of the Crystal Proteins. Appl Environ Microbiol. 1994 Oct;60(10):3840–3846. doi: 10.1128/aem.60.10.3840-3846.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Garczynski S. F., Crim J. W., Adang M. J. Identification of putative insect brush border membrane-binding molecules specific to Bacillus thuringiensis delta-endotoxin by protein blot analysis. Appl Environ Microbiol. 1991 Oct;57(10):2816–2820. doi: 10.1128/aem.57.10.2816-2820.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Hermiston M. L., Gordon J. I. In vivo analysis of cadherin function in the mouse intestinal epithelium: essential roles in adhesion, maintenance of differentiation, and regulation of programmed cell death. J Cell Biol. 1995 Apr;129(2):489–506. doi: 10.1083/jcb.129.2.489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hermiston M. L., Wong M. H., Gordon J. I. Forced expression of E-cadherin in the mouse intestinal epithelium slows cell migration and provides evidence for nonautonomous regulation of cell fate in a self-renewing system. Genes Dev. 1996 Apr 15;10(8):985–996. doi: 10.1101/gad.10.8.985. [DOI] [PubMed] [Google Scholar]
  11. Hofmann C., Vanderbruggen H., Höfte H., Van Rie J., Jansens S., Van Mellaert H. Specificity of Bacillus thuringiensis delta-endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7844–7848. doi: 10.1073/pnas.85.21.7844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Höfte H., Van Rie J., Jansens S., Van Houtven A., Vanderbruggen H., Vaeck M. Monoclonal Antibody Analysis and Insecticidal Spectrum of Three Types of Lepidopteran-Specific Insecticidal Crystal Proteins of Bacillus thuringiensis. Appl Environ Microbiol. 1988 Aug;54(8):2010–2017. doi: 10.1128/aem.54.8.2010-2017.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Höfte H., Whiteley H. R. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev. 1989 Jun;53(2):242–255. doi: 10.1128/mr.53.2.242-255.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Knight P. J., Knowles B. H., Ellar D. J. Molecular cloning of an insect aminopeptidase N that serves as a receptor for Bacillus thuringiensis CryIA(c) toxin. J Biol Chem. 1995 Jul 28;270(30):17765–17770. doi: 10.1074/jbc.270.30.17765. [DOI] [PubMed] [Google Scholar]
  16. Kogan P. H., Blissard G. W. A baculovirus gp64 early promoter is activated by host transcription factor binding to CACGTG and GATA elements. J Virol. 1994 Feb;68(2):813–822. doi: 10.1128/jvi.68.2.813-822.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Lee M. K., Dean D. H. Inconsistencies in determining Bacillus thuringiensis toxin binding sites relationship by comparing competition assays with ligand blotting. Biochem Biophys Res Commun. 1996 Mar 27;220(3):575–580. doi: 10.1006/bbrc.1996.0445. [DOI] [PubMed] [Google Scholar]
  19. Lee M. K., Milne R. E., Ge A. Z., Dean D. H. Location of a Bombyx mori receptor binding region on a Bacillus thuringiensis delta-endotoxin. J Biol Chem. 1992 Feb 15;267(5):3115–3121. [PubMed] [Google Scholar]
  20. Lee M. K., You T. H., Young B. A., Cotrill J. A., Valaitis A. P., Dean D. H. Aminopeptidase N purified from gypsy moth brush border membrane vesicles is a specific receptor for Bacillus thuringiensis CryIAc toxin. Appl Environ Microbiol. 1996 Aug;62(8):2845–2849. doi: 10.1128/aem.62.8.2845-2849.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Li J. D., Carroll J., Ellar D. J. Crystal structure of insecticidal delta-endotoxin from Bacillus thuringiensis at 2.5 A resolution. Nature. 1991 Oct 31;353(6347):815–821. doi: 10.1038/353815a0. [DOI] [PubMed] [Google Scholar]
  22. Martínez-Ramírez A. C., González-Nebauer S., Escriche B., Real M. D. Ligand blot identification of a Manduca sexta midgut binding protein specific to three Bacillus thuringiensis CryIA-type ICPs. Biochem Biophys Res Commun. 1994 Jun 15;201(2):782–787. doi: 10.1006/bbrc.1994.1769. [DOI] [PubMed] [Google Scholar]
  23. Masson L., Lu Y. J., Mazza A., Brousseau R., Adang M. J. The CryIA(c) receptor purified from Manduca sexta displays multiple specificities. J Biol Chem. 1995 Sep 1;270(35):20309–20315. doi: 10.1074/jbc.270.35.20309. [DOI] [PubMed] [Google Scholar]
  24. Sangadala S., Walters F. S., English L. H., Adang M. J. A mixture of Manduca sexta aminopeptidase and phosphatase enhances Bacillus thuringiensis insecticidal CryIA(c) toxin binding and 86Rb(+)-K+ efflux in vitro. J Biol Chem. 1994 Apr 1;269(13):10088–10092. [PubMed] [Google Scholar]
  25. Steller H. Mechanisms and genes of cellular suicide. Science. 1995 Mar 10;267(5203):1445–1449. doi: 10.1126/science.7878463. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Valaitis A. P., Lee M. K., Rajamohan F., Dean D. H. Brush border membrane aminopeptidase-N in the midgut of the gypsy moth serves as the receptor for the CryIA(c) delta-endotoxin of Bacillus thuringiensis. Insect Biochem Mol Biol. 1995 Dec;25(10):1143–1151. doi: 10.1016/0965-1748(95)00050-x. [DOI] [PubMed] [Google Scholar]
  29. Van Rie J., Jansens S., Höfte H., Degheele D., Van Mellaert H. Specificity of Bacillus thuringiensis delta-endotoxins. Importance of specific receptors on the brush border membrane of the mid-gut of target insects. Eur J Biochem. 1989 Dec 8;186(1-2):239–247. doi: 10.1111/j.1432-1033.1989.tb15201.x. [DOI] [PubMed] [Google Scholar]

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