Skip to main content
SpringerLink
Log in

Formation of Macromolecule Complex with Bacillus thuringiensis Cry1A Toxins and Chlorophyllide Binding 252-kDa Lipocalin-Like Protein Locating on Bombyx mori Midgut Membrane

  • Published:
The Journal of Membrane Biology Aims and scope Submit manuscript

Abstract

P252, a 252-kDa Bombyx mori protein located on the larval midgut membrane, has been shown to bind strongly with Bacillus thuringiensis Cry1A toxins (Hossain et al. Appl Environ Microbiol 70:4604–4612, 2004). P252 was also shown to bind chlorophyllide (Chlide) to form red fluorescence–emitting complex Bm252RFP with significant antimicrobial activity (Pandian et al. Appl Environ Microbiol 74:1324–1331, 2008). In this article, we show that Cry1A toxin bound with Bm252RFP and Bm252RFP–Cry1A macrocomplex, with both antimicrobial and insecticidal activities, was formed. The insecticidal activity of Bm252RFP–Cry1Ab was reduced from an LD50 of 1.62 to 5.05 μg, but Bm252RFP–Cry1Aa and Bm252RFP–Cry1Ac did not show such reduction. On the other hand, the antimicrobial activity of Bm252RFP–Cry1Ab was shown to retain almost the same activity as Bm252RFP, while the other two complexes lost around 30% activity. The intensity of photo absorbance and fluorescence emission of Bm252RFP–Cry1Ab were significantly reduced compared to those of the other two complexes. Circular dichroism showed that the contents of Cry1Ab α-helix was significantly decreased in Bm252RFP–Cry1Ab but not in the other two toxins. These data suggested that the reduction of contents of α-helix in Cry1Ab affected the insecticidal activity of the macrocomplex but did not alter the antimicrobial moiety in the macrocomplex of Bm252RFP–Cry1Ab.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Angelucci C, Aarrett-Wilt BG, Hunt DF, Akhurst RJ, East PD, Gordon KH, Campbell PM (2008) Diversity of aminopeptidases, derived from four lepidopteran gene duplications, and polycalins expressed in the midgut of Helicoverpa armigera: identification of proteins binding the delta-endotoxin, Cry1Ac of Bacillus thuringiensis. Insect Biochem Mol Biol 38:685–696

    Article  CAS  PubMed  Google Scholar 

  • Bergman P, Johansson L, Wan H, Jones A, Gallo RL, Gudmundsson GH, Hökfelt T, Jonsson AB, Agerberth B (2006) Induction of the antimicrobial peptide CRAMP in the blood–brain barrier and meninges after meningococcal infection. Infect Immun 74:6982–6991

    Article  CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Bravo A, Soberón M, Gill SS (2005) Bacillus thuringiensis: mechanisms and use. In: Gilbert LI, Iatrou K, Gill SS (eds) Comprehensive molecular insect science, vol 6. Elsevier, Amsterdam, pp 175–205

    Chapter  Google Scholar 

  • Bravo A, Gill SS, Soberón M (2007) Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49:423–435

    Article  CAS  PubMed  Google Scholar 

  • Coleman RA, Pugh BF (1997) Slow dimer dissociation of the TATA binding protein dictates the kinetics of DNA binding. Proc Natl Acad Sci USA 94:7221–7226

    Article  CAS  PubMed  Google Scholar 

  • Federici B (2003) Effects of Bt on non-target organisms. In: Metz M (ed) Bacillus thuringiensis: a cornerstone of modern agriculture. Food Products Press, Binghamton, NY, pp 11–30

    Google Scholar 

  • Ferré J, Van Rie J (2002) Biochemistry and genetics of insect resistance to Bacillus thuringiensis. Annu Rev Entomol 47:501–533

    Article  PubMed  Google Scholar 

  • Flo TH, Smith KD, Sato S, Rodriguez DJ, Holmes MA, Strong RK, Akira S, Aderem A (2004) Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature 432:917–921

    Article  CAS  PubMed  Google Scholar 

  • Gill SS, Cowles EA, Francis V (1995) Identification, isolation, and cloning of a Bacillus thuringiensis CryIAc toxin-binding protein from the midgut of the lepidopteran insect Heliothis virescens. J Biol Chem 270:27277–27282

    Article  CAS  PubMed  Google Scholar 

  • Griffitts JS, Aroian RV (2005) Many roads to resistance: how invertebrates adapt to Bt toxins. Bioessays 27:614–624

    Article  PubMed  Google Scholar 

  • Heckel DG (1994) The complex genetic basis of resistance to Bacillus thuringiensis toxin in insects. Biocontrol Sci Technol 4:405–417

    Article  Google Scholar 

  • Hossain DM, Shitomi Y, Hayakawa T, Higuchi M, Mitsui T, Sato R, Hori H (2004) Characterization of a novel plasma membrane protein, expressed in the midgut epithelia of Bombyx mori that binds to Cry1A toxins. Appl Environ Microbiol 70:4604–4612

    Article  CAS  PubMed  Google Scholar 

  • Hossain DM, Shitomi Y, Nanjo Y, Takano D, Nishiumi T, Hayakawa T, Mitsui T, Sato R, Hori H (2005) Localization of a novel 252-kDa plasma membrane protein that binds Cry1A toxins in the midgut epithelia of Bombyx mori. Appl Entomol Zool 40:125–135

    Article  CAS  Google Scholar 

  • Ihara H, Uemura T, Masuhara M, Ikawa S, Sugimoto S, Wadano A, Himeno M (1998) Purification and partial amino acid sequences of the binding protein from Bombyx mori for CryIAa δ-endotoxin of Bacillus thuringiensis. Comp Biochem Physiol B 120:197–204

    Article  CAS  PubMed  Google Scholar 

  • Indrasith LS, Ogiwara K, Minami M, Iwasa T, Maruyama T, Suzuki N, Asano S, Sakanaka K, Hori H (1991) Processing of delta endotoxin from Bacillus thuringiensis subspp. Kurstaki HD-1 and HD-73 by immobilized trypsin and chymotrypsin. Appl Entomol Zool 26:485–492

    CAS  Google Scholar 

  • Janmaat AF, Myers J (2003) Rapid evolution and the cost of resistance to Bacillus thuringiensis in greenhouse populations of cabbage loopers, Trichoplusia ni. Proc R Soc Lond B Biol Sci 270:2263–2270

    Article  Google Scholar 

  • Kim YS, Kanda K, Kato F, Murata A (1998) Effect of the carboxyl terminal portion of Cry1Ab in Bacillus thuringiensis on toxicity against the silkworm, Bombyx mori. Appl Entomol Zool 33:473–477

    CAS  Google Scholar 

  • Kishimoto T, Hori H, Takano D, Nakano Y, Watanabe M, Mitsui T (2001) Rice α-mannosidase digesting the high mannose glycopeptide of glutelin. Physiol Plant 112:15–24

    Article  CAS  PubMed  Google Scholar 

  • Knight PJ, Crickmore N, Ellar DJ (1994) The receptor for Bacillus thuringiensis Cry1A(c) δ-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N. Mol Microbiol 11:429–436

    Article  CAS  PubMed  Google Scholar 

  • Kough J (2003) The safety of Bacillus thuringiensis for human consumption. In: Metz M (ed) Bacillus thuringiensis: a cornerstone of modern agriculture. Food Products Press, Binghamton, NY, pp 1–10

    Google Scholar 

  • Mauchamp B, Royer C, Garel A, Jalabert A, Rocha MD, Grenier AM, Labas V, Vinh J, Mita K, Kadono K, Chavancy G (2006) Polycalin (chlorophyllide A binding protein), a novel, very large fluorescent lipocalin from the midgut of the domestic silkworm Bombyx mori L. Insect Biochem Mol Biol 36:623–633

    Article  CAS  PubMed  Google Scholar 

  • Moonsom S (2007) Characterization of Bacillus thuringiensis Cry4Ba -Binding proteins from midgut epithelium and peritrophic membrane of Aedes aegypti larvae. PhD thesis, Mahidol University, Thailand.

  • Nagamatsu Y, Toda S, Koike T, Miyoshi Y, Shigematsu S, Kogure M (1998) Cloning, sequencing, and expression of the Bombyx mori receptor for Bacillus thuringiensis insecticidal CryIA (a) toxin. Biosci Biotechnol Biochem 62:727–734

    Article  CAS  PubMed  Google Scholar 

  • Nair MS, Dean DH (2008) All domains of Cry1A toxins insert into insect brush border membranes. J Biol Chem 283:26324–26331

    Article  CAS  PubMed  Google Scholar 

  • Ong ST, Ho JZ, Ho B, Ding JL (2006) Iron-withholding strategy in innate immunity. Immunobiology 211:295–314

    Article  CAS  PubMed  Google Scholar 

  • Pandian NG, Ishikawa T, Togashi M, Shitomi Y, Haginoya K, Yamamoto K, Nishiumi T, Hori H (2008) Bombyx mori midgut membrane protein P252 which binds to Cry1A of Bacillus thuringiensis is a chlorophyllide binding protein and its resulting complex has antimicrobial activity. Appl Environ Microbiol 74:1324–1331

    Article  CAS  PubMed  Google Scholar 

  • Pigott CR, Ellar DJ (2007) Role of receptors in Bacillus thuringiensis crystal toxin activity. Microbiol Mol Biol Rev 71:255–281

    Article  CAS  PubMed  Google Scholar 

  • Russel RM, Robertson JL, Savin NE (1977) POLO: a new computer program for probit analysis. Bull Entomol Soc Am 23:209–213

    Google Scholar 

  • Sangadala S, Walters FS, English LH, Adang MJ (1994) 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 269:10088–10092

    CAS  PubMed  Google Scholar 

  • Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775–806

    CAS  PubMed  Google Scholar 

  • Shaw J, Beadle LC (1949) A simplified ultra-micro Kjeldahl method for the estimation of protein and total nitrogen in fluid samples of less than 1.0 μl. J Exp Biol 26:15–23

    CAS  PubMed  Google Scholar 

  • Shelton AM, Roberson JL, Tang JD, Perez C, Eigenbrode SD, Preisler HK, Wilsey WK, Cooley RJ (1993) Resistance of diamondback moth (Lepidoptera: Plutellidae) to Bacillus thuringiensis subspecies in the field. J Econ Entomol 8:697–705

    Google Scholar 

  • Shitomi Y, Hayakawa T, Hossain DM, Higuchi M, Miyamoto K, Nakanishi K, Sato R, Hori H (2006) A novel 96-kDa aminopeptidase localized on epithelial cell membranes of Bombyx mori midgut, which binds to Cry1Ac toxin of Bacillus thuringiensis. J Biochem 139:223–233

    Article  CAS  PubMed  Google Scholar 

  • Suzuki M, Hori H, Ogiwara K, Asano S, Sato R, Ohba M, Iwahana H (1992) Insecticidal spectrum of a novel isolate of Bacillus thuringiensis serovar japonensis. Biol Control 2:138–142

    Article  Google Scholar 

  • Tabashnik BE (1994) Evolution of resistance to Bacillus thuringiensis. Annu Rev Entomol 39:47–79

    Article  Google Scholar 

  • Tapaneeyakorn S, Pornwiroon W, Katzenmeier G, Angsuthanasombat C (2005) Structural requirements of the unique disulphide bond and the proline-rich motif within the α4–α5 loop for larvicidal activity of the Bacillus thuringiensis Cry4Aa δ-endotoxin. Biochem Biophys Res Commun 330:519–525

    Article  CAS  PubMed  Google Scholar 

  • Tomimoto K, Hayakawa T, Hori H (2006) Pronase digestion of brush border membrane-bound Cry1Aa shows that almost the whole activated Cry1Aa molecule penetrates into the membrane. Comp Biochem Physiol B 144:413–422

    Article  PubMed  Google Scholar 

  • Vadlamudi RK, Weber E, Ji I, Ji TH, Bulla LA Jr (1995) Cloning and expression of a receptor for an insecticidal toxin of Bacillus thuringiensis. J Biol Chem 270:5490–5494

    Article  CAS  PubMed  Google Scholar 

  • Valaitis AP, Lee MK, Rajamohan F, Dean DH (1995) Brush border membrane aminopeptidase-N in the midgut of the gypsy moth serves as the receptor for the CryIA(c) δ-endotoxin of Bacillus thuringiensis. Insect Biochem Mol Biol 25:1143–1151

    Article  CAS  PubMed  Google Scholar 

  • Van Rie J, McGaughey WH, Johnson DE, Barnett BD, Van Mellaert H (1990) Mechanism of insect resistance to the microbial insecticide Bacillus thuringiensis. Science 247:72–74

    Article  PubMed  Google Scholar 

  • Yang JT, Wu CS, Martinez HM (1986) Calculation of protein conformation from circular dichroism. Methods Enzymol 130:208–269

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported, in part, by research grants from the Ministry of Education, Culture, Sports, Science and Technology (13306006 and 12558069 to H. H.) and a grant for the promotion of the Niigata University Research Project (2004) (also to H. H.). We also thank the student scholarships awarded to G. P. by the Ministry of Education, Culture, Sports, Science and Technology. C. A. visited H. H.’s laboratory to discuss this research in the short-range exchange program for foreign researchers of Japan Society for the Promotion of Science (S-08207).

Author information

Authors and Affiliations

  1. Graduate School of Science and Technology, Niigata University, Niigata, 950-2181, Japan

    Ganesh N. Pandian, Toshiki Ishikawa, Thangavel Vaijayanthi, Delwar M. Hossain, Shuhei Yamamoto, Tadayuki Nishiumi, Kohsuke Haginoya, Toshiaki Mitsui & Hidetaka Hori

  2. Institute of Molecular Biosciences, Mahidol University, Nakornpathom, 73170, Thailand

    Chanan Angsuthanasombat

  3. Laboratories of Applied Biosciences, Graduate School of Science and Technology, Niigata University, Niigata, 950-2181, Japan

    Hidetaka Hori

Authors
  1. Ganesh N. Pandian
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Toshiki Ishikawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Thangavel Vaijayanthi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Delwar M. Hossain
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shuhei Yamamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tadayuki Nishiumi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Chanan Angsuthanasombat
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Kohsuke Haginoya
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Toshiaki Mitsui
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Hidetaka Hori
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hidetaka Hori.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pandian, G.N., Ishikawa, T., Vaijayanthi, T. et al. Formation of Macromolecule Complex with Bacillus thuringiensis Cry1A Toxins and Chlorophyllide Binding 252-kDa Lipocalin-Like Protein Locating on Bombyx mori Midgut Membrane. J Membrane Biol 237, 125–136 (2010). https://doi.org/10.1007/s00232-010-9314-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00232-010-9314-x

Keywords

Search

Navigation