Pronase digestion of brush border membrane-bound Cry1Aa shows that almost the whole activated Cry1Aa molecule penetrates into the membrane

Kazuya Tomimoto, Tohru Hayakawa, Hidetaka Hori

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Bacillus thuringiensis insecticidal proteins, Cry toxins, following ingestion by insect larvae, induce insecticidal effect by penetrating the brush border membranes (BBM) of midgut epithelial cells. Purified, activated B. thuringiensis Cry1Aa bound to Bombyx mori BBMV or unbound Cry1Aa were vigorously digested with Pronase. Both digests were compared by Western blotting. Free Cry1Aa was digested to α-helix and/or to amino acids at 1 mg Pronase/mL within 2.4 h at 37 °C. Whereas, BBMV-bound Cry1Aa was very resistant to Pronase digestion and even at 2 mg for 24 h, 7.5 kDa and ∼30 kDa peptide were detected by α-2,3 antiserum, and α-4,5 and α-6,7 antisera, respectively. Another ∼30 kDa peptide was also detected by β-6-11 and domain III antisera. These fragments are believed either to be embedded in or to strongly interact with the BBMV. The 7.5 and former ∼30 kDa peptides are thought to be derived from α-2,3 helix and stretch of α-4 to α-7 helices. Furthermore the latter ∼30 kDa was thought to include the stretch of β-6 to domain III. Moreover, the embedded Cry1Aa molecule appears to be segregated in some areas of β-1-5 sheets, resulting in the above two ∼30 kDa peptides. From these digestion patterns, we proposed new membrane insertion model for single Cry1Aa molecule. On the other hand, in digestion of BBMV-bound Cry1Aa, 15 kDa peptide which was recognized only by α-4,5 antiserum was observed. This fragment must be dimeric α-4,5 helices and we discussed the origin of this peptide.

Original languageEnglish
Pages (from-to)413-422
Number of pages10
JournalComparative Biochemistry and Physiology - B Biochemistry and Molecular Biology
Volume144
Issue number4
DOIs
Publication statusPublished - Aug 2006
Externally publishedYes

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Keywords

  • α-Helix
  • Bacillus thuringiensis
  • BBMV
  • Bombyx mori
  • Cry toxin insertion
  • Cry1Aa
  • Plasma membrane
  • Pore formation
  • Protease digestion

ASJC Scopus subject areas

  • Biochemistry
  • Physiology

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