Pronase digestion of brush border membrane-bound Cry1Aa shows that almost the whole activated Cry1Aa molecule penetrates into the membrane.
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 alpha-helix and/or to amino acids at 1 mg Pronase/mL within 2.4 h at 37 degrees C. Whereas, BBMV-bound Cry1Aa was very resistant to Pronase digestion and even at 2 mg for 24 h, 7.5 kDa and approximately 30 kDa peptide were detected by alpha-2,3 antiserum, and alpha-4,5 and alpha-6,7 antisera, respectively. Another approximately 30 kDa peptide was also detected by beta-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 approximately 30 kDa peptides are thought to be derived from alpha-2,3 helix and stretch of alpha-4 to alpha-7 helices. Furthermore the latter approximately 30 kDa was thought to include the stretch of beta-6 to domain III. Moreover, the embedded Cry1Aa molecule appears to be segregated in some areas of beta-1-5 sheets, resulting in the above two approximately 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 alpha-4,5 antiserum was observed. This fragment must be dimeric alpha-4,5 helices and we discussed the origin of this peptide.
Tomimoto
Kazuya
K
Laboratory of Molecular Life Sciences, School of Science and Technology, Niigata University, Niigata, Japan.
Hayakawa
Tohru
T
Hori
Hidetaka
H
D000818:Animals; D001413:Bacillus thuringiensis; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism; D012831:Bombyx / Q000378:metabolism* / Q000648:ultrastructure; D002462:Cell Membrane / Q000378:metabolism; D004731:Endotoxins / Q000737:chemistry* / Q000378:metabolism; D041981:Gastrointestinal Tract / Q000378:metabolism / Q000648:ultrastructure; D006460:Hemolysin Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism; D011402:Pronase / Q000737:chemistry*; D017433:Protein Structure, Secondary
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D008565:Membrane Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D011402:Pronase