Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle

Bacillus thuringiensis (Bt) Cry8D insecticidal proteins are unique among Cry8 family proteins in terms of its insecticidal activity against adult Scarab beetles, such as Japanese beetle (Popillia japonica Newman). From the sequence homology with other Bt Cry proteins especially those active against beetles, such as Cry3Aa whose 3D structure is available, the structure of the Cry8D protein has been predicted to be a typical three-domain Cry protein type. In addition, the activation process of Cry8D in gut juice of susceptible insects is presumed to be similar to that of Cry3A (Yamaguchi et al., 2008). In this study, the activation process of Cry8Da in insect gut juice was closely examined. Japanese beetle gut juice proteases digested the 130 kDa Cry8Da protein to produce a 64 kDa protein. This 64 kDa protein was active against both adult and larval Japanese beetle and considered to be an activated toxin. N-terminal sequencing of this 64 kDa protein revealed that the Cry8Da leader sequence consisting of 63 amino acid residues from M¹ to F⁶³ was removed. As in the case of Cry3Aa, the proteases further digested the 64 kDa protein to two 8 kDa and 54 kDa fragments. N-terminal amino acid analysis of these smaller fragments indicated that the proteases digested the loop between Alpha Helix (Alpha for short) 3 and Alpha 4. This means that the 8 kDa fragment consists of Alpha 1-3 of Domain I and that the 54 kDa fragment contains the remaining Domain I and full Domain II and Domain III. Size exclusion chromatography and anion exchange chromatography could not separate these 64, 54 and 8 kDa proteins suggesting that the 54 kDa and 8 kDa fragments are still forming the toxin complex equivalent to the 64 kDa protein by size and ionic charge. The sequencing and chromatography results suggest that the gut juice proteases merely nicked the loop between Alpha 3 and Alpha 4. This nicking process appeared to be essential for receptor binding of the Cry8Da toxin. BBMV binding assay revealed that the Cry8Da toxin bound to BBMV preparations from both adult and larval Japanese beetle only after the loop was nicked. Only the 54 kDa fragment bound to the BBMV preparations but not the 64 kDa protein. Ligand blot showed that the protease activated Cry8Da toxin, presumably the 54 kDa fragment, bound to specific BBMV proteins, one or more of those would be receptor(s). The sizes and binding affinities of these Cry8Da-bound proteins of Japanese beetle BBMV differed between larvae and adults.