Proteolytic processing of native Cry1Ab toxin by midgut extracts and purified trypsins from the Mediterranean corn borer Sesamia nonagrioides.
The proteolytic processing of native Cry1Ab toxin by midgut extracts from the Mediterranean corn borer, Sesamia nonagrioides, takes place in successive steps. Several cuts occur until a 74 kDa protein is obtained; this is further digested to give rise to an active form of 69 kDa, which can be again processed to fragments of 67, 66 and 43 kDa. We have shown that three different trypsins (TI, TIIA and TIII) purified from the S. nonagrioides midgut were able to digest Cry1Ab protoxin to obtain the active form of 69 kDa. Interestingly, TI and TIII further hydrolyzed the 69 kDa protein to a fragment of slightly lower molecular mass (67 kDa), while TIIA was able to continue digestion to give fragments of 46 and 43 kDa. These results contrast with those obtained using bovine trypsin, in which the main product of Cry1Ab digestion is a 69 kDa protein. The digestion of the toxin with a "non-trypsin" fraction from S. nonagrioides midgut lumen, mostly containing chymotrypsins and elastases and free of trypsin-like activity, resulted in a different processing pattern, yielding fragments of 79, 77, 71, 69 and 51 kDa. Our results indicate that trypsins and other proteases are involved in the first steps of protoxin processing, but trypsins play the most important role in obtaining the 74 and 69 kDa proteins. All the digestion products, including the proteins of 46 and 43 kDa obtained from the digestion of Cry1Ab by TIIA, were toxic to neonate larvae, indicating that none of the tested proteases contribute to toxin degradation in a significant manner.
Díaz-Mendoza
Mercedes
M
Dpto. de Biología de Plantas, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Farinós
Gema P
GP
Castañera
Pedro
P
Hernández-Crespo
Pedro
P
Ortego
Félix
F
D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D004731:Endotoxins / Q000378:metabolism*; D041981:Gastrointestinal Tract / Q000201:enzymology; D006460:Hemolysin Proteins / Q000378:metabolism*; D007306:Insecticides / Q000378:metabolism*; D007814:Larva / Q000201:enzymology / Q000378:metabolism; D009036:Moths / Q000201:enzymology / Q000378:metabolism*; D014020:Tissue Extracts / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism*; D003313:Zea mays / Q000469:parasitology
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; D014020:Tissue Extracts; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014357:Trypsin