Unusual proteolysis of the protoxin and toxin from Bacillus thuringiensis

Abstract

Trypsin is shown to generate an insecticidal toxin from the 130-kDa protoxin of Bacillus thuringiensis subsp. kurstaki HD-73 by an unusual proteolytic process. Seven specific cleavages are shown to occur in an ordered sequence starting at the C-terminus of the protoxin and proceeding toward the N-terminal region. At each step, C-terminal fragments of approximately 10 kDa are produced and rapidly proteolyzed to small peptides. The sequential proteolysis ends with a 67-kDa toxin which is resistant to further proteolysis. However, the toxin could be specifically split into two fragments by proteinases as it unfolded under denaturing conditions. Papain cleaved the toxin at glycine 327 to give a 34.5-kDa N-terminal fragment and a 32.3-kDa C-terminal fragment. Similar fragments could be generated by elastase and trypsin. The N-terminal fragment corresponds to the conserved N-terminal domain predicted from the gene-deduced sequence analysis of toxins from various subspecies of B. thuringiensis, and the C-terminal fragment is the predicted hypervariable sequence domain. A double-peaked transition was observed for the toxin by differential scanning calorimetry, consistent with two or more independent folding domains. It is concluded that the N-and C-terminal regions of the protoxin are two multidomain regions which give unique structural and biological properties to the molecule.Bacillus thuringiensis is an insect pathogen with an unusual but highly specific mode of action. During the sporulation cycle it lays down a parasporal protein crystal which is rendered toxic on ingestion by susceptible insect larvae. The major component of crystals toxic to lepidoptera is a protein (protoxin) with a molecular mass of approximately 130 kDa [l -31. Treatment with thiol reagents at basic pH solubilizes the protoxin by cleaving the disulfide bonds which stabilize the crystal. Incubation of the solubilized protoxin with proteolytic enzymes or insect gut juice produces a 58 -70-kDa proteinaseresistant toxin derived from the N-terminal portion of the molecule [4,5]. The toxin then binds to receptors in the midgut epithelium, causing cell lysis and eventual larval death [6 -81. The details of the lytic mechanism are not yet established but it appears that the toxin generates small pores or localized perturbations in the plasma membrane, causing disruption of homeostatic ion regulation [9].Large proteins are generally organized into distinct structural units referred to as domains or subdomains, but the criteria used for this classification is somewhat subjective. It is clear that the protoxin is divided into at least two major domains: the carboxyl half of the molecule which is readily attacked by proteinases, and the toxin derived from the Nterminal half which is proteinase-resistant. The toxins from

Production and Characterization of Bacillus thuringiensis Cry1Ac-Resistant Cotton Bollworm Helicoverpa zea (Boddie)

“…The fact that total cross-resistance does not even extend to protoxin forms of Cry1Ac (MVP II) and Cry1Ab (Bt corn powder) might be indicative of a differential activation of protoxin in the insect midgut, as opposed to in vitro bovinetrypsin activation (25,38). Alternatively, the C-terminal end of the protoxin may protect the active toxin from the degradative action of midgut proteases, resulting in a higher yield of the fully active toxin (7,32).…”

Protease Inhibitors Fail To Prevent Pore Formation by the Activated Bacillus thuringiensis Toxin Cry1Aa in Insect Brush Border Membrane Vesicles

“…Midgut proteases play an essential role in the activation of B. thuringiensis toxins (1,6,13). In the case of Cry1A toxins, the first 28 amino acid residues are removed from the N terminus and approximately half of the protoxin residues are removed from the C terminus (6).…”

Role of Proteolysis in Determining Potency of Bacillus thuringiensis Cry1Ac δ-Endotoxin

“…It has been postulated that the C-terminal region that is lost through activation directs assembly of the crystal and facilitates efficient solubilization at alkaline pH values (42). It is interesting to note that Cry1Ac activation proceeds through seven specific cleavages, starting from the C terminus and perhaps involving a sequence of conformational changes, to remove the C-terminal half of the protoxin (8). The resulting 10-to 35-kDa protoxin-derived fragments are themselves rapidly proteolysed into peptides, and they apparently play no further role in toxicity.…”