RESUMO
A novel crystal protein that exhibited potent cytotoxicity against human leukemic T-cells was cloned from the Bacillus thuringiensis TK-E6 strain. The protein, designated as parasporin-2Ab (PS2Ab), was a polypeptide of 304 amino acid residues with a predicted molecular weight of 33,017. The deduced amino acid sequence of PS2Ab showed significant homology (84% identitiy) to parasporin-2Aa (PS2Aa) from the B. thuringiensis A1547 strain. Upon processing of PS2Ab with proteinase K, the active form of 29 kDa was produced. The activated PS2Ab showed potent cytotoxicity against MOLT-4 and Jurkat cells and the EC(50) values were estimated as 0.545 and 0.745 ng/mL, respectively. The cytotoxicity of PS2Ab was significantly higher than that of PS2Aa reported elsewhere. Although both cytotoxins were structurally related, it was thought that the minor differences found were responsible for the different cytotoxicities of PS2Ab and PS2Aa.
Assuntos
Antineoplásicos/toxicidade , Bacillus thuringiensis , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Endotoxinas/toxicidade , Sequência de Aminoácidos , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Sequência de Bases , Endotoxinas/genética , Endotoxinas/isolamento & purificação , Humanos , Células Jurkat , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/toxicidade , Análise de Sequência de DNA , Células Tumorais CultivadasRESUMO
The 70-kDa protoxin of Cry11A, a dipteran-specific insecticidal protein, was processed by trypsin into 36- and 32-kDa fragments. To investigate the potent function of the two processed fragments, a GST (Glutathione-S-transferase) fusion protein of each polypeptide was constructed. While neither the 36- nor the 32-kDa fragment was toxic to Culex pipiens larvae, coexpression of the two fragments restored the insecticidal activity. Furthermore, the coprecipitation experiment demonstrated that the 36-kDa fragment was associated with the 32-kDa fragment. It was, therefore, shown that the coexistence of the two processed fragments of Cry11A was essential for the toxicity. The mutant of the 36-kDa fragment lacking the region from Gly(257) to Arg(360) bound to the 32-kDa fragment but the coexpression with the 32-kDa fragment resulted in no toxicity, suggesting that this region was involved in insecticidal activity.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidade , Culex/efeitos dos fármacos , Endotoxinas/química , Endotoxinas/toxicidade , Inseticidas/química , Inseticidas/toxicidade , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Bioensaio , Endotoxinas/genética , Glutationa Transferase/análise , Glutationa Transferase/química , Glutationa Transferase/genética , Proteínas Hemolisinas , Larva/efeitos dos fármacos , Mutação , Mapeamento de Peptídeos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/toxicidade , Tripsina/químicaRESUMO
A novel cytotoxic protein was isolated from the crystal produced by Bacillus thuringiensis subsp. coreanensis A1519 strain. Upon treatment of the crystal proteins by proteinase K, the significant cytotoxicity toward the leukemic T cell, MOLT-4, was exhibited. The microscopic observation indicated that the cell death was accompanied by no extensive rupture of the cell membrane. It was, therefore, suggested that the cell death of MOLT-4 was induced through a mechanism other than the colloid-osmotic swelling and cell lysis as caused by hitherto known B. thuringiensis crystal proteins. The 29-kDa polypeptide proved to be an active component of the proteinase K-digested A1519 crystal proteins. EC(50) of the purified 29-kDa polypeptide was 0.078 microg/ml. The N-terminal amino acid sequence of the 29-kDa polypeptide shared no significant homology with all the known proteins, suggesting that this polypeptide belong to a new family of B. thuringiensis crystal proteins. In the ligand blotting analysis, specific binding proteins for the 29-kDa polypeptide were detected from the cell membrane of MOLT-4.