Investigation of a Novel Bacillus thuringiensis Gene Encoding a ...

Biosci. Biotechnol. Biochem., 70 (12), 2935–2941, 2006

Investigation of a Novel Bacillus thuringiensis Gene Encoding a Parasporal Protein, Parasporin-4, That Preferentially Kills Human Leukemic T Cells Hiroyuki S AITOH,1; y Shiro O KUMURA,1 Tomoyuki I SHIKAWA,1 Tetsuyuki A KAO,1 Eiichi M IZUKI,1 and Michio O HBA2 1

Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, 1465-5 Aikawa-machi, Kurume, Fukuoka 839-0861, Japan 2 Graduate School of Agriculture, Kyushu University, Fukuoka 812-8581, Japan Received June 23, 2006; Accepted August 7, 2006; Online Publication, December 7, 2006 [doi:10.1271/bbb.60352]

A novel gene encoding a leukemic cell-killing parasporal protein, designated parasporin-4, was cloned from an isolate of Bacillus thuringiensis serovar shandongiensis. The amino acid sequence of the parasporin4, as deduced from the gene sequence, had low-level homologies of 4 times more susceptible than the latter. As Fig. 3 shows, the protein of wild-type inclusions of strain A1470 was more toxic than the recombinant E. coli protein. For example, when tested on MOLT-4 cells, the EC50 value of the wild-type protein was 0.137 mg/ml, while the value of the recombinant protein was 0.472 mg/ml.

Discussion The present study indicates that the 27 kDa leukemic cell-killing protein, occurring in parasporal inclusions of B. thuringiensis serovar shandongiensis strain A1470, is a novel toxin unrelated to the insecticidal parasporal proteins of this bacterium. This protein shared little amino acid sequence homology (< 30%) with any other proteins, including B. thuringiensis Cry and Cyt proteins. Obviously, it constitutes a new class of Cry

Novel Cytotoxic Protein from B. thuringiensis

MOLT-4

HeLa

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Normal T

D

G

B

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H

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F

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Mock-inoculated

Parasporin (Cry45Aa)

A1470

A

Fig. 2. Cytopathic Effect of Proteinase K-Treated Inclusion Proteins on MOLT-4, HeLa, and Normal T Cells. Inclusion proteins: A, D, and G, Bacillus thuringiensis strain A1470; B, E, and H, Escherichia coli BL21(DE3)(pSHAN27.2); C, F, and I, mock-inoculated control. Phase-contrast microscopic observation was performed at 24 h post-administration. Bar ¼ 50 mm.

120 100 80 60 40

MOLT–4

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20 0 120 100 80 60 40

HeLa

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Fig. 3. Dose-Response Studies of Cytotoxic Effects of Inclusion Proteins on MOLT-4, HeLa, and Normal T Cells. The proteins were alkali-solubilized and proteinase K-activated: open square, Bacillus thuringiensis strain A1470; closed circle, Escherichia coli BL21(DE3)(pSHAN27.2). The level of cytotoxicity was assessed with MTT assays at 20 h post-administration.

protein, designated Cry45Aa by the Bacillus thuringiensis delta-endotoxin nomenclature committee (see N. Crickmore’s nomenclature website at http://www.biols. susx.ac.uk/home/Neil Crickmore/Bt/index.html).

Previously, Mizuki et al.9) established the name of parasporin (PS) for a functional protein group of B. thuringiensis parasporal proteins that have in vitro cytotoxic activities preferential for certain human cancer cells. Recently, we identified four parasporins from noninsecticidal B. thuringiensis strains.20) Of these, PS-1, PS-2, and PS-3 correspond respectively to Cry31Aa,9) Cry46Aa,10) and Cry41Aa.11) The 27 kDa protein (Cry45Aa) obtained here can be allocated to parasporin-4 (PS-4). Obviously, the protein cloned in this study is identical to the 27 kDa protein present in proteinase K-activated inclusion proteins of strain A1470. This is fully supported by the following evidence: (i) these two activated proteins had the same electrophoretic mobility, (ii) there was no difference in N-terminal or internal sequences between the two, (iii) polyclonal antibodies against the cloned protein reacted strongly with the activated protein of strain A1470. In immunoblotting analysis of intact inclusion protein, antibodies directed at the cloned protein recognized proteins of 31 kDa from strain A1470 as well as recombinant E. coli. It is clear from this result that the 31 kDa protein is a protoxin, which is converted to the 27 kDa toxin upon proteolytic processing. The 31 kDa protein was previously estimated to be 34 kDa.21) The antibodies also bound to a minor protein of 28 kDa present in the A1470 inclusions. It is likely that the 28 kDa protein is a proteolysis product from the 31 kDa protein generated by the action of endogenous protease.

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It is of interest to note that a substantial difference was evident in the level of cytotoxic activity between the wild-type inclusions of strain A1470 and the recombinant E. coli inclusions. Comparative dose-response studies provided evidence that the activity associated with the wild-type inclusions is significantly higher than that of recombinant inclusions. SDS–PAGE analysis indicated that the A1470 inclusion contains multiple protein components. Hence, it is very likely that the higher activity of the wild-type inclusion is due to the coexistence of multiple toxins. In fact, our previous study strongly suggested that at least two cytotoxic moieties are involved in the activity of the A1470 (89-T34-22) inclusions when partially separated by anionexchange chromatography.12) Another interesting item in our results is that the 27 kDa protein, PS-4, exhibited preferential and rapid cytotoxic activities: it was highly toxic to leukemic T cells (MOLT-4) at around 1 h after administration and slightly toxic to normal T cells, but non-toxic to uterus cervix cancer cells (HeLa). Mizuki et al.9) reported that the first parasporin, PS-1, exhibited strong cytotoxicity for both MOLT-4 and HeLa cells but not normal T cells. Recently, Ito et al.10) determined that PS-2 is cytotoxic to MOLT-4 and normal T cells but not to HeLa, and the rapid cytotoxic effect is non-apoptotic. Thus, PS-4 might be rather similar to PS-210) in terms of activity spectrum and mechanism, although a difference was evident in activity levels between these two parasporins. These findings, coupled with the fact that there are few homologies between the four parasporins, suggest the hypothesis that the difference in specific receptors might lead to varied cytotoxic activities and different levels of cytotoxicity. Future work will include identification and characterization of the receptor, if any, responsible for preferential cytotoxic activity of parasporin-4.

Acknowledgments We thank D. R. Zeigler of the Bacillus thuringiensis delta-endotoxin nomenclature committee for invaluable advice on the designation of the Cry protein classes. This study was supported by Special Coordination Funds for the Promotion of Science and Technology (Leading Research Utilizing Potential Science and Technology) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of the Japanese Government.

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