!vlemoirs of the Facultr of Engineering. Okarama Universitr. VoL38. Nos.l&2. pp.97-100. ~'Iarch 200~1

The cytotoxicity of Bacillus thuringiensis subsp.coreanensis A2316 strain against the human leukemic T cell

Masashi YAMAGIWADepartment of Bioscience and Biotechnology

Okayama UniversityOkayama 700-8530, Japan

Tetsuyuki AKAOBiotechnology and Food Research Institute

Fukuoka Industrial Technology Center

Michio OHBAGraduate School ofAgriculture

Kyushu UniversityFukuoka,812-8581,Japan

Taichi HlRAODepartment of Bioscience and Biotechnology

Okayama UniversityOkayama 700-8530, Japan

Eiichi MlZUKlBiotechnology and Food Research Institute

Fukuoka Industrial Technology Center

Hiroshi SAKAI*Department of Bioscience and Biotechnology

Okayama UniversityOkayama 700-8530, Japan

(Received February 13. 2004)

Bacillus lhuringiensis subsp. coreanensis A23 I6 is a newly isolated strain fromYonakunijima Island in Japan. It produces the proteinaceous inclusion body (crystal)which has no insecticidal and hemolytic activities. When the crystal proteins were digestedby proteinase K, they exhibited the strong cytotoxicity against human leukemic T cell,MOLT-4. The proteinase K-digested A2316 crystal proteins have little damage upon thecell membrane of MOLT-4, suggesting that the cell death of MOLT-4 was induced througha mechanism other than the colloid-osmotic swelling and cell lysis as caused by hithertoknown B. Ihuringiensis crystal proteins. The 29-kDa polypeptide proved to be an activecomponent of the proteinase K-digested A23 I6 crystal proteins. EC,o of the purified 29­kDa polypeptide was 0.0579 Ilglml. The N-terminal amino acid sequence of the 29-kDapolypeptide was identical with that of p29 produced by B. Ihuringiensis A 15 I9 strain 'andshared no significant homology with all the known proteins, suggesting that this polypeptidebelong to a new family of B. Ihuringiensis crystal proteins.

1. INTRODUCTIONBacillus Ihuringiensis produces crystalline

inclusions consisting of highly specific insecticidalproteins called Ii-endotoxins during sporulation,which are toxic to the larvae of lepidopteran, dipteran,and coleopteran insects, and are currently classifiedinto two families, Cry and Cyt proteins [I].However it was also reported that non-insecticidal B.Ihuringiensis occurs in natural environments morewidely than insecticidal ones [2, 3]. In 1999, anovel activity, the cytotoxicity against leukemia T*E-mail sakahrsh@biotech.okayama-u.ac.jp

97

cells and other human cancer cells, was found inparasporal inclusions of non-insecticidal and non­haemolytic B. Ihuringiensis isolates [4]. Amongthese isolates, the strain 84-HS-1-11 producesparasporal inclusions containing an 8 I-kDa proteinwhich shows, upon activation by trypsin orproteinase K, a strong cytocidal activity againsthuman leukemic T cells (MOLT-4) and human uteruscervix cancer cells (HeLa) but not against nonnal Tcells. The ay31Aal gene encoding this 81-kDaprotein designated as parasporin was cloned and the

98 Nlasashi YA?I.'IAGlvVA et 'II.

deduced amino acid sequence contained the fiveconserved blocks, but shared very low homologieswith the known classes of CJy and Cyt proteins [5].Lee et al. reported that the 28-kOa polypeptideproduced, upon proteinase K digestion, from thecrystal protein of B. thllringiensis subsp.shandongiensis 89-T-34-22 strain exhibited thehuman leukemic cell-specific cytotoxicity [6, 7].Furthermore, the 64-kDa major polypeptide produced,upon proteinase K digestion, ITom the crystalproteins of the B. thllr;ngiensis isolate 89-T-26-17exhibited cytotoxicity against MOLT-4 and HeLacells, but showed no cytotoxicity to nonnal T cells[8].

In this study the clystal proteins produced byB. thl/ringiensis subsp. coreanensis A2316 strainwere examined for the functional propertiesregarding the cytotoxicity toward MOLT-4, theleukemic T cell. It was suggested that thecytopathic effect by the A2316 crystal proteins withIinle damage to the cell membrane was differentITom the colloid-osmotic swelling and cell lysisinduced by Cry or Cyt proteins of B. thllringiensis.We purified the 29-kDa active component from theproteinase K-digested A2316 crystal proteins, whichis likely to be a member of a new family of B.thuringiensis crystal proteins.

2. MATERIALS AND METHODS2.1 Preparation, solubilization, and digestion ofcrystal proteins

Purification of crystal proteins was done asdescribed previously [9]. The crystal wassolubilized in 100 mM Na,C03 (pH 10.5)/ 10 mMOIT at 3TC for 1 hr. The solubilized crystalproteins were digested by proteinase K (Roche) ortrypsin (Roche) at 3TC for I hr. At the end ofincubation, PMSF, phenylmethylsulfonyl fluoride(Sigma) was added at a concentration of I mM.

2.2 MIT assayThe cytotoxicity was estimated by MTT assay.

Ninety microliters of a cell suspension (5x I0'cells/ml) and 10 III solution of the crystal proteinswere placed in each well of a 96-well microplate, andincubated at 3TC for 3 hr. Then 10111 solution of0.5 Ilg/id of MIT, 3-(4,5-dimethyl-2-thiazoyl)-2,5­diphenyl-2H-tetrazolium bromide (Sigma), wasadded and incubated for 3 hr. After centrifugation,the supernatant was removed, and the precipitate ofthe converted dye was solubilized with acidicisopropanol. The absorbance of the converted dyewas measured at 570 nm and the survival rate of thecells was calculated. The average absorbance ofmock-inoculated negative controls was taken as a

lvID..I.FAC.ENG.OKA.UNI. Vo1.38. Nos.l &2

high value (100% cell survival), and that of Triton­XI00 (Nacalai Tesque) -inoculated positive controlswas taken as a low value (0% cell survival).

2.3 Protein sequencingThe proteinase K-digested A2316 crystal

proteins were fractionated in SOS-14%-PAGE andtransferred to a PVOF membrane (Bio-Rad). TheN-terminal amino acid residues of the 29-kDapolypeptide were sequenced using a model 491protein sequencer of Applied Biosystems.

3. RESULTS3.1 Processing of A2316 crystal proteins

The purified A2316 crystal proteins wereprocessed in vitro by proteinase K and analyzed bySOS-PAGE (Fig. IA). Through the proteinase Kdigestion, the 150-kDa band disappeared and bandsof about 60 kDa were generated as the ratio ofproteinase K to the crystal protein was increased.The other polypeptides were also processed into thesmaller ones, and finally only polypeptides smallerthan 30 kOa were detected at high ratios ofproteinase K to the crystal protein. The cytotoxicityof the A2316 crystal proteins against MOLT-4 cellswas analyzed by MTT assay, showing that thesolubilized crystal proteins exhibited no detectablecytotoxicity (data not shown) and that the fullyprocessed crystal proteins induced the cell death ofMOLT-4 (Fig. IB). We confinned that proteinaseK itself induced no detectable cell death of MOLT-4at the concentrations indicated in Fig. I (data notshown).

3.2 The cytopathic effect of A2316 crystal proteinstoward MOLT-4 cells

The cytopathic effect of the proteinase K­digested A2316 crystal proteins toward MOLT-4cells was observed under a light microscopy (Fig. 2).The proteinase K-digested A2316 crystal proteinscaused the nuclear condensation and the cell swellingI hr after the toxin administration, but cell lysis wasnot observed even after the prolonged incubation for24 hr. It was suggested that the cytopathic effect ofthe A2316 crystal proteins was different from thecolloid-osmotic swelling and cell lysis caused by sofar known B. thllringiensis Cry or Cyt toxins.

3.3 Purification of the active component from theproteinase K-digested A2316 crystal proteins

To identify the active component responsiblefor the cytotoxicity against MOLT-4 cells, gelfiltration chromatography of the proteinase K­digested A2316 crystal proteins was done, and thecytotoxicity of each fraction was monitored by MTT

The cytotoxicity of Bacillus thuringiensis subsp. coreanensis A2316 strain against the human leukemic T cell 99

assay with MOLT-4 cells. The fractions thatcontained the cytotoxic activity were found andanalyzed hy SDS-PAGE, manifesting a handcorresponding to a 29-kDa polypeptide (Fig. 3A and3B).

Alberatio of proteinase K against crystci proteins

M S 10-5 10-4 10-3 10-2 10- 1 Wi

The N-terrninal antino acid sequence of the29-kDa polypeptide was proved DVIREYLMFNELSALSS(T/S)PE, which was identical with that of p29produced by Al5l9 strain [10] and shared nohomology with those of all the known proteinsincluding Cry or Cyt proteins of B. thuringiensis.Based on the obtained data of MTT assay, EC,o ofthe purified 29-kDa polypeptide to MOLT-4 cellswas estimated to be 0.0579 flg/ml (95% confidencelimits; 0.0557-0.0602) by Probit analysis.

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4. DISCUSSIONIn this study, we investigated the cytotoxicity

against human leukemic cells of the crystal proteinsproduced by B. thuringiensis subsp. coreanensisA23l6 strain. We found that the proteinase K­digested crystal proteins induced the specific celldeath of MOLT-4 through a mechanism other thanthe colloid-osmotic swelling and cell lysis that werecaused by hitherto known Cry or Cyt proteins of B.thuringiensis, and that the 29-kDa fragment of theproteinase K-digested crystal proteins was the activecomponent against MOLT-4. The purified 29-kDapolypeptide had a cytotoxic activity similar to p29from Al5l9 strain; EC,o of the 29-kDa polypeptideagainst MOLT-4 was 0.0579 flg/mI, whereas that ofp29 was 0.Q78 flg/mi [10]. The N-terrninal 20­antino acid sequence of the 29-kDa polypeptide wasidentical with that of p29 produced by Al5l9 strain[10] and shared no significant homology with all theknown proteins including Cry or Cyt toxins.

B

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The ratio of proteinase K against. crystal proteins

Fig. 1 The processing and the cytotoxicity of theA2316 crystal proteins.(A) The purified A2316 crystal proteins were solubilized andprocessed by proteinase K at the indicated ratio of proteinaseK to the crystal proteins. Ten micrograms of each digestwere analyzed by SDS-14o/~PAGE followed by eBB staining.The arrowhead indicates proteinase K. M, molecular sizemarker; S, the solubilized crystal proteins. (B) Tbecytotoxicity against MOLT-4 of the proteinase K-digestedcrystal proteins was estimated by MIT assay.

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Fig. 2 The cytopathic elleet of the proteinase K­digested A2316 crystal proteins against MOLT-4.MOLT-4 cells were incubated in the presence of 10 ~g/ml ofthe proteinase K-digested A2316 crystal proteins. Thecytopathic effect on the cells was observed under a lightmicroscopy I hr, 3 hr, 6 hr, I2 hr or 24 hr after applying thecrystal proteins.

100 M.s.shi YAMAGIWA et .1. MEM.FAC.ENG.OKA.UNI. Vo1.38. Nos.l&2

B(kD.) M

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M Ac 170 172 174 176178 180 182 184 186 188 (min)

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Fig.3 Purification ofthe 29-kDa polypeptide active toward the MOLT-4 cells.(A) Gel filtration chromatography of the proteinase K-digested A2316 crystal pmteins was done using the superdex 200 pg(Amersham Pharrnacia Biotech) column. The eluate from the column was pooled in 2-ml aliquots. and was analyzed by MITassay for the cytotoxicity against MOLT-4 cells. The arrowhead indicates the 29-kDa polypeptide cytotoxic against MOLT-4cells. +. cytotoxic against MOLT-4; -. not cytotoxic against MOLT-4. M. molecular size marker; Ac, the proteinase «-digestedA2316 crystal proteins. (B) One microgram of the purified 29-kDo polypeptide was analyzed by SDS-14% PAGE followed bysilver staining. M. molecular size marker.

Since the 29-kDa polypeptide from A2316strain had the same N-tenninaJ amino acid sequenceas p29 from A1519 strain, il was suggested that boththe polypeptides were very similar to each other.Recently we have found that p29 from A1519 straininduce apoptosis in Jurkat, another human leukemicT cell line (manuscript in preparation). Therefore, itis reasonable to assume that the 29-kDa polypeptidefrom A2316 strain would also induce the apoptoticcell death.

ACKNO~EDGEMENTS

This study was performed through SpecialCoordination Funds for Promoting Science andTechnology (Leading Researcb Utilizing Potential ofRegional Science and Technology) of the Ministry ofEducation, Culture, Sports, Science and Technologyof the Japanese Government.

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