Proteolytic Activation of Bacillus thuringiensis Cry2Ab through a Belt

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Proteolytic activation of Bacillus thuringiensis Cry2Ab through a belt-and-braces approach Lian Xu, Zhi-Zhen Pan, Jing Zhang, Bo Liu, Yu-Jing Zhu, and Qing-Xi Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03111 • Publication Date (Web): 06 Sep 2016 Downloaded from http://pubs.acs.org on September 7, 2016

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Journal of Agricultural and Food Chemistry

Proteolytic activation of Bacillus thuringiensis Cry2Ab through a belt-and-braces approach Lian Xu †, #, Zhi-Zhen Pan ‡, #, Jing Zhang †, Bo Liu ‡, Yu-Jing Zhu ‡, *, Qing-Xi Chen †,*

†

State Key Laboratory of Cellular Stress Biology, Key Laboratory of the Ministry of

Education for Coastal and Wetland Ecosystems, School of Life Sciences, Xiamen University, Xiamen 361005, China. ‡

Agricultural Bio-Resources Research Institute, Fujian Academy of Agricultural

Sciences, Fuzhou 350003, China.

#

The authors contribute equally in this work.

*

Corresponding author.

(Qing-Xi Chen) Tel: 0592-2185487/13459256685, Fax: 0592-2185487, E-mail: [email protected]. (Yu-Jing Zhu) E-mail: [email protected].

ACS Paragon Plus Environment


1

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ABSTRACT

2

Proteolytic processing of Bacillus thuringiensis (Bt) crystal toxins by insect

3

midgut proteases play an essential role in their insecticidal toxicities against target

4

insects. In present study, proteolysis of Bt crystal toxin Cry2Ab by Plutella

5

xylostella L. midgut proteases (PxMJ) was evaluated. Both trypsin and chymotrypsin

6

were identified involving in the proteolytic activation of Cry2Ab, and cleaving

7

Cry2Ab at Arg139 and Leu144, respectively. Three Cry2Ab mutants (R139A, L144A

8

and R139A-L144A) were constructed by replacing residues Arg139, Leu144 and

9

Arg139-Leu144 with alanine. Proteolysis assays revealed that mutants R139A and

10

L144A but not R139A-L144A could be cleaved into 50 kDa activated-toxins by

11

PxMJ. Bioassays showed that mutants R139A and L144A were high toxic against P.

12

xylostella larvae, while mutant R139A-L144A was almost non-insecticidal. Those

13

results demonstrated that proteolysis by PxMJ was associated with Cry2Ab’s

14

toxicity against P. xylostella. It also revealed that either trypsin or chymotrypsin was

15

enough to activate Cry2Ab protoxin. This characteristic was regarded as a

16

belt-and-braces approach and might contribute to control of resistances development

17

in target insects. Our studies characterized the proteolytic processing of Cry2Ab and

18

provided a new insight into the activation of this Bt toxin.

19 20

KEYWORDS:

Bacillus

21

Insecticidal activity.

thuringiensis,

Cry2Ab,

Proteolysis,

22


Cleavage-site,

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23

INTRODUCTION

24

The bacterium Bacillus thuringiensis (Bt) is the most successful and commercial

25

bio-insecticides against various agriculture pests around the world.1 The pesticidal

26

activities of Bt toxins mainly attribute to sorts of δ-endotoxins synthesized during

27

the sporulation stage. Among all δ-endotoxins, insecticidal crystal proteins (Cry

28

toxins) have been widely used as part of spray products or expressed in transgenic

29

crops to control agricultural pests such as lepidopterans, coleopterans, hemipterans,

30

dipterans as well as some nematodes.2, 3

31

The mode of action of Cry toxins has been characterized principally in

32

lepidopteran insects using Cry1A toxins as models.4, 5 In most cases, Cry toxins were

33

produced in insoluble and inactive forms which were called protoxins.6 When

34

ingested by susceptible insects, Cry protoxins dissolved in the alkaline environment

35

of midgut and were further processed into activated-toxins by target insect midgut

36

proteases.7 Those activated-toxins sequential interacted with special receptors

37

inlaying in the brush border membranes vesicles (BBMV) of insect midgut

38

epithelium,8,

39

membrane to form pores,10 eventually those actions ended in the death of insects.

9

assembled pre-pore oligomeric structures and inserted into cell

40

The Cry toxins comprise at least 73 subgroups and more than 700 members are

41

identified according to similarity of amino acid sequences.11 Among them, Cry2A

42

toxins families (principally found in B. thuringiensis subsp. kurstaki HD-1) are

43

characterized by their high pathogenicity against lepidopteran insects such as

44

Plutella xylostella L. and Helicoverpa armigera (Hübner) that are the worldwide



45

agricultural pests.12 Up to date, twelve Cry2A subtypes including Cry2Aa to Cry2Al

46

have been identified and reported.11 The phylogenetic analysis indicates that Cry2A

47

evolves as a separate group which is far from other Cry toxins families.13 The

48

homology of Cry2A and other Bt toxins are less than 20% in amino acid

49

sequences.14 As a result, Cry2A exhibits lower level of cross-resistance with other Bt

50

toxins such as Cry1A or Vip3A, and is lethal to insects that are resistant against

51

Cry1A or Vip3A.15,

52

co-expressed Cry1Ac and Cry2Ab, greatly prolong the efficacy of Bt toxins and

53

provide a new strategy for the insects resistance management.17, 18

16

The second generation Bt-transgenic crops, which

54

Among all subtypes of Cry2A toxins, Cry2Aa is undoubtedly one of the most

55

significant Cry toxins and is devoted considerable studies since its importance in

56

pest control. Based on the crystal structure of Cry2Aa, Morse et al. suggested that

57

N-terminus cleavage of Cry2Aa might unmask a hydrophobic patch which was

58

involved in toxin-receptor or toxin-membrane interaction.

59

demonstrated that cleavage of Cry2Aa protoxin by midgut proteases occurred at

60

Try49 and Leu144, and resulted in a 58 kDa and a 50 kDa fragments, separately.20

61

Further researches by Ohsawa et al. showed that the 50 kDa fragment of Cry2Aa was

62

toxic to Bombyx mori L. and Lymantria dispar L.21 The homology of amino acid

63

sequences in Cry2Aa and other Cry2A toxins were less than 90%. The diversity in

64

amino acid sequences suggested the proteolysis mode of Cry2Aa might differ from

65

other Cry2A toxins. However, the proteolysis of other Cry2A toxins such as Cry2Ab

66

was still unevaluated. Furthermore, only chymotrypsin was documented involving in


19

Audtho et al. firstly

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67

the proteolysis of Cry2A protoxins.20- 22 Other proteases such as trypsin were also

68

found in lepidopteran insect midguts, and whether those proteases contributed to the

69

processing of Cry2A were unreported yet.

70

In present work, we revealed a novel proteolytic pattern of Cry2Ab which was

71

regarded as a belt-and-braces approach in the activation of this Cry toxin. Proteolysis

72

of Cry2Ab protoxin occurred at Arg139 and Leu144, these two residues were regarded

73

as trypsin and chymotrypsin cleavage-site, separately. Furthermore, Cry2Ab mutants

74

by substituting Arg139 and Leu144 with alanine were constructed. Proteolysis assay

75

and bioassay on wild type and mutants Cry2Ab were further evaluated. Those

76

studies provided a new insight into the proteolytic activation of Cry2Ab toxin.

77 78

MATERIALS AND METHODS

79

Insects.

80

A laboratory population of P. xylostella larvae was kindly provided by

81

Bio-Pesticide Engineering Research Center, Wuhan of Hubei Province, China. P.

82

xylostella larvae were fed with artificial diet under the conditions of 27 ± 2℃, 70%

83

humidity and photoperiod of 14:10 h (light: dark).

84 85

P. xylostella midgut protease.

86

The P. xylostella midgut proteases were prepared as previous described.23 Briefly,

87

twenty midguts were extracted from 3rd instar larvae of P. xylostella and followed by

88

two washes with cold sodium chloride (128 mM). Midgut tissues were homogenized



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89

with 200 µL PBS buffer (50 mM, pH 7.4), centrifuged at 25,000 g for 30 min at 4℃.

90

The supernatants which contained midgut proteases were labeled as P. xylostella

91

midgut juice (PxMJ) and quantified by BCA Protein Assay Kit (BEYOTIME,

92

CHINA) according to the manufacturers’ instructions. PxMJ were stored at -80℃

93

until used.

94 95

Preparation of Cry2Ab.

96

Escherichia Coli BL21 (DE3) cells harbouring pET30a-cry2Ab (NCBI accession

97

number: EU623976) were grown in LB medium with 35 µg/ mL kanamycin.24

98

Expression of Cry2Ab was induced overnight at 16 ℃

99

isopropyl-B-D-thiogalactopyranoside (IPTG) after OD600

nm

with 0.2 mM

reached 0.6. Cry2Ab

100

protein was purified by the method of Pan et al.24 using a Ni-IDA Prepacked Column

101

(Sangon, CHINA). Purified Cry2Ab was detected by SDS-PAGE electrophoresis and

102

western blotting using anti-Cry2Ab antibody. The concentration of Cry2Ab was

103

quantified by BCA Protein Assay Kit (BEYOTIME, CHINA).

104 105

Proteolysis assay.

106

Proteolysis assay was conducted to evaluate the proteolytic kinetics of Cry2Ab

107

processed by PxMJ. 15 µg Cry2Ab protoxin was mixed with PxMJ (150 ng) in 60

108

µL final volume of sodium carbonate buffer (50 mM, pH 9.5). The mixtures were

109

incubated at 30℃ for different incubation times, with constant shaking (about 40

110

rpm). The proteolytic reactions were stopped by boiling for 10 min. SDS-PAGE


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111

electrophoresis and western blotting were used to assess the proteolytic activation of

112

Cry2Ab protoxin.

113 114

Edman degradation sequencing analysis.

115

N-terminal sequencing was performed to determine the cleavage site and involved

116

proteases of Cry2Ab processed by PxMJ. Briefly, after PxMJ treatment, 500 µg

117

Cry2Ab activated-toxin was separated by SDS-PAGE electrophoresis and further

118

transferred onto a PVDF membrane (Millipore, GERMANY). The Cry2Ab

119

activated-toixn band was excised according to prestained protein molecular weight

120

marker (Thermo Scientific, AMERICA) and submitted for amino acid sequencing

121

using SHIMADZU automated protein/peptide sequencer (PPSQ-333A, JAPAN).25

122

PeptideCutter program (http://web.expasy.org/peptide_cutter/)26 was used to identify

123

the potential protease responsible for the proteolytic processing of Cry2Ab.

124 125

Proteolytic inhibition assay.

126

Proteolytic inhibition assay was performed as Freedman et al. described,27 PxMJ

127

was incubated with excess specific protease inhibitors including serine protease

128

inhibitor (SPI), cysteine proteinase inhibitor (CPI), aspartic proteinase inhibitor (API)

129

and metalloproteinase inhibitor (MPI) for 60 min at 30℃. After inhibitor treatment,

130

150 ng PxMJ was incubated with 15 µg Cry2Ab protoxin for proteolytic processing.

131

Subsequently, the proteolytic reactions were stopped by boiling for 10 min.

132

SDS-PAGE electrophoresis was used to evaluate the proteolysis of Cry2Ab



133

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protoxin.

134 135

Site-directed mutagenesis.

136

The pET30a-cry2Ab plasmid which contained a 1902 bp of cry2Ab DNA sequence

137

was selected as template for site-directed mutagenesis. Three cry2Ab mutant

138

plasmids

139

(chymotrypsin-site) and Arg139-Leu144 (both trypsin-site and chymotrypsin-site) with

140

alanine (R139A, L144A, and R139A-L144A), using Fast Site-Directed Mutagenesis

141

Kit (TIANGEN, CHINA) according to instruction manual. The mutation primers

142

were shown in Table 1. All the mutant plasmids were confirmed by sequencing and

143

positive clones were transformed into Escherichia coli BL21 cells. The expression

144

and purification of mutants Cry2Ab were conducted as previously described.24 The

145

purified mutants Cry2Ab were detected both by SDS-PAGE electrophoresis and

146

western blotting using anti-Cry2Ab antibody.

had

been

generated

by

replacing

Arg139 (trypsin-site),

Leu144

147 148

Toxicity assays.

149

Bioassays were executed with 3rd instar larvae of P. xylostella according to Pan et

150

al.28 2 mL artificial diet was added to 6-well polystyrene plates (Sangon, CHINA)

151

and air-dried. Wild type, R139A, L144A and R139A-L144A Cry2Ab toxins were

152

diluted in sodium carbonate buffer (50 mM, pH 9.5). Five concentrations of wild

153

type and mutants Cry2Ab toxins from 0.032 to 20 µg/cm2 were set up. Insects tested

154

with sodium carbonate buffer (50 mM, pH 9.5) were served as negative control.


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155

Fifteen 3rd instar larvae of P. xylostella were used in each concentration assay and

156

three independent replicates were carried out. Observations were recorded at 72

157

hours and the LD50 value was analyzed by SPSS 17.0 (Statistical Product and

158

Service Solutions) using PROBIT analysis.29

159 160

RESULTS

161

Cry2Ab preparation and identification.

162

The purity and identity of Cry2Ab were accessed by SDS-PAGE electrophoresis

163

and western blotting using anti-Cry2Ab antibody. As shown in Fig 1A, a clear single

164

protein band was purified using Ni-IDA Prepacked Column and further confirmed to

165

be Cry2Ab protein by anti-Cry2Ab antibody (Fig 1B). The purified Cry2Ab was

166

about 65 kDa in molecular weight, which was in accordance with the reports by Jain

167

et al.30

168 169

Proteolytic kinetics of Cry2Ab by PxMJ.

170

Time course of proteolytic processing of Cry2Ab was examined ranging from 0 to

171

180 min. This proteolysis was detected both by SDS-PAGE electrophoresis with

172

Coomassie staining (Fig 2A) and western blotting (Fig 2B). During the treatment

173

with PxMJ, two protein bands corresponding to Cry2Ab protoxin (65 kDa) and

174

Cry2Ab activated-toxin (50 kDa) were constantly detected. As time went by, the

175

Cry2Ab protoxin band gradually decreased while the Cry2Ab activated-toxin band

176

continuously increased. The Cry2Ab activated-toxin band was detectable from the



177

first 1 min and complete presented after 60 min. Observation at 180 min revealed

178

that Cry2Ab activated-toxin was not further degraded and remained 50 kDa form.

179

This result suggested this 50 kDa fragment was resistant to PxMJ.

180 181

Edman degradation analysis revealed two cleavage-sites on Cry2Ab.

182

Five cycles of Edman degradation reactions were executed to determine the

183

N-terminal sequence of Cry2Ab activated-toxin. Strangely, two kinds of amino acids

184

were identified in each round of Edman degradation reactions (Fig S1). The

185

identified amino acids in first round were asparagine (N) and serine (S), the second

186

were alanine (A) and isoleucine (I), the third were threonine (T) and valine (V), the

187

fourth were serine (S) and proline (P), the identified amino acids in last round were

188

serine (S) and leucine (L) (Fig S1). When compared with Cry2Ab protein sequences,

189

those ten amino acids could be exactly divided into two groups: NAVPL and SITSS,

190

which completely matched the sequences of 140NAVPL144 and 145SITSS149 in Cry2Ab

191

sequences. Those findings suggested that there were two cleavage-sites existed on

192

the Cry2Ab. After proteolytic processing by PxMJ, two kinds of N-terminus

193

products corresponding to 140NAVPL144 and 145SITSS149 were generated.

194 195

Trypsin and chymotrypsin were both involved in the proteolysis of Cry2Ab.

196

Based on N-terminal sequences of Cry2Ab activated-toixn, we concluded that

197

proteolysis by PxMJ cleaved Cry2Ab at Arginine (Arg139) and Leucine (Leu144),

198

separately (Fig 3A). The amino acid Arg139 was predicted as a putative trypsin


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199

cleavage-site by PeptideCutter program. Similarly, the residue Leucine (Leu144),

200

preceding 145SITSS149, was determined as a putative chmyotrypsin cleavage-site. The

201

Cry2Ab 3D structure was built by I-TASSER servers31 and viewed by PyMOL

202

program. Based on its protein structure, proteolysis of Cry2Ab removed three

203

α-helices (α1 to α3) in Domain Ι and processed Cry2Ab protoxin into Cry2Ab

204

activated toxin (Fig 3B and 3C). PyMOL revealed that both Arg139 and Leu144 were

205

located at the loop structure between helices α3 and α4 in Domain Ι of Cry2Ab (Fig

206

3D).

207

Edman degradation assays indicated that both trypsin and chymotrypsin were

208

involved in the proteolytic activation of Cry2Ab. Our proteolytic assays confirmed

209

this hypothesis with the results that both commercial trypsin and chymotrypsin could

210

cleave Cry2Ab protoxin into activated-toxin, which was similar to the treatment of

211

PxMJ (Fig 3E). Trypsin and chymotrypsin both belonged to serine proteases, so we

212

supposed that inhibiting serine proteases in PxMJ could block the proteolysis of

213

Cry2Ab. As expected, the proteases inhibition assay demonstrated that only PMSF,

214

which served as a serine protease inhibitor, could seriously block the proteolytic

215

cleavage of Cry2Ab into 50 kDa activated toxin (Fig 3F). Collectively, those results

216

illustrated that serine proteases including trypsin and chymotrypsin in PxMJ played

217

an essential role in proteolytic activation of Cry2Ab.

218 219

Mutations both on trypsin-site and chymotrypsin-site blocked the proteolytic

220

processing of Cry2Ab.



221

To further evaluate the proteolytic activation of Cry2Ab, three mutants R139A

222

(replaced Arg139 with Alanine), L144A (replaced Leu144 with Alanine) and

223

R139A-L144A (replaced Arg139 and Leu144 with Alanine) were generated by

224

site-directed mutagenesis (Fig 4A). The production and purification of mutants

225

Cry2Ab were performed as Pan et al.24 described. SDS-PAGE electrophoresis

226

revealed that R139A, L144A and R139A-L144A mutants Cry2Ab were approximate

227

65 kDa, which was similar to wild type Cry2Ab in molecular sizes. Furthermore,

228

R139A, L144A and R139A-L144A mutants Cry2Ab could be detected by

229

anti-Cry2Ab antibody, suggesting that these mutations did not cause major structural

230

disturbance of Cry2Ab toxins (Fig 4B).

231

We further carried out proteolysis assay to assess the proteolytic activation of wild

232

type, R139A, L144A and R139A-L144A Cry2Ab protoxins. The results

233

demonstrated that proteolysis of wild type, R139A and L144A Cry2Ab were similar.

234

Those three Cry2Ab protoxins gradually processed into activated-toxins under the

235

treatment of PxMJ. The 50 kDa activated-toxin bands were detectable within 20 min

236

and completely generated at 60 min (Fig 4C- 4E). In contrary, the proteolytic pattern

237

of R139A-L144A was totally different when compared to other three Cry2Ab toxins.

238

A smaller protein band with molecular weight of 63 kDa rather than 65 kDa was

239

observed during the proteolysis of R139A-L144A Cry2Ab. However, the 50 kDa

240

activated-toxin band wasn’t generated even detected at 60 min (Fig 4F). Those

241

results suggested that proteolytic processing of wild type or single-site mutation

242

Cry2Ab (R139A or L144A) could produce the 50 kDa activated-toxin as well. Only


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243

when Arg139 and Leu144 were both mutated, this proteolysis could be blocked.

244 245

Mutations both on trypsin-site and chymotrypsin-site linked to the deactivation of

246

insecticidal activity in Cry2Ab.

247

Bioassays were performed to further evaluate the mutation of cleavage-sites on

248

Cry2Ab insecticidal activities. The results indicated that mortalities of P. xylostella

249

were dose-related and increased with the dose rise of Cry2Ab. However, the

250

mortality caused by mutant R139A-L144A was significant lower contrasted to wild

251

type, R139A and L144A Cry2Ab (Fig 5). Correlation with the mortalities results, the

252

LD50 value of mutant R139A-L144A was more than 20 µg/cm2, much higher when

253

compared to wild type, R139A and L144A Cry2Ab, with LD50s 1.801, 2.134 and

254

1.579 µg/cm2 respectively (Table 2). Based on mortalities and LD50 values, we

255

concluded that P. xylostella was less susceptible to R139A-L144A but not to R139A

256

or L144A. This results revealed that single-site mutation on Cry2Ab (R139A or

257

L144A) had no distinct influence on insecticidal activity of Cry2Ab. However, the

258

mutation of Cry2Ab both on Arg139 and Leu144 resulted in the great loss of toxicity

259

against P. xylostella.

260 261

DISSCUSSION

262

Proteolysis of Cry toxins by insect midgut proteases was crucial for their

263

insecticidal actions.5 Here we demonstrated that proteolytic processing of Cry2Ab

264

occurred at Arg139 and Leu144, respectively (Fig S1). Interestingly, the positions of



265

those two cleavage sites were adjacent and both of them located on the loop structure

266

connecting helices α3 and α4 in Domain Ι of Cry2Ab (Fig 3D). PyMOL program

267

revealed that the state of this loop structure was almost solvent-exposed. This

268

peculiarity ensured midgut proteases could easily get into, recognize specific

269

residues, and further execute cleavage processing (Fig 3B and 3D). The cleavage

270

N-terminal peptide (including α1 to α3 helices) covered the surface of Cry2Ab and

271

might prevent Cry2Ab from exposing the activated region which was similar to the

272

reports that N-terminus of Cry2Aa acted as a masked to block the activation of

273

Cry2Aa toxin.19 So proteolysis of Cry2Ab to remove its N-terminal sequences, was

274

essential for its toxicity activation.

275

Proteolytic processing of Cry2Ab directly cleaved into a 50 kDa activated-toxin

276

(Fig 2), while proteolysis of Cry2Aa, which occurred at Tyr49 and Leu144, firstly

277

processed into a 58 kDa toxin, and further cleaved into a 49 kDa fragment.20, 21

278

Those results suggested that proteolysis of Cry2Aa and Cry2Ab might be different.

279

We further demonstrated that trypsin and chymotrypsin were both involved in the

280

proteolysis of Cry2Ab (Fig 3E). Chymotrypsin was reported took part in the

281

activation of Cry2Aa,20- 22 while identification of trypsin and chymotrypsin both

282

involved in the proteolysis of Cry2Ab was for the first time. The proteases inhibition

283

assays confirmed that serine proteases were the functional proteases during the

284

proteolysis of Cry2Ab protoxin (Fig 3F). This result was consistent with the reports

285

that serine proteases were the main digestive proteases in lepidopteran insects. 32 It

286

could also explain why Cry2Ab was high lethal to lepidopteran but not coleopteran


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287

insects, for the main digestive proteases in coleopteran insects were cysteine

288

protease.32 Taken together, those findings made it apparent that serine proteases

289

(which included trypsin and chymotrypsin) in PxMJ played a functional role in the

290

processing and activation of Cry2Ab.

291

Previous studies provided considerable insights into the proteolysis of Cry toxins.

292

For example, Lebel et al. demonstrated that Cry1Aa with no cleavage of helix α1

293

failed to assemble pre-pore oligomeric structures.33 It was also documented that a

294

Cry1Ac mutant that retained the N-terminus after trypsin treatment bound

295

non-specifically to Manduca sexta L. BBMV.34 Those reports suggested that

296

proteolysis of Cry toxins by midgut protease was an indispensable step for activating

297

their pesticidal activities. In current study, proteolysis results together with bioassay

298

results indicated that proteolytic processing of Cry2Ab was associated with its

299

insecticidal action. Mutant R139A-L144A could not be cleaved by PxMJ and was

300

low toxic against P. xylostella larvae (Fig 4F and Table 2). This results strongly

301

suggested that proteolysis was critical for Cry2Ab insecticidal activity against

302

P.xylostella. Furthermore, we found an interesting phenomenon that both mutant

303

R139A (only recognized by chymotrypsin and cleaved at Leu144) and L144A (only

304

recognized by trypsin and cleaved at Arg139) killed P. xylostella larvae as well. The

305

LD50 values of these two toxins against P. xylostella were on the same level (Table

306

2). Those results revealed that cleavage at Arg139 (by trypsin) or Leu144 (by

307

chymotrypsin) was enough to activate Cry2Ab. In other words, either trypsin or

308

chymotrypsin was sufficient for invoking the toxicity of Cry2Ab. This characteristic



309

seemed to be a belt-and-braces approach for proteolysis of Cry2Ab. In most cases,

310

mode of action of Cry toxins in lepidopteran insects began with the proteolytic

311

processing by midgut serine protease such as trypsin and chymotrypsin. Those two

312

proteases were not only the primary digestive enzymes in lepidopteron but also

313

involved in the activation of Cry toxins.32 Liu et al. documented that Cry1Ac

314

resistance to H. armigera was associated with down-regulations of trypsin R gene.35

315

More recently, a significant decrease of trypsin-like proteases were observed in

316

Cry1Ac-resistant larvae in P. xylostella when compared to Cry1Ac-susceptible

317

larvae.36 Those results indicated that reduction or mutation of serine proteases in

318

lepidopteran insect midguts might result in improper processing of Cry toxins and

319

emphasized the importance of midgut serine proteases during the mode of Cry

320

toxins.37,

321

protease seemed less likely occurred in Cry2Ab since it had two cleavage sites

322

corresponding to trypsin and chymotrypsin. Cleavage could take place as long as

323

either trypsin or chymotrypsin existed. Only when trypsin and chymotrypsin were

324

absent, the proteolysis could be blocked. This belt-and-braces characteristic made

325

Cry2Ab less likely for development of resistance in target insects.

38

However, Cry resistance caused by reduction expression of serine

326

In summary, we systematically evaluated the proteolytic processing of Cry2Ab

327

and proved that trypsin and chymotrypsin were both involved in the proteolysis of

328

Cry2Ab. The characteristic that either trypsin or chymotrypsin was enough to

329

activate Cry2Ab was regarded as a belt-and-braces approach and might slow down

330

insect resistances development. However, more studies should be investigated to


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331

further illuminate the insecticidal mechanism of this Cry toxin. Our findings pointed

332

out new directions for pest resistance control and provided new insight into the

333

proteolytic activation of Cry toxins.

334 335

ABBREVIATIONS USED

336

Bt, Bacillus thuringiensis; PxMJ, Plutella xylostella midgut proteases; BBMV, brush

337

border membranes vesicles; kDa, kilodalton; SPI, serine protease inhibitor; CPI,

338

cysteine

339

metalloproteinase inhibitor. PVDF, Polyvinylidene fluoride; LD50, median lethal

340

dose; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEM,

341

standard error of mean; CL, confidence level.

proteinase

inhibitor;

API,

aspartic

proteinase

inhibitor;

MPI,

342 343

ASSOCIATED CONTENT

344

Supporting Information

345

N-terminal sequencing identified cleavage-sites of Cry2Ab processed by PxMJ (Fig

346

S1). (A) Spectrum of 19 PTH-amino acids standards; (B)-(F) N-terminal amino acid

347

identification of Cry2Ab activated-toxin. This material is available free of charge via

348

the Internet at http://pubs.acs.org.

349 350 351

Funding

352

This work was supported by the National Natural Science Foundation of China



353

(31371999), the Natural Science Foundation of Fujian Province, China

354

(2016J01130).

355

Notes

356

The authors declare no competing financial interest.


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Page 19 of 38


RFFERENCES 1.

Bravo, A.; Likitvivatanavong, S.; Gill, S. S.; Soberón, M. Bacillus thuringiensis: a story of a successful bioinsecticide. Insect Biochem. Mol. Biol. 2011, 41, 423-431.

2.

Schnepf, E.; Crickmore, N. V.; Van Rie, J.; Lereclus, D.; Baum, J.; Feitelson, J.; Dean, D. H.. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 1998, 62, 775-806.

3.

Bravo, A.; Gómez, I.; Porta, H.; García-Gómez, B. I.; Rodriguez-Almazan, C.; Pardo, L.; Soberón, M.. Evolution of Bacillus thuringiensis Cry toxins insecticidal activity. Microb. Biotechnol. 2013, 6, 17-26.

4.

Pardo-Lopez, L.; Soberon, M.; Bravo, A. Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection. FEMS Microbiol. Lett. 2013, 37, 3-22.

5.

Bravo, A.; Gill, S. S.; Soberon, M. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon. 2007, 49, 423-435.

6.

Vachon, V.; Laprade, R.; Schwartz, J. L. Current models of the mode of action of Bacillus thuringiensis insecticidal crystal proteins: a critical review. J. Invertebr. Pathol. 2012, 111, 1-12.

7.

Palma, L.; Muñoz, D.; Berry, C.; Murillo, J.; Caballero, P. Bacillus thuringiensis toxins: an overview of their biocidal activity. Toxins. 2014, .6, 3296-3325.

8.

Pigott, C. R.; Ellar, D. J. Role of receptors in Bacillus thuringiensis crystal toxin activity. Microbiol. Mol. Biol. Rev. 2007, 71, 255-281.

9.

Likitvivatanavong, S.; Chen, J.; Evans, A. M.; Bravo, A.; Soberon, M.; Gill, S. S. Multiple



Page 20 of 38

receptors as targets of Cry toxins in mosquitoes. J. Agric. Food Chem. 2011, 59, 2829-2838. 10. Gómez, I.; Sánchez, J.; Muñoz-Garay, C.; Matus, V.; Gill, S. S.; Soberón, M.; Bravo, A. Bacillus thuringiensis Cry1A toxins are versatile proteins with multiple modes of action: two distinct pre-pores are involved in toxicity. Biochem. J. 2014, 459, 383-396. 11. Crickmore, N.; Baum, J.; Bravo, A.; Lereclus, D.; Narva, K.; Sampson, K.; Schnepf, E.; Sun, M.;

Zeigler,

D.

R.

Bacillus

thuringiensis

toxin

nomenclature.

2016.

(http://wwwlifescisussexacuk/home/Neil_Crickmore/Bt/) (July 15, 2016). 12. Widner, W. R.; Whiteley, H. R. Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities. J. Bacteriol. 1989,171, 965-974. 13. de Maagd, R. A.; Bravo, A.; Crickmore, N. How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet. 2001, 17, 193-199. 14. Höfte, H.; Whiteley, H. R. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol. Rev. 1989, 53, 242-255. 15. Caccia, S.; Hernández-Rodríguez, C. S.; Mahon, R. J.; Downes, S.; James, W.; Bautsoens, N.; Ferre, J. Binding site alteration is responsible for field-isolated resistance to Bacillus thuringiensis Cry2A insecticidal proteins in two Helicoverpa species. PLoS One. 2010, 5, e9975. 16. Mahon, R. J.; Downes, S. J.; James, B. Vip3A resistance alleles exist at high levels in Australian targets before release of cotton expressing this toxin. PLoS One. 2012, 7, e39192. 17. Hamilton, K. A.; Pyla, P. D.; Breeze, M.; Olson, T.; Li, M.; Robinson, E.; Chen, Y.. Bollgard II cotton: compositional analysis and feeding studies of cottonseed from insect-protected


Page 21 of 38


cotton (Gossypium hirsutum L.) producing the Cry1Ac and Cry2Ab2 proteins. J. Agric. Food Chem. 2004, 52, 6969-6976. 18. Yang, Y.; Chen, X.; Cheng, L.; Cao, F.; Romeis, J.; Li, Y.; Peng, Y. Toxicological and biochemical analyses demonstrate no toxic effect of Cry1C and Cry2A to Folsomia candida. Sci. Rep. 2015, 5. 19. Morse, R. J.; Yamamoto, T.; Stroud, R. M.; Structure of Cry2Aa suggests an unexpected receptor binding epitope. Structure. 2001, 9, 409-417. 20. Audtho, M.; Valaitis, A. P.; Alzate, O.; Dean, D. H, Production of chymotrypsin-resistant Bacillus thuringiensis Cry2Aa1 δ-endotoxin by protein engineering. Appl. Environ. Microbiol. 1999, 65, 4601-4605. 21. Ohsawa, M.; Tanaka, M.; Moriyama, K.; Shimazu, M.; Asano, S. I.; Miyamoto, K.; Hori, H. A 50-kilodalton Cry2A peptide is lethal to Bombyx mori and Lymantria dispar. Appl. Environ. Microbiol. 2012, 78, 4755-4757. 22. Nouha, A.; Sameh, S.; Fakher, F.; Slim, T.; Souad, R. Impact of Q139R substitution of MEB4-Cry2Aa toxin on its stability, accessibility and toxicity against Ephestia kuehniella. Int. J. Biol. Macromol. 2015, 81, 701-709. 23. Li, H.; Chougule, N. P.; Bonning, B. C. Interaction of the Bacillus thuringiensis delta endotoxins Cry1Ac and Cry3Aa with the gut of the pea aphid, Acyrthosiphon pisum (Harris). J. Invertebr. Pathol. 2011, 107, 69-78. 24. Pan, Z. Z.; Xu, L.; Zhu, Y. J.; Shi, H.; Chen, Z.; Chen, M. C.; Liu, B. Characterization of a new cry2Ab gene of Bacillus thuringiensis with high insecticidal activity against Plutella xylostella L. World J. Microbiol. Biotechnol. 2014, 30, 2655-2662.



25. Wang, X.; Xiao, B.; Zhang, J.; Chen, D.; Li, W.; Li, M.; Luo, S. Identification and Characterization of a Cleavage Site in the Proteolysis of Orf Virus 086 Protein. Front Microbiol. 2016,7. 26. Lafarga, T.; O’Connor, P.; Hayes, M. Identification of novel dipeptidyl peptidase-IV and angiotensin-I-converting enzyme inhibitory peptides from meat proteins using in silico analysis. Peptides. 2014, 59, 53-62. 27. Freedman, J. C.; Li, J.; Uzal, F. A.; McClane, B. A. Proteolytic processing and activation of Clostridium perfringens epsilon toxin by caprine small intestinal contents. MBio. 2014, 5, e01994-14. 28. Pan, Z. Z.; Zhu, Y. J.; Chen, Z.; Ruan, C. Q.; Xu, L.; Chen, Q. X.; Liu, B. A protein engineering of Bacillus thuringiensis δ-endotoxin by conjugating with 4 ″-O-succinoyl abamectin. Int. J. Biol. Macromol. 2013, 62, 211-216. 29. Finney, D. J. Probit Analysis: 3d Ed. Cambridge University Press. 1971. 30. Jain, D.; Udayasuriyan, V.; Arulselvi, P. I.; Dev, S. S.; Sangeetha, P. Cloning, characterization, and expression of a new cry2Ab gene from Bacillus thuringiensis strain 14-1. Appl. Biochem. Biotechnol. 2006, 128, 185-194. 31. Yang, J.; Yan, R.; Roy, A.; Xu, D.; Poisson, J.; Zhang, Y. The I-TASSER Suite: protein structure and function prediction. Nat. Methods. 2015, 12, 7-8. 32. Bravo, A.; Gill, S. S.; Soberón, M. Bacillus thuringiensis mechanisms and use. Comprehensive Molecular Insect Science. 2005, 6, 175-205. 33. Lebel, G.; Vachon, V.; Préfontaine, G.; Girard, F.; Masson, L.; Juteau, M.; Schwartz, J. L. Mutations in domain I interhelical loops affect the rate of pore formation by the Bacillus


Page 22 of 38

Page 23 of 38


thuringiensis Cry1Aa toxin in insect midgut brush border membrane vesicles. Appl. Environ. Microbiol. 2009, 75, 3842-3850. 34. Bravo, A.; Sa´nchez, J.; Kouskoura, T.; Crickmore, N. N-terminal activation is an essential early step in the mechanism of action of the B. thuringiensis Cry1Ac insecticidal toxin. J. Biol. Chem. 2002, 277, 23985-23987. 35. Liu, C.; Xiao, Y.; Li, X.; Oppert, B.; Tabashnik, B. E.; Wu, K. Cis-mediated down-regulation of a trypsin gene associated with Bt resistance in cotton bollworm. Sci. Rep. 2014, 4. 36. Xia, J.; Guo, Z.; Yang, Z.; Zhu, X.; Kang, S.; Yang, F.; Wu, Q.; Wang, S.; Xie, W.; Xu, W.; Zhang, Y.. Proteomics-based identification of midgut proteins correlated with Cry1Ac resistance in Plutella xylostella (L.).Pestic Biochem Physiol. 2016, 32, 108-117. 37. Wu, Y. Detection and mechanisms of resistance evolved in insects to Cry toxins from Bacillus thuringiensis. Adv. Insect Physiol. 2014, 47, 297-342. 38. Ferré, J.; Van, Rie. J. Biochemistry and genetics of insect resistance to Bacillus thuringiensis. Annu. Rev. Entomol. 2002, 47, 501-533.



Figure Captions

Fig 1. Purification and identification of Cry2Ab. The purity and identity of Cry2Ab were assessed by SDS-PAGE electrophoresis followed by Coomassie blue staining (A) and western blotting using anti-Cry2Ab antibody (B).

Fig 2. Time course of proteolytic processing of Cry2Ab by PxMJ. Cry2Ab protoxin (65 kDa) was cleaved into Cry2Ab activated-toixn (50 kDa), the proteolysis was detected both by SDS-PAGE electrophoresis (A) and western blotting using anti-Cry2Ab antibody (B).

Fig 3. Identification of trypsin and chymotrypsin involved in the proteolysis of Cry2Ab. (A) Verification of N-terminal sequences of Cry2Ab activated-toxin by Edman degradation sequencing analysis. The identified amino acid sequences were underlined. (B) Protein 3D structure of Cry2Ab protoxin showed in cartoon mode by PyMOL program. 0-144 amino acid sequences was colored in gray. The cleavage-sites Arg139 and Leu144 residues were colored in red and showed in sticks mode. (C) Protein 3D structure of Cry2Ab activated-toxin, which removed 0-144 amino acid sequences from Cry2Ab protoxin. (D) Closer look of Domain I in Cry2Ab. This domain was composed by seven α-helices and residues Arg139 and Leu144 both occurred at the loop structure between helices α3 and α4. (E) Proteolysis of Cry2Ab by commercial trypsin or chymotrypsin and detected by SDS-PAGE electrophoresis. (F)


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The effects of protease inhibitors on proteolytic processing of Cry2Ab. SPI, serine protease inhibitors; API, aspartic protease inhibitor; MPI, metalloproteinase inhibitor; CPI, cysteine protease inhibitor.

Fig 4. The proteolytic processing of wild type, R139A, L144A and R139A-L144A Cry2Ab. (A) Design and construction of mutant Cry2Ab based on Arg139 and Leu144 residues. R139A, replaced Arg139 with Alanine; L144A, replaced Leu144 with Alanine; R139A-L144A, replaced Arg139 and Leu144 with Alanine. (B) SDS-PAGE electrophoresis and western blotting analysis of wild type, R139A, L144A and R139A-L144A Cry2Ab protoxins. Proteolysis of wild type (C), R139A (D), L144A (E) and R139A-L144A (F) Cry2Ab by PxMJ.

Fig 5. The susceptibility of 3rd instar larvae of P. xylostella to wild type, R139A, L144A and R139A-L144A Cry2Ab toxins. Data were showed as mean ± SEM (n=3).

Fig S1. N-terminal sequencing identified cleavage-sites of Cry2Ab processed by PxMJ. (A) Spectrum of 19 PTH-amino acids standards; (B)-(F) N-terminal amino acid identification of Cry2Ab activated-toxin.



Page 26 of 38

Table 1. Primer sequences used for generation of R139A, L144A and R139A-L144A mutants Cry2Ab. Primers

Primers sequences

Products

R139AF

5’-TTGAACCCTAACGCAAACGCTGTTCCTTTATCAATAACTTC-3’

R139A

R139A R

5’-AGAAGTTATTGATAAAGGAACAGCGTTTGCGTTAGGGTTCAA-3’

L144A F

5’-TTGAACCCTAACCGAAACGCTGTTCCTGCATCAATAACTTCT-3’

L144A R

5’-AGAAGTTATTGATGCAGGAACAGCGTTTCGGTTAGGGTTCAA-3’

R139A-L144A F

5’-TTGAACCCTAACGCAAACGCTGTTCCTGCATCAATAACTTCT-3’

R139A-L144A R

5’-AGAAGTTATTGATGCAGGAACAGCGTTTGCGTTAGGGTTCAA-3’


L144A

R139A-L144A

Page 27 of 38


Table 2. The LD50 of wild type, R139A, L144A and R139A-L144A Cry2Ab to 3rd instar larvae of P. xylostella. Cry2Ab protoxins

LD50 (µg/cm2)

95% CL (µg/cm2)

wild type

1.801

0.996-3.571

R139A

2.134

1.095-4.877

L144A

1.579

0.907-2.937

R139A-L144A

> 20

no avail



Fig 1.


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Fig 2.



Fig 3.


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Fig 4.



Fig 5.


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TOC Graphic



Fig 1. 87x99mm (300 x 300 DPI)


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Fig 2. 76x46mm (300 x 300 DPI)



Fig 3. 153x185mm (300 x 300 DPI)


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Fig 4. 155x190mm (300 x 300 DPI)



Fig 5. 53x36mm (300 x 300 DPI)


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Proteolytic Activation of Bacillus thuringiensis Cry2Ab through a Belt

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