A novel peptidase Kunitz inhibitor from Platypodium elegans seeds is

Subscriber access provided by READING UNIV

Article

A novel peptidase Kunitz inhibitor from Platypodium elegans seeds is active against Spodoptera frugiperda larvae Suellen Rodrigues Ramalho, Cezar da Silva Bezerra, Daniella Gorete Lourenço de Oliveira, Letícia Souza Lima, Simone Maria Neto, Caio Fernando Ramalho de Oliveira, Newton Valério Verbisck, and Maria Lígia Rodrigues Macedo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04159 • Publication Date (Web): 14 Dec 2017 Downloaded from http://pubs.acs.org on December 14, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 46

Journal of Agricultural and Food Chemistry

1

A novel peptidase Kunitz inhibitor from Platypodium elegans seeds is active

2

against Spodoptera frugiperda larvae

3 4

Suellen Rodrigues Ramalho1,3, Cézar da Silva Bezerra1,3, Daniella Gorete Lourenço de

5

Oliveira3, Letícia Souza Lima3, Simone Maria Neto3, Caio Fernando Ramalho de

6

Oliveira3, Newton Valério Verbisck4, Maria Lígia Rodrigues Macedo3*

7 8

1

9

da Saúde, Universidade Federal do Mato Grosso do Sul. Campo Grande, MS, Brazil.

Programa de Pós-Graduação em Biologia Vegetal, Centro de Ciências Biológicas e

10

2

11

SBBq, Campo Grande, MS, Brazil.

12

3

13

Ciências Biológicas e da Saúde, Universidade Federal do Mato Grosso do Sul, 79070-

14

900, Campo Grande, MS, Brazil.

15

4

16

*Corresponding author: Maria Lígia Rodrigues Macedo. Phone +55 67 33457612, e-

17

mail: [email protected]

Programa Multicêntrico de Pós-Graduação em Bioquímica e Biologia Molecular,

Laboratório de Purificação de Proteínas e suas Funções Biológicas, Centro de

Embrapa Gado de Corte, 79106-550, Campo Grande, MS, Brazil.

18

ACS Paragon Plus Environment


19

Abstract

20

A novel Kunitz-type inhibitor from Platypodium elegans seeds (PeTI) was purified and

21

characterized. The mass spectrometry analyses of PeTI indicated an intact mass of

22

19,701 Da, and a partial sequence homologous to Kunitz inhibitors. PeTI was purified

23

by ion exchange and affinity chromatographies. A complex with a 1:1 ratio was

24

obtained only for bovine trypsin, showing a Ki = 0.16 nM. Stability studies showed that

25

PeTI was stable over a wide range of temperature (37–80 °C) and pH (2–10). The

26

inhibitory activity of PeTI was affected by dithiothreitol (DTT). Bioassays of PeTI on

27

Spodoptera frugiperda showed negative effects on larval development and weight gain,

28

besides to extend the insect life cycle. The activities of digestive enzymes, trypsin and

29

chymotrypsin, were reduced by feeding larvae with 0.2% PeTI in an artificial diet. In

30

summary, we describe a novel Kunitz inhibitor with promising biotechnological potential

31

for pest control.

32 33

Keywords: Kunitz-type inhibitor, Fabaceae, pest insect, trypsin inhibitor, Spodoptera

34

frugiperda.

35 36


Page 2 of 46

Page 3 of 46

37


Introduction

38

Insects are the most dispersed group of organisms. Part this success is associated with

39

the ability to obtain food from different sources. This adaptation to a wide variety of food

40

sources (e.g. plants) created a high insect diversity1. Among the insects that evolved

41

phytophagous habits, a complex interface has been originated during millions of years of

42

plant-insect interaction2. Some beneficial interactions such as pollination emerged, while

43

negative ones, including herbivory, imposed to plants risks of primary and secondary

44

attacks since insects’ wounding and chewing allow the occurrence of infections provoked

45

by opportunistic microrganisms3,

46

biochemical arsenal against these attacks, which have been refined in tissue-specific

47

responses, triggered by chemical stimuli or genetic factors5, 6.

4

. Along with this co-evolution, plants evolved a

48

Peptidase inhibitors (PIs) are multifunctional proteins, attending both storage protein

49

and plant defense. When ingested by insects, the PIs form tight complexes with cognate

50

peptidases compromising the larval digestive process. These anti-nutritional molecules are

51

widely distributed in the Fabaceae, Solanaceae, and Poaceae families and have been

52

extensively studied7, 8. The biological activity of PIs has been explored for pest control.

53

Insects fed with artificial diet containing PIs of the main class of their gut peptidases show

54

negative effects on larval development9-11. Currently, the use of PIs-coding genes is been

55

investigated as a biotechnological tool for crop protection12.

56

The PIs have traditionally been classified into families known as Kunitz, Bowman-Birk,

57

Potato, Pumpkin, Barley, Cystatin and others. This classification is based on molecular

58

weight, number of polypeptide chains and number of reactive sites, type of inhibition and

59

other features. According to MEROPS database, the homologous sets of peptidases and

60

PIs are grouped into protein species, families and subfamilies. In this method of

61

classification, Kunitz inhibitors from animal sources are grouped into family I2 (Kunitz-A)



62

and Kunitz inhibitors from plants (Kunitz-P) are grouped into family I313. Kunitz inhibitors

63

are composed of a heterogeneous group of proteins with respect to the number of

64

polypeptide chains, reactive sites, and disulfide bridges. They are active against serine

65

peptidases such as trypsin, chymotrypsin, and elastase, and some of them show activity

66

against peptidases from other families14.

67

Herein, we describe the purification and characterization of Platypodium elegans

68

trypsin inhibitor – PeTI, obtained from P. elegans seeds (Fabaceae: Caesalpinioideae). P.

69

elegans is also known as ‘Uruvalheira’ and is found in the Brazilian cerrado and transition

70

areas between the cerrado and seasonal forest15. Our group is located in the Brazilian

71

Midwest, enabling us to explore the biodiversity of the cerrado in search for new

72

molecules. Cerrado is a Brazilian biome that covers over 2 million km2 of the national

73

territory and presents a high degree of plant endemism16. There are no reports on the

74

previous application of this species in folk medicine. Furthermore, we evaluated the effects

75

of PeTI on the fall armyworm development (Spodoptera frugiperda), with a view to defining

76

its biotechnological application. The fall armyworm is an important pest of different crops,

77

including corn, soybean, and cotton, and has developed resistance against Bt-crops and

78

chemical insecticides17-19. These facts reinforce the necessity to search for new

79

alternatives to control this pest.

80

Material and Methods

81

Materials

82

P. elegans seeds were obtained from the ArboCenter company (registration at

83

Brazilian Institute of Environment and Renewable Natural Resources, IBAMA, 2082600).

84

The P. elegans seeds were collected near the city of Birigui, located in the northwest of the

85

São Paulo state (under the latitude 21º17'19" S and longitude 50º20'24" W). Bovine trypsin

86

and α-chymotrypsin, bovine serum albumin (BSA), N-α-benzoyl-DL-arginine-p-nitroanilide


Page 4 of 46

Page 5 of 46


87

(BAPNA), N-α-benzoyl-DL-tyrosyl-p-nitroanilide (BTPNA), dithiothreitol (DTT), Succinyl-

88

alanyl-alanyl-propyl-phenyl-alanyl-p-nitroanilide

89

chloromethyl ketone (TLCK), electrophoresis and mass spectrometry reagents the

90

matrices Dihydroxybenzoic Acid (DHB) and Sinapinic Acid were purchased from Sigma

91

(St. Louis, MO, USA). Chromatography supports were acquired from GE Life Sciences

92

and Waters. Sequencing grade modified trypsin was purchased from Promega. All other

93

chemicals and reagents used were of analytical grade.

94

Insects

(SAAPFPNA),

N-p-tosyl-lysine

95

The insects were housed and maintained under laboratory conditions at the

96

Insectary-LPPFB (Laboratório de Purificação de Proteínas e suas Funções Biológicas),

97

Universidade Federal do Mato Grosso do Sul. Campo Grande, MS, Brazil. The colonies of

98

Anagasta kuehniella (Lepidoptera: Pyralidae) and Corcyra cephalonica (Lepidoptera:

99

Pyralidae) were housed under standard conditions at 25 °C ± 2 °C, 60 – 70% relative

100

humidity (RU), 14:10 h (light:dark) and maintained on an artificial diet (wheat

101

germ:wholemeal flour; 3:2 ratio). Aedes aegypti (Diptera: Culicidae) eggs were hatched in

102

dechlorinated water and maintained at 25 °C ± 2 °C, with 70 - 80% RU and a 12:12 h

103

photoperiod with cat food (Whiskas®) in the water. S. frugiperda (Lepidoptera: Noctuidae)

104

were reared individually on the artificial diet (white bean, wheat germ, brewer’s yeast

105

powder, agar, ascorbic acid, sorbic acid, casein, nipagin, vitamin complex, tetracycline,

106

40% formaldehyde and distilled water) and maintained under standard conditions at 25 °C

107

± 2 °C, 60 – 70% RU, 14:10 h photoperiod.



108

Methods

109

Isolation of P. elegans trypsin inhibitor (PeTI)

110

P. elegans seeds, integument-free, were dried at room temperature and stored at -

111

20 °C until use. The seeds were ground and defatted using hexane for the removal of lipid

112

content. Three changes of hexane were carried out until the liquid showed a clear aspect.

113

The defatted flour was used for crude extract preparation, obtained by extraction of this

114

meal (100 g) with 100 mM potassium phosphate pH 7.6 buffer (1:10; w/v) overnight at 4

115

ºC, with subsequent centrifugation at 13,500 g at 4 ºC for 30 min. The supernatant was

116

dialyzed against distilled water at 4 °C and lyophilized. For PeTI purification, the crude

117

extract (150 mg) was dissolved in a 50 mM Tris-HCl buffer pH 8.0, and loaded onto a

118

DEAE-Sepharose column (2 × 20 cm) equilibrated with the same buffer. Proteins were

119

eluted in 0.25-0.6 mM NaCl in 50 mM Tris-HCl buffer, pH 8.0 at a flow rate of 40 mL/h. The

120

fractions containing inhibitory activity were pooled, dialyzed and lyophilized. For the next

121

purification step, the peak containing inhibitory activity was loaded onto a trypsin-

122

Sepharose 4B column (2 × 10 cm), equilibrated with 100 mM sodium phosphate buffer, pH

123

7.6 containing 100 mM NaCl. The elution of PeTI was performed with 50 mM HCl at a flow

124

rate of 30 mL/h. The PeTI was subjected to a Reverse Phase High Performance Liquid

125

Chromatography (RP-HPLC), using the Waters µBondaparkTM C18 column (3.9 × 300

126

mm). The column was previously equilibrated with 0.1% (v/v) trifluoroacetic acid (solvent

127

A), followed by a linear gradient from 0-95% (v/v) of acetonitrile in 0.1% (v/v) aqueous

128

trifluoroacetic acid (solvent B) at a flow rate of 1 mL/min. All purification steps were

129

monitored at 280 nm.


Page 6 of 46

Page 7 of 46

130


Protein quantification

131

Protein concentration was determined by Coomassie brilliant blue G-250 staining,

132

as described by Bradford20. Bovine serum albumin (BSA) was used as standard protein to

133

obtain a standard calibration curve.

134

Polyacrylamide gel electrophoresis

135

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was

136

performed according to Laemmli21 using low range molecular weight standards (14.4 –

137

97.4 kDa). The proteins were separated on the gels and subsequently detected by staining

138

the gel with 0.1% (w/v) Coomassie Brilliant Blue R-250 and removal of excess staining

139

carried out with a solution of distilled water, methanol and acetic acid (5:4:1 – v/v/v).

140

Protein digestion, mass spectrometry and amino acid sequencing

141

The PeTI HPLC fraction was freeze-dried and dissolved in 1% (v/v) TFA. The matrices,

142

2,5-dihydroxybenzoic acid (20 mg/mL) and sinapinic acid (10 mg/mL), were mixed

143

individually in a 1:1 proportion with sample solutions. The intact protein was analyzed in

144

linear mode by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

145

(MALDI-TOF-MS) on an Autoflex III Smartbeam (Bruker Daltonics) apparatus. Ions were

146

generated by 1000 to 2000 laser shots with a 20 kV accelerating voltage applied. For

147

calibration, protein standards from the MSCAL1 135 Calibration Kit (Sigma- Aldrich) were

148

used. FlexAnalysis v.3.3 software (Bruker Daltonics) was used for data analyses. Peaks

149

were smoothed and the baseline correction was applied to the spectra.

150

The sample obtained from the HPLC was also submitted to SDS-PAGE (17%) under

151

reducing conditions and stained with colloidal Coomassie Blue G-250. The PeTI band was

152

excised and treated for in-gel digestion, as described by Hellman22. In brief, the gel pieces

153

were unstained by consecutive washes in 50% (v/v) acetonitrile solution containing 50 mM



154

ammonium bicarbonate for 15 min at room temperature, vortexing every 5 min. The

155

washes were repeated until complete gel discoloration. The washing solution was replaced

156

by 100% acetonitrile with vertical shaking for 5 min. Gel pieces were air-dried and then

157

incubated in 30 µL of 10 mM dithiothreitol in 100 mM ammonium bicarbonate solution

158

under stirring for 30 min at room temperature, for reduction of disulfide bonds. To this

159

solution, 30 µL of 50 mM iodoacetamide in 100 mM ammonium bicarbonate solution was

160

added, in the dark, for alkylation of reduced cysteine residues. The gel pieces were

161

washed with 100 mM ammonium bicarbonate solution, dehydrated in 100% acetonitrile,

162

and air-dried. In-gel protein digestion was carried out with Sequencing Grade Modified

163

Trypsin solution (Promega), protein ratio of 1:20 (w/w). Buffered trypsin (1 µg) was added

164

to the gel tube and incubated on ice for 1 h. Next, 30 µL of 50 mM ammonium bicarbonate

165

was added and incubated at 37 ºC for 16 h. The digested supernatant was collected,

166

freeze-dried and re-suspended in 5 µL of 0.1% TFA in 50% (v/v) acetonitrile. The sample

167

was mixed in a 1:1 ratio with the matrix solution (10 mg/mL α-cyano-4-hydroxycinnamic

168

acid, 1% (v/v) TFA and 70% (v/v) acetonitrile) and 2 µL applied onto a MALDI target plate.

169

After crystallization at room temperature, the peptides derived from tryptic digestion were

170

analyzed using an UltraFlex Extreme MALDI-TOF/TOF (BrukerDaltonics), precisely

171

calibrated with Tryptic Digest of Bovine Serum Albumin (BrukerDaltonics). The mass

172

spectrometer was operated in reflector mode for MS acquisitions and LIFT mode for

173

tandem MS (MS/MS). Protein identification was carried out by peptide de novo

174

sequencing. Sequenced peptides were directly correlated to NCBI non-redundant protein

175

database using the BLASTp search system23. The sequences that showed the highest

176

identities from different organisms were selected and submitted to signal peptide predictor

177

using Phobius web serve24. After signal peptide removal, the sequences were aligned

178

using the CLUSTAL O (1.2.4) software with the multiple alignment tools.


Page 8 of 46

Page 9 of 46

179 180


Midgut preparation The larvae were cold immobilized, and midguts were dissected, according to 25

181

Macedo, et al.

. The midguts were then homogenized with a hand-held Potter-Elvehjem

182

in isosmotic solution (150 mM NaCl in 50 mM Tris-HCl, pH 8.0) and centrifuged at 6 000 g

183

for 20 min at 4°C. The supernatants were then collected and used for measuring their

184

protein concentrations for enzymatic activity assays or stored at -20°C.

185

Enzymatic assays

186

For inhibitory activity assays, PeTI was assayed against the bovine trypsin and

187

chymotrypsin using the substrates BAPNA and BTPNA, respectively26. For trypsin- and

188

chymotrypsin-like activity from larval midgut, we used BAPNA and SAAPFPNA as

189

substrates, respectively27. A stock solution of BAPNA (100 mM), SAAPFPNA (100 mM)

190

and BTPNA (100 mM) was prepared in DMSO. All substrates were diluted in a working

191

solution (1 mM) in 50 mM Tris-HCl buffer, pH 8.0. The SAAPFPNA working solution buffer

192

was prepared in 50 mM Tris-HCl buffer, pH 8.0 containing 20% (v/v) dimethylformamide.

193

Aliquots of 4 µL of bovine trypsin (0.25 mg/mL) were incubated with PeTI in 50 mM Tris–

194

HCl buffer, pH 8.0. After a 5 min incubation at 30 °C, 200 µL of 1 mM BAPNA were added

195

(final 270 µL assay volume). The total time of assays was 30 min. For the chymotrypsin

196

assay, 5 µL of bovine chymotrypsin (1 mg/mL) were incubated with PeTI in 50 mM Tris–

197

HCl buffer, pH 8.0. After 5 min of incubation at 30 °C, we added 100 µL of 1 mM BTPNA

198

(final 120 µL assay volume). The total time of the assay was 5 min. The substrate

199

hydrolysis was monitored by changes in absorbance at 410 nm, after subtracting the

200

blank, using a Multiskan Microplate Reader (Thermo Scientific), and the results showed as

201

specific activity (ng substrate/min/µg protein). One unit of inhibitory activity was defined as

202

the amount of inhibitor responsible to decrease 0.01 absorbance unit at 280 nm.



203

For measuring the trypsin-like activity assay from larval midgut enzymes, samples of 1

204

µg of larval midgut homogenate were incubated with the assay buffer, and then 200 µL of

205

BAPNA were added to a final volume of 270 µL. The assay was conducted for 30 min at

206

30 °C. For the chymotrypsin-like activity assay, samples of 1 µg of larval midgut

207

homogenate were incubated with the assay buffer, and 20 µL of SAAPFPNA was then

208

added to a final volume of 120 µL. The assay was carried out for 5 min at 30 °C. To

209

analyze the sensitivity of insect trypsin to inhibition by PeTI, an inhibition curve was made

210

with increasing concentrations of PeTI (0–0.5 µg), and the inhibitory activity measured as

211

described above.

212

Ki determination and stoichiometry inhibition

213

Bovine trypsin was incubated with different concentrations of PeTI and residual

214

inhibitory activity was recorded. To determine the dissociation constant (Ki), the slow tight-

215

binding inhibition method, described by Morrison

216

of PeTI (0; 0.3; 0.15 nM) were added to a fixed concentration of trypsin (4.2 nM), and the

217

residual enzymatic activity was determined. The Ki value was determined as the average

218

of three independent assays and obtained with the aid of a Sigma Plot (Systat Software

219

Inc.).

220

Stability of inhibitory activity

28

, was used. Increasing concentrations

221

Stability studies were performed as described by Oliveira, et al.26. The inhibitor

222

sample (0.3 mg/mL in 50 mM Tris−HCl buffer, pH 8.0) was heated for 30 min at different

223

temperatures (30−100 °C), and then submitted to inhibitory activity assays. To measure

224

pH stability, a solution of PeTI (0.3 mg/mL) was diluted with an equal volume of various

225

buffers (100 mM): sodium citrate (pH 2−4), sodium acetate (pH 5), sodium phosphate (pH

226

6−7), Tris−HCl (pH 8) and sodium bicarbonate (pH 9−10). After incubation in each buffer


Page 10 of 46

Page 11 of 46


227

for 1 h at 30°C, the pH was adjusted to pH 8.0 and the inhibitory activity against trypsin

228

was assayed. PeTI was incubated with DTT at final concentrations of 1, 10 and 100 mM

229

for 15–120 min at 30°C. The reaction was completed by adding iodoacetamide at twice the

230

amount of each DTT concentration, and the residual inhibitory activity of trypsin was

231

determined. All experiments were carried out in triplicate.

232

Screening assay for inhibition of insect trypsins

233

The ability of PeTI to inhibit trypsin from different insect pests was evaluated in vitro.

234

The fourth-instar larvae of A. kuehniella, C. cephalonica, A. aegypti and S. frugiperda were

235

used for obtaining the midgut extract containing digestive enzymes. Samples of 2 µg of

236

midgut extract were incubated with increasing concentrations of PeTI (0 – 0.2 µg) for 10

237

min at 30 ºC, and then 200 µL of BAPNA was added to a final volume of 270 µL. The

238

assay was conducted for 30 min at 30 °C, monitoring the absorbance at 410 nm. All

239

assays were carried out in triplicate. The activity of the positive control, midgut samples

240

without PeTI, was set as 100% of trypsin activity.

241

Bioassays

242

S. frugiperda was the species chosen further bioassays. Neonate larvae were

243

individually placed in glass tubes (8.5 x 2 cm) containing artificial diet (control) or a diet

244

supplemented with 0.2% PeTI (w/w). The larvae were kept on these respective diets until

245

reaching the fifth instar, after approximately 11 days, under standard conditions. Each

246

treatment was composed of 40 larvae. Subsequently, the larvae were dissected for

247

obtaining the midgut extract. To determine effects on the insect life cycle, the same

248

bioassays were performed. In these bioassays, the larvae were fed until pupation. The

249

number of emerging moths was counted and the moths were kept in cages while



250

remaining alive, to evaluate the possibility of total lifetime changes after PeTI exposure. All

251

experimental results represent the average of three independent bioassays.

252

Digestion of PeTI by S. frugiperda midgut enzymes

253

PeTI was incubated with S. frugiperda larval midgut homogenate at 30 °C in a ratio

254

of 1:5 (inhibitor: midgut homogenate) for 0, 1, 3, 6 and 12 h. The digestion was stopped by

255

immersing the tubes in boiling water for 2 min. The degradation of BSA was used as a

256

positive control for proteolytic activity and the digestion time intervals of 0, 0.5, 1, 2, 3, 4

257

and 5 h were used. After digestion, the samples were separated by SDS-PAGE 15%.

258

Statistical analysis

259

Data were analyzed using Student's t-test for comparing two sets of data. The one-

260

way analysis of variance (ANOVA) with Tukey’s post hoc test was used for multiple

261

comparisons. A p-value of 70 °C), we observed a significant decrease in inhibitory activity (Table 3).

306

At 100 ºC, the inhibitory activity was reduced by 75%. Different pH values, ranging from

307

2.0 to 10.0, exerted no effect on PeTI activity (Table 3). In regard to DTT incubation, all

308

concentrations evaluated led to reductions in inhibitory activity, but during different

309

incubation periods. At 1 mM, DTT promoted a significant reduction in inhibition after 60

310

min of incubation. For higher DTT concentrations, the reduction in inhibitory activity was

311

seen at all incubation times: 15, 30, 60 and 120 min (Table 4).

312

Screening assay for inhibition of insect trypsins

313

The midgut extracts from 4 insects were incubated with different PeTI concentrations

314

and a gradual inhibition of the trypsin was observed (Figure 5). PeTI displayed a strong

315

inhibition of trypsin enzymes from A. kuehniella and C. cephalonica, even at low inhibitor

316

concentration (0.025 µg). At a higher concentration (0.2 µg), PeTI almost completely

317

inhibited the trypsin activity of A. kuehniella (95%), C. cephalonica (90%), A. aegypti (82%)

318

and S. frugiperda (95%). Based in this result, we further investigated the effects of PeTI on

319

S. frugiperda development.


Page 14 of 46

Page 15 of 46

320


Effects of PeTI on S. frugiperda development

321

An artificial diet containing 0.2% PeTI (w/w) was used in bioassays. At the fifth-instar,

322

the average larval weight in PeTI-fed larvae decreased by 55% (Figure 6A). In comparison

323

with larvae from the control group, no significant difference in survival rate was observed in

324

PeTI-fed larvae (Figure 6B). The duration of larval stage was significantly extended (Table

325

5), while the pupal and adult stages were not altered. Anyway, the Total Development

326

Time (TDT) was increased, indicating that the exposition of S. frugiperda larvae to PeTI

327

prompts the extension of S. frugiperda life cycle.

328

Effects of PeTI on S. frugiperda enzymes

329

The ingestion of PeTI affected the activity of digestive enzymes of S. frugiperda larvae.

330

The trypsin and chymotrypsin activities in PeTI-fed larvae were reduced by 59 and 17%,

331

respectively (Figure 7A and 7B). In order to verify whether the S. frugiperda trypsins

332

remained sensitive to PeTI after chronic exposure, the midgut extract was incubated with

333

increasing concentrations of inhibitor to compare the residual trypsin activities. Results of

334

this assay indicated that the trypsins from PeTI-fed larvae became resistant to inhibition

335

(Figure 7C).

336

PeTI digestion by S. frugiperda midgut enzymes

337

As the ingestion of PeTI triggered the change in trypsin composition, we evaluated the

338

capacity of midgut enzymes to digest PeTI during in vitro incubation. The band of 20 kDa,

339

corresponding to PeTI, remained intact after incubation in both midgut samples at all

340

intervals, indicating PeTI resistance to proteolysis by S. frugiperda midgut enzymes

341

(Figure 8A). The positive control (BSA) was completely digested within 1 h while the

342

complete digestion of BSA by midgut sample from PeTI-fed larvae occurred between 1

343

and 3 h (Figure 8B).



344

Discussion

345

PeTI, a trypsin inhibitor, was purified using a two-step procedure: ion exchange and

346

bioaffinity chromatographies. This procedure has been shown to effectively purify PIs from

347

legume seeds and other source29,

348

showed a single band in SDS–PAGE and was denominated PeTI. A series of experimental

349

data allowed us to conclude that PeTI is a Kunitz inhibitor, as indicated by its molecular

350

mass, obtained by MALDI-TOF spectrometry, and finally, partial sequencing by mass

351

spectrometry.

30

. The fraction obtained from Trypsin-Sepharose

352

Multiple sequence alignment methods are important for identifying highly conserved

353

residues that are essential for stability or function of the protein. There are many

354

conserved regions in plant-Kunitz type inhibitors; such as Asp4, Gly7, Gly13, Tyr-Tyr16,17,

355

Pro20, Arg-Gly23, 24, Gly-Gly-Gly26-28, Gly53, Pro55 (PeTI numbering) (Figure 4). The

356

RNG domain is present in the partial primary amino acid sequence of PeTI. The conserved

357

Asn12 residue (PeTI numbering) contained in this domain modulate the inhibitory activity

358

by stabilizing the peptidase-inhibitor complex. According to Iwanaga, et al. 31, Asn residues

359

provide structural support in N-terminal region for stabilization of the primary binding loop

360

via hydrogen bonds.

361

Partial PeTI sequence obtained by mass spectrometry revealed a specific region

362

containing arginine–glycine-rich (RGG) motifs, as well as observed in [XP_006372631.1]

363

and [ADW95389.1] sequences, suggesting a wide network of protein-protein interactions

364

through the RGG motif (Figure 4). Arg/Gly-rich motifs play an important role in several

365

physiological processes via the arginine methylation of RGG/RG protein motifs32. The

366

RGD motif was described in peptidase inhibitor from Bauhinia rufa seeds (BrTI)33 and

367

cardosin A, an aspartic peptidase inhibitor from Cynara cardunculus34, 35.


Page 16 of 46

Page 17 of 46


368

Kinetic studies showed that PeTI inhibits both trypsin and chymotrypsin, but the

369

inhibitory activity for chymotrypsin is weaker (Table 2). From this analysis, we observed

370

that the equimolar amount of PeTI necessary to inhibit 100% of trypsin activity inhibited

371

only 30% of chymotrypsin activity. PIs can inhibit specifically trypsin, such as

372

Caesalpinia echinata trypsin inhibitor (CeKI)36, as also inhibit only chymotrypsin37, or

373

both enzyme38. Based on stoichiometric studies, we demonstrated that PeTI is a

374

single-headed Kunitz inhibitor with higher activity against trypsin than chymotrypsin.

375

This difference in affinity is due to the reactive site composition, as observed in

376

soybean inhibitors. We also suggest that binding of chymotrypsin to PeTi has a non-

377

specific component, explaining the weak inhibition observed, since both enzymes

378

belong to the same family and have similar catalysis mechanisms, with singular

379

peculiarities. In stoichiometric studies, PeTI inhibited trypsin at a molar ratio of 1:1

380

(inhibitor: enzyme) (Figure 2), confirming the presence of a single reactive site for

381

trypsin. Similar results were reported from inhibitors isolated from Leucaena

382

leucocephala39, Poincianella pyramidalis40 and Pithecellobium dulce41. The PeTI Ki

383

value for bovine trypsin was 0.16 nM, indicating a high affinity. The deconvolution of

384

data revealed that PeTI is a competitive inhibitor, similarly to PIs from Putranjiva

385

roxburghii42. Additionally, PeTI is very potent inhibitor because it inhibits hydrolysis of

386

trypsin at low concentrations.

387

PIs exhibit considerable stability to temperature, pH, denaturing agents and

388

proteolysis26,

29, 38, 43

389

bonds and other non-covalent interactions44. PeTI also showed these properties, being

390

active and stable after incubation up to 70 ºC and over a range of pH, as seen for other

391

PIs from other species of legumes, including Moringa oleifeira29 and Piptadenia

392

moniliformis45.In contrast, the PeTI inhibitory activity was sensitive to all concentrations

. These stabilities may be attributed to the presence of disulfide



Page 18 of 46

393

of DTT after 30 min of incubation. A similar result was reported for ILTI46, a trypsin

394

inhibitor from Inga laurina. The loss of inhibitory activity is attributed to reduction of

395

disulfide bonds, with reflections on other non-covalent interactions that stabilize the

396

reactive site of the inhibitor47. According to Joshi and collaborators44, disulfide bonds

397

are collectively responsible for the functional activity of PIs by maintaining the

398

conformational rigidity and function of PIs. Other authors, however, have reported PIs

399

where the disulfide bridges are more involved in the maintenance of native folding than

400

reactive site structure26.

401

PIs have been identified as potential candidates for the development of insect-

402

resistant transgenic crops, targeting digestive enzymes of pests5,

403

inhibitory activities of PIs on insect enzymes are variable, according to their structures

404

and specificities, which makes in vitro preliminary screening necessary to select better

405

targets, as evaluated in bioassays45,

406

bovine trypsin enzyme, we verified its inhibitory activity against midgut trypsins from the

407

insect orders Lepidoptera and Diptera. In vitro, PeTI strongly inhibited the trypsin-like

408

activity of the four economically important pests (Figure 5). S. frugiperda trypsins were

409

successfully inhibited and, hence, selected for further assays, based on the possibility

410

of controlling this pest using transgenic plants.

49

30, 48

. However, the

. Based on PeTI effectiveness in suppressing

411

An essential aspect of the successful use of PIs in pest control constitutes the

412

prevention of their inactivation in the insect midgut. The high alkalinity of the

413

lepidopteran midgut environment and the activity of several peptidases are one of the

414

main barriers that an ingested inhibitor must overcome50. In our study, PeTI maintained

415

inhibitory activity over a wide range of pH (Table 3). Some insects are able to degrade

416

the PIs using constitutively expressed51 or induced52 peptidases as an adaptive

417

strategy to overcome plant defenses. S. frugiperda midgut peptidases that were either


Page 19 of 46


418

constitutively expressed or induced were unable to degrade PeTI, even during long

419

incubation periods (Figure 8A). Additionally, BSA (used as a positive control) was

420

readily degraded in control-fed larvae (Figure 8B), while a longer incubation period was

421

necessary to complete degradation using PeTI-fed larvae as an enzyme source (Figure

422

8B), signifying that the ability of PeTI to bind to digestive enzymes might impair protein

423

digestion. As such, PeTI represents a good candidate for further studies as an anti-

424

nutritional protein for insect control management.

425

Bioassays demonstrated that the ingestion of PeTI led to changes in S. frugiperda

426

development and digestive physiology. Although PeTI does not induce larval mortality,

427

there was a meaningful reduction in larval weight gain (Figure 6A), beyond the

428

extension of the larval stage (Table 5), and probably associated with a reduced amino

429

acid availability due to the inhibition of the trypsin and chymotrypsin enzymes (Figure

430

7). The rapid growth rate of lepidopteran larvae depends on the efficient acquisition

431

and utilization of essential amino acids acquired from the diet53. Therefore, the

432

inhibition of proteolytic enzymes by PeTI causes an amino acid deficiency due to the

433

disruption of dietary protein assimilation, which leads to significant delays on S.

434

frugiperda growth and development, as reported in other studies9, 30, 43. These delays

435

may be an important strategy for pest control, since it may increase the exposure time

436

of larvae to their natural enemies (predators and pathogens), contributing to integrated

437

pest management54, 55.

438

It is known, however, that S. frugiperda possesses adaptive mechanisms against

439

PIs, such as the overexpression of peptidases resistant to inhibition27. We, herein,

440

observed that the trypsin enzymes from PeTI-fed larvae presented insensitivity to

441

inhibition by PeTI (Figure 7C), but no increase in enzymatic activity was noted,

442

suggesting a changing of peptidase profile expression (sensitive to resistant trypsins),



443

maintaining the same level of midgut proteolysis. The synthesis of PeTI-insensitive

444

trypsin is a costly process, and the energy spent from this process possibly caused the

445

delay observed in S. frugiperda development. Moreover, the ingestion of PIs may affect

446

not only protein metabolism, but other pathways. Kuwar and collaborators56 showed

447

that the ingestion of soybean Kunitz trypsin inhibitor (SKTI) by Helicoverpa armigera

448

affected not only peptidase genes, but also affected genes associated with stress,

449

carbohydrate metabolism, lipid metabolism, immune defense and detoxification

450

pathways. Therefore, it would be interesting to investigate the influence of PeTI on

451

other metabolic pathways further.

452

In conclusion, this is the first report of a PI isolated from P. elegans seeds. PeTI is a

453

single-headed Kunitz inhibitor composed by a single polypeptide chain, active against

454

trypsin enzymes and moderately active against chymotrypsin. PeTI presents

455

moderately stability to temperature (up to 80 °C), resistant to proteolysis and pH

456

ranging, but is inactivated by longer exposition to heat, suggesting that its use in a

457

transgenic crop would not affect the final consumer. Bioassays showed that PeTI was

458

able to inhibit the trypsin enzymes from four important pests and impaired S. frugiperda

459

development via a reduction in weight gain and an extension of the life cycle. These

460

findings indicate that PeTI should be further investigated in order to compose an

461

alternative molecule for biotechnological studies, with a view to crop protection or other

462

research purposes, since all organisms possess peptidases involved in critical

463

pathways and the inhibition of these pathways could be explored in various

464

applications for pest control.

465 466

Abbreviations Used


Page 20 of 46

Page 21 of 46


467

BAPNA,

N-benzoyl-DL-arginine-p-nitroanilide;

468

nitroanilide;

469

TLCK, N-p-tosyl-lysine chloromethyl ketone; DHB, Dihydroxybenzoic Acid; DTT,

470

dithiothreitol; BSA, bovine serum albumin; RU, Relative humidity. H, hours; TIU,

471

Trypsin inhibitor units.

SAAPFPNA,

BTPNA,

N-benzoyl-L-tyrosyl-p-

Succinyl-alanyl-alanyl-propyl-pheny-lalanyl-p-nitroanilide;

472 473

Acknowledgments

474

We are grateful to Nicola Conran for English proofreading.

475 476

Funding Sources

477

This

478

(23/200.591/2012).

work

was

supported

by

CNPq

(407127/2013-5)

and

FUNDECT

479 480

Notes

481

Conflict of interests the authors declare that they have no competing interests.

482 483

Supporting Information Available: Description

484

Supplementary Figures 1-3.

485



486

Reference

487

1. Futuyma, D. J.; Agrawal, A. A., Macroevolution and the biological diversity of plants and

488

herbivores. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 18054-18061.

489

2. Scriber, J. M., Integrating ancient patterns and current dynamics of insect–plant

490

interactions: Taxonomic and geographic variation in herbivore specialization. Insect Sci.

491

2010, 17, 471-507.

492

3. Franco, F. P.; Moura, D. S.; Vivanco, J. M.; Silva-Filho, M. C., Plant–insect–pathogen

493

interactions: a naturally complex ménage à trois. Curr. Opin. Microbiol. 2017, 37, 54-60.

494

4. Agrawal, A. A., Current trends in the evolutionary ecology of plant defence. Funct. Ecol.

495

2011, 25, 420-432.

496

5. Dang, L. Y.; Van Damme, E. J. M., Toxic proteins in plants. Phytochemistry 2015, 117,

497

51-64.

498

6. Kant, M. R.; Jonckheere, W.; Knegt, B.; Lemos, F.; Liu, J.; Schimmel, B. C. J.; Villarroel,

499

C. A.; Ataide, L. M. S.; Dermauw, W.; Glas, J. J.; Egas, M.; Janssen, A.; Van Leeuwen, T.;

500

Schuurink, R. C.; Sabelis, M. W.; Alba, J. M., Mechanisms and ecological consequences

501

of plant defence induction and suppression in herbivore communities. Ann. Bot. 2015, 115,

502

1015-1051.

503

7. Oliva, M. L. V.; Silva, M. C.; Sallai, R. C.; Brito, M. V.; Sampaio, M. U., A novel

504

subclassification for Kunitz proteinase inhibitors from leguminous seeds. Biochimie 2010,

505

92, 1667-1673.


Page 22 of 46

Page 23 of 46


506

8. Oliva, M. L. V.; Ferreira, R. S.; Ferreira, J. G.; de Paula, C. A. A.; Salas, C. E.; Sampaio,

507

M. U., Structural and Functional Properties of Kunitz Proteinase Inhibitors from

508

Leguminosae: A Mini Review. Curr. Protein Pept. Sci. 2011, 12, 348-357.

509

9. Bezerra, C. S.; Oliveira, C. T.; Macedo, M. L. R., Inga vera trypsin inhibitor interferes in

510

the proteolytic activity and nutritional physiology of Ephestia kuehniella larvae. Entomol.

511

Exp. Appl., n/a-n/a.

512

10. Machado, S. W.; Oliveira, C. F. R.; Bezerra, C. S.; Freire, M. G. M.; Kill, M. R.;

513

Machado, O. L. T.; Marangoni, S.; Macedo, M. L. R., Purification of a Kunitz-type inhibitor

514

from Acacia polyphylla DC seeds: Characterization and insecticidal properties against

515

Anagasta kuehniella Zeller (Lepidoptera: Pyralidae). J. Agric. Food Chem. 2013.

516

11. Jamal, F.; Pandey, P. K.; Singh, D.; Ahmed, W., A Kunitz-type serine protease inhibitor

517

from Butea monosperma seed and its influence on developmental physiology of

518

Helicoverpa armigera. Process Biochem. 2015, 50, 311-316.

519

12. Dunse, K. M.; Stevens, J. A.; Lay, F. T.; Gaspar, Y. M.; Heath, R. L.; Anderson, M. A.,

520

Coexpression of potato type I and II proteinase inhibitors gives cotton plants protection

521

against insect damage in the field. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 15011-5.

522

13. Rawlings, N. D.; Barrett, A. J.; Finn, R., Twenty years of the MEROPS database of

523

proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 2016, 44, D343-

524

D350.

525

14. Migliolo, L.; Oliveira, A. S.; Santos, E. A.; Franco, O. L.; Sales, M. P., Structural and

526

mechanistic insights into a novel non-competitive Kunitz trypsin inhibitor from Adenanthera

527

pavonina L. seeds with double activity toward serine- and cysteine-proteinases. J. Mol.

528

Graph. Model. 2010, 29, 148-156.



529

15. Lorenzi, H., Árvores brasileiras: manual de identificação e cultivo de plantas arbóreas

530

nativas do Brasil. vol. 2. Nova Odessa, Brazil: Instituto Plantarum de Estudos da Flora

531

Ltda. 352p.-col. illus.. ISBN 8586714070 Por Icones. Geog 1998, 4.

532

16. Furley, P. A., The nature and diversity of neotropical savanna vegetation with particular

533

reference to the Brazilian cerrados. Global Ecol. Biogeogr. 1999, 8, 223-241.

534

17. Carvalho, R. A.; Omoto, C.; Field, L. M.; Williamson, M. S.; Bass, C., Investigating the

535

molecular mechanisms of organophosphate and pyrethroid resistance in the fall armyworm

536

Spodoptera frugiperda. PLoS One 2013, 8, e62268.

537

18. Ríos-Díez, J.; Saldamando-Benjumea, C., Susceptibility of Spodoptera frugiperda

538

(Lepidoptera: Noctuidae) strains from central Colombia to two insecticides, methomyl and

539

lambda-cyhalothrin: a study of the genetic basis of resistance. J. Econ. Entomol. 2011,

540

104, 1698-1705.

541

19. Santos-Amaya, O. F.; Rodrigues, J. V.; Souza, T. C.; Tavares, C. S.; Campos, S. O.;

542

Guedes, R. N.; Pereira, E. J., Resistance to dual-gene Bt maize in Spodoptera frugiperda:

543

selection, inheritance, and cross-resistance to other transgenic events. Sci. Rep. 2015, 5.

544

20. Bradford, M., A rapid and sensitive method for the quantitation of microgram quantities

545

of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-54.

546

21. Laemmli, U., Cleavage of structural proteins during the assembly of the head of

547

bacteriophage T4. Nature 1970, 227, 680-685.

548

22. Hellman, U., Sample preparation by SDS/PAGE and in-gel digestion. In Proteomics in

549

Functional Genomics: Protein Structure Analysis, Jollès, P.; Jörnvall, H., Eds. Birkhäuser

550

Basel: Basel, 2000; pp 43-54.


Page 24 of 46

Page 25 of 46


551

23. Altschul, S. F.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J., Basic local alignment

552

search tool. J. Mol. Biol. 1990, 215, 403-410.

553

24. Käll, L.; Krogh, A.; Sonnhammer, E. L. L., A Combined Transmembrane Topology and

554

Signal Peptide Prediction Method. J. Mol. Biol. 2004, 338, 1027-1036.

555

25. Macedo, M. L. R.; Diz Filho, E. B.; Freire, M. G.; Oliva, M. L.; Sumikawa, J. T.;

556

Toyama, M. H.; Marangoni, S., A Trypsin Inhibitor from Sapindus saponaria L. Seeds:

557

Purification, Characterization, and Activity Towards Pest Insect Digestive Enzyme. Protein

558

J. 2011, 30, 9-19.

559

26. Oliveira, C. F. R.; Vasconcelos, I. M.; Aparicio, R.; Freire, M. d. G. M.; Baldasso, P. A.;

560

Marangoni, S.; Macedo, M. L. R., Purification and biochemical properties of a Kunitz-type

561

trypsin inhibitor from Entada acaciifolia (Benth.) seeds. Process Biochem. 2012, 47, 929-

562

935.

563

27. Oliveira, C. F. R.; Paula Souza, T.; Parra, J. R. P.; Marangoni, S.; Castro Silva-Filho,

564

M.; Macedo, M. L. R., Insensitive trypsins are differentially transcribed during Spodoptera

565

frugiperda adaptation against plant protease inhibitors. Comp. Biochem. Physiol., Part B:

566

Biochem. Mol. Biol. 2013, 165, 19-25.

567

28. Morrison, J. F., The slow-binding and slow, tight-binding inhibition of enzyme-catalysed

568

reactions. Trends Biochem. Sci. 1982, 7, 102-105.

569

29. Bijina, B.; Chellappan, S.; Basheer, S. M.; Elyas, K. K.; Bahkali, A. H.;

570

Chandrasekaran, M., Protease inhibitor from Moringa oleifera leaves: Isolation,

571

purification, and characterization. Process Biochem. 2011, 46, 2291-2300.



572

30. Jamal, F.; Pandey, P.; Singh, D.; Khan, M. Y., Serine protease inhibitors in plants:

573

nature’s arsenal crafted for insect predators. Phytochem. Rev. 2013, 12, 1-34.

574

31. Iwanaga, S.; Yamasaki, N.; Kimura, M.; Kouzuma, Y., Contribution of Conserved Asn

575

Residues to the Inhibitory Activities of Kunitz-Type Protease Inhibitors from Plants. Biosci.

576

Biotechnol. Biochem. 2005, 69, 220-223.

577

32. Thandapani, P.; O’Connor, T. R.; Bailey, Timothy L.; Richard, S., Defining the

578

RGG/RG Motif. Mol. Cell 2013, 50, 613-623.

579

33. Nakahata, A. M.; Bueno, N. R.; Rocha, H. A. O.; Franco, C. R. C.; Chammas, R.;

580

Nakaie, C. R.; Jasiulionis, M. G.; Nader, H. B.; Santana, L. A.; Sampaio, M. U.; Oliva, M. L.

581

V., Structural and inhibitory properties of a plant proteinase inhibitor containing the RGD

582

motif. Int. J. Biol. Macromol. 2006, 40, 22-29.

583

34. Frazão, C.; Bento, I.; Costa, J.; Soares, C. M.; Verıś simo, P.; Faro, C.; Pires, E.;

584

Cooper, J.; Carrondo, M. A., Crystal Structure of Cardosin A, a Glycosylated and Arg-Gly-

585

Asp-containing Aspartic Proteinase from the Flowers of Cynara cardunculus L. J. Biol.

586

Chem. 1999, 274, 27694-27701.

587

35. Simões, I.; Mueller, E.-C.; Otto, A.; Bur, D.; Cheung, A. Y.; Faro, C.; Pires, E.,

588

Molecular analysis of the interaction between cardosin A and phospholipase Dα. FEBS J.

589

2005, 272, 5786-5798.

590

36. Cruz-Silva, I.; Gozzo Andrezza, J.; Nunes Viviane, A.; Carmona Adriana, K.; Faljoni-

591

Alario, A.; Oliva Maria Luiza, V.; Sampaio Misako, U.; Sampaio Claudio, A. M.; Araujo

592

Mariana, S., A proteinase inhibitor from Caesalpinia echinata (pau-brasil) seeds for plasma

593

kallikrein, plasmin and factor XIIa. In Biol. Chem., 2004; Vol. 385, p 1083.


Page 26 of 46

Page 27 of 46


594

37. Kouzuma, Y.; Suetake, M.; Kimura, M.; Yamasaki, N., Isolation and primary structure

595

of proteinase inhibitors from Erythrina variegata (Linn.) var. Orientalis seeds. Bioscienci

596

Biotechnol Biochem 1992, 56, 1819-1824.

597

38. Bhattacharyya, A.; Babu, C., Exploring the protease mediated conformational stability

598

in a trypsin inhibitor from Archidendron ellipticum seeds. Plant Physiol. Biochem. 2006, 44,

599

637-44.

600

39. Oliva, M. L. V.; Souza-Pinto, J. C.; Batista, I. F. C.; Araujo, M. S.; Silveira, V. F.;

601

Auerswald, E. A.; Mentele, R.; Eckerskorn, C.; Sampaio, M. U.; Sampaio, C. A. M.,

602

Leucaena leucocephala serine proteinase inhibitor: primary structure and action on blood

603

coagulation, kinin release and rat paw edema. Biochim. Biophys. Acta 2000, 1477, 64-74.

604

40. Guimarães, L. C.; Oliveira, C. F. R.; Marangoni, S.; Oliveira, D. G. L.; Macedo, M. L.

605

R., Purification and characterization of a Kunitz inhibitor from Poincianella pyramidalis with

606

insecticide activity against the Mediterranean flour moth. Pestic. Biochem. Physiol. 2015,

607

118, 1-9.

608

41. Delgado-Vargas, F.; López-Valdés, H. E.; Valdés-Rodríguez, S.; Blanco-Labra, A.;

609

Chagolla-López, A.; López-Valenzuela, E. d. J., Isolation and properties of a Kunitz-type

610

protein inhibitor obtained from Pithecellobium dulce seeds. J. Agric. Food Chem. 2004, 52,

611

6115-6121.

612

42. Chaudhary, N.; Shee, C.; Islam, A.; Ahmad, F.; Yernool, D.; Kumar, P.; Sharma, A.,

613

Purification and characterization of a trypsin inhibitor from Putranjiva roxburghii seeds.

614

Phytochemistry 2008, 69, 2120-6.

615

43. Bezerra, C. S.; Oliveira, C. F. R.; Machado, O. L. T.; Mello, G. S. V.; Pitta, M. G. R.;

616

Rêgo, M. J. B. M.; Napoleão, T. H.; Paiva, P. M. G.; Ribeiro, S. F. F.; Gomes, V. M.; Silva,



617

O. N.; Maria-Neto, S.; Franco, O. L.; Macedo, M. L. R., Exploiting the biological roles of the

618

trypsin inhibitor from Inga vera seeds: A multifunctional Kunitz inhibitor. Process Biochem.

619

2016, 51, 792-803.

620

44. Joshi, R. S.; Mishra, M.; Suresh, C. G.; Gupta, V. S.; Giri, A. P., Complementation of

621

intramolecular interactions for structural–functional stability of plant serine proteinase

622

inhibitors. Biochim. Biophys. Acta, Gen. Subj. 2013, 1830, 5087-5094.

623

45. Cruz, A. C. B.; Massena, F. S.; Migliolo, L.; Macedo, L. L. P.; Monteiro, N. K. V.;

624

Oliveira, A. S.; Macedo, F. P.; Uchoa, A. F.; Grossi de Sá, M. F.; Vasconcelos, I. M.;

625

Murad, A. M.; Franco, O. L.; Santos, E. A., Bioinsecticidal activity of a novel Kunitz trypsin

626

inhibitor from Catanduva (Piptadenia moniliformis) seeds. Plant Physiol. Biochem. 2013,

627

70, 61-68.

628

46. Macedo, M. L. R.; Garcia, V. A.; Freire, M. G. M.; Richardson, M., Characterization of a

629

Kunitz trypsin inhibitor with a single disulfide bridge from seeds of Inga laurina (SW.) Willd.

630

Phytochemistry 2007, 68, 1104-1111.

631

47. QI, R. F.; SONG, Z. W.; CHI, C. W., Structural Features and Molecular Evolution of

632

Bowman‐Birk Protease Inhibitors and Their Potential Application. Acta Biochim. Biophys.

633

Sin. 2005, 37, 283-292.

634

48. Mithofer, A.; Boland, W., Plant Defense Against Herbivores: Chemical Aspects. Annu.

635

Rev. Plant Biol. 2012, 63, 431-450.

636

49. Rufino, F. P. S.; Pedroso, V. M. A.; Araujo, J. N.; França, A. F. J.; Rabêlo, L. M. A.;

637

Migliolo, L.; Kiyota, S.; Santos, E. A.; Franco, O. L.; Oliveira, A. S., Inhibitory effects of a

638

Kunitz-type inhibitor from Pithecellobium dumosum (Benth) seeds against insect-pests'

639

digestive proteinases. Plant Physiol. Biochem. 2013, 63, 70-76.


Page 28 of 46

Page 29 of 46


640

50. Terra, W. R.; Ferreira, C., 11 - Biochemistry and Molecular Biology of Digestion. In

641

Insect Molecular Biology and Biochemistry, Gilbert, L. I., Ed. Academic Press: San Diego,

642

2012; pp 365-418.

643

51. Yang, L.; Fang, Z.; Dicke, M.; van Loon, J. J.; Jongsma, M. A., The diamondback

644

moth, Plutella xylostella, specifically inactivates Mustard Trypsin Inhibitor 2 (MTI2) to

645

overcome host plant defence. Insect Biochem. Mol. Biol. 2009, 39, 55-61.

646

52. Zhu-Salzman, K.; Koiwa, H.; Salzman, R. A.; Shade, R. E.; Ahn, J. E., Cowpea bruchid

647

Callosobruchus maculatus uses a three-component strategy to overcome a plant

648

defensive cysteine protease inhibitor. Insect Mol. Biol. 2003, 12, 135-145.

649

53. Neal, J. J., Brush border membrane and amino acid transport. Arch. Insect Biochem.

650

Physiol. 1996, 32, 55-64.

651

54. Ordóñez-García, M.; Rios-Velasco, C.; Berlanga-Reyes, D. I.; Acosta-Muñiz, C. H.;

652

Salas-Marina, M. Á.; Cambero-Campos, O. J., Occurrence of Natural Enemies of

653

Spodoptera frugiperda (Lepidoptera: Noctuidae) in Chihuahua, Mexico. Fla. Entomol.

654

2015, 98, 843-847.

655

55. Yuan, J. S.; Köllner, T. G.; Wiggins, G.; Grant, J.; Degenhardt, J.; Chen, F., Molecular

656

and genomic basis of volatile-mediated indirect defense against insects in rice. Plant J.

657

2008, 55, 491-503.

658

56. Kuwar, S. S.; Pauchet, Y.; Vogel, H.; Heckel, D. G., Adaptive regulation of digestive

659

serine proteases in the larval midgut of Helicoverpa armigera in response to a plant

660

protease inhibitor. Insect Biochem. Mol. Biol. 2015, 59, 18-29.

661



Figure captions Figure 1. PeTI purification steps. A) DEAE-Sepharose chromatogram of Platypodium elegans CE. (B) Trypsin-Sepharose chromatogram of D-III fraction. Inset: 12.5% SDSPAGE of purificated fractions. MW: molecular weight standard; CE: Crude Extract; D-III: DEAE-Sepharose fraction; S-II: Trypsin-Sepharose fraction. Horizontal bars indicate fractions with inhibitory activity. (C) RP-HPLC analysis onto C-18 column at a flow rate of 1 mL/min-1. D) Intact mass of PeTI determined by MALDI-TOF.

Figure 2. Increasing concentrations of PeTI were added to a fixed concentration of trypsin enzyme. Stoichiometry of the PeTI peptidase interaction. Bar indicates standard deviation from triplicate determinations.

Figure 3. Lineweaver–Burk plots are showing simple competitive inhibition of trypsin by PeTI inhibitor. The BAPNA hydrolysis for a constant amount of trypsin (4.2 nM) was measured with increasing concentrations of substrate in the absence or presence of the PeTI (■, 0 nM; ●, 0.15nM; ▼, 0.3nM).

Figure 4. Amino-terminal sequence alignment of PeTI with other Kunitz-type proteinase inhibitors. Consensus symbols below the protein sequences denote fully conserved residue (*), strongly conservation of similar properties (:), weakly conservation of similar properties (.). ªAccession number from NCBI database. PeTI has an RGG/RG motif (highlighted in grey). The multiple alignment shows a glycine-rich region with arginine– glycine-rich (RGG) underlined at the amino (N)-terminal partial of the PeTI. Amino acids Asn12 position (RNG boxes in grey) are double underline (PeTI numbering).


Page 30 of 46

Page 31 of 46


Figure 5. Inhibitory activity of PeTI against trypsin-like enzymes from the midgut of the fourth instar larvae of pest insects. The assays were performed using BAPNA as substrate. Values are mean ± standard error of triplicates (N= 5).

Figure 6. Effect of artificial diets supplemented with PeTI on Spodoptera frugiperda larvae development of fifth instar. (A) Larval weight and (B) survival of S. frugiperda larvae. Inset: size difference in the (1) control-fed larvae and (2) PeTI-fed larvae, bar = 1 cm. Values are mean ± standard error for three independent bioassays (N= 30). Statistical analyses were performed by one-way ANOVA with post-hoc Tukey's multiple comparison. Different letters above columns denote a significant difference between the treatments (p < 0.05).

Figure 7. Effect of PeTI on proteolytic activity in the midgut lumen of fifth instar larvae of Spodoptera frugiperda. (A) Trypsin-like activity and (B) chymotrypsin-like activity from control- and PeTI-fed larvae using BAPNA and SAAPFPNA as substrates, respectively. (C) Inhibition by PeTI of trypsin-like enzymes from control-fed and PeTI-fed larvae, using BAPNA as a substrate. Experiments were carried out in triplicate. The statistical analyses were performed by one-way ANOVA with post-hoc Tukey's multiple comparison. Different letters above columns denote a significant difference between the treatments (p < 0.05).

Figure 8. Digestibility of (A) PeTI and (B) BSA by midgut proteases from control- and PeTI-fed larvae of Spodoptera frugiperda. PeTI and BSA were incubated at a ratio of 1:5 (inhibitor: midgut extract) at 30 °C for different times. Digestion products were visualized by SDS-PAGE (15%).



Page 32 of 46

Tables Table 1. Purification steps of PeTI Mass

Total protein

Total activity

Purification

Yield

(mg)

(mg)

(TUI/µg) x 10

(Fold)

(%)

Crude Extract

2,000

908

47.22

0.52

1

100

DEAE-Sepharose

399

105.33

10.60

11.01

1.94

22.45

Trypsin-Sepharose

39

7.02

1.60

2.28

4.40

3.40

Steps

6

Specific activity (TUI/mg) x 10


5

Page 33 of 46


Table 2. Inhibitory activity of PeTI toward serine peptidases

Enzyme

Source

PeTI (0.1 µg) residual activity % ± SE*

Trypsin

Bovine pancreas

30 ± 0.33a

Chymotrypsin Bovine pancreas

98 ± 0.79b

* Values are mean ± standart error (each average is three replicates). Different letters denote a significant difference between the treatments (p < 0.05).



Table 3. Stability studies of PeTI. Temperature stability after incubation 30 min at the indicated temperature. pH stability of the inhibitor after incubation at the indicated pH for 1h at 37ºC. Trypsin residual activity

Temperature Trypsin residual activity pH (ºC)

(%) *

(%)*

30

100 ± 4.69a

2

97.84 ± 0.06a

37

100 ± 4.88a

3

95.61 ± 1.09a

40

99.55 ± 5.38a

4

95.82 ± 0a

50

100 ± 5.86a

5

98.35 ± 0a

60

99.82 ± 6.03a

6

96.80 ± 1.79a

70

100 ± 6.80a

7

97.93 ± 0.46a

80

72 ± 5.93b

8

98.40 ± 0.99a

90

68.78 ± 6.39b

9

97.41 ± 0.26a

100

25.26 ± 8.71c

10

98.21 ± 0.19a



Page 34 of 46

Page 35 of 46


Table 4. Effect of DTT on the stability of PeTI. The inhibitor was treated with different final concentrations of DTT (1, 10 e 100 mM) for 15 – 120 min at 30 ºC.

Reduncing Agent

Trypsin Residual Activity

(DTT)

(%)*

Time

Control

1mM

10mM

100mM

15

96.60 ± 0.90a 96.71 ± 0.93a 51.69 ± 5.44b 52.79 ± 1.03b

30

97.97 ± 0.39a 96.61 ± 0.78a 47.64 ± 4.88b 49.44 ± 5.11b

60

98.64 ± 2.45a 88.91 ± 2.96b 44.21 ± 0.25c 40.33 ± 2.73c

120

99.89 ± 0.35a 83.67 ± 1.98b 36.55 ± 1.80c 27.52 ± 3.12d




Page 36 of 46

Table 5. Effect of PeTI on Spodoptera frugiperda development. Total development

Stage (days)* Emergence*

time* Larval

Pupal

Adult

(%) (TDT)

18.00 ±

11.06 ±

6.67 ±

Control

0.2% PeTI

0.47a

0.68a

0.50a

21.36 ±

11.56 ±

6.20 ±

1.08b

0.63a

1.23a

100a

35.10 ± 2.33a

100a

39.10 ± 1.52b

* Values are mean ± standart error (each average is three replicates). Different letters denote a significant difference between the treatments (p < 0.05)


Page 37 of 46


Table 6. Amino acid similarity of PeTI with Kunitz trypsin inhibitors from different plant sources. Accession: NCBI Reference Sequence.

Query Accession

Protein

E-value Identity (%) cover

BAA82254.1

Kunitz trypsin inhibitor p20 [Glycine max]

95%

3e-10

55%

BAB82379.1

Mcp20 [Matricaria chamomilla]

95%

3e-10

55%

P83036.2

Kunitz-type trypsin inhibitor LlTI [Leucaena leucocephala]

97%

5e-08

55%

P09941.1

Trypsin inhibitor DE5 alpha chain [Adenanthera pavonina]

97%

9e-08

52%

XP_020224523.1 Kunitz-type trypsin inhibitor KTI1-like [Cajanus cajan]

84%

1e-07

68%

XP_006372631.1 Trypsin protein inhibitor 3 [Populus trichocarpa]

95%

1e-07

54%

ADW95389.1

Kunitz-type trypsin inhibitor [Populus nigra]

95%

1e-07

54%

P83594.1

Factor Xa inhibitor BuXI [Bauhinia ungulata]

97%

1e-07

48%

XP_015938771

Kunitz-type trypsin inhibitor KTI1-like [Arachis duranensis]

95%

3e-07

53%

4J2K_A

Trypsin Inhibitor Ecti [Enterolobium contortisiliquum]

84%

4e-07

61%



Figures Figure 1


Page 38 of 46

Page 39 of 46


Figure 2



Figure 3


Page 40 of 46

Page 41 of 46


Figure 4

Protein

Accession numberª

PeTI

1 FVVDTDGDPLRNGGSYYILPVFRGRGGGIEQAAIG------------SEVSKGLPLR

Kunitz trypsin inhibitor p20

BAA82254.1

2 IVFDTEGNPIRNGGTYYVLPVIRGKGGGIEFAKTETETCPLTVVQSPFEVSKGLPLI

Mcp20

BAB82379.1

2 IVFDTEGNPIRNGGTYYVLPVIRGKGGGIEFAKTETETCPLTVVQSPFEVSKGLPLI

Kunitz-type trypsin inhibitor LlTI

P83036.2

2 ILVDLDGDPLYNGMSYYILPVARGKGGGLELARTGSESCPRTVVQTRSETSRGLPAR

Trypsin inhibitor DE5 alpha chain

P09941.1

2 ELLDVDGNFLRNGGSYYIVPAFRGKGGGLELARTGSETCPRTVVQAPAEQSRGLPAR

Kunitz-type trypsin inhibitor KTI1-like

XP_020224523.1

1 DVLDTDGKLLRNGGSYYVVPVKRGSGGGIELAATGNETCPLTVVQSPNKASKGNPCL

Trypsin protein inhibitor 3

XP_006372631.1

2 PVLDIDGEKLVAGTEYYILPVFRGRGGGITMASN-KTSCPLAVVQDRLEVSKGLPLT

Kunitz-type trypsin inhibitor

ADW95389.1

2 PVLDIDGEKLVAGTKYYILPVFRGRGGGITMASN-KTSCPLAVVQDRLEVSKGLPLT

Factor Xa inhibitor BuXI

P83594.1

2 IVLDTDGKPVNNGGQYYIIPAFRGNGGGLELTRVGRETCPHTVVQASSEISNGLPVM

Kunitz-type trypsin inhibitor KTI1-like

XP_015938771

1 ELVDTDGNLIRNGGLYYILPVFRGNGGGIGRTSTGNETCPLTVVQQRSEVDNGLPII

Trypsin Inhibitor Ecti

4J2K_A

2 ELLDSDGDILRNGGTYYILPALRGKGGGLELAKTGDETCPLNVVQARSETKRGRPAI :.* :*. :


*

**::*. ** ***:

:

..* *


Figure 5


Page 42 of 46

Page 43 of 46


Figure 6



Figure 7


Page 44 of 46

Page 45 of 46


Figure 8



TOC graphic


Page 46 of 46

A novel peptidase Kunitz inhibitor from Platypodium elegans seeds is

Recommend Documents