Novel Peptidase Kunitz Inhibitor from Platypodium elegans Seeds Is

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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

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Journal of Agricultural and Food Chemistry

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A novel peptidase Kunitz inhibitor from Platypodium elegans seeds is active

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against Spodoptera frugiperda larvae

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

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

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Oliveira3, Newton Valério Verbisck4, Maria Lígia Rodrigues Macedo3*

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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

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2

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SBBq, Campo Grande, MS, Brazil.

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3

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

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900, Campo Grande, MS, Brazil.

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

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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.

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ACS Paragon Plus Environment


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Abstract

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

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

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

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

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

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

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

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

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

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

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

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for pest control.

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

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frugiperda.

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Introduction

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

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

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

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

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

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

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

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by opportunistic microrganisms3,

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

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

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. Along with this co-evolution, plants evolved a

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

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

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

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

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

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

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

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investigated as a biotechnological tool for crop protection12.

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

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

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

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

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

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



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

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

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

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

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against peptidases from other families14.

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Herein, we describe the purification and characterization of Platypodium elegans

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

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

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

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

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

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

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

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

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

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

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

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alternatives to control this pest.

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Material and Methods

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Materials

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

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

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

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

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


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

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

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

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

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

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

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chemicals and reagents used were of analytical grade.

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Insects

(SAAPFPNA),

N-p-tosyl-lysine

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The insects were housed and maintained under laboratory conditions at the

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

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

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

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

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

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

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

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

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

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

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

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



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Methods

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Isolation of P. elegans trypsin inhibitor (PeTI)

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

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

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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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monitored at 280 nm.


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Protein quantification

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

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

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obtain a standard calibration curve.

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Polyacrylamide gel electrophoresis

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

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

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

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

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

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Protein digestion, mass spectrometry and amino acid sequencing

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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Macedo, et al.

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

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

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

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

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Enzymatic assays

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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

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described above.

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Ki determination and stoichiometry inhibition

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Bovine trypsin was incubated with different concentrations of PeTI and residual

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

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binding inhibition method, described by Morrison

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

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

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

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Inc.).

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Stability of inhibitory activity

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, was used. Increasing concentrations

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

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

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

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

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

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


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

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

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

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

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determined. All experiments were carried out in triplicate.

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Screening assay for inhibition of insect trypsins

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

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

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

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

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

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

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

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

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Bioassays

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

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

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

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

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

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

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

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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

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experimental results represent the average of three independent bioassays.

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Digestion of PeTI by S. frugiperda midgut enzymes

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

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

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

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

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

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Statistical analysis

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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

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

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

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

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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

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

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Screening assay for inhibition of insect trypsins

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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

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

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

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S. frugiperda development.


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Effects of PeTI on S. frugiperda development

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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

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

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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.

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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

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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).


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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%).



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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

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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



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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




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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)


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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


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Figure 2



Figure 3


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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


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Figure 6



Figure 7


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Figure 8



TOC graphic


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Novel Peptidase Kunitz Inhibitor from Platypodium elegans Seeds Is

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