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Journal of Stored Products Research 45 (2009) 133–138

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Journal of Stored Products Research

journal homepage: www.elsevier .com/locate/ jspr

Partial characterization of a chymotrypsin-like protease in the larger grain borer(Prostephanus truncatus (Horn)) in relation to activity of Hyptis suaveolens (L.)trypsin inhibitor

Cesar Aguirre a, Jose L. Castro-Guillen b, Lucrecia Contreras b, Elizabeth Mendiola-Olaya b,Luis Gonzalez de la Vara b, Alejandro Blanco-Labra b,*

a Instituto Tecnologico de Roque, Division de Estudios de Posgrado e Investigacion. km 8 Carretera Celaya-Juventino Rosas, C.P. 38110, Celaya, Gto., Mexicob Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional, Unidad Irapuato, Dpto. de Biotecnologıa y Bioquımica, km 9.6 Libramiento Norte CarreteraIrapuato-Leon, C.P. 36821. Irapuato, Gto., Mexico

a r t i c l e i n f o

Article history:Accepted 23 November 2008

Keywords:Protease inhibitorsProstephanus truncatusInsect resistanceArtificial seedsInsect proteasesChymotrypsin-like proteases

* Corresponding author. Fax: þ52 462 624 5996.E-mail address: ablanco@ira.cinvestav.mx (A. Blan

0022-474X/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.jspr.2008.11.001

a b s t r a c t

Hyptis suaveolens trypsin inhibitor (HSTI) inhibits, in vitro, most of the trypsin-like serine proteasespresent in the gut of Prostephanus truncatus (Horn), the larger grain borer. In order to measure its in vivoeffectiveness, different doses of the purified HSTI were incorporated into artificial seeds to measure theireffects on the growth of P. truncatus larvae and pupae. Results showed that this protease inhibitor had nosignificant effect in vivo. Consequently, we investigated, identified, and partially characterized aninhibitor-insensitive protease that could explain the growth of P. truncatus on diet containing HSTI. Thisprotease had an apparent molecular mass of 31 kDa, an optimum pH of 7.0, and an activity rangeextending from pH 6.0 to 8.0, which would allow activity in the gut of P. truncatus. This enzyme could beone of the physiological mechanisms in P. truncatus that allow the species to avoid the adverse effects oftrypsin-like protease inhibitors present in the normal diet.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Insects utilize a diversity of proteolytic enzymes with a pHactivity range from acidic to alkaline in order to digest dietaryproteins (Berenbaum, 1980; Terra and Ferreira, 1994; Nation, 2002).Prostephanus truncatus (Horn), the larger grain borer, has a gut pHgradient from 4.2 to 6.2 (Vazquez-Arista et al., 1999). The diversityof its proteases may be a characteristic of non-specialized digestionin this cosmopolitan stored-product pest which enables efficientutilization of a wide variety of diets. Insect larvae reared on dietswith different protease inhibitors (PIs) have been shown to exhibitdifferences in digestive proteases. For instance, the resistancestrategy adopted by Spodoptera frugiperda (Smith), the fall army-worm, consists, in part, of the production of a suite of digestiveenzymes that may minimize impact of PIs in their diets (Jongsmaet al., 1996; Mazumdar-Leighton and Broadway, 2001; Oppert et al.,2005; Brioschi et al., 2007).

Plant PIs could aid in plant defense against herbivorous insectsby reducing their digestive capacity through specific inhibitionof digestive proteases, thereby arresting insect growth and

co-Labra).

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development (Broadway and Duffey, 1986). These inhibitors can beused as an alternative strategy to control insect pests, but theirpotential activity against a specific insect depends on the quantityand quality of the enzymes present in the target insect’s midgut(Elden, 1995). In the case of Hyptis suaveolens (L.) trypsin inhibitor(HSTI), we previously demonstrated its ability to inhibit all but one ofthe trypsin-like proteases present in the gut of P. truncatus (Aguirreet al., 2004).

In most cases, the effect of PIs on selected insects has beendetermined by measuring their in vitro activity in an insect guthomogenate or by measuring insect growth on artificial diets(Murdock et al., 1987; Oppert et al., 1993), or after PIs genes havebeen expressed in the insect host plant. Although results of some invivo feeding experiments have been inconclusive (Jongsma andBolter, 1997), feeding assays are nevertheless necessary to supportthe evidence of in vitro assays and to provide a more valid esti-mation of the effectiveness of a specific inhibitor, particularly if it isto be considered as an alternative for the production of transgenicplants with higher resistance to that particular insect.

Most studies on artificial diet trials have been performed usingcrude extracts of the PI. In some of these trials, PIs reduced growthand increased insect mortality (Rodrigues et al., 2002). However,insects may overcome the effects of PIs (Girard et al., 1998; Ba�zoket al., 2005) by using different mechanisms, such as the presence or

0

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Ctrl 0.001 0.010 0.100 0.150HSTI in diet (% w/w)

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up

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HSTI in diet (% w/w)

Pu

pae w

eig

ht (g

)

Ctrl 0.001 0.010 0.100 0.150

A B

Fig. 1. Effect of HSTI doses on average number and weight of P. truncatus pupae. Larvae were grown for 28 days in artificial seeds containing the indicated amounts of HSTI. (A)Pupae content of each treatment. Linear regression analysis shows no correlation between the number of pupae and the HSTI content of the diet (R2 ¼ 6 � 10�5). (B) Average weightof pupae from different treatments. Linear regression analysis shows no correlation between average weight of pupae and the HSTI content of the diet (R2 ¼ 3.5 � 10�2).

0.00.20.40.60.81.01.21.41.61.82.0

0 20 40 60 80 100Fraction number

012345678910

Ab

s 280 n

m[N

aC

l]

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Pro

tease A

ctivity (µ

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021 31 41 51Fraction number

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2.0

2.5

1 61

A

Time (min)

30.80 60

0.5

0

0.25A

bs 220 n

m

C

*

Fig. 2. Purification of chymotrypsin-like protease from P. truncatus third-instar larvae.(A) Gel filtration chromatography after (NH4)2SO4 precipitation. The line representsprotein absorbance at 280 nm and diamonds the protease activity using SAAPFpNA assubstrate. (B) Ion exchange chromatography of a protease-rich fraction obtained by gelfiltration. Absorbance at 280 nm (line), chymotrypsin-like activity (diamonds), andNaCl gradient (broken line). (C) HPLC of the fraction previously purified by ion-exchange chromatography. Asterisk indicates the peak with protease activity.

C. Aguirre et al. / Journal of Stored Products Research 45 (2009) 133–138134

expression of inhibitor-resistant proteases or through de novosynthesis of resistant enzymes (Bolter and Jongsman, 1995;Broadway, 1997; Volpicella et al., 2003; Bown et al., 2004). Anothermechanism is the long-term down-regulation of the expression ofthose proteases that are most sensitive to the inhibitor, or by directdigestion of the inhibitor (Bown et al., 2004). The effect of the PIsalso depends on different factors, such as PI concentration and itskinetic properties, as well as the stability of the PI in the insect gut(Jongsma and Bolter, 1997; Oppert et al., 2003). Recently, thestructural basis of inhibitor-resistance of an insect protease wasdescribed by Bayes et al. (2007). The objective of our study was toinvestigate whether HSTI was active in in vivo assays and to look forthe presence of an insensitive protease in the gut of P. truncatusfeeding on diets without HSTI.

2. Materials and methods

2.1. Chemicals and biological materials

The reagents used were obtained from Baker and Merck(Darmstadt, Germany). Bovine pancreas trypsin (type I; EC3.4.21.4), N-a-benzoyl-L-arginine ethyl ester (BAEE), N-a-benzoyl-L-tyrosine ethyl ester (BTEE), N-benzoyl-L-tyrosine p-nitroanilide(BTpNA), N-benzoyl-L-arginine p-nitroanilide (BApNA), andN-succinyl Ala-Ala-Pro-Phe p-nitroanilide (SAAPFpNA) were fromSigma–Aldrich (St. Louis, MO, USA). Sephadex G-75 and molecularmass markers were from Pharmacia (Uppsala, Sweden). All chem-icals were analytical grade. Reagents for SDS–PAGE and Econo-Pac�

High Q cartridge were from Bio-Rad (Hercules, CA, USA). Maize var.cacahuazintle was obtained from a local market to produce artificialseeds. Chan seeds were provided by Dr. Martha Vergara-Santana(Herbarium, University of Colima, Mexico). From them, HSTI wasextracted and purified according to Aguirre et al. (2004).

2.2. Insects and artificial diet

Adult insects were grown on maize seeds at 28 � 2 �C and 60%relative humidity (r.h.) under a photoperiod of light/darkness(12:12 h). Larvae were obtained by crushing and sieving infestedseeds, whereas the eggs were removed from the exterior of thekernel under a stereomicroscope. Artificial seeds (2 g averageweight), were prepared according to Shade et al. (1986), withmodifications. Maize seeds were milled and passed through a 40

mesh screen; this flour was blended with deionized water at 1:1.2(w/v) ratio producing a smooth paste to produce the artificial seeds.Semi-purified (after gel filtration chromatography) HSTI was thenincorporated into the paste. HSTI concentrations used were 0.001%,0.010%, 0.100% and 0.150% (w/w) with respect to the whole seed.This paste was then transferred to a syringe and injected directlyinto each mold of a Corning plate. Once all molds were filled, they

66200

45000

31000

21500

31 kDa

M A

Fig. 3. Apparent molecular mass determination on SDS–PAGE (10%) of the HPLCpurified protease. M, molecular mass markers; A, HPLC purified protease.

C. Aguirre et al. / Journal of Stored Products Research 45 (2009) 133–138 135

were immersed in liquid nitrogen for 5 s and then dried at 55 �C for13 h. Seeds were incubated for 24 h at 27 �C with 60% r.h. to checkfor contamination.

Feeding assays were performed by perforating three small holeson the surface of each artificial seed, and one egg was placed in eachof the holes. Seven seeds were used for each treatment. After eggshad been introduced, the holes were filled with the correspondingflour and then they were incubated for 25 days at 28 �C and 60% r.h.Each treatment was undertaken in triplicate. After the incubationperiod, each one of the seeds was broken open and larvae werecollected, separated, and weighed.

2.3. Protease purification and characterization

Both insect and bovine chymotrypsin activity were measuredusing the substrates SAAPFpNA, BTpNA (Gervaix et al., 1991), andBTEE (Kang and Morton, 1973). Trypsin and trypsin-like activitywere determined using BApNA (Erlanger et al., 1961) and BAEE(Schwertz and Takenaka, 1955) as substrates. Chymotrypsin-likeprotease was extracted by stirring a suspension of ground wholethird-instar-larvae of P. truncatus (20 g) in 0.01 M Tris–HCl, pH 7.5(1:5 w/v) for 1 h at 4 �C, since Vazquez-Arista et al. (1999) deter-mined that this extract was similar in activity to that obtained

Table 1Prostephanus truncatus chymotrypsin purification.

Procedure Totalactivity(katals)

Totalprotein(mg)

Specific activity(katals protein mg�1)

Yield(%)

Purificationfold

Crude extract 1473258 138.74 10618 100 1G-75 filtration

fraction708861 13.38 52968 48.1 4.98

Ion-exchangefraction

33660 0.35 95897 2.3 9.03

HPLC fraction 6270 0.0462 135714 0.42 12.78

directly from the dissected intestine. Insoluble material wasremoved by centrifugation at 10,000 � g for 60 min. The crudeextract was precipitated with ammonium sulfate at 70% saturationand the precipitate was then separated and dialyzed against water.After dialysis, the concentrated solution of P. truncatus chymo-trypsin-like protease was passed through a G-75 Sephadex gelfiltration column (2.25 � 167 cm) equilibrated with 0.02 Mammonium bicarbonate, pH 7.8. Elution was completed at a flowrate of 0.3 ml min�1 collecting 4 ml fractions. Those showingchymotrypsin-like activity were pooled and concentrated by ultra-filtration. This fraction was further purified by ion-exchange chro-matography in an Econo-Pac� high Q cartridge column (1 � 5 cm)equilibrated with 0.01 M Tris–HCl, pH 8.0. Elution was carried outusing a 0.0 to 0.25 M NaCl linear gradient in 0.01 M Tris–HCl buffer(pH 7.5), with a flow rate of 1 ml min�1, collecting 2 ml fractions.

Fractions showing protease activity were pooled, dialyzedagainst deionized water, and concentrated by ultra filtration.Concentrated enzyme solution was further purified by high-performance liquid chromatography (HPLC) using a Superose12 FPLC (Amersham) column equilibrated with 0.02 M Tris–HCl pH8.0, with a flow rate of 0.4 ml min�1. Peak fractions were concen-trated by ultrafiltration. Fractions with protease activity wereevaluated by electrophoresis and those containing a single bandwere used for protein characterization. Protein concentration wasdetermined according to Bradford (1976), using bovine serumalbumin (BSA) as standard.

Apparent molecular mass was determined by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using 13%separating gel (Schagger and von Jagow, 1987). Bovine serumalbumin (66,200 Da), ovalbumin (45,000 Da), carbonic anhydrase(31,000 Da), and trypsin inhibitor (21,500 Da) were used asmolecular mass markers. Optimal pH activity was determinedusing the following buffer solutions: citrate buffer pH 3.0, succi-nate buffer pH 5.0, phosphate buffers pH 7.0 and 7.5, Tris–HClbuffers pH 8.0 and 8.5, and carbonate buffers pH 9.2 and 10.7,keeping their ionic strength constant at 0.15. Optimal temperatureactivity was measured at its optimal pH of 7.0. The temperaturesused ranged from 0 to 94 �C with 10 �C intervals, incubating theenzyme for 10 min at each temperature, before determining itsactivity in an Ultramark Microplate Manager instrument (Bio-Rad).The isoelectric point was determined by isoelectric focusing (IEF)in a Pharmacia LKB electrophoresis PhastSystem using a Phastgel3-9. The kinetic constants Km and Vmax values were obtained usingSAAPFpNA as a substrate. Assays were performed at roomtemperature (25 �C), analyzing the results with a non-linearregression program using the software Origin v6.1. Proteaseconcentration used for insect enzyme determination was9 pmol ml�1 and 20 pmol ml�1 for the bovine chymotrypsin.

3. Results

Modifications made to the method of Shade et al. (1986)produced an appropriate seed consistency that was necessary formaking three holes in seeds into which P. truncatus eggs wereplaced. After egg hatching, larvae and pupae were counted after28 days. At this time, no adults were present, and approximately70% of individuals were larvae and pupae. The pupae were used forstatistical analysis (Fig. 1). Average pupal weight varied from 3.6 to4.05 mg. Regression analysis showed a non-significant effect ofHSTI dosage on the number of pupae. Mortality for controls, 0.010%and 0.150% HSTI treatments was 2.1%, 10.0%, and 10.9% respectively;and there was no mortality in the 0.001% and 0.100% HSTItreatments.

A chymotrypsin-like protease from the third-instar-larvae waspurified to homogeneity using a combination of ammonium sulfate

Pro

tease activity (m

kat x 10

-4)

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tease activity (%

o

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axim

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A B

Fig. 4. Optimal pH and temperature of the chymotrypsin-like protease from P. truncatus. (A) Protease activity determined at the indicated pH values, at 30 �C and a constant ionicstrength of 0.15. (B) Protease activity at temperatures from 0 to 95 �C with 10 �C intervals at pH 7.0.

C. Aguirre et al. / Journal of Stored Products Research 45 (2009) 133–138136

precipitation, G-75 gel filtration chromatography, and ion-exchange chromatography. In this chromatography three peakswith chymotrypsin-like activity were detected, of which the leastsensitive one to HSTI was selected and purified further using HPLC(Fig. 2). Neither trypsin-like activity nor cysteine-like activity wasdetected. This enzyme had an apparent molecular mass of 31 kDa,as determined by SDS–PAGE (Fig. 3), and was purified 12.8 times(Table 1). The enzyme isoelectric point was 3.5 (data not shown),whereas its optimal pH for activity was 7.0, with an activity rangeextending from 6.0 to 8.0, and an optimal temperature at 60 �C(Fig. 4).

A Km value of 560 mM for the purified enzyme was calculatedusing SAAPFpNA as substrate (Fig. 5). The enzyme failed to hydro-lyze the common substrates for chymotrypsin: BTEE and BTpNA,and for trypsin: BApNA and BAEE.

0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.20.00

0.02

0.04

0.06

0.08

0.10

SAAPFpNA (mM)

Vmax = 0.106 ± 0.009 nmol min-1Km = 0.560 ± 0.117 mM

R2= 0.962

-1

Pro

tease activity (n

mo

l m

in

-1)

A B

Fig. 5. Determination of kinetic constants for (A) chymotrypsin-like protease from P. truncatboth cases, protease activity was measured at room temperature (25 �C), with SAAPFpNA a

The kcat (enzymatic overall catalytic rate constant) found for thisenzyme was 3533 s�1 and its efficiency (kcat/Km) was6.3 � 106 M�1 s�1. These values are similar to those obtained withbovine chymotrypsin for this substrate (kcat 315 s�1 and efficiency9 � 106 M�1 s�1).

4. Discussion

The development of a system that mimics P. truncatus normalgrowing conditions was required to measure the in vivo effect ofHSTI (Aguirre et al., 2004). The artificial seeds reported herepermitted the incorporation of different inhibitor doses. Resultsshowed no significant differences in the number or weight ofpupae of insects fed on diets with different amounts of HSTI. Onepossible reason for this negative result is that P. truncatus has

Pro

tease activity (n

mo

l m

in

)

0 40 80 120 160 200 2400.000

0.004

0.008

0.012

0.016

0.020

SAAPFpNA ( M)

R2 = 0.938Vmax = 0.021 ± 0.002 nmol min-1Km = 35.114 ± 11.080 µM

us, using 9 pmol ml�1 of enzyme, and (B) bovine chymotrypsin, using 20 pmol ml�1. Ins substrate.

C. Aguirre et al. / Journal of Stored Products Research 45 (2009) 133–138 137

developed a tolerance to these inhibitors, so that it can exist ona wide range of diets. Neither trypsin-like activity nor cysteineprotease activity was found in the purified sample, but at leasttwo different chymotrypsin-like proteases were present. The onewe studied could be actively present in the gut of P. truncatus, andits kinetic constants (Km, kcat and efficiency), were similar to thosecalculated for bovine chymotrypsin. However, this enzyme differsfrom the bovine enzyme because it did not hydrolyze the twomost characteristic substrates for that enzyme (BTEE and BTpNA).Its optimal temperature was 60 �C, similar to that which has beenreported for other proteases (Siringan et al., 2007). Results indi-cate that simple inhibition of the insect gut trypsin-like serineprotease activity found in in vitro assays is not sufficient evidenceto predict resistance in P. truncatus. Broadway (1997) found thatthe presence of an inhibitor-resistant protease in the guts ofHelicoverpa zea (Boddie), the corn earworm, Agrotis ipsilon (Huf-nagel), the black cutworm, and Trichoplusia ni (Hubner), thecabbage looper, was responsible for the null effect of the PI on itsgrowth. Brioschi et al. (2007) showed that adaptations in trypsinand chymotrypsin proteases could also be responsible for resis-tance to soybean PI in Spodoptera frugiperda.

According to previous studies on P. truncatus (Vazquez-Aristaet al., 1999), both adult and larval guts have two main pH values:6.2 for the midgut and fore-hindgut (ileum) and 4.2 to 4.6 in partof the midgut and in the hind-hindgut. Therefore, the enzymedescribed here would only be active in part of the midgut and inthe fore-hindgut. However, in the regions with a pH between 4.2and 4.6, this enzyme would be practically inactive, with less than10% activity. The optimal pH of 7.0 for this enzyme is alsodifferent from other insect chymotrypsin-like enzymes, such asone described from Tenebrio molitor L., the yellow mealworm,which had an optimal pH of 9.5 (Elpidina et al., 2005). Also theisoelectric point (pI) for this chymotrypsin-like enzyme, 3.5, wasvery low in comparison with the pI values of bovine chymo-trypsin (9.1), bovine trypsin (8.7), or the chymotrypsin-likeenzyme from T. molitor (8.4), as reported by Elpidina et al.(2005).

When comparing the kinetic constants of this enzyme withthose of bovine chymotrypsin, the main difference was that the kcat

(Vmax/[E]t) for the insect protease (3533 s�1) was over ten timeshigher than the corresponding kcat for the bovine chymotrypsinenzyme (315 s�1). This difference was even higher with the kcat of T.molitor protease: 36.5 s�1 (Elpidina et al., 2005), using the samesubstrate. Finally, HSTI did not inhibit this enzyme at concentra-tions that completely inhibited most of the trypsin-like enzymespresent in the gut of P. truncatus.

In summary, the characterized enzyme showed differencesfrom other enzymes of the same type found in other organisms.The diversity of protease content in the insect gut of P. truncatusseems to provide this species with the necessary enzyme activityto overcome its inhibited enzymes (trypsin-like enzymes) whenfed on foods with high PI content. Although inhibitors of thistype efficiently inhibit proteases of some insects, this does notguarantee that those inhibitors would be efficient in controllingthe growth of P. truncatus. It is therefore necessary to measure itseffect directly in in vivo assays, using artificial seeds whenneeded.

Acknowledgehments

We thank Javier Luevano Borroel, Yolanda Rodrıguez, andMa. Isabel Cristina Elizarraraz for their valuable technicalassistance.

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