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The component of

Carica papaya

seed toxic to

A. aegypti

and the identification of tegupain, the enzyme that generates it

Natalia N. dos S. Nunes

a

, Lucimeire A. Santana

a

, Misako U. Sampaio

a

, Francisco J.A. Lemos

b

,

Maria Luiza Oliva

a,

aDepartamento de Bioquímica, Universidade Federal de São Paulo, 04044-020, São Paulo, SP, Brazil

bLaboratório de Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil

h i g h l i g h t s

"A new cysteine proteinase, named tegupain, was purified fromCarica papayategument. "The enzyme tegupain efficiently hydrolysed the substrate Z-Phe-Arg-pNan (Km58.8lM).

"Tegupain is responsible for the generation of toxic compound present in the cotyledon ofCarica papayaagainstAedes aegypti.

a r t i c l e

i n f o

Article history:

Received 13 August 2012

Received in revised form 19 December 2012 Accepted 31 December 2012

Available online 10 February 2013

Keywords: Aedes aegypti Carica papaya Cysteine proteinase Dengue

Plant enzyme

a b s t r a c t

AsAedes aegyptitransmits the etiologic agents of both yellow and dengue fever; vector control is consid-ered essential to minimise their incidence. The aim of this work was to identify the component ofCarica papayaseed toxic toA. aegypti,and the identification of tegupain, the enzyme that generates it. Aqueous extracts (1%, w/v) of the seed tegument and cotyledon ofC. papayaare not larvicidal isolately. However, a mixture of 17lg mL 1tegument extract and 27lg mL 1cotyledon extract caused 100% larval mortality

in a bioassay. The mixture was no longer larvicidal after the tegument extract was pre-treated at 100°C for 10 min. The enzyme tegupain efficiently hydrolysed the substrate Z-Phe-Arg-pNan (Km58.8lM, Kcat

28020 s1, K

cat/Km5108M 1s 1), and its activity increased with 2 mM dithiothreitol (DTT), at 37°C, pH 5.0. The chelating agent EDTA did not modify the enzyme activity. Inhibition of tegupain by cystatin (Kiapp2.43 nM), E64 (3.64 nM, 83% inhibition), and the propeptide N-terminal sequence indicate that the

toxic activity is due to a novel cysteine proteinase-like enzyme, rendered active upon the hydrolysis of a cotyledon component ofC. papayaseeds.

Ó2013 Elsevier Ltd. All rights reserved.

1. Introduction

Aedes aegypti(Diptera: Culicidae) is a major public health chal-lenge because it acts as the vector of malaria, filariasis, Japanese encephalitis, dengue fever, chikungunya, and yellow fever in

trop-ical and subtroptrop-ical zones (Sá et al., 2009; Kovendan et al., 2011).

Dengue fever can be caused by four serotypes of the dengue arbo-virus. Clinically, it can occur in asymptomatic forms, classic dengue fever, haemorrhagic dengue and other more severe forms. World-wide, 2.5 billion people are at risk of acquiring the disease and 50 million are infected every year; for these pandemic figures there

is no available vaccine (World Health Organization, 2002).

Currently, only insecticides and larvicides can control epidemic events. Moreover, the indiscriminate use of insecticides has

increased the resistance of the vector, thus reducing the efficiency

of conventional control methods (Gluber and Clark, 1995;

Halstead, 1997). Hence, there has been an increasing interest in the development of alternative methods less hazardous to humans and other organisms. In this regard, plant-derived compounds have emerged as good candidates, not only as effective new tools in vector management but also as environmentally safer agents (Garcez et al., 2009).

Proteinases are enzyme that catalyse the degradation of peptides and proteins and play a significant role in physiology. Proteinases are involved in the activation of proenzymes, blood coagulation, the digestion of fibrin clots, the processing and mem-brane transport of secretory proteins, germination, senescence, the defence against plant pathogens (especially fungi and insects), and

the acquisition of nutrients and apoptosis (Liggieri et al., 2009).

In plants, cysteine proteinases are widely distributed through-out various tissues responsible for protein metabolism. They participate in a fundamental network of reactions required during

0045-6535/$ - see front matterÓ2013 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.chemosphere.2012.12.078

⇑ Corresponding author. Address: Universidade Federal de São Paulo, Escola Paulista de Medicina, Departamento de Bioquímica, Rua Três de Maio, 100, 04044-020 São Paulo, SP, Brazil. Tel.: +55 1155764444.

E-mail address:olivaml.bioq@epm.br(M.L. Oliva).

Contents lists available atSciVerse ScienceDirect

Chemosphere

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the life cycle and are involved in physiological events including germination, senescence and environmental stress responses. Pa-pain-like cysteine proteinases are often found in senescing organs, particularly the leaves, flowers, and the legume nodules, as well as in germinating seeds. Cysteine proteinases have been intensively studied, with expression patterns reported in various stages of plant development. Equally well-studied is the role of cysteine pro-teinases in the processing and degradation of seed proteins, in the development of the legume nodule in response to stresses such as

wounding, cold and drought, and in programmed cell death (Salas

et al., 2008; Andrade et al., 2011).

Carica papayabelongs to the Caricaceae family, several species

of which have been used therapeutically (Kovendan et al., 2011).

Originating in the lowlands of Eastern Central America from Mex-ico to Panama, papaya seeds were taken to the Caribbean and South East Asia during the Spanish exploration of the 16th century,

and they then spread rapidly to India, Africa and other places.C.

papayanow grows in all tropical countries and in many subtropical

regions of the world (Ezike et al., 2009).

C. papaya is a lactiferous plant and contains specialised cells known as laticifers that secrete latex and are dispersed throughout most of the plant tissues. Papaya latex is well known as a rich source of the four cysteine proteinases: papain, chymopapain, glycyl endopeptidase and caricain; the levels of these proteinases

vary in the fruit, leaves and roots (Anuar et al., 2008).

This report addresses the potential larvicidal activity of

C. papayaseeds againstA. Aegypti, and the characterisation of the tegument enzyme responsible for the toxic activity.

2. Materials and methods

2.1. C. papaya seeds

Commercially available ripe C. papaya seeds were manually

separated from the pulp, washed with water and dried at room temperature. The tegument was separated from the cotyledon, and each part was ground with distilled water at 1% (w/v) and

centrifuged at 11 410gfor 10 min at 4°C. The supernatants were

separated and further processed for purification.

2.2. Insect rearing tests in C. papaya crude extracts

The bioassay to evaluate the effect of papaya crude extracts on

larval development was adapted from theWorld Health

Organiza-tion (2005). Three groups of twenty 3rd and 4th instar larvae were

maintained in (A) 0.5 mL of crude tegument extract (668

l

g mL 1)

and 0.5 mL of crude cotyledon extract (1100

l

g mL 1), (B) 0.5 mL

crude tegument extract (668

l

g mL 1), or (C) 0.5 mL crude

cotyle-don extract (1100

l

g mL 1). The final volume was brought to

20 mL with water. The effect on larvae mortality (%) was determined after 24 h incubation. Each treatment was performed in duplicate.

2.3. Thermal treatment of the aqueous extracts of C. papaya teguments and cotyledons

The cotyledon and tegument extracts were pre-treated at

100°C for 10 min before the temperature was slowly returned to

room temperature (27°C), and the larvicidal activity was

determined as described above. The bioassay was composed of four groups: (A) neither extract heat-treated, (B) heat-treated tegument extract and untreated cotyledon extract; (C) heat-treated cotyledon extract and untreated tegument extract; (D) both

ex-tracts heat-treated at 100°C. All experiments were performed in

duplicate, and the results reported represent the mean value of two independent tests.

2.4. LD50of C. papaya extracts on A. aegypti larvae

A linear regression analysis was used to determine the response ofA. aegyptito the wholeC. papayaseed extracts (1:10 w/v). Lethal

doses (LD50) were calculated using varying concentrations ofC.

pa-payain terms of the seed weight (0.25 mg mL 1, 0.375 mg mL 1,

0.625 mg mL 1, 1.25 mg mL 1, 2.5 mg mL 1 and 5 mg mL 1) in

20 mL, containing 30 larvae (3rd and 4th instar). The mortality (%) was determined after 24 h. The bioassay was performed in duplicate, and the probit function of StatPlus 2009 Software was used to calculate the concentration that caused 50% mortality (LD50) (Chowdhury et al., 2008).

2.5. Effect of E-64 in the generation of the larvicidal component

About 200

l

L of tegument extract (985

l

g mL 1) was activated

for 10 min at 37°C with 100

l

L 0.05 M sodium citrate buffer, pH

5.0 containing 5 mM DTT and 300 mM NaCl. After incubation

100

l

L of E-64 20

l

M (BACHEM, USA) was added reaching a final

concentration of 5

l

M. This solution was maintained for 2 h at

37°C, and after this period the bioassay was performed as

described.

The bioassay was composed of three groups of a mixture: (A)

100

l

L tegument of activated extract and 1000

l

L cotyledon

ex-tract (729

l

g mL 1); (B) 100

l

L tegument of activated extract and

E-64 (5

l

M) with 1000

l

L cotyledon extract (729

l

g mL 1); (C)

The anti-larval effect of E64 was evaluated by incubating 100

l

L

sodium citrate buffer, pH 5.0 containing 5 mM DTT and 300 mM

NaCl and 5

l

M E-64. All experiments were performed in duplicate.

The final volume was brought to 20 mL with water. The effect on larvae mortality (%) was monitored every hour for 5 h.

2.6. Enzyme purification

The tegument extract was resolved by chromatography over a

Sephadex G-25 column (29.5 cm1.43 cm) equilibrated in

Milli-Q water at a flow rate of 1.0 mL min 1 and monitored by

absorbance at 280 nm; the sample was eluted under these same conditions. The enzyme activity was monitored using Z-Phe-Arg-pNan as the substrate, and also the generation of the larvicidal component, as described elsewhere. The enzymatically active fractions were pooled and then resolved by chromatography over a size exclusion Superdex 75 10/300 GL column (GE Healthcare/ New Jersey, USA) using an ÄKTA Purifier (GE Healthcare) in 0.05 M Tris/HCl, pH 8.0. The column was washed with the same

solution at a flow rate of 0.5 mL min 1and monitored by

absor-bance at 215 and 280 nm. Enzymatically active proteins were separated for further characterisation.

Molecular weight of enzyme was obtained from calibration curves using Aprotinin, Ribonuclease A, Carbonic anhydrase,

Ovalbumin and Conalbumin all 0.1 mg mL 1.

The total protein contents of the crude extract and the chro-matographic fractions were determined by the Folin Ciocalteu

assay (Lowry et al., 1951) using a bovine serum albumin (BSA)

standard curve at 0–500

l

g mL 1. The enzyme obtained was

named tegupain.

2.7. The N-terminal amino acid sequence

Purified tegupain was denatured and reduced by the addition of

200

l

L 50 mM Tris/HCl buffer, pH 8.5, containing 6.0 M

guanidi-nium HCl, 1.0 mM EDTA, and 5.0 mM dithiothreitol for 3 h at

37°C. TheS-pyridylethylation of the cysteines was achieved by

the addition of 5

l

L 4-vinylpyrimidine for 3 h at 37°C in the dark

under a nitrogen atmosphere. The excess reagents were removed

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conditions described previously. The N-terminal amino acid sequences were determined by Edman degradation using a PPSQ-23 Model Protein Sequencer (Shimadzu, Tokyo, Japan). Phen-ylthiohydantoin-derived amino acids were identified.

2.8. Enzyme activity

Proteinase activity was measured using Z-Phe-Arg-pNan (Calbiochem Ltda, Darmstadt, Germany) as the substrate. Tegupain

was incubated at 37°C in a microplate assay in a 250

l

L final

vol-ume of 0.05 M sodium citrate buffer, pH 6.0. The reaction was monitored for 30–120 min, and the reaction was stopped by

add-ing 30

l

L 30% acetic acid (v/v). Substrate hydrolysis was monitored

by measuring the absorbance of releasedp-nitroaniline at 405 nm

in a Packard™ spectrophotometer.

2.9. The effects of activators on enzyme activity

Tegupain was preincubated for 10 min with varying

concentra-tions of DTT orL-cysteine in 0.05 M sodium citrate buffer, pH 6.0 at

37°C for 120 min. The enzyme activity was determined as

de-scribed, using Z-Phe-Arg-pNan (0.4 mM) as the substrate.

2.10. The effects of DTT on enzyme activity

Tegupain was preincubated for 10 min at 37°C with varying

concentrations of DTT (1–10 mM). The enzyme activity was determined as described, using Z-Phe-Arg-pNan (0.4 mM) as the substrate.

2.11. The influence of pH on enzyme activity

The hydrolysis of the substrate Z-Phe-Arg-pNan (0.4 mM) by

100

l

L tegupain (4

l

g) was determined in 0.05 M sodium citrate

buffer (pH 4.0–6.0) or 0.05 M sodium phosphate buffer (pH 6.5–

8.0) in a final volume of 250

l

L.

2.12. The effect of NaCl on enzyme activity

Tegupain (4

l

g) was incubated for 120 min at 37°C in 0.05 M

sodium citrate buffer, pH 5.0, containing 5 mM DTT and increasing concentrations of NaCl (0.70–700 mM) before the addition of the substrate Z-Phe-Arg-pNan (0.4 mM). The effect of NaCl on the en-zyme activity was determined by comparing the rate of substrate hydrolysis with that in the absence of salt.

2.13. The influence of temperature on enzyme activity

Heat stability was determined by incubating tegupain (4

l

g) at

various temperatures (0°C, 25°C, 37°C, 40°C, 60°C and 80°C) for 10 min and then cooling it in an ice bath. The activity of the enzyme was then assayed by incubation it with Z-Phe-Arg-pNan in 0.05 M sodium citrate buffer, pH 5.0, 300 mM NaCl, 5 mM DTT at 37°C for 2 h.

2.14. The effect of EDTA on enzyme activity

Tegupain (4

l

g) was preincubated for 10 min at 37°C with

1 mM, 5 mM, 10 mM or 15 mM EDTA in 0.05 M sodium citrate buf-fer, pH 5.0 containing 5 mM DTT and 300 mM NaCl. The activity was determined by incubation with the substrate Z-Phe-Arg-pNan

(0.4 mM) at 37°C in final volume 250

l

L. The effect on the enzyme

activity was determined by comparing the rate of substrate hydro-lysis with that in the absence of EDTA.

2.15. The effects of metal ions on enzyme activity

The effects of Ba2+, Ca2+, Mg2+, Mn2+, Zn2+ions were determined

by incubating tegupain (4

l

g) with each metal ion at 0.8 mM in

0.05 M sodium citrate buffer, pH 5.0, containing 5 mM DTT and 300 mM NaCl. The enzymatic activity assay was performed as

de-scribed. In detail, the effect of Zn2+and Hg2+on tegupain activity

was assayed by incubating the enzyme in increasing ion concentra-tions (0.2–1.0 mM) under the same condiconcentra-tions described earlier.

Particularly in the case of Hg2+, the enzyme was activated by

5 mML-cysteine, as a precipitate is formed in the mixture of HgCl2

and DTT.

2.16. The determination of kinetic parameters

To determine the kinetic parameters, 2

l

g tegupain was

as-sayed by incubation it with varying concentrations of the substrate

Z-Phe-Arg-pNan (10–700

l

M) in 0.05 M sodium citrate buffer, pH

5.0, 5 mM DTT and 300 mM NaCl for 2 h. After the incubation

per-iod, the reaction was stopped with 30

l

L 30% acetic acid. Substrate

hydrolysis was monitored by absorbance at 405 nm. Thekm and

Vmaxvalues were determined according to the Michaelis–Menten

model by the computer program Curve Expert 1.3 (Linewever

and Burk, 1934).

2.17. Inhibition studies

Tegupain was incubated with E-64 (l-trans-epoxysuccinyl-leu-cylamido [4-guanidino] butane) (1.24–3.64 nM), benzamidine

(43.4

l

M, 108.0

l

M and 217.3

l

M) and iodoacetamide (14.5

l

M,

43.4

l

M, 72.4

l

M and 101.3

l

M) in 0.05 M sodium citrate buffer,

pH 5.0, containing 0.5 mM DTT and 300 mM NaCl for 10 min at

37°C, before the addition of the substrate Z-Phe-Arg-pNan

(0.4 mM).

The proteinase inhibitor cystatin from chicken egg white was

pre-incubated at 2–15 nM with the enzyme for 10 min at 37°C

in the assay buffer. Enzyme activity was expressed as the percent residual activity toward Z-Phe-Arg-pNan compared with that of the control.

2.18. Statistical analysis

Differences between mean values were analysed using a one-way ANOVA followed by Tukey’s test. Values were considered to

be significant whenp< 0.05 the data represent means ± SD.

3. Results

3.1. The characterisation of the crude extract

Although separated aqueous crude extracts (1:10, w/v) of the

cotyledon and the tegument of theC. papayaseeds did not exhibit

larvicidal activity, a mixture of both extracts was lethal to the lar-vae. The most potent toxicity (100% larval mortality) was achieved

by mixing 17

l

g mL 1tegument protein content with 27

l

g mL 1

cotyledon protein content (Fig. 1a). LD50 of 1.13 mg mL 1 was

determined using the dry seed weight (w/v) (Fig. 1b). The larvicidal

effect of the mixture lost its activity when the tegument extract,

but not the cotyledon extract, was pre-treated at 100°C (Fig. 1c)

and also when the extract mixture was extensively dialysed. The seed coat extract pre-treatment with the cysteine protease inhibitor E-64 decreased approximately 50% the generation of the

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3.2. Extraction and purification of tegupain, a proteinase from the C. papaya tegument

To isolate and purify the enzyme (tegupain) from the

C. papaya tegument, the aqueous extract was subjected to size exclusion chromatography over a Sephadex G-25 column (Fig. 2a). The fraction exhibiting enzyme activity toward Z-Phe-Arg-pNan was pooled and concentrated by lyophilisation. Further purification was achieved using the size exclusion chromatogra-phy over a Superdex 75 column, which resolved the proteins into two peaks, being the enzyme activity detected in the first peak (Fig. 2b), which also showed to be responsible for the generation of the larvicidal component present in the cotyledon. The appar-ent MW was determined as 38 kDa based in the column

devel-oped with standard proteins (Fig. 2b, insert). The purification

steps are summarized inTable 1. To determine the N-terminus,

chromatography over a C18 reverse phase was performed (data not shown).

3.3. The N-terminal sequence

The N-terminal sequences of the reduced and pyridylethylat-ed enzyme determinpyridylethylat-ed by automatpyridylethylat-ed Edman degradation al-lowed the identification of the first 20 amino acid residues

(AVPSNKKLLIPAACVLLGQP). BLASTNCBI Protein Blast (http://

blast.ncbi.nlm.nih.gov/Blast.cgi) revealed the similarity of tegu-pain to the propeptide region of the C1 family of cysteine

pro-teinases that includes caricain (Dubois et al., 1988), papain

(Drent et al., 1968; Hunsain and Lowe, 1970), chymopapain (Watson et al., 1990), and papaya proteinase (Linn and Yaguchi, 1979) (Fig. 2c).

3.4. Proteolytic activity characterisation

3.4.1. The effect of sulfhydryl groups and sodium chloride on enzyme activity

The hydrolytic activity toward Z-Phe-Arg-pNan was measured in the presence of reducing agents. The enzyme activity was

en-hanced by the sulfhydryl groupsL-cysteine (data not shown) and

DTT (Fig. 3a), and slightly stimulated (20%) by 300 mM NaCl

(Fig. 3b). Due to this effect, 2.0 mM DTT and 300 mM NaCl were added to the buffer in all subsequent assays.

3.4.2. The effect of pH on enzyme activity

The hydrolytic activity of the enzyme persists after its preincu-bation at varying conditions of pH (4.0–8.0, at 0.5 intervals). As the

maximum activity was achieved at pH 5.0 (Fig. 3c), the following

assay buffer was used in subsequent kinetic studies: 0.05 M so-dium citrate buffer, pH 5.0 containing 0.5 mM DTT and 300 mM NaCl.

3.4.3. The influence of temperature on enzyme activity

Compared to the value at 37°C, the enzyme activity was lower

at 0°C, 25°C and 40°C. However, the enzyme activity only, not its

function, is significantly reduced because the enzyme activity is recovered when the incubation is subsequently performed at

37°C. Conversely, at temperatures above 40°C, both the enzyme

activity and its function are significantly reduced, decreasing 93% after treatment at 80°C (Fig. 3d).

3.4.4. The determination of kinetic parameters

Tegupain activity at various concentrations of the substrate

Z-Phe-Arg-pNan is shown inFig. 4. The linearity of the plots of

contr ol

0.025 0.375 0.625 1.25 2.5 5.0

0 50 100 150

*** ***

Extract (mg/mL)

Mortality (%)

1 2 3 4 5

0 10 20 30 40 50

Control E-64

*** ***

***

Time (hours)

Mortality (%)

(a)

(b)

(c)

(d)

***

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the initial concentrations of the enzyme indicates first order

kinet-ics with respect to the substrate and the enzyme, resulting inKm

58.8

l

M,Kcat28 020 s 1andKcat/Km5108M 1s 1, respectively.

3.4.5. The effects of sulfhydryl reagents, selective inhibitors and metal ions on proteinase activity

The enzyme activity was assessed in the presence of cystatin, E-64, iodoacetamide, benzamidine and EDTA. The enzyme activity was not altered neither by benzamidine, an inhibitor of serine proteinase, nor by the chelating agent EDTA, an inhibitor of metalloproteinases. However, in the presence of the cysteine proteinase inhibitor E-64, the enzyme activity was blocked. The inhibition by E-64 and its concentration ratio dependence is shown inFig. 5a, based on an IC50value of 1.4 nM. The enzyme was also

inhibited by the alkyl halide iodoacetamide,Fig. 5b, with an IC50

of 72

l

M, and cystatin,Fig. 5c, withKiapp2.4 nM.

Zn2+ and Hg2+ reduce tegupain activity in a dose-dependent

manner, leading to a loss of approximately 50% and 71% of the enzyme’s activity at 1 mM Zn2+(Fig. 6b) and Hg2+(Fig. 6c),

respec-tively. Ca2+and Ba2+ions also reduced the enzyme activity (Fig. 6a).

4. Discussion

As mentioned in the introduction, dengue epidemics occur

fre-quently, and it is a major problem to controlA. Aegyptias the

mor-tality of the adult mosquito is temporary and inefficient, and the pesticide approach to control the insect population is hazardous

to the environment (Howard et al., 2007; Kyle and Harris, 2008).

The most effective strategy against dengue is to control the larvae, since applied locally, a specific biological larvicide is less pollutant.

Bacillus thuringiensis(Bt) larvicide protein used to controlA. ae-gypti is efficient because of its specificity and its non-toxicity to vertebrates. However, there are reports of resistance to Bt among

Lepidoptera larvae (Pérez et al., 2005). Thus, the identification of

potential larvicides from plants may provide alternatives to control

insect populations (Prajapati et al., 2005).

Natural compounds have been used in several studies. For example, concanavalin A lectin has been used as a pesticide that binds to the glycosylated receptor of the epithelial cell surface of the Acyrthosiphon pisumnymphs stomach (Sauvion et al., 2004).

The effect of a water soluble lectin derived fromMoringa oleifera

(WSMol) against A. aegypti is also under investigation (Coelho

et al., 2009).

An interesting aspect of the current work was the observation

that the tegument extract of C. papayaseeds must be in contact

with the cotyledon to produce the low molecular weight

insecti-cidal compound that is active againstA. aegyptilarvae. This activity

is lost when filtered through a 10 kDa membrane (data not shown), indicating the low molecular weight of the larvicidal component, and the insecticidal effect is not observed in the separated compo-nents of the seeds.

The identification of the insecticidal properties of C. papaya

seeds againstA. aegyptilarvae should be considered as significant

because papaya is widely consumed in Brazil and other countries, and the seeds could have a useful application instead of being

1 22

Tegupain . . A V P S N K K L L I P A A C V L L G Q P

Papain M A M I P S I S K L L F V A I C L F V Y M G

Caricain M A M I P S I S K L L F V A I C L F V H M S

proteinase M A M I P S I S K L L F V A I C L F V H M S 0 5 10 15 20 25 30 35 40 45 50 55 60

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

mL

A 2

8

0

0 5 10 15 20 25 30 35 40

0 100 200 300 400 500

mL

A

280

(a)

(b)

(c)

Fig. 2.Purification of the extract from the tegument of theCarica papayaseed. (a) Size exclusion molecular chromatography of theC. papayaseed tegument extract over a Sephadex G-25 column. Elution: Milli Q water. Flow rate: 24 mL h1. (b) Size exclusion molecular chromatography over a Superdex 75 column in an AKTA system purifier. The tegupain fraction obtained from the Sephadex G-25 chromatography was resolved over a Superdex 75 column. Eluting buffer: 50 mM Tris/HCl, pH 8.0 containing 150 mM NaCl. Flow rate: 30 mL h1. The arrow indicates activity toward Z-Phe-Arg-pNan (0.4 mM). Insert: Estimation of tegupain molecular weight by the equation y= 0.1453x+ 3.1796 (R2= 0967) obtained with standard MW proteins. (c) The N-terminal sequence of the tegupain proenzyme in comparison to other cysteine proteinases.

Table 1

Purification of the cysteine proteinase from theCarica papayaseed tegument.

Steps Volume (mL) Protein (mg mL 1) Total protein U(mL)a TotalU Specific activity (U mg 1) Purif. Yield (%)

Crude extract 40 0.67 26.8 0.6610 3 0.03 9.8510 4 1 100

Sephadex G-25 11 0.18 1.98 0.610 3 6.610 3 3.3410 3 3.4 22

Superdex 75 3.5 0.03 0.10 1.7510 3 6.1210 3 0.060 60.9 20.4

Enzyme activity was defined using 0.4 mM Z-Phe-Arg-pNan as the substrate. The protein concentration was determined by the Lowry assay. aU – unit of enzyme activity.U= 1

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discarded. Moreover, as the larvicidal activity of the described mix-ture is very effective, robust and heat resistant, its application may take place at relatively high temperatures in warm regions.

With regard to insect control,Farias et al. (2007)isolated a

pro-tein fromC. papayaseeds that inhibits the digestive enzyme

a

-amylase ofCallosobruchus maculatus in vitro.In vivo, this inhibitor

reduces the longevity and fecundity of adults and causes larval

mortality. The larvicidal activity againstCulex quinquefasciatusof

C. papayaseed aqueous extract was also reported (Rawani et al., 2009).

The establishment of the seed amount (LD501.13 mg mL 1) that

acts as an effective larvicide is extremely important to apply this knowledge to public health. Unsurprisingly, when using the

puri-fied fractions, the dose necessary to achieve the LD50is decreased

to 0.680 mg mL 1 cotyledon extract and 1.08 mg mL 1 tegument

extract.

The larvicidal agent present in the cotyledon has a low molecu-lar weight and is heat-resistant; however, it is produced by a ther-molabile substance of the tegument that is lost after heating. After

the purification and characterisation of the cotyledon activity, achieved by conventional chromatographic methods, including size exclusion chromatography over Sephadex G-25 and Superdex 75 columns, we identified the component as an enzyme that hydrolysed Bz-Arg-pNa and the specific cysteine proteinase syn-thetic substrate Z-Phe-Arg-pNan even more efficiently [data not shown]. A major portion of the extract pigment was separated using Sephadex G-25 chromatography, and high-resolution chro-matography systems allowed the isolation of the enzyme at a yield of approximately 20%.

The enzyme remained active at temperatures of 37°C and 40°C,

losing almost 93% of its activity at 80°C. The activity was enhanced

20% by NaCl (300 mM), similarly to what is observed for enzymes

such as cathepsin L (Barros et al., 2004). The interference of ZnCl2

can be attributed to the precipitation of the enzyme by salt or even by conformational change.

In contrast to benzamidine and EDTA, which do not interfere

with the hydrolytic enzyme activity,L-cysteine and dithiothreitol

potentiate it, indicating that sulfhydryl groups are involved in en-zyme activity. This hypothesis was confirmed by the enen-zyme

inac-tivation by E-64 (IC50 1.4 nM) and iodoacetamide (IC50 72

l

M);

both these agents block sulfhydryl groups, confirming the presence of a thiol group at the catalytic centre, suggesting that the protein belongs to the cysteine proteinase class [34]. Indeed, the impair-ment of anti-larval activity by E-64 treatimpair-ment indicates that tegu-pain is involved in the generation of the larvicidal component. In

addition, as observed with papain (Sueyoshi et al., 1988), a strong

inhibition of tegupain was also achieved by cystatin, a classical inhibitor of cysteine proteinases that contains the sequence motif

QXVXG (Colman and Schmaier, 1997), which is reported to inhibit

cysteine proteinases of the papain family (Gzonka et al., 2001;

Andrade et al., 2011). These results support the classification of tegupain as a cysteine proteinase.

contr

ol 1.0 2.0 4.0 8.0 10.0

0.00 0.05 0.10 0.15 0.20

*** ***

*** *** ***

DTT (mM)

A 405

Contr

ol 0.07 0.15 0.3 0.4 0.5 0.6 0.7

0.0 0.1 0.2 0.3

NaCl (M)

A 405

4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0

0.00 0.05 0.10 0.15 0.20

pH

A 405

0 25 37 40 60 80

0.00 0.05 0.10 0.15

Temperature (°C)

A 405

(a)

(b)

(c)

(d)

Fig. 3.Proteolytic Activity Characterisation. (a) Tegupain was pre-incubated with increasing concentrations of DTT in 0.5 M sodium citrate buffer, pH 5.0. The activity was defined as the hydrolysis of Z-Phe-Arg-pNan (0.4 mM) at 37°C for 2 h. (b) The enzyme was incubated with increasing concentrations of NaCl. The enzymatic activity was defined as the hydrolysis of Z-Phe-Arg-pNan (0.4 mM) in sodium citrate buffer 50 mM, pH 5.0 and 5 mM DTT at 37°C for 2 h. (c) The effect of pH on enzyme activity was defined as the hydrolysis of Z-Phe-Arg-pNan (0.4 mM) at 37°C in buffer with at varying pH values for 2 h. (d) In the influence of temperature the enzyme was pre-incubated at varying temperatures for 10 min. After this period, the sample was kept at 37°C and the activity was defined as the hydrolysis of Z-Phe-Arg-pNan (0.4 mM) in 50 mM sodium citrate buffer, pH 5.0, containing 300 mM NaCl and 5 mM DTT for 2 h.p-Value < 0.001 (). The data represent means ± SDs,n= 2.

S = 0.95954431 r = 0.96581658

1/Z-Phe-Arg-pNan (µM)

1/

V

0 (µ

M

/mi

n

)

-0.03 -0.01 0.01 0.03 0.05 0.07 0.09 0.11 0.00

4.00 8.00 12.00 16.00 20.00

(7)

Contr ol

1.24 1.64 2.04 2.44 2.84 3.24 3.64 0.0

0.5 1.0 1.5

*** *** ***

*** *** *** ***

E-64 (nM)

Re

si

d

u

a

l Ac

ti

v

it

y

(

%

)

0

14.5 43.4 72.4 101.3 0.0

0.5 1.0 1.5

**

*** ***

Re

si

d

u

a

l Ac

ti

v

it

y

(

%

)

(a)

(b)

(c)

Fig. 5.Action of cysteine proteinases inhibitors. (a) Increasing concentrations of E-64 incubated with tegupain (2lg). (b) Increasing concentrations of iodoacetamide incubated with the enzyme (2lg). (c) The enzyme was incubated with increasing concentrations of cystatin. The activity was defined as the hydrolysis of Z-Phe-Arg-pNan (0.4 mM) in 50 mM sodium citrate buffer, pH 5.0, 300 mM NaCl, 5 mM DTT at 37°C for 2 h.p-Value < 0.001 () andp-value < 0.01 (). The data represent means ± SDs, n= 2.

0.0 0.1 0.2 0.3

***

** *

Ion concentration 0.8 mM

A

405

Contr

ol 0.2 0.4 0.6 0.8 10

0.00 0.05 0.10 0.15 0.20 0.25

***

*** *** *** ***

A

40

5

Contr

ol 0.2 0.4 0.6 0.8 1.0

0.00 0.05 0.10 0.15 0.20 0.25

** **

*** ***

***

A 4

0

5

(a)

(b)

(c)

(8)

The optimum pH for tegupain activity, 5.0, is compatible with the pH range for high hydrolytic activity of the cysteine protein-ases (4.0–6.5), and the very low activity observed at extremely acidic and basic pH values observed with papain-like cysteine

pro-teinases (Gzonka et al., 2001). This optimum pH value is favourable

for a larvicidal compound againstA. aegyptibecause mosquito eggs

are layed in water with pH generally lower than 7.0. Therefore, the enzyme is active under the appropriate conditions to produce, by proteolysis, a larvicidal fragment.

The tegupain N-terminal region is similar to the propeptide re-gion of the CA family members. However, despite some identity in sequence, the differences indicate that tegupain is a novel cysteine proteinase. Tegupain is not papain because commercial papain

preparations are not toxic towardA. aegyptiin the same manner

that tegupain is (data not shown).

In conclusion, this work demonstrates the presence of an

en-zyme activity in the tegument ofC. papayaseeds that is very

sim-ilar in many respects to the activity of cysteine proteinases. The similarities include the optimum pH for the hydrolysis of the sub-strate Z-Phe-Arg-pNan, the activation by reducing agents such as

DTT andL-cysteine, the inhibition by E-64 and cystatin, and the

Kmvalue of 58.8

l

M, Kcat28 020 s 1andKcat/Km 5108M 1s 1.

The results of this investigation established the proteolytic effect of tegupain on endogenous proteins in the cotyledon and the

gen-eration of a fragment withA. aegypti larvicidal activity. Further

experiments are required to elucidate the nature of the insecticidal

compound in C. papaya, as well as the mechanism of action

involved.

Acknowledgment

We are grateful to FAPESP (2009/17058-6 and 2009/53766-5), CAPES and CNPq for providing financial support.

References

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