Abstract
Anticarsia gemmatalis Hübner, 1818 (Lepidoptera) is a major pest of soybean in the Brazil. It is known that the reduction of proteolytic activity by the ingestion of protease inhibitors reduces digestion and larval development of the insects. Control via inhibition of the digestive enzymes necessitates deeper knowledge of the enzyme kinetics and the characterization of the inhibition kinetics of these proteases, for better understanding of the active centers and action mechanisms of this enzyme. Trypsin-like proteases found in the gut of Anticarsia gemmatalis were purified in a p-aminobenzamidine agarose column. Kinetic characterization showed KM 0.503 mM for the L-BApNA substrate; Vmax= 46.650 nM s-1; Vmax/[E]= 9.256 nM s-1 mg L-1 and Vmax/[E]/KM= 18.402 nM s-1 mg L-1 mM. The Ki values for the inhibitors benzamidine, berenil, SKTI and SBBI were 11.2 µM, 32.4 µM, 0.25 nM and 1.4 nM, respectively, and all revealed linear competitive inhibition. The SKTI showed the greatest inhibition, which makes it a promising subject for future research to manufacture peptide mimetic inhibitors.
Key words
Enzyme kinetics; inhibitor competitive; inhibition kinetics; velvet bean caterpillar
INTRODUCTION
Soybean plants can be damaged by insects throughout their whole cycle, with Anticarsia gemmatalis (Lepidoptera: Noctuidae) being one of this crop main pest (Bernardi et al. 2012BERNARDI O, MALVESTITI G, DOURADO P, OLIVEIRA W, MARTINELLI S, BERGER G, HEAD G & OMOTO C. 2012. Assessment of the high-dose concept and level of control provided by MON 87701×MON 89788 soybean against Anticarsia gemmatalis and Pseudoplusia includens (Lepidoptera: Noctuidae) in Brazil. Pest Manag Sci 68: 1083-1091.). This insect reduces production quantities and requires rapid control to prevent economic losses (Vianna et al. 2011VIANNA UR, PRATISSOLI D, ZANUNCIO JC, ALENCAR JRC & ZINGER FD. 2011. Espécies e/ou linhagens de Trichogramma spp. (Hymenoptera: Trichogrammatidae) para o controle de Anticarsia gemmatalis (Lepidoptera: Noctuidae). Arq Inst Biol 78: 81-87.).
The study of the endogenous resistance mechanisms of plants against insect herbivores promises a safe strategy for pest control as an alternative to the use of chemical insecticides that affect other non-target organisms and cause environmental impact (Gatehouse 2002GATEHOUSE JA. 2002. Plant resistance towards insect herbivores: a dynamic interaction. New Phytol 156: 145-169., Ferry et al. 2006FERRY N, EDWARDS MG, GATEHOUSE JA, CAPELL T, CHRISTOU P & GATEHOUSE AMR. 2006. Transgenic plants for insect pest control: a forward looking scientific perspective. Transgenic Res 15: 13-19., Scott et al. 2010SCOTT IM, THALER JS & SCOTT JG. 2010. Response of a generalist herbivore Trichoplusia ni to jasmonate-mediated induced defense in tomato. J Chem Ecol 36: 490-499., Mills & Kean 2010MILLS N & KEAN J. 2010. Behavioral studies, molecular approaches, and modeling: Methodological contributions to biological control success. Biol Cont 52: 255-262., Patarroyo-Vargas et al. 2017PATARROYO-VARGAS AM, MERIÑO-CABRERA YB, ZANUNCIO JC, ROCHA F, CAMPOS WG & OLIVEIRA MGA. 2017. Kinetic Characterization of Anticarsia gemmatalis digestive serine proteases and the inhibitory effect of synthetic peptides. Protein Pept Lett 24: 1-8.). The inhibition mechanism of the digestive enzymes is an alternative strategy for pest control, although the structure and function of these macromolecules must be studied.
Plants possess protease inhibitor (PI), which are important multi-mechanistic components of defense against pests (Silva-Filho & Falco 2000SILVA-FILHO MC & FALCO MC. 2000. Interação planta inseto-adaptação dos insetos aos inibidores de proteinases produzidas pelas plantas. Biotecnolog Cienc Desenvolv 12: 38-42.). PI are generally small and stable molecules, specific for the major digestive proteases (serine, cysteine, and aspartyl proteases) of pest herbivores (Jamal et al. 2012JAMAL F, PANDEY P, SINGH D & KHAN M. 2012. Serine protease inhibitors in plants: Nature’s arsenal crafted for insect predators. Phytochem Rev 12: 1-34.). The serine proteases, trypsin and chymotrypsin, are the major digestive enzymes occurring in the midgut of Lepidoptera (Terra & Ferreira 1994TERRA WR & FERREIRA C. 1994. Insect digestive enzymes: compartimentalization and function. Comp Biochem Physiol Part B Biochem Mol Biol 109: 1-62., Shi et al. 2013SHI M, ZHU N, YI Y & CHEN X. 2013. Four serine protease cDNAs from the midgut of Plutella xylostella and their proteinase activity are influenced by the endoparasitoid, Cotesia vestalis. Arch Insect Biochem Physiol 83: 101-114.). PI ingestion reduces the proteolytic activity, and consequently, the larval digestion, growth and development, as well as adult fertility and fecundity (Mahdavi et al. 2013MAHDAVI A, GHADAMYARI M, SAJEDI R, SHARIFI M & KOUCHAKI B. 2013. Identification and partial characterization of midgut proteases in the lesser mulberry pyralid, Glyphodes pyloalis. J Insect Sci 13: 1-11., Shi et al. 2013SHI M, ZHU N, YI Y & CHEN X. 2013. Four serine protease cDNAs from the midgut of Plutella xylostella and their proteinase activity are influenced by the endoparasitoid, Cotesia vestalis. Arch Insect Biochem Physiol 83: 101-114.). However, insects have developed defense mechanisms for protection against the deleterious effects of PI. These defenses include increasing the enzymatic synthesis of the class that is being inhibited, as well as of enzymes insensitive to the inhibitors, in response to the inhibitory effect (Oliveira et al. 2005OLIVEIRA MGA, DE SIMONE SG, XAVIER LP & GUEDES RNC. 2005. Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis. Comp Biochem Physiol Part B Biochem Mol Biol 140: 369-380., Pilon et al. 2009PILON AM, OLIVEIRA MGA, PILON FM, GUEDES RNC, OLIVEIRA JA & FAZOLLO A. 2009. Adaptação da lagarta-da-soja Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) ao inibidor de protease benzamidina. Rev Ceres 56: 744-748., Scott et al. 2010SCOTT IM, THALER JS & SCOTT JG. 2010. Response of a generalist herbivore Trichoplusia ni to jasmonate-mediated induced defense in tomato. J Chem Ecol 36: 490-499., Oliveira et al. 2013OLIVEIRA CFR, SOUZA TP, PARRA JRP, MARANGONI S, SILVA-FILHO MC & MACEDO MLR. 2013. Insensitive trypsins are differentially transcribed during Spodoptera frugiperda adaptation against plant protease inhibitors. Comp Biochem Physiol Part B Biochem Mol Biol 165: 19-25., Wielkopolan et al. 2015WIELKOPOLAN B, WALCZAK F, PODLEŚNY A, NAWROT R & OBRĘPALSKA-STĘPLOWSKA A. 2015. Identification and partial characterization of proteases in larval preparations of the cereal leaf beetle (Oulema melanopus, Chrysomelidae, Coleoptera). Arch Insect Biochem Physiol 88: 192-202.).
Deeper knowledge of the enzyme and the inhibition kinetics of the A. gemmatalis digestives proteases allows for a better understanding of the active centers, of the action mechanisms of these enzymes and the protein and/or synthetic inhibitors that need to be applied as inhibitors of the complex systems of the insect’s digestive proteases.
The objective of this work was to study the inhibition kinetics of the intestinal serine proteases of Anticarsia gemmatalis Hübner, 1818 (Lepidoptera: Noctuidae) using synthetic and protein proteases inhibitors. This is a pioneer study on the characterization of the inhibition model of the trypsin-like enzyme in the midgut of A. gemmatalis and will enable mapping of the active centers of these enzymes. In addition, it will provide a basis for applied ecological studies of how insect resistance occurs to plants of interest. They may also demonstrate how plant-herbivore interactions should be considered in strategies for Integrated Pest Management.
MATERIALS AND METHODS
The experiments were performed in the Laboratory of Enzymology, Biochemistry of Proteins and Peptides of the Institute of Biotechnology Applied to Agriculture and Laboratory of Insect Rearing of the Department of Biochemistry and Molecular Biology at the Federal University of Viçosa in Viçosa, Minas Gerais State, Brazil.
Anticarsia gemmatalis
The fourth and fifth instar larvae of A. gemmatalis were reared in the Laboratory of Insect Rearing of Department of Biochemistry and Molecular Biology of Federal University of Viçosa on an artificial diet (Hoffman-Campo et al. 1985HOFFMAN-CAMPO CB, OLIVEIRA EB & MOSCARDI F. 1985. Criação massal de lagarta-da-soja (Anitcarsia gemmatalis). EMBRAPA-CNPSO, Documentos 10. Londrina, 23 p.), and maintained in climatized chambers with 25 ± 2ºC, relative humidity 70 ± 10% and photoperiod 14 hours.
Crude midgut extract preparation
The larvae were immobilized on ice. Their guts were removed and placed in 10-3M HCl solution, in the proportion of five guts/mL solution at -20°C. The crude enzyme extract was obtained by cell disruption after passing through nine cycles of freezing in liquid nitrogen followed by thawing in a water bath at 37°C. The suspension obtained was centrifuged at 100,000 rpm for 45 minutes at 4°C (Oliveira et al. 2005OLIVEIRA MGA, DE SIMONE SG, XAVIER LP & GUEDES RNC. 2005. Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis. Comp Biochem Physiol Part B Biochem Mol Biol 140: 369-380.).
Enzyme purification
The crude enzyme extract was then subjected to affinity chromatography on a p-aminobenzamidine agarose column (2.5 mL) (Sigma®) balanced with Tris-HCl buffer 0.05 M at pH 7.5 containing NaCl 0.5 M. Proteins were eluted under continuous flow of 1 mL/min of glycine buffer 0.05 M at pH 3.0. The fractions corresponding to the activity peak were pooled and stored at -20°C for future use during enzyme kinetic assays.
Protein assays
The protein concentrations of the samples of crude and purified enzymatic extracts were measured using the ABS 0.2 mg mL-1 as the standard (Bradford 1976BRADFORD M. 1976. A rapid and sensitive method for the quantification of microgram quantities of proteins utilizing the principle of protein dye binding. Analyt Biochem 72: 248-254.).
Enzymatic activity
The activity of trypsin-like serine proteases was determined in Tris-HCl buffer 0.1 M at pH 8.0 containing CaCl2 20 mM with the chromogenic substrate L-BApNA 1.2 mM (Erlanger et al. 1961ERLANGER B, KOKOWSKY N & COHEN W. 1961. The preparation and properties of two newchromogenic substrates of trypsin. Arch Biochem Biophys 95: 271-278.). The initial rates of the product p-nitroanilide were determined at 410 nm in function of time (2.5 min) using the molar extinction coefficient of 8800 M-1cm-1 for the product. The experiment was conducted in a series of three repetitions (Oliveira et al. 2005OLIVEIRA MGA, DE SIMONE SG, XAVIER LP & GUEDES RNC. 2005. Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis. Comp Biochem Physiol Part B Biochem Mol Biol 140: 369-380.).
Kinetic characterization
The KM and Vmax were determined using the Tris-HCl buffer 0.1 M at pH 8.0 containing CaCl2 20 mM and the purified enzyme at a final concentration of 5.04 µg mL-1 and increasing the concentrations of the L-BApNA chromogenic substrate (from 0.1 mM to 1.0 mM). The initial rates were determined as described above.
The Ki were determined with Tris-HCl buffer 0.1 M at pH 8.0 containing CaCl2 20 mM in the presence of the chromogenic substrate L-BApNA in a concentration range of 0.1 mM to 0.4 mM, used in all the analyses. Synthetic and protein inhibitors and their concentrations were as follows: synthetic inhibitors benzamidine (10 μM to 50 μM) and berenil (20 μM to 100 μM), protein inhibitors SKTI (0.1 nM to 1.0 nM) and SBBI (0.5 nM to 4.0 nM). The initial rates were determined as described earlier. The kinetic parameters were calculated by nonlinear regression using the software Sigma Plot 10.0, adopting the simple uni reaction kinetic model.
The general equation rate for this model is:
The linear method used was the double reciprocal graph of Lineweaver-Burk in which the linearization was represented by the equation:
The Ki of the inhibitors were obtained by the intersection of the lines corresponding to the substrate concentrations (Dixon et al. 1979DIXON M, WEBBER RC, THERONE CJR & TRIPTON KF. 1979. Inhibition and activation in enzymes. Acad Press NY: 332-381.). Equation (3) represents the Dixon plot, followed by the inhibition kinetics model:
The competitive inhibition model was determined using the double reciprocal graph methods of Lineweaver-Burk (Equation 4) and the double reciprocal graph of the slopes (Equation 5):
RESULTS
Partial purification of the serine proteases
The crude enzyme extract from the guts of the A. gemmatalis larvae was subjected to affinity chromatography on a p-aminobenzamidine agarose column with fractions containing the activity of the trypsin-like serine proteases, eluted with glycine buffer 0.05 M, corresponding to the second absorbance peak (Figure 1). Proteins that did not adhere to the p-aminobenzamidine agarose column were identified in the first absorbance peak. This procedure resulted in a purification factor of 6.0 and an original activity yield of trypsin-like serine proteases of approximately 124% (Table I). The partially purified sample showed two bands with an approximate molecular mass of 66 kDa and 35 kDa on 15% polyacrylamide gels (data not shown).
Chromatographic elution profile of trypsin-like serine proteases of Anticarsia gemmatalisis (Lepidoptera: Noctuidae) in p-aminobenzamidine agarose column. Absorbance of 280 nm (●), enzymatic activity of μM s-1 (■).
Partial purification of digestive serine proteases from Anticarsia gemmatalis (Lepidoptera: Noctuidae).
Determination of the kinetic parameters
The kinetic parameters estimated for the serine proteases were apparent because the system used was partially purified (Table II). Data were compared against those of the bovine β-trypsin as standard.
The amidolytic activity generated a concentration-speed hyperbolic curve following the Michaelis-Menten kinetic model for the substrate concentration range used (Figure 2). The KMapp and Vmaxapp were confirmed by linearization of the Michaelis-Menten curve generating the double reciprocal graph of Lineweaver-Burk with an R2 value of 0.9860 (inset of Figure 2).
The partially purified serine proteases show a faster transformation rate of approximately 5.6 times more than that of β-trypsin (Table II).
Michaelis-Menten graph for the hydrolysis of L-BApNA catalyzed by the enzyme. The substrate concentrations ranged from 0.1 mM to 1 mM. The points are experimental. Insertion: graph of the double reciprocal Lineweaver-Burk. The line drawn was calculated by linear regression with R2 of 0.9860.
Kinetic parameters of the substrate hydrolysis L-BApNA by trypsin of the digestive serine proteases from Anticarsia gemmatalis (Lepidoptera: Noctuidae).
Determination of the inhibition constant
The Lineweaver-Burk graphs (Figure 3) were constructed utilizing variable concentrations of the inhibitor and substrate to determine the inhibition of the serine proteases trypsin-like partially purified. The four Lineweaver-Burk graphs showed inhibition of the competitive type. The increase in the slopes of the lines with rising concentrations of the inhibitor indicates that the enzyme is distributed in larger quantities in the form of the EI complex. The presence of the inhibitor did not change the value of Vmax where the intersection of the line in axis 1/V corresponds to 1/Vmax in the Lineweaver-Burk graphs.
Lineweaver-Burk graphic of the inhibition (I) of the intestinal trypsin-like of Anticarsia gemmatalis (Lepidoptera: Noctuidae) by the inhibitors (a) benzamidine, (b) berenil, (c) SKTI and (d) SBBI in the presence of L-BApNA substrate. The lines were calculated by linear regression using the parameters of Table II. The points are experimental. I1 (▲), I2 (□), I3 (■), I4 (○), I5 (●).
The slopes graph obtained from the reciprocal plots of the data (Figure 4) show a significant linear curve. This confirms that the pattern of inhibition of the amidolytic activity of the partially purified enzyme against the inhibitors benzamidine, berenil, SKTI and SBBI is of the competitive type, where an inhibitor molecule binds to the enzyme forming a type of EI binary complex.
Graphic slopes of the Lineweaver-Burk graphic versus concentration of I for the hydrolysis of L-BApNA by partially purified trypsin-like in the presence of the inhibitors (a) benzamidine, (b) berenil, (c) SKTI and (d) SBBI.
The data from the Dixon plot, fixing the concentration of the tested inhibitors showed that an increase in the substrate concentration triggered a decrease in the degree of inhibition; however, fixing the substrate concentration and increasing the inhibitor concentration increased the degree of inhibition (Figure 5).
Dixon’s plot of the inhibition of intestinal trypsin-like of Anticarsia gemmatalis (Lepidoptera: Noctuidae) by the inhibitors (a) benzamidine, (b) berenil, (c) SKTI and (d) SBBI in the presence of L-BApNA substrate (S). The lines were calculated by linear regression using the parameters of Table II. The points are experimental. S1 (▲), S2 (♦), S3 (■).
The calculated inhibition constants by the Dixon et al. (1979)DIXON M, WEBBER RC, THERONE CJR & TRIPTON KF. 1979. Inhibition and activation in enzymes. Acad Press NY: 332-381. method of the inhibitors benzamidine, berenil, SKTI and SBBI were obtained (Table III) and the data compared with the β-trypsin.
Inhibition constant of inhibitors of serine proteases of Anticarsia gemmatalis (Lepidoptera: Noctuidae) in the presence of L-BApNA substrate.
The Ki of benzamidine, SKTI and SBBI for the partially purified enzyme were lower than the Ki for the β-trypsin. This shows the higher affinity of these inhibitors for the active center of the partially purified trypsin-like protease of A. gemmatalis compared with the β-trypsin. The berenil showed a higher Ki, suggesting a lower affinity of this inhibitor for the active center of the partially purified trypsin-like enzyme.
DISCUSSION
In order to promote a plant defense against pests by eliminating the use of pesticides, we have been working to obtain a protein molecule, which is own from physiological environment. Therefore, this protein molecule needs have an inhibitory action over the proteases in the intestines of insects.
To construct a peptide or a mimetic peptide with great potential to use in the protection of plants is necessary to know the kinetic model of inhibition of insect pest gut proteases.
In this sense, we performed the kinetic characterization of the trypsin-like inhibition of A. gemmatalis to understand the inhibition from the point of view of structure/physiological function. From the kinetic model it is possible to propose an inhibitor molecule ecologically acceptable for the control of agricultural pests by lipoxygenase pathway.
The benzamidine, a potent competitive inhibitor of the trypsin-like serine proteases, occupies the S1 subsite of this enzyme, which is the specificity site. The first eluting peak probably corresponds to the cysteine proteases present in the midgut enzyme extract of A. gemmatalis, the enzymes lacking a p-aminobenzamidine affinity but capable of hydrolyzing the substrate L-BApNA (Terra & Ferreira 1994TERRA WR & FERREIRA C. 1994. Insect digestive enzymes: compartimentalization and function. Comp Biochem Physiol Part B Biochem Mol Biol 109: 1-62.). The increase in the total activity of the trypsin-like enzyme following affinity chromatography (Table I) is due to the separation of its probable inhibitors present in the crude extract during the purification process. This was also reported for the purification of the trypsins of Bombyx mori Lineu, 1758 (Eguchi & Kuriyama 1985EGUCHI M & KURIYAMA K. 1985. Purification and characterization of membrane-bound alkaline proteases from the midgut tissue of the silk-worm Bombyx mori. J Biochem 97: 1437-1445.), Locusta migratoria (Linnaeus, 1758) (Lam et al. 2000LAM W, COAST GM & RAYNE RC. 2000. Characterization of multiple trypsins from the midgut of Locusta migratoria. Insect Biochem Mol Biol 30: 85-94.) and Heliothis virescens (Fabricius, 1781) (Brito et al. 2001BRITO LO, LOPES AR, PARRA JR, TERRA WR & SILVA-FILHO MC. 2001. Adaptation of tobacco budworm Heliothis virescens to proteinase inhibitors may be mediated by the synthesis of new proteinases. Comp Biochem Physiol Part B Biochem Mol Biol 128: 365-375.). Purification of the midgut extract of A. gemmatalis in the p-aminobenzamidine agarose column showed a better yield (124.3%) than the aprotinin-agarose column (66.7%) (Oliveira et al. 2005OLIVEIRA MGA, DE SIMONE SG, XAVIER LP & GUEDES RNC. 2005. Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis. Comp Biochem Physiol Part B Biochem Mol Biol 140: 369-380.).
The band with mass close to 35 kDa of the soluble trypsin-like enzyme from A. gemmatalis is found to be consistent with the masses of the trypsins of most insects, ranging between 20 kDa and 35 kDa (Terra & Ferreira 1994TERRA WR & FERREIRA C. 1994. Insect digestive enzymes: compartimentalization and function. Comp Biochem Physiol Part B Biochem Mol Biol 109: 1-62.), similar to those isolated from other Lepidoptera: 27 kDa and 24 kDa of Sesamia nonagrioides (Lefèbvre, 1827) (Novillo et al. 1999NOVILLO C, CASTAÑERA P & ORTEGO F. 1999. Isolation and characterization of two digestive trypsin-like proteinases from larvae of the stalk corn borer, Sesamia nonagroides. Insect Biochem Mol Biol 29: 177-184.), 26 kDa and 29 kDa of Helicoverpa armigera (Hübner, 1805) (Telang et al. 2005TELANG MA, GIRI AP, SAINANI MN & GUPTA VS. 2005. Characterization of two midgut proteinases of Helicoverpa armigera and their interection with proteinase inhibitors. J Insect Physiol 51: 513-522.) and 28.7 kDa of Diatrea saccharalis (Fabricius, 1794) (Lopes et al. 2006LOPES AR, JULIANO MA, MARANA SR, JULIANO L & TERRA WR. 2006. Substrate specificity of insect trypsins and the role of their subsites in catalysis. Insect Biochem Mol Biol 36: 130-140.). Multiple trypsins commonly occur in the intestine of Lepidoptera and are usually related to the adaptability of the insect to plant protease inhibitors (Brito et al. 2001BRITO LO, LOPES AR, PARRA JR, TERRA WR & SILVA-FILHO MC. 2001. Adaptation of tobacco budworm Heliothis virescens to proteinase inhibitors may be mediated by the synthesis of new proteinases. Comp Biochem Physiol Part B Biochem Mol Biol 128: 365-375., Volpicella et al. 2003VOLPICELLA M, CECI LR, CORDEWENER J, AMERICA T, GALLERANI R, BODE W, JONGSMA MA & BEEKWILDER J. 2003. Properties of purified gut trypsin from Helicoverpa zea, adapted to proteinase inhibitors. FEBS J 270: 10-19., Budatha et al. 2008BUDATHA M, MEUR G & DUTTA-GUPTA A. 2008. Identification and characterization of midgut proteases in Achaea janata and their implications. Biotechnol Lett 30: 305-310.). The band with a mass of 66 kDa is consistent with the molecular mass of 67 kDa and 70 kDa of H. virescens (Brito et al. 2001BRITO LO, LOPES AR, PARRA JR, TERRA WR & SILVA-FILHO MC. 2001. Adaptation of tobacco budworm Heliothis virescens to proteinase inhibitors may be mediated by the synthesis of new proteinases. Comp Biochem Physiol Part B Biochem Mol Biol 128: 365-375.). Proteins reported with a molecular mass of 66 kDa to 91 kDa represented either the clustering of the molecules of trypsin-like proteases or an evolutionary adaptation of A. gemmatalis due to the constant exposure to protease inhibitors (Oliveira et al. 2005OLIVEIRA MGA, DE SIMONE SG, XAVIER LP & GUEDES RNC. 2005. Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis. Comp Biochem Physiol Part B Biochem Mol Biol 140: 369-380.). The molecular mass of a band, practically the double of the other, indicates the enzyme dimerization (Oliveira et al. 2005OLIVEIRA MGA, DE SIMONE SG, XAVIER LP & GUEDES RNC. 2005. Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis. Comp Biochem Physiol Part B Biochem Mol Biol 140: 369-380.).
The similar KM value of the partially purified trypsin-like serine proteases and that of the β-trypsin in the presence of L-BApNA (Table II) shows the similar interaction of this substrate with the active center of both enzymes. The kinetic parameters of the trypsin-like serine proteases from A. gemmatalis partially purified on a column of aprotinin showed a KM of 0.32 mM for the substrate L-BApNA (Oliveira et al. 2005OLIVEIRA MGA, DE SIMONE SG, XAVIER LP & GUEDES RNC. 2005. Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis. Comp Biochem Physiol Part B Biochem Mol Biol 140: 369-380.). The transformation rate in the semi-pure systems can be expressed as Vmax/[E] and specificity constant by Vmax/[E]/KM. The faster rate of product formation by the action of the trypsin-like serine proteases on the substrate L-BApNA does not correspond to a greater interaction by the substrate because the KM values were similar for the enzymes compared. The partially purified trypsin-like enzyme showed a greater specificity constant than the β-trypsin, and therefore, was more efficient in the formation of the ES complex. The semi-purified enzyme showed the greater adaptation between the substrate and the active center in the transition stage, which indicates the better catalytic efficiency of the trypsin-like enzymes from A. gemmatalis.
Benzamidine, berenil, SKTI and SBBI acted as pure competitive inhibitors of the partially purified trypsin-like serine proteases in the analyzed concentration ranges of S and I. The competitive inhibitor increased the KM of the substrate, requiring a higher concentration of the substrate for the enzyme to reach any fraction of Vmax. The factor can be considered an important statistical dependent factor on [I] which describes the free form of enzyme distribution and in the EI form. The highest concentration of the inhibitor increases the KM and the slope of the line by the factor . The inhibitor and the substrate are mutually exclusive, for competing for the same binding site in the region of the active center of the enzyme.
The characterization of three different types of graphic profiles shows that the inhibitors analyzed with the pure competitive inhibition of the trypsin-like enzyme in the concentration range of the inhibitors and substrates analyzed. This is the first time that an inhibition model of the trypsin-like enzyme of the midgut from A. gemmatalis is characterized and, therefore, deserves special attention given to the standardization of the model. The characterization of an inhibition kinetic model requires more than one type of plot adjusted to more than one kinetic equation, because different kinetic models can present the same profile of a given graph. The Lineweaver-Burk graph of the bovine β-azotrypsin with partially hyperbolic competitive inhibition showed the same profile of pure competitive inhibition (Oliveira et al. 1993OLIVEIRA MGA, ROGANA E, ROSA JC, REINHOLD BB, ANDRADE MH, GREENE LJ & MARES-GUIA M. 1993. Tyrosine 151 is partof the substrate activation binding site of bovine trypsin. J Biol Chem 268: 26893-26903.). The characterization of the inhibition of the bovine trypsin that exhibits the partially parabolic competitive inhibition graph showed the Lineweaver-Burk graph with the same profile of pure competitive inhibition (Junqueira et al. 1992JUNQUEIRA RG, SILVA E & MARES-GUIA M. 1992. Partial competitive parabolic inhibition of bovine trypsin by bis-benzamidines: a general model for the trypsin-like family of proteases. Braz J Med Biol Res 25: 873-887.).
The values of the inhibition constants can be explained by the chemical and structural characteristics of the inhibitors tested. The benzamidine, with Ki 18.4 µM, is a synthetic aromatic amide that competitively inhibits the trypsin, electrostatically interacting with the Asp189 of the trypsin and hydrophobically with specificity site of the active center of this enzyme (Oliveira et al. 1993OLIVEIRA MGA, ROGANA E, ROSA JC, REINHOLD BB, ANDRADE MH, GREENE LJ & MARES-GUIA M. 1993. Tyrosine 151 is partof the substrate activation binding site of bovine trypsin. J Biol Chem 268: 26893-26903., Patarroyo-Vargas et al. 2017PATARROYO-VARGAS AM, MERIÑO-CABRERA YB, ZANUNCIO JC, ROCHA F, CAMPOS WG & OLIVEIRA MGA. 2017. Kinetic Characterization of Anticarsia gemmatalis digestive serine proteases and the inhibitory effect of synthetic peptides. Protein Pept Lett 24: 1-8.). The inhibition of the serine proteases partially purified by benzamidine indicates that they are trypsin-like proteases. The berenil interacts electrostatically with the Asp189 of the trypsin and hydrophobically with the specificity site of the enzyme active center and strongly with the secondary binding site in a way similar to the Asp17 of the bovine pancreatic trypsin inhibitor (BPTI) (Oliveira et al. 1993OLIVEIRA MGA, ROGANA E, ROSA JC, REINHOLD BB, ANDRADE MH, GREENE LJ & MARES-GUIA M. 1993. Tyrosine 151 is partof the substrate activation binding site of bovine trypsin. J Biol Chem 268: 26893-26903.). The S2’ region is very close to the active site of the trypsin and presents the residues Tyr39, His40 and Tyr151. The residue, Tyr151 modification in the S2’ subsite of the bovine trypsin shows that the tyrosine residue participates in the activation of the enzyme making it permanently more active (Oliveira et al. 1993OLIVEIRA MGA, ROGANA E, ROSA JC, REINHOLD BB, ANDRADE MH, GREENE LJ & MARES-GUIA M. 1993. Tyrosine 151 is partof the substrate activation binding site of bovine trypsin. J Biol Chem 268: 26893-26903.).
The lower Ki values for the inhibitors SKTI and SBBI compared with the Ki of synthetic inhibitors allows them to occupy all the binding sites available in the active center of the enzyme and thus an optimized adjustment occurs in the EI complex formation. The synthetic inhibitors, being smaller, do not occupy all the available sites of the enzyme, forming a little less stable EI complex. The protein inhibitors reveal Ki in the same order of magnitude; however, the highest affinity of the SKTI to the active center of the enzymes can be explained by the differences in the architecture of these inhibitors (Volpicella et al. 2003VOLPICELLA M, CECI LR, CORDEWENER J, AMERICA T, GALLERANI R, BODE W, JONGSMA MA & BEEKWILDER J. 2003. Properties of purified gut trypsin from Helicoverpa zea, adapted to proteinase inhibitors. FEBS J 270: 10-19.). The Kunitz-type inhibitor interacts with the enzyme through the amino acid residue at the position P1, arginine or lysine, and this direct interaction between the reactive site residues of the inhibitor with the catalytic site of the enzyme characterizes a competitive mechanism of inhibition. The canonical conformation of the reactive site loop of SKTI interacts with the reactive site of the enzyme through the electrostatic links and hydrogen interactions (Bode & Huber 1992BODE W & HUBER R. 1992. Natural protein inhibitors and their interactions with proteinases. Eur J Biochem 204: 433-451.), forming a stable EI complex. The Bowman-Birk inhibitors also present the canonical loop conformation that interacts with the active center of the enzyme. The presence of seven disulfide bonds stabilizes the structure of the inhibitor, making it less flexible to adapt to the partially purified trypsin-like enzyme when compared with SKTI. Another hypothesis is that through evolution, the Lepidopteran trypsins showed increased hydrophobicity of the subsites of the active site by ingesting plant protease inhibitors. These inhibitors possess, in the active region, a hydrophilic sequence that does not adapt to the hydrophobic trypsin sites. The change in the primary specificity of the trypsin in Lepidoptera is an evolutionary adaptation in resistance to the plant trypsin inhibitors (Lopes et al. 2006LOPES AR, JULIANO MA, MARANA SR, JULIANO L & TERRA WR. 2006. Substrate specificity of insect trypsins and the role of their subsites in catalysis. Insect Biochem Mol Biol 36: 130-140.). This makes it difficult for the interaction between the inhibitor SBBI and the partially purified enzymes to occur.
Inhibition kinetic studies are tools for understanding the multi-mechanistic system of enzymes. Our study demonstrated, for the first time, the adaptation of trypsin-like enzymes in the gut of A. gemmatalis against different inhibitors. The SKTI soybean inhibitor showed the greatest inhibition of these enzymes. This is the reason underlying the aim of studying the production of peptide-mimetic inhibitors that may be produced by the plant or applied to it, to act as inhibitors of the complex system of the insect’s digestive proteases. However, the adaptive mechanisms of the insect should be carefully considered. The possibility of producing resistant plants expressing PI makes the development of new studies necessary to increase the chances of success of this method (Chitgar et al. 2013CHITGAR MG, GHADAMYARI M & SHARIFI M. 2013. Identification and characterization of gut proteases in the fig tree skeletoniser moth Choreutis nemorana Hübner (Lepidoptera: Choreutidae). Plant Prot Sci 49: 19-26.). In future, these studies should include the expression levels of the PI, its inhibition constant, the stability of the PI in the gut and the adaptation ability of the insect to the inhibitors via alteration in gene expression. Besides the important economic benefits, these studies may also provide environmental and social benefits by enabling the possibility of developing cultivars resistant to the insect pests and thus significantly reduce the use of pesticides. They, also, provide the basis for applied ecological studies on how insect resistance occurs in plants of interest. This also demonstrates how the plant-herbivore interactions need to be considered in the strategies for Integrated Pest Management. This knowledge will be of great importance for plant breeding programs aiming producing cultivars resistant to insect pests.
ACKNOWLEGMENTS
This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Instituto Nacional de Ciência e Tecnologia em Interações Planta-Praga (INCTIPP).
REFERENCES
- BERNARDI O, MALVESTITI G, DOURADO P, OLIVEIRA W, MARTINELLI S, BERGER G, HEAD G & OMOTO C. 2012. Assessment of the high-dose concept and level of control provided by MON 87701×MON 89788 soybean against Anticarsia gemmatalis and Pseudoplusia includens (Lepidoptera: Noctuidae) in Brazil. Pest Manag Sci 68: 1083-1091.
- BODE W & HUBER R. 1992. Natural protein inhibitors and their interactions with proteinases. Eur J Biochem 204: 433-451.
- BRADFORD M. 1976. A rapid and sensitive method for the quantification of microgram quantities of proteins utilizing the principle of protein dye binding. Analyt Biochem 72: 248-254.
- BRITO LO, LOPES AR, PARRA JR, TERRA WR & SILVA-FILHO MC. 2001. Adaptation of tobacco budworm Heliothis virescens to proteinase inhibitors may be mediated by the synthesis of new proteinases. Comp Biochem Physiol Part B Biochem Mol Biol 128: 365-375.
- BUDATHA M, MEUR G & DUTTA-GUPTA A. 2008. Identification and characterization of midgut proteases in Achaea janata and their implications. Biotechnol Lett 30: 305-310.
- CHITGAR MG, GHADAMYARI M & SHARIFI M. 2013. Identification and characterization of gut proteases in the fig tree skeletoniser moth Choreutis nemorana Hübner (Lepidoptera: Choreutidae). Plant Prot Sci 49: 19-26.
- DIXON M, WEBBER RC, THERONE CJR & TRIPTON KF. 1979. Inhibition and activation in enzymes. Acad Press NY: 332-381.
- EGUCHI M & KURIYAMA K. 1985. Purification and characterization of membrane-bound alkaline proteases from the midgut tissue of the silk-worm Bombyx mori. J Biochem 97: 1437-1445.
- ERLANGER B, KOKOWSKY N & COHEN W. 1961. The preparation and properties of two newchromogenic substrates of trypsin. Arch Biochem Biophys 95: 271-278.
- FERRY N, EDWARDS MG, GATEHOUSE JA, CAPELL T, CHRISTOU P & GATEHOUSE AMR. 2006. Transgenic plants for insect pest control: a forward looking scientific perspective. Transgenic Res 15: 13-19.
- GATEHOUSE JA. 2002. Plant resistance towards insect herbivores: a dynamic interaction. New Phytol 156: 145-169.
- HOFFMAN-CAMPO CB, OLIVEIRA EB & MOSCARDI F. 1985. Criação massal de lagarta-da-soja (Anitcarsia gemmatalis). EMBRAPA-CNPSO, Documentos 10. Londrina, 23 p.
- JAMAL F, PANDEY P, SINGH D & KHAN M. 2012. Serine protease inhibitors in plants: Nature’s arsenal crafted for insect predators. Phytochem Rev 12: 1-34.
- JUNQUEIRA RG, SILVA E & MARES-GUIA M. 1992. Partial competitive parabolic inhibition of bovine trypsin by bis-benzamidines: a general model for the trypsin-like family of proteases. Braz J Med Biol Res 25: 873-887.
- LAM W, COAST GM & RAYNE RC. 2000. Characterization of multiple trypsins from the midgut of Locusta migratoria. Insect Biochem Mol Biol 30: 85-94.
- LOPES AR, JULIANO MA, MARANA SR, JULIANO L & TERRA WR. 2006. Substrate specificity of insect trypsins and the role of their subsites in catalysis. Insect Biochem Mol Biol 36: 130-140.
- MAHDAVI A, GHADAMYARI M, SAJEDI R, SHARIFI M & KOUCHAKI B. 2013. Identification and partial characterization of midgut proteases in the lesser mulberry pyralid, Glyphodes pyloalis. J Insect Sci 13: 1-11.
- MARES-GUIA M & SHAW E. 1965. Studies on the active center of trypsin the binding of amidines and guanidines as models of the substrate side chain. J Biol Chem 240: 1579-1585.
- MILLS N & KEAN J. 2010. Behavioral studies, molecular approaches, and modeling: Methodological contributions to biological control success. Biol Cont 52: 255-262.
- NAKATA H & ISHII SI. 1972. Substrate activation of trypsin and acethyl-trypsin caused by N-α-benzoyl-L-arginine-p-nitroanilide. J Biochem 72: 281-290.
- NOVILLO C, CASTAÑERA P & ORTEGO F. 1999. Isolation and characterization of two digestive trypsin-like proteinases from larvae of the stalk corn borer, Sesamia nonagroides. Insect Biochem Mol Biol 29: 177-184.
- OLIVEIRA CFR, SOUZA TP, PARRA JRP, MARANGONI S, SILVA-FILHO MC & MACEDO MLR. 2013. Insensitive trypsins are differentially transcribed during Spodoptera frugiperda adaptation against plant protease inhibitors. Comp Biochem Physiol Part B Biochem Mol Biol 165: 19-25.
- OLIVEIRA MGA, DE SIMONE SG, XAVIER LP & GUEDES RNC. 2005. Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis. Comp Biochem Physiol Part B Biochem Mol Biol 140: 369-380.
- OLIVEIRA MGA, ROGANA E, ROSA JC, REINHOLD BB, ANDRADE MH, GREENE LJ & MARES-GUIA M. 1993. Tyrosine 151 is partof the substrate activation binding site of bovine trypsin. J Biol Chem 268: 26893-26903.
- PATARROYO-VARGAS AM, MERIÑO-CABRERA YB, ZANUNCIO JC, ROCHA F, CAMPOS WG & OLIVEIRA MGA. 2017. Kinetic Characterization of Anticarsia gemmatalis digestive serine proteases and the inhibitory effect of synthetic peptides. Protein Pept Lett 24: 1-8.
- PILON AM, OLIVEIRA MGA, PILON FM, GUEDES RNC, OLIVEIRA JA & FAZOLLO A. 2009. Adaptação da lagarta-da-soja Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) ao inibidor de protease benzamidina. Rev Ceres 56: 744-748.
- SCOTT IM, THALER JS & SCOTT JG. 2010. Response of a generalist herbivore Trichoplusia ni to jasmonate-mediated induced defense in tomato. J Chem Ecol 36: 490-499.
- SHI M, ZHU N, YI Y & CHEN X. 2013. Four serine protease cDNAs from the midgut of Plutella xylostella and their proteinase activity are influenced by the endoparasitoid, Cotesia vestalis. Arch Insect Biochem Physiol 83: 101-114.
- SILVA-FILHO MC & FALCO MC. 2000. Interação planta inseto-adaptação dos insetos aos inibidores de proteinases produzidas pelas plantas. Biotecnolog Cienc Desenvolv 12: 38-42.
- TELANG MA, GIRI AP, SAINANI MN & GUPTA VS. 2005. Characterization of two midgut proteinases of Helicoverpa armigera and their interection with proteinase inhibitors. J Insect Physiol 51: 513-522.
- TERRA WR & FERREIRA C. 1994. Insect digestive enzymes: compartimentalization and function. Comp Biochem Physiol Part B Biochem Mol Biol 109: 1-62.
- VIANNA UR, PRATISSOLI D, ZANUNCIO JC, ALENCAR JRC & ZINGER FD. 2011. Espécies e/ou linhagens de Trichogramma spp. (Hymenoptera: Trichogrammatidae) para o controle de Anticarsia gemmatalis (Lepidoptera: Noctuidae). Arq Inst Biol 78: 81-87.
- VOLPICELLA M, CECI LR, CORDEWENER J, AMERICA T, GALLERANI R, BODE W, JONGSMA MA & BEEKWILDER J. 2003. Properties of purified gut trypsin from Helicoverpa zea, adapted to proteinase inhibitors. FEBS J 270: 10-19.
- WIELKOPOLAN B, WALCZAK F, PODLEŚNY A, NAWROT R & OBRĘPALSKA-STĘPLOWSKA A. 2015. Identification and partial characterization of proteases in larval preparations of the cereal leaf beetle (Oulema melanopus, Chrysomelidae, Coleoptera). Arch Insect Biochem Physiol 88: 192-202.
- ZOLLNER H. 1999. Handbook of enzyme inhibitor, 3rd ed., New York: Wiley-VCH, 583 p.
Publication Dates
-
Publication in this collection
01 June 2020 -
Date of issue
2020
History
-
Received
16 May 2018 -
Accepted
26 Mar 2019