Protease Inhibitors: Potential and Constraints

Protease inhibitors as biopesticides: Potential and constraints

Neha Khandelwal, Rakesh S. Joshi, Vidya S. Gupta and Ashok P. Giri*

Plant Molecular Biology Unit, Division of Biochemical Sciences, National Chemical Laboratory (CSIR)

Corresponding Author: Ashok P. Giri

Abstract: Plant protease inhibitors are the well studied class of plant defensive proteins. High level of up-regulation upon insect damage, significantly elevated levels in reproductive and storage organs as well as specificity to pest proteases ascertain their defensive function. In the present chapter, we introduced molecular basis of plant-insect interactions and focused on different classes of protease inhibitors based on their target protease(s). In particular serine protease inhibitors are well-studied in plants and the effort to deploy them for plant protection methods has been discussed. Wound-induced serine protease inhibitors Pin-I and Pin-II type are in major focus due their unique features such as high binding specificity to target proteases, multi-domain structure, stability and processing in digestive tract of insects. Numerous efforts by different research groups all over the world for their application for insect control have been discussed emphasizing the merit and constraints. It is known that insect respond to protease inhibitors by expressing variants of insensitive proteases which nullify the effect of protease inhibitors. However, there are several unanswered questions that needs to be addressed for the successful use of proteinase inhibitors (i) What are the protein engineering and molecular biology approaches for their competent use against insect pests? (ii) How different strategies can be applied for the successful implementation of protease inhibitors commercially? (iii) How to explore selective use of broad spectrum or specific protease inhibitors? etc. These issues are discussed throughout the review by emphasizing more on limitations of protease inhibitors as bio-pesticide agents to date.

Keywords: Biopesticides, Lepidopteran insects, Protease inhibitors and Transgenic plants

Insect pests: A major challenge in sustainable agriculture

Agriculture production in India significantly plays major role in country’s economic development by accounting over 28% of largest global pulse production. India is the second largest producer of vegetables, cotton, wheat and rice. Agriculture in India has gone through immense transitions ranges from low productivity due to pest attack and high productivity by the use of chemical pesticides. About 3% of the total pesticides used in the world are utilized in India. The used pattern of pesticides reflects that cotton crop alone consumes 44.5% pesticides estimating USD 250 million for the control of bollworms which is followed by rice, accounting 22.8% of pesticide consumption for the control of yellow stem borer. These two crops consume more than two thirds of the total quantity of the pesticides used in the country (Report by Dileep K. Singh). Other key pests of similar importance are stem borers of sorghum and maize, fruit and shoot borer of brinjal, fruit and pod borer of tomato and chickpea and diamond back moth of cruciferous crops, cabbage and cauliflower. These pests are perennial and persistently causing losses to these economically important crops (Reddy and Zehr, 2004) An average estimation of the damage caused by insects throughout the year has been shown in terms of percentages (figure 1). Past fifty years the usage of chemical insecticides and pesticides is at their peak for pest control in various crop species. Around 50 per cent of the total insecticide consumption is that of organophosphates, followed by the synthetic pyrethroids (19%), organochlorines (18%), carbamates (4%) and biopesticides (1%). Most of the chemical pesticides become detrimental to human health as well as raises environmental concern when used above threshold limit and results in eradication of beneficial insects. In addition, most of the agronomically important pests have developed resistance towards it which causes the use of such insecticides inefficient at moderate level and demands for their increased use on agricultural crops. Sustainable agricultural practices are thus of global concern today which takes maximum utilization of environment without causing any harm to it.

Despite the use of various protective measures, pests have remained as a potential threat to agricultural crops. Insects that cause serious economic loss belong to lepidopteran, coleopteran, hemipteran, orthopteran and dipteran families. As pollinators of many plants, lepidopteran adult moths and butterflies are usually beneficial insects that feed on nectar. Caterpillar however consists of chewing mouth parts which allow them to feed on plant parts causing extensive damage. Unlike lepidopteran, coleopteran beetles have been known to cause damage usually in their adult stage, for instance Caryedon serratus (Olivier) and Phyllotreta cruciferae infects groundnut and seedling phase of cabbage (Ranga Rao et al., 2010; K Mayuri and G. Mikunthan, 2009). Much of the emphasis has been given on lepidopterans which represent the most damaging class of insects that feeds on cereals, pulses, vegetables, and oil seeds. Research in last few decades are much focused in studying about the interaction between plants and insects to develop plant protection strategy against them. Crop biotechnology, which broadly includes areas of development of transgenic crops, structural and functional genomics and marker-assisted breeding, could provide us with the vital breakthroughs to achieve improvements in both quality and quantity in a sustainable manner (Manju Sharma et al., 2003).

Plant-insect interaction

In natural ecosystem, plants and insects are in constant interaction with each other. This complex and dynamic interaction between plants and insects are being studied from molecular, ecological and evolutionary perspective. Insects, as pollinators and predators of undesirable pests provide beneficial role, though many insects share same life cycle (figure 2), but many have emerged as harmful due to their feeding habit on enormous number of plant species. The number of plant attackers is seemed to be almost limitless whose knowledge is essential in respect to develop effective strategy for the protection of crop plants. Insects that feed on plants are categorized into monophagous, oligophagous and polyphagous depending upon the number of species or families they feed upon.

A. Monophagous or specialized pests - Insects which are known to feed on single species of plant and during the absence of their main host plants they shift to some other related host species temporarily. Examples includes Pink bollworm (Pectinophora gossypiella), rice stem borer (Scirpophaga incertulas), brinjal shoot and fruit borer (Leucinodes orbonalis) (Hanur, 2008)
B. Oligophagous pests - Insects that confine their feeding activity belonging to one family, a well known example is cabbage diamondback moth (Plutella xylostella L.; Plutellidae, Lepidoptera)
C. Polyphagous or generalized pests - feeds on various species of plants belonging to more than one taxonomic group or family e.g. cotton bollworm or pod borer (Helicoverpa armigera), Tobacco cut worm (Spodoptera litura)

Polyphagous insects are considered as the most threatening among above categories due to their adaptation to various host plants posing problem in controlling them by employing same strategy with different crops. Spodoptera exigua for example feeds on approximately 50 plant species including pigweed, Amaranthus hybridus, and cotton, Gossypium hirsutum (Showler et al., 2001). Similarly Heliothis virescens mostly feed on cotton, soybean, flax, alfalfa but found at high density on tobacco being it the most preferable crop (Tilmann, 2006). Polyphagous pests possess the ability to adapt their digestive system in response to diverse type of host plant they feed upon, by switching on/ off genes involved in the metabolism of the host plant components. The well known example is the Helicoverpa armigera (Lepidoptera: Noctuidae) which has been widely studied for its adaptability on various host plants. Voracious caterpillars of H. armigera feeds on approximately 200 plant species including number of forest and fruit trees along with agronomically important crops such as cotton, chickpea, pigeon pea, tomato, maize, okra etc. by metabolizing nutrients for their growth and development. In order to grow on varied type of host plants these insects bring about molecular changes in their gut complement (Patankar et al, 2001). These molecular flexibilities enable insect’s digestive system to react efficiently upon exposure to different crops by synthesizing more isoforms of proteases, if feeding on protein rich diet and amylases if feeding on starch rich diet. Thus insects are capable of surviving on enormous number of plants due to which it has attained the status of notorious pest. Several studies have been attempted in order to control H. armigera, which alone is responsible for causing heavy losses of economically important crops ranging from minimum 14% to maximum loss of 100%. Currently, control of insect pests is mainly dependent on the use of chemical insecticides which are commercially available in large numbers. Insect pest management by chemicals obviously has brought about considerable protection to crop yields over the past five decades. Pesticides such as lambda cyhalothrin, cypermethrin, and quinalphos are amongst few chemicals which have been formulated and utilized in pest management. Though they have been found to be efficient in reducing insect proliferation, their detrimental effect on ecosystem has to be considered. In search of environmentally benign approach transgenic plants have been emerged as an efficient strategy for the pest control management. Bt transgenics were successfully implemented for the control of major field pest, its implications and constrains are discussed partly in the following sections.

Plant defense system against insects

Since years of interaction plants and insects, both have coevolved in order to survive in their changing niches. Insects gradually adapt to take maximum benefit from the host while plants on the other hand have evolved to protect themselves. Being sedentary in nature plants have developed the ability to react spontaneously against insect attack by synthesizing number of defensive molecules. These responses of plants have been extensively studied to exploit them for crop protective measures. Plants show different types of defence responses against pathogens and pests which are categorized into direct and indirect responses (figure 3). Direct mode includes production of toxic, repellent or anti digestive molecules like cyanogenic glucosides, glucosinolates, alkaloids, phenolics, proteinase inhibitors etc, along with this they also pose some physical barriers (e.g. Cuticle, Trichomes, Thorns etc.) which play a role in primary protection against infestation (Bennett et al., 1994). In indirect mode, plant emits some volatiles which produce the extra floral nectar that attracts predators of insect herbivorous to gain indirect means of protection (Kessler and Baldwin, 2001; 2002).

Wounding by insect herbivory switches on different signalling reflexes in plants, leads to induction of different defence mechanisms (figure 4). When an insect feeds on plant, some elicitor molecules are transferred through the saliva of insect. These elicitor molecules cause local secretion of some peptide hormones like prosystemin, threonin deaminase etc. (Ferry et al., 2004; Kang et al., 2006). Prosystemin undergoes proteolytic cleavage to produce active systemin. Production of systemin switches the Octadeconide (OD) pathway, which catalyzes the breakdown of linolenic acid and the formation of jasmonic acid (JA) (Ferry et al., 2004). Jasmonic acid reacts with free isoleucine, which is produce active signalling molecule. On cleavage threonine gives isoleucine (react with jasmonic acid to gives JA-Ile) and a keto butyrate (Toxic to insect) this reaction is catalyzed by threonin deaminase (Kang et al., 2006). JA-Ile switches on early genes in vascular bundle which cause activation of NADPH oxidase. NADPH oxidase catalyzes the production of hydrogen peroxide (H2O2) (Orozco-Cardenas M and Ryan, 1999). H2O2 is transfer to mesophyll cells, where they activate late gene responsible for production of defence molecule like proteinase inhibitor (PIs). In other way of defence Jasmonic acid induces the release of some volatiles that attract natural predators of the insect pest.

Jasmonic acid on méthylation produces methyl jasmonate. Methyl Jasmonate acts as a mediator molecule to give plant-plant interaction (Orozco-Cardenas M et al., 2001; Rojo et al., 2003).

Role of protease inhibitors in plants

PIs are of common occurrence in the plant kingdom and initially were found to be abundant in storage tissues such as tubers and seeds. They protect storage organs from predators by inhibiting hydrolytic activity of target proteases and play developmental role by the regulation of protein turnover. Later on their presence has also been detected in the aerial parts of plants where their expression is induced in response to pest attack (figure 5). Protease inhibitors mostly have been characterized from Fabaceae (legumes), Poaceae (cereals) and Solanaceae families. Since last decade insects digestive proteolytic enzymes have come under intensive investigation thus major attention has been given in exploitation of defensive role of protease inhibitors which induced upon insect attack for the purpose of crop protection. Thorough understanding of protease inhibitors from its induction to its inhibitory effect on insects has been determined in various studies to characterize their potential role in controlling pest population. PIs are responsible in causing reduction in the availability of amino acids by blocking the hydrolytic activity of proteases and were first shown as plant defensive proteins in 1972 when their induction in potato and tomato was observed due to wounding and insect herbivory. Subsequently Gatehouse et al and co­workers demonstrated the resistance of a cowpea variety to a bruchid beetle (Coleoptera) due to the elevated trypsin inhibitor (Donald Boutler et al., 1990). PIs act on insects by interfering with the metabolic breakdown of the protein in the insect midgut thereby arresting their catabolism that leads to the accumulation of the protein causing physiological burden on insects. Most of the lepidopteran insects prefers protein rich diet hence employment of protease inhibitor have been proven to be the most promising approach for the development of effective strategy against insect pest. Natural mutants of trypsin inhibitors in wild tobacco plants recently provided the first true evidence that the inhibitors exert a certain level of control even against their natural ‘adapted’ pests (Jongsma and Beekwilder, 2008). In addition to resistance from insects, PIs are also known to confer resistance from abiotic stresses such as salt stress, change in pH and osmolarity (Tamura et al., 2003). Strategically use of protease inhibitors in integrated crop management requires the knowledge of the synthesis and role of PIs in plant system in response to insect attack.

Mode of action of protease inhibitor against insect proteases

The different classes of inhibitors are distinguished by the structure of their polypeptide backbone. However the mechanism of binding of the plant protease inhibitors to the insect proteases appears to be similar with all the four classes of inhibitors. Interaction between enzyme and inhibitor occurs in canonical (substrate like) manner. The inhibitor binds to the active site on the enzyme to form a complex with a very low dissociation constant (10 to 10 M at neutral pH values), thus effectively blocking the active site. For serine proteinase inhibitors, most residues interacting with the proteases are located on a single loop, to which the P1 residue is central. The P1 residue of protease substrates is the one on the amino-side of the hydrolyzed bond, and is often crucial for substrate recognition. The surrounding residues (P2, P3 etc. at its N-terminus, and P1’, P2’ etc. at its C-terminus) play secondary roles in the interaction of proteases and their substrates. Both in inhibitors and in substrates, the P1 residue fits into the S1 substrate binding site of the proteinase. Unlike normal peptide substrates, the inhibitor residues around P1 interact with the enzyme through complementary polar and hydrophobic interactions, and are held in position by strong bonds with the inhibitor scaffold. This prevents immediate dissociation of the complex, thus keeping the enzyme inactive. P1 residue in reactive loop of inhibitor is primary determinant of inhibitor specificity. If P1 residue is positively charged like Arg and Lys they are act as trypsin inhibitor. In case of chymotrypsin inhibitor P1 is hydrophobic in nature (Leu, Ile, Trp or Phe). The inhibitor residues around P1 residue are from gene family which more hypervariable in nature relative to most of protein.

Distinct classes of protease inhibitors

Protease inhibitors are categorized into four classes on the basis of the mechanistic classes of the proteases they inhibit: serine PIs, cysteine PIs, asparatate PIs and metallo proteases PIs (figure 5). Serine PIs have a widespread distribution in plant kingdom and are extensively studied as most of the lepidopteran insects utilize active serine proteases for their nutrient requirement by metabolic breakdown of the protein.

Cysteine PIs represent the second most well studied class of protease inhibitors whereas little is known about asparatate and metallo protease inhibitors.

Serine protease inhibitors

Plant serine PIs acts against proteases like trypsin, chymotrypsin, elastases and subtilisin (bacterial enzyme). Trypsin- and chymotrypsin-like enzymes are predominantly present in the midgut of lepidopteran insects. These enzymes are active in the alkaline pH range from 9 to 11 of gut proteolytic environment. Protease function of cleaving proteins at specific sites, enable insects to utilize them for their nutrient enrichment. Trypsin cleaves at the C-terminal residue of lysine or arginine containing basic side chains whereas chymotrypsin cuts at the C-terminal residue of phenylalanine, trypsin and leucine, all containing hydrophobic side chain. Various researchers have identified the presence of serine protease inhibitors in plants for their defensive role and divided into various families of which four major types are: Kunitz, Bowman birk, squash and wound inducible type. Latter is further categorized in to two families namely Potato inhibitor type 1 (Pin I) and potato inhibitor type 2 (Pin II). Different families of serine PIs were recognized based on their distribution in plant kingdom, sequence and functional divergence.

As wounding induces high accumulation of the PIs in plant parts, one might ask about their consequential effect on native proteinases of the plants. Major digestion of protein in plants occurs by the proteases which contain cysteine residues at their active site rather than serine residue thus the significant amount of serine PIs in plants does not affect plants metabolic function and cannot be used for regulating endogenous proteases. This clearly reflects the presence of different type of proteinase inhibitors for the regulation of protein turnover in plants. However few reports have emerged describing endogenous function of serine proteinase inhibitors in plants (Hartl et al., 2010). Moreover, many plants express a number of different PIs and it was unknown if these proteins work synergistically as defenses or if they also have other functions. In case of serine PIs, gene- and tissue-specific expression patterns suggest multiple functions in generative tissues, including a possible involvement in development. Role of PIs have been characterized from various plants towards their defensive function against pests. From example, recently, protease inhibitors from Capsicum annuum plant were also accessed for their expression in different parts of plants. Single and double inhibitory domain proteins were found to be strongly expressed in stems, whereas multi IRD protein expression was restricted to leaves which clearly distinguish their role in defence from endogenous function (Tamhane et al, 2009). Role of plant serine PIs in defence has been well documented as they were shown to inhibit insect digestive enzymes leading to retardation in their growth and development. Broadway and Duffey in their studies have demonstrated the antinutritional effect of serine protease inhibitors which showed inhibition of Heliothis Zea and Spodoptera exigua after incorporated in artificial diet (Broadway and Duffer, 1986). In another similar study PIs of tobacco (Nicotiana alata) have shown to affect H. punctigera larval and Black field cricket (Teleogryllus commodus) growth (Heath et al., 1997). Following this several serine protease inhibitors from non host plants were investigated for their potential in conferring resistance to plants and attempts were made to develop transgenic plants.

Cysteine Protease inhibitors

Cysteine protease inhibitors are also called plant cystatins or phytocystatins and are divided into two groups one group containing single inhibitory domain whereas second group includes PIs carrying multiple domains. Cysteine PIs have been characterized from several plants viz. cowpea, potato, cabbage, ragweed, carrot, papaya, apple fruit, avocado, chestnut and various crop plants like sunflower, rice, wheat, maize, soybean and sugarcane (Shu-Guo FAN and Guo-Jiang WU, 2005). Cysteine PIs acts as an antagonist for cysteine proteases which are active in the insect midgut of pH ranges from 5 to 7. Cysteine proteases are usually found in coleopteran order of insects such as Cowpea weevil (C. maculates), Bruchid Zabrates subfaceatus, Flour beetle (Tribolium castaneum), Mexican beetle (Epilachna varivestis) and the Bean weevil Ascantho sc elides beetles. Studies revealed that in few insects cysteine proteases also get inhibited by serine protease inhibitor in addition to cysteine PIs for eg. C. maculatus proteolytic enzymes get inhibited by Cowpea and soybean Trypsin inhibitor (serine PI) (Lawrence, 2002). It could be possible if they share high sequence homology between cysteine and serine protease inhibitors and thus cross interaction might occur. Oryzacystatin from rice have been well characterized among other cysteine PIs for its role in plant protection and attempts were also made to develop transgenic plants. Oryzacystatin contains highly conserved motifs glu-val- val-ala-gly (QVVAG) and pro-trp (PW) of which the former is the primary region which involve in interaction with the enzyme and PW motif acts like a cofactor (Arai et al., 1991). Other than inhibitory effect on herbivory, cysteine proteases and their inhibitors are known to participate in program cell death according to the study conducted on soybean cells. Endogenous cysteine protease inhibitors may function in modifying PCD that is activated during oxidative stress and pathogen attack.

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