Phylogenetic and structural comparisons of phytocystatins

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CHAPTER2 Characterization of the digestive proteases in the banana weevil gut and the effects of recombinant phytocystatins on early larval growth and development


It is well-documented that insects posses different protease forms used to digest dietary proteins. Therefore, studies to characterize the forms of protease are important to provide the basis for selecting appropriate protease inhibitors likely to be effective in a transgenic approach. In this study the protease activity in the gut of banana weevil was analysed in order to determine the potential of phytocystatins (OC-I and papaya cystatin) for the control of the banana weevil Cosmopolites sordidus G. (Coleoptera: Curculionidae). Extracts from complete weevil larvae guts were found to hydrolyse casein at an acidic pH optimum (pH 5.5). Lesser activity was also detected at alkaline pH conditions (pH 8.0). Cathepsin L and B like cysteine proteases were found in the larval gut as shown by hydrolysis of the specific substrates Z-Phe-Arg-MCA and Z-Arg-Arg-MCA, respectively. In addition, activity of trypsin and chymotrypsin-like serine proteases were also detected using the specific substrates Bz-Arg-MCA and N-Suc-Ala-Ala-Pro-Phe-MCA, respectively. OC-I and papaya cystatin produced as a His-tagged fusion protein in Escherichia coli and purified by affinity chromatography inhibited cysteine protease activity in the banana weevil gut homogenates by 66.2 and 81.6% and LD50’s of 1×10-5ng/ml and 2.1×10-5ng/ml, respectively. A new bioassay was applied to evaluate the effect of OC-I on early growth and development of the larvae. After banana stem disks were vacuum infiltrated with purified OC-I., weight gain per day of larvae was inhibited by 77% at an inhibitor concentration of 0.6mg of cystatin/g fresh weight. This part of the study demonstrated that the banana weevil uses cysteine proteases similar to cathepsin L and B for protein digestion and metabolism in the gut while phytocystatins are potential control agents for banana weevil growth.


Numerous protease inhibitors have been isolated from numerous plants species and there is evidence that they contribute to the natural defense against insect and pathogen attack (Green and Ryan, 1972; Jacinto et al., 1998). Several studies have already demonstrated the effectiveness of protease inhibitors for the control of various pests when engineered into transgenic plants. Lecardonnel et al. (1999) found increased resistance to the Colorado potato beetle (Leptinotarsa decemlineata) by developing transgenic potatoes expressing OC-I. Furthermore, Newell et al. (1995) developed sweet potato plants expressing cowpea trypsin inhibitors and found resistance to the West Indian sweet potato weevil (Euscepes postfasciatus).
There are generally two major protease classes in the digestive systems of phytophagus insects, either the serine or the cysteine class. Serine protease activity is characteristic of Lepidoptera, Dictyoptera and Hymenoptera while the cysteine class is characteristic of Odoptera and Hemiptera. Initial investigations had concluded that Coleopteran insects mainly use cysteine proteases (Gatehouse at al., 1985; Murdock et al., 1988). However, from more recent work it appears that a combination of both serine and cysteine proteases is active in this more advanced order (Mochizuki, 1998) suggesting a higher diversity of proteases in these insects.
The objectives of this study were to identify the major classes of proteolytic activity in the gut of banana weevil larvae using in-vitro and in-vivo assays in order to determine the protease classes present in the weevil. A further objective was to evaluate the potential of OC-I and papaya cystatin to control growth of banana weevil larvae by targeting the cysteine proteases in the weevil gut.

Materials and methods


Azocasein, N- Z-Arg-Arg-7-amido-4-methylcoumarin hydrochloride (Z-Arg-Arg-MCA), Z-Phe-Arg-7-amido-4-methylcoumarin hydrochloride (Z-Phe-Arg-MCA), Z-L-arginine-4-methyl-7-coumarinylamide hydrochloride (Z-Arg-MCA), N-Succinyl-Ala-Ala-Pro-Phe7-amido-4-methylcoumarin hydrochloride (N-Suc-Ala-Ala-Pro-Phe-MCA), Benzoyl-L-arginine-7-amido-4-methylcoumarin hydrochloride (Bz-Arg-MCA), bovine serum albumin (BSA), trans-epoxysucci-nyl-L-leucylamido-(4-guanidino) butane (E-64), gelatin (porcine type A), Triton X-100, phenylmethylsulfonyl fluoride, ethylenediamine tetra acetic acid (EDTA), Phenylmethanesulfonyl fluoride (PMSF), trypsin-chymotrypsin inhibitor from Glycine max (Soybean) (SBTi) and aprotinin were purchased from Sigma (Aston Manor, South Africa). Recombinant OC-I and OC-II, corn cystatin-II (CC-II), stefin-A from human (HSA) were a gift from Prof. D. Michaud, who expressed them using the S-transferase (GST) gene fusion system (Michaud et al., 1994; Brunelle et al., 1999).

 Insect colony and maintenance

Adult banana weevils were collected from banana growers in Kwazulu Natal Province (South Africa) and maintained in the greenhouse at the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa. The weevils were kept in 10 liter plastic buckets and provided with fresh banana stem (pseudostem and corm) material to oviposit. After two days, weevils were moved to a different container to allow development of laid eggs. After one week, corms were dissected to collect 3th to 4th instar larvae. These were quickly stored at –20oC until required.

Gut extractions and protein concentration determination

Frozen larvae were thawed on ice and dissected in cold distilled water under a stereomicroscope to remove whole guts. The guts were then homogenized in liquid nitrogen followed by addition of 0.15M calcium chloride buffer containing 0.1% Triton X-100 at a tissue to buffer ratio of 0.2g/ml of buffer. The mixture was incubated on ice for 30min and then centrifuged at 15,000rpm for 10min. The clear supernatant was collected into fresh tubes and stored at -20oC. For extracts to be used in gelatin SDS-PAGE (see below), guts were homogenized directly in 100µl gelatin-PAGE sample loading buffer (62.5mM Tris-HCl pH 8.0, 2% sucrose, and 0.001% bromophenol blue). The protein concentration of both types of extracts was determined using the Bio-Rad protein assay kit (Bio-Rad, UK), which is based on the Bradford method with bovine serum albumin as the standard.

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Determination of pH optima

To determine the pH optima of the crude larvae extracts, protease activity of the extracts was determined using azocasein as a protein substrate as described by Michaud et al. (1995). Basically, 50µl (50µg total soluble protein) of a gut extract were mixed with 450µl of assay buffer (0.1M citrate phosphate buffer for pH 4.0; pH 4.5; pH 5.0; pH 5.5; pH 6.0; pH 6.5 and pH 7.0; 0.1M Tris-HCl buffer for pH 7.5; pH
8.0; pH 8.5 and pH 9; 0.1M glycine buffer for pH 9.0, 9.5 and pH 10). All buffers were made to contain 5mM L-cysteine before use. After pre-activating proteases by incubating the mixture for 10min at 37°C, an equal volume of 2% azocasein (in the respective assay buffer) was added and the complete mixture incubated at 37°C for 3hrs. To stop the reaction, 100µl of 10% (w/v) trichloro-acetic acid was added to the mixture and the mixture incubated for 30min at 4°C. Residual azocasein was removed by centrifugation at 12000rpm for 5min at 4°C. To 1.0ml of the supernatant, 1.0ml of 1N NaOH was added to precipitate the hydrolysed azocasein and finally the absorbance of this solution was determined at 440nm in a spectro-photometer. At this wavelength, one unit of protease activity is defined to be the amount of enzyme required to produce an absorbance change of 1.0 in a 1cm cuvette under the conditions of the assay (Sarath, 1989). Reactions were performed in triplicate on a micro-titre plate.

Fluorometric assay

Protease specific proteolytic activity and inhibition by specific inhibitors were investigated using the substrates Z-Arg-Arg-MCA (specific to cathepsin B), Z-Phe-Arg-MCA (specific to cathepsin L), Z-Arg-MCA (specific to cathepsin H), Bz-Arg-MCA (specific to trypsin) and N-Suc-Ala-Ala-Pro-Phe-MCA (specific to chymotrypsin). These are highly sensitive fluorometric substrates. When hydrolyzed by their specific proteases, bound α-amino 4-methylcoumarin (MCA) is released, which is highly florescent and MCA release is determined using fluorescence spectro-photometry.
Hydrolysis of the specific substrates by the gut extract was monitored using hydrolysis progress curves as described by Salvesen and Nagase (1989). For detection of cathepsin B, L and H like activity, reaction mixtures contained 10µl (10µg total soluble protein) of the gut extract, 1µl (1%) substrate solution in DMSO dissolved in 89µl reaction buffer; 0.1M citrate phosphate buffer pH 6.0 with 5mM L-cysteine freshly added for cysteine like activity or 0.1M Tris-HCL pH 8.0 for trypsin and chymotrypsion like activity. Hydrolysis was monitored at room temperature using a spectro-fluorometer (BMG FluoStar Galaxy) with excitation and emission at 360nm and 450nm, respectively. Reaction rates represented by the slope of the curve were recorded as Fluoresence Units (FU) per unit time. All reactions were performed in triplicate.
Inhibitors for the different protease classes were used to evaluate inhibition of their activity. For that, 1µl of a 1% inhibitor solution (E-64, OCI, OCII, CCII, HSA, STBi, aprotinin and PMSF) prepared in the same reaction buffer was introduced into the protease reaction monitored in the spectro-fluorometer. The reactions were briefly mixed and detection of protease reaction continued until a steady rate was reached. Slope values were determined before addition and after addition of the inhibitor.

CHAPTER 1 Introduction: The banana weevil and protease inhibitors
1.1 Plant improvement and Africa
1.2 The banana weevil
1.2.1 Weevil resistance.
1.2.2 Weevil resistance screening
1.2.3 Resistance mechanisms
1.2.4 Resistance breeding
1.3 Protease/protease inhibitor system
1.3.1 Insect proteases
1.3.2 Plant protease inhibitors.
1.3.3 Regulation of protease inhibitors.
1.3.4 Structure of protease inhibitor genes
1.3.5 Protease inhibitors and insect control
1.3.6 Engineering of protease inhibitors
1.4 Study hypothesis aim and objectives
CHAPTER 2 Characterization of the digestive proteases in the banana weevil gut and the effects of recombinant phytocystatins on early larval growth and development
2.1 Abstract
2.2 Introduction
2.3 Material and methods

2.3.1 Reagents
2.3.2 Insect colony and maintenance
2.3.3 Gut extractions and protein concentration determination.
2.3.4 Determination of pH optima
2.3.5 Fluorometric assay
2.3.6 Gelatin SDS-polyacrylamide gel electrophoresis
2.3.7 Cloning of OC-I and PC genes
2.3.8 Protein expression and purification
2.3.9 In-vitro assays with recombinant phytocystatins
2.3.10 Infiltration of banana stem with phytocystatin
2.4 Results
2.4.1 pH optima
2.4.2 Fluorometric assays
2.4.3 Gelatin SDS-polyacrylamide gel electrophoresis
2.5 Discussion .
CHAPTER 3 Phylogenetic and structural comparisons of phytocystatins: A bioinformatics approach
3.1 Abstract
3.2 Introduction
3.3 Materials and methods
3.4 Results
3.5 Discussion
CHAPTER 4 Engineering of a papaya cystatin using site – directed mutagenesis to improve its activity against papain and weevil digestive cysteine proteases 
4.1 Abstract
4.2 Introduction
4.3 Materials and methods
4.4 Results
4.5 Discussion
CHAPTER 5 General discussion and future outlook
5.1 Summary
5.2 Future outlook

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