Partial N-terminal Amino Acid Sequence Research Articles

Purification and characterization of a serine protease from the fruit of Ficus carica cultivar Masui Dauphine.

Ficus carica produces, in addition to the cysteine protease ficin, a serine protease (FSP). Here, we purified FSP to homogeneity from the fruit of F. carica cultivar Masui Dauphine. An 81-fold enrichment in specific activity of FSP with 2.1% recovery was attained. Three protein bands (70, 62, and 60kDa) were identified on SDS-PAGE. Each band was identified as a subtilisin-like protease (661 amino acids) by trypsin digestion and LC-MS/MS analysis, and the partial N-terminal amino-acid sequence analysis. Gelatin zymography revealed that the active FSP exists as a dimer. The optimum hydrolysis pH of FSP was 7.5, and the pHs at which the enzyme retained its initial activity by 70% in 24h were 8.0-11.0. The optimum hydrolysis temperature of FSP was 50-60ºC, and the temperature required to reduce the initial activity by 50% in 15min was 70ºC. These results will inform the industrial use of FSP.

Isolation and partial structural characterization of new Kunitz-type trypsin inhibitors from the pike cestode Triaenophorus nodulosus

The inhibitors produced by the parasitic worms successfully protect them from the host’s proteases and are supposed to underlie the host-parasite specificity. Our previous study has shown that the extracts from the pike tapeworm Triaenophorus nodulosus inhibit host proteinases and commercial trypsin. We aimed to isolate and identify the components responsible for trypsin inactivation. After a two-step separation the molecular masses were measured by SE-HPLC. The sample proved to contain four fractions represented by polypeptides (1–45 kDa) and low-molecular hydrophobic compounds. According to SDS-PAGE analysis, the major polypeptides in the fractions displaying the highest inhibition had masses of 14.4 kDa. The study culminated in partial N-terminal amino acid sequence analysis with a further search for homology. The research revealed two novel Kunitz-type proteins potentially responsible for the inhibitory capacity of the tapeworms against trypsin. Our findings extend the list of cestodes relying on Kunitz-type proteins in the host-parasite molecular cross-talk.

Physicochemical Properties and Biological Activity of the Peptide–Protein Complex from Bovine Scleral Tissue

The peptide–protein complex from bovine sclera was studied. It is shown that it contained a protein with a molecular weight of 66387 Da with the partial N-terminal amino acid sequence DTHKSEIAHRFKDLG-, which is homologous to the mature molecule of bovine serum albumin, and polypeptides with molecular weights of 1300–5080 Da. With a model of the organotypic cultivation of posterior eye tissues of the newt Pleurodeles waltl in vitro, it was shown that the effect of this peptide–protein complex in low doses increased the viability of scleral fibroblasts.

Leucocin C-607, a Novel Bacteriocin from the Multiple-Bacteriocin-Producing Leuconostoc pseudomesenteroides 607 Isolated from Persimmon.

Leuconostoc pseudomesenteroides 607, isolated from persimmon fruit, was found to have high inhibitory activity against Listeria monocytogenes and several other Gram-positive bacteria. Inhibitory substances were purified from culture supernatant by ion-exchange chromatography, Sep-Pak C18 cartridge, and reverse-phase high-performance liquid chromatography (RP-HPLC). Two antibacterial peptides were observed during the purification procedures. One of these peptides had a molecular size of 4623.05Da and a partial N-terminal amino acid sequence of NH2-KNYGNGVHxTKKGxS, in which the YGNGV motif is specific for class IIa bacteriocins. A BLAST search revealed that this bacteriocin was similar to leucocin C from Leuconostoc mesenteroides. Leucocin C-specific primers were designed and a single PCR product was amplified. Analysis of the nucleotide sequence has revealed a putative peptide differing by only one amino acid residue from the sequence of leucocin C. No identical peptide or protein has been reported in the literature, and this peptide, termed leucocin C-607, was therefore considered to be a new variant of leucocin C produced by Leuc. pseudomesenteroides 607. Another antibacterial peptide purified from the same culture supernatant had a molecular size of 3007.7 or 3121.97Da. However, detailed information regarding this second peptide remains to be determined. Distinct characteristics, such as heat stability and inhibitory spectrum, were observed for the two bacteriocins produced by Leuc. pseudomesenteroides 607. These results suggested that Leuc. pseudomesenteroides 607 produces leucocin C-607 along with another unknown bacteriocin.

Structural elucidation and molecular characterization of Marinobacter sp. α-amylase

ABSTRACTHalophiles have been perceived as potential source of novel enzymes in recent years. The interest emanates from their ability to catalyze efficiently under high salt and organic solvents. Marinobacter sp. EMB8 α-amylase was found to be active and stable in salt and organic solvents. A study was carried out using circular dichroism (CD), fluorescence spectroscopy, and bioinformatics analysis of similar protein sequence to ascertain molecular basis of salt and solvent adaptability of α-amylase. Structural changes recorded in the presence of varying amounts of NaCl exhibited an increase in negative ellipticity as a function of salt, confirming that salt stabilizes the protein and increases the secondary structure, making it catalytically functional. The data of intrinsic and extrinsic fluorescence (using 1-anilinonaphthalene 8-sulfonate [ANS] as probe) further confirmed the role of salt. The α-amylase was active in the presence of nonpolar solvents, namely, hexane and decane, but inactivated by ethanol. The decrease in the activity was correlated with the loss of tertiary structure in the presence of ethanol. Guanidine hydrochloride and pH denaturation indicated the molten globule state at pH 4.0. Partial N-terminal amino acid sequence of the purified α-amylase revealed the relatedness to Pseudoalteromonas sp. α-amylase. “FVHLFEW” was found as the N-terminal signature sequence. Bioinformatics analysis was done using M. algicola α-amylase protein having the same N-terminal signature sequence. The three-dimensional structure of Marinobacter α-amylase was deduced using the I-TASSER server, which reflected the enrichment of acidic amino acids on the surface, imparting the stability in the presence of salt. Our study clearly indicate that salt is necessary for maintaining the secondary and tertiary structure of halophilic protein, which is a necessary prerequisite for catalysis.

Isolation, characterization, kinetics, and enzymatic and nonenzymatic microbicidal activities of a novel c-type lysozyme from plasma of Schistocerca gregaria (Orthoptera: Acrididae).

A protein, designated as Sgl, showing a muramidase lytic activity to the cell wall of the Gram-positive bacterium Micrococcus lysodeikticus was isolated for the first time from plasma of Escherichia coli-immunized fifth instar Schistocerca gregaria. The isolated Sgl was detected as a single protein band, on both native- and SDS-PAGE, has a molecular weight of ∼15.7 kDa and an isoelectric point (pI) of ca 9.3 and its antiserum has specifically recognized its isolated form. Fifty-nine percentage of Sgl lytic activity was recovered in the isolated fractions and yielded ca 126-fold increase in specific activity than that of the crude. The partial N-terminal amino acid sequence of the Sgl has 55 and 40% maximum identity with Bombyx mori and Gallus gallus c-type lysozymes, respectively. The antibacterial activity against the Gram-positive and the Gram-negative bacteria were comparatively stronger than that of the hen egg white lysozyme (HEWL). The detected Sgl poration to the inner membrane that reach a maximum ability after 3 h was suggested to operate as a nonenzymatic mechanism for Gram-negative bacterial cell lysis, as tested in a permease-deficient E. coli, ML-35 strain. Sgl showed a maximal muramidase activity at pH 6.2, 30–50°C, and 0.05 M Ca2+ or Mg2+; and has a Km of 0.5 μg/ml and a Vmax of 0.518 with M. lysodeikticus as a substrate. The Sgl displayed a chitinase activity against chitin with a Km of 0.93 mg/ml and a Vmax of 1.63.

A protein from Pleurotus eryngii var. tuoliensis C.J. Mou with strong removal activity against the natural steroid hormone, estriol: Purification, characterization, and identification as a laccase

A protein with strong removal activity against the natural estrogen estriol was purified from a culture supernatant of Pleurotus eryngii var. tuoliensis C.J. Mou. The protein was characterized as a laccase and had a molecular mass of 60kDa on SDS-PAGE. The enzyme was most active at pH 7.0 and 50°C. The partial N-terminal amino acid sequence of the enzyme showed homology with laccases from mushrooms, such as Pleurotus ostreatus, Coriolus versicolor (current name: Trametes versicolor), Pycnoporus cinnabarinus, and P. eryngii. A recombinant yeast assay confirmed that laccase treatment was very efficient for removing the estrogenic activity of steroid estrogens. Our results suggest that the enzyme may be applicable as a potential factor for removing natural steroid hormones.

Antioxidant peptides from Cicer arietinum of Xinjiang, China

Four peptides of molecular weights 1.148, 4.68, 5.41, and 9.086 kDa with antioxidant activity were isolated from chickpea sprout using two-step ion-exchange chromatographic and HPLC techniques for the first time. The partial N-terminal amino acid sequences of the peptide named YZDBCM-1 (9.086 kDa) was determined as H2N1-15-H2N-Ala-Ile-Thr-Cys-Gly-Arg-Val-Ser-Ala-Ala-Leu-Ala-Pro-Pro-Leu using the Edman automated sequencing apparatus, which was a new peptide rich in alanine. It was shown that the antioxidant activity of the peptide YZDBCM-1 was IC50 156.2 mu g/mL (17.2 mu mol/L) to the free radical of ABTS.

IN VITRO ANTIVIRAL ACTIVITY OF AN ALKALINE TRYPSIN FROM THE DIGESTIVE JUICE OF Bombyx mori LARVAE AGAINST NUCLEOPOLYHEDROVIRUS

Alkaline trypsin protein of molecular mass 25,436 Da purified from the digestive juice of Bombyx mori larvae indicated strong antiviral activity against Bombyx mori nucleopolyhedrovirus (BmNPV) under in vitro conditions. Partial N-terminal amino acid sequence of the protein was determined and the cDNA was cloned based on the amino acid sequence. A homology search of the deduced amino acid sequence of the cDNA showed 55% identity with Helicoverpa armigera trypsin and the active site of this protein was completely conserved. Hence, the protein was designated B. mori trypsin (Bmtryp). The results suggest that Bmtryp, an insect digestive enzyme, can be a potential antiviral factor against BmNPV at the initial site of viral infection.

Circulating salmon 28- and 22-kDa insulin-like growth factor binding proteins (IGFBPs) are co-orthologs of IGFBP-1

Circulating insulin-like growth factor binding proteins (IGFBPs) play pivotal roles in stabilizing IGFs and regulating their availability to target tissues. In the teleost circulation, three major IGFBPs are typically detected by ligand blotting with molecular masses around 20–25, 28–32 and 40–45 kDa. However, their identity is poorly established and often confused. We previously identified salmon 22- and 41-kDa forms as IGFBP-1 and -2b, respectively. In the present study, we cloned the cDNA of 28-kDa IGFBP from Chinook salmon ( Oncorhynchus tshawytscha) as well as rainbow trout ( Oncorhynchus mykiss) based on the partial N-terminal amino acid sequence of purified protein and identified it as an ortholog of IGFBP-1. Structural and phylogenetic analyses revealed that the 28-kDa IGFBP is more closely related to human IGFBP-1 and zebrafish IGFBP-1a than the previously identified salmon IGFBP-1 (i.e. 22-kDa IGFBP). We thus named salmon 28- and 22-kDa forms as IGFBP-1a and -1b, respectively. Salmon IGFBP-1a contains a potential PEST region involved in rapid protein turnover and phosphorylation sites typically found in mammalian IGFBP-1, although the PEST and phosphorylation scores are not as high as those of human IGFBP-1. There was a striking difference in tissue distribution patterns between subtypes; Salmon igfbp-1a was expressed in a variety of tissues while igfbp-1b was almost exclusively expressed in the liver, suggesting that IGFBP-1a has more local actions. Direct seawater exposure (osmotic stress) of Chinook salmon parr caused increases in both IGFBP-1s in plasma, while IGFBP-1b appeared to be more sensitive. The presence of two co-orthologs of IGFBP-1 in the circulation in salmon, and most likely in other teleosts, provides a good opportunity to investigate subfunction partitioning of duplicated IGFBP-1 during postnatal growth.

Antibacterial Peptides of The Ovine Reproductive Tract*

The female sheep reproductive tracts were freshly collected from a local meat processing plant and used as experimental materials.Two antibacterial peptides were isolated and characterized from female sheep reproductive tracts by two consecutive chromatographic steps.The peptide isolation procedures included acetic acid extraction,dialyzed,gel filtration chromatography on Sephadex G-50,and reverse phase high-performance liquid chromatography (RP-HPLC).Their molecular mass were 4 820.47 u and 4 012.5 u,respectively,analyzed by MALDI-TOF-MS.The partial N-terminal amino acid sequences of two peptides were determined as AYVLDEPKP and YDSGA,respectively,by Edman degradation.The antimicrobial activity was tested during each purification step by the radial diffusion plate assay and broth microdilution method.These two peptides showed good antimicrobial activities against reference strains of G + (S.aureus ATCC2592 and Streptococcu ATCC55121),G (E.coli ATCC25922) and fungi(C.albicans ATCC2002).The peptides did not show active hemolytic activity against rabbit blood red cells and had no significant effects on human blood coagulation system.The discovery of antibacterial peptides in sheep reproductive system reveals that antibacterial peptides may play a role in innate immunity against microorganisms in a wide range of animal species.

Purification and characterization of cysteine proteinase inhibitors from crucian carp Carassius auratus eggs

Two cystatins (cst-I and cst-II) were purified from crucian carp eggs by acidification and subsequent ion exchange and molecular sieve chromatography. The molecular masses of cst-I and cst-II analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis were 11.9 and 14.4 kDa, respectively, under reducing conditions and 13.5 and 12.7 kDa, respectively, under non-reducing conditions. The cst-I and cst-II molecules were stable after 30 min of incubation at 60 and 50°C, respectively. There was no significant loss in the inhibitory activity of either cst in the pH range 4–11. These two cystatins were able to affect the proteolysis of papain, cathepsin L, and bromelain, but they were unable to inhibit cathepsin B and trypsin. The partial N-terminal amino acid sequences of both cst inhibitors were homologous and that of cst-I was recognized as NH2-AGIPGGLVDADINDADVQ. This latter fragment shared 88.9% identity to common carp cystatin and 44.4–55.6% to cystatins of other aquatic animals. Based on these results, we conclude that the two cst inhibitors are members of family II cystatin.

A new cytolytic protein from the sea anemone Urticina crassicornis that binds to cholesterol- and sphingomyelin-rich membranes

A new pore-forming cytolytic protein was isolated from the Northern red sea anemone, Urticina crassicornis. Its biochemical properties were characterized and partial N-terminal amino acid sequence was determined. The cytolysin, named UcI, has a molecular mass of around 30 kDa and lacks phospholipase A 2 activity. UcI lyses bovine erythrocytes at nanomolar concentrations. Hemolysis is a result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores and can be prevented by osmotic protectants of size >600 Da. The functional radius of an average pore was estimated to be about 0.66 nm. A more detailed study of the cytolytic activity of UcI was performed with lipid vesicles and monolayers. The toxin binds to monolayers and efficiently permeabilizes small lipid vesicles composed of sphingomyelin and cholesterol. However, the cytolytic activity is not prevented by preincubation with either pure cholesterol or sphingomyelin dispersions. We conclude that the presence of both sphingomyelin and cholesterol, key components of lipid rafts, greatly enhances toxin binding to membranes and probably facilitates pore formation. Alignment of the toxin partial amino acid sequence with sequences of cytolysins belonging to the actinoporin family reveals no sequence homology. We conclude that partial sequence of UcI resembles only the N-terminal part of UpI, a cytolytic protein isolated from a related sea anemone species, Urticina piscivora. The two proteins most probably belong to a separate family of sea anemone cytolysins that are worthy of further characterization.

A novel anti-plant viral protein from coelomic fluid of the earthworm Eisenia foetida: Purification, characterization and its identification as a serine protease

A novel protein showing strong antiviral activities against cucumber mosaic virus (CMV) and tomato mosaic virus (TMV) was purified from the coelomic fluid of the earthworm Eisenia foetida. The protein was characterized as a cold-adapted serine protease. Its molecular weight was estimated to be 27,000 by SDS-PAGE. The enzyme was most active at pH 9.5 and 40–50 °C. The protease activity at 4 °C was 60% of that obtained at the optimal temperature. The activity was suppressed by various serine protease inhibitors. Partial N-terminal amino acid sequence of the enzyme showed homology with serine proteases of earthworms, E. foetida and Lumbricus rubellus previously studied. Our results suggest that the enzyme can be applicable as a potential antiviral factor against CMV, TMV, and other plant viruses.

Two L‐amino acid oxidase isoenzymes from Russell’s viper (Daboia russelli russelli) venom with different mechanisms of inhibition by substrate analogs

Two isoforms, L(1) and L(2), of L-amino acid oxidase have been isolated from Russell's viper venom by Sephadex G-100 gel filtration followed by CM-Sephadex C-50 ion exchange chromatography. The enzymes, with different isoelectric points, are monomers of 60-63 kDa as observed from size exclusion HPLC and SDS/PAGE. Partial N-terminal amino acid sequencing of L(1) and L(2) showed significant homology with other snake venom L-amino acid oxidases. Both the enzymes exhibit marked substrate preference for hydrophobic amino acids, maximum catalytic efficiency being observed with L-Phe. Inhibition of L(1) and L(2) by the substrate analogs N-acetyltryptophan and N-acetyl-L-tryptophan amide has been followed. The initial uncompetitive inhibition of L(1) followed by mixed inhibition at higher concentrations suggested the existence of two different inhibitor-binding sites distinct from the substrate-binding site. In the case of L(2), initial linear competitive inhibition followed by mixed inhibition suggested the existence of two nonoverlapping inhibitor-binding sites, one of which is the substrate-binding site. An inhibition kinetic study with O-aminobenzoic acid, a mimicking substrate with amino, carboxylate and hydrophobic parts, indicated the presence of three and two binding sites in L(1) and L(2), respectively, including one at the substrate-binding site. An inhibitor cross-competition kinetic study indicated mutually excluding binding between N-acetyltryptophan, N-acetyl-L-tryptophan amide and O-aminobenzoic acid in both the isoforms, except at the substrate-binding site of L(1). Binding of substrate analogs with different electrostatic and hydrophobic properties provides useful insights into the environment of the catalytic sites. Furthermore, it predicts the minimum structural requirement for a ligand to enter and anchor at the respective functional sites of LAAO that may facilitate the design of suicidal inhibitors.

Functional Interaction of Neuronal Cav1.3 L-type Calcium Channel with Ryanodine Receptor Type 2 in the Rat Hippocampus

Neuronal L-type Ca(2+) channels do not support synaptic transmission, but they play an essential role in synaptic activity-dependent gene expression. Ca(v)1.2 and Ca(v)1.3 are the two most widely expressed L-type Ca(2+) channels in neurons and have different biophysical and subcellular distributions. The function of the Ca(v) 1.3 L-type Ca(2+) channel and its cellular mechanisms in the central nervous system are poorly understood. In this study, using a yeast two-hybrid assay, we found that the N terminus of the rat Ca(v)1.3 alpha(1) subunit interacts with a partial N-terminal amino acid sequence of ryanodine receptor type 2 (RyR2). Reverse transcription-PCR and Western blot assays revealed high expression of both Ca(v)1.3 and RyR2 in the rat hippocampus. We also demonstrate a physical association of Ca(v)1.3 with RyR2 using co-immunoprecipitation assays. Moreover, immunocytochemistry revealed prominent co-localization between Ca(v)1.3 and RyR2 in hippocampal neurons. Depolarizing cells by an acute treatment of a high concentration of KCl (high-K, 60 mm) showed that the activation of L-type Ca(2+) channels induced RyR opening and led to RyR-dependent Ca(2+) release, even in the absence of extracellular Ca(2+). Furthermore, we found that RyR2 mRNA itself is increased by long term treatment of high-K via activation of L-type Ca(2+) channels. These acute and long term effects of high-K on RyRs were selectively blocked by small interfering RNA-mediated silencing of Ca(v)1.3. These results suggest a physical and functional interaction between Ca(v)1.3 and RyR2 and important implications of Ca(v)1.3/RyR2 clusters in translating synaptic activity into alterations in gene expression.

The P-Glycoprotein (ABCB1) Linker Domain Encodes High-Affinity Binding Sequences to α- and β-Tubulins

P-Glycoprotein (or ABCB1) has been shown to cause multidrug resistance in tumor cell lines selected with lipophilic anticancer drugs. ABCB1 encodes a duplicated molecule with two hydrophobic and hydrophilic domains linked by a highly charged region of approximately 90 amino acids, the "linker domain" with as yet unknown function(s). In this report, we demonstrate a role for this domain in binding to other cellular proteins. Using overlapping hexapeptides that encode the entire amino acid sequence of the linker domain of human ABCB1, we show a direct and specific binding between sequences in the linker domain and several intracellular proteins. Three different polypeptide sequences [617EKGIYFKLVTM627 (LDS617-627), 657SRSSLIRKRSTRRSVRGSQA676 (LDS657-676), and 693PVSFWRIMKLNLT705 (LDS693-705)] in the linker domain interacted tightly with several proteins with apparent molecular masses of approximately 80, 57, and 30 kDa. Interestingly, only the 57 kDa protein (or P57) interacted with all three different sequences of the linker domain. Purification and partial N-terminal amino acid sequencing of P57 showed that it encodes the N-terminal amino acids of alpha- and beta-tubulins. The identity of the P57 interacting protein as tubulins was further confirmed by Western blotting using monoclonal antibodies to alpha- and beta-tubulin. Taken together, the results of this study provide the first evidence for ABCB1 protein interaction mediated by sequences in the linker domain. These findings are likely to provide further insight into the functions of ABCB1 in normal and drug resistant tumor cells.

Identification of insect-selective and mammal-selective toxins from Parabuthus leiosoma venom

Venoms were collected from two scorpion species: Parabuthus leiosoma and Parabuthus pallidus from Kenya. Subcutaneous injection and oral toxicity tests of crude and pure fractions of scorpion venoms were done in Mus musculus (mice), Chilo partellus and Busseola fusca. The highest activity against C. partellus was found in P. leiosoma venom (LC 50 0.689 mg/50 mg body weight). Bioassay-guided purification by a combination of cation-exchange (CE) and reverse-phase high-performance liquid chromatography (RP-HPLC) led to the isolation of three toxic peptides. A lepidopteran-selective toxin ( P. leiosoma insect toxin, Plit) was isolated, and the partial N-terminal amino acid sequence (-KDGYPVDNANCKYE-) plus the molecular weight (6688.5 Da) determined. A peptide with significant insect toxicity coupled with mild effects on mice ( P. leiosoma toxin, Plt) was isolated, and the partial N-terminal amino acid sequence (-LCEKFKVQRLVELNCVD-) plus the molecular weight (6742.5 Da) was determined. Another toxin with anti-mammalian activity ( P. leisoma mammal-selective toxin, Plmt), and N-terminal partial amino acid sequence of ADVPGNYPLDKNGNRYY- plus a molecular weight of 7145.5 Da was also isolated. Comparison of the partial N-terminal amino acid sequences with other toxins revealed that Plit shows high homology to other known insect toxins. Similarly, Plmt shows high homology with several birtoxin-like anti-mammalian toxins. Plt does not exhibit homology with any known scorpion toxin with combined anti-insect and anti-mammalian activity.

Purification, characterization and comparison of reptile lysozymes

Cation exchange column chromatography and gel filtration chromatography were used to purify four reptile lysozymes from egg white: SSTL A and SSTL B from soft shelled turtle ( Trionyx sinensis), ASTL from Asiatic soft shelled turtle ( Amyda cartilagenea) and GSTL from green sea turtle ( Chelonia mydas). The molecular masses of the purified reptile lysozymes were estimated to be 14 kDa by SDS-PAGE. Enzyme activity of the four lysozymes could be confirmed by gel zymograms and showed charge differences on native-PAGE. SSTL A, SSTL B and ASTL had sharp pH optima of about pH 6.0, which contrasts with that of GSTL, which showed dual pH optima at about pH 6.0 and pH 8.0. The activities of the reptile lysozymes rapidly decreased within 30 min of incubation at 90 °C except for ASTL, which was more stable. Partial N-terminal amino acid sequencing and peptide mapping strongly suggested that the enzymes were C-type lysozymes. Interestingly, the mature SSTL lysozymes show an extra Gly residue at the N-terminus, which was previously found in soft-shelled turtle lysozyme. The reptile lysozymes showed lytic activity against several species of bacteria, such as Micrococcus luteus and Vibrio cholerae, but showed only weak activity to Pseudomonas aeruginosa and lacked activity towards Aeromonas hydrophila.

Molecular modeling and inhibitory activity of cowpea cystatin against bean bruchid pests

Plant cystatins show great potential as tools to genetically engineer resistance of crop plants against pests. Two important potential targets are the bean weevils Acanthoscelides obtectus and Zabrotes subfasciatus, which display major activities of digestive cysteine proteinases in midguts. In this study a cowpea cystatin, a cysteine proteinase inhibitor found in cowpea (Vigna unguiculata) seeds, was expressed in Escherichia coli and purified with a Ni-NTA agarose column. It strongly inhibited papain and proteinases from midguts of both A. obtectus and Z. subfasciatus bruchids, as seen by in vitro assays. When the protein was incorporated into artificial seeds at concentrations as low as 0.025%, and seeds were consumed by the bruchids larva, dramatic reductions in larval weight, and increases in insect mortality were observed. Molecular modeling studies of cowpea cystatin in complex with papain revealed that five N-terminal residues responsible for a large proportion of the hydrophobic interactions involved in the stabilization of the enzyme-inhibitor complex are absent in the partial N-terminal amino acid sequencing of soybean cystatin. We suggest that this structural difference could be the reason for the much higher effectiveness of cowpea cystatin when compared to that previously tested phytocystatin. The application of this knowledge in plant protein mutation programs aiming at enhancement of plant defenses to pests is discussed.