Bowman-Birk Type Protease Inhibitor Research Articles

Combining elicitor treatment of chitosan, methyl jasmonate, and cyclodextrin to induce the generation of immune response bioactive peptides in peanut hairy root culture

The endogenous peptides from peanut hairy root culture were induced upon elicitor treatment with chitosan (CHT), methyl jasmonate (MeJA), and cyclodextrin (CD): CHT+MeJA+CD. The peptides secreted into the liquid culture medium play an important role in plant signaling and stress responses. By performing gene ontology (GO) analysis, a number of plant proteins involved in biotic and abiotic defense responses were identified, such as endochitinase, defensin, antifungal protein, cationic peroxidase and Bowman-Birk type protease inhibitor A-II. The bioactivity of 14 peptides synthesized from secretome analysis was determined. Peptide BBP1–4, derived from the diverse region of Bowman-Birk type protease inhibitor, displayed high antioxidant activity and mimicked the property of chitinase and β-1,3-glucanase enzymes. The antimicrobial activity against S. aureus, S. typhimurium, and E. coli was evidenced with different peptide concentrations. Additionally, peptide BBP1–4 has the potential to be a useful candidate for an immune response property, as it was found to increase the expression of some pathogenesis-related (PR) proteins and stilbene biosynthesis genes in peanut hairy root tissues. The findings indicate that secreted peptides may play a role in plant responses to both abiotic and biotic stresses. These peptides, which possess bioactive properties, could be considered as potential candidates for use in the pharmaceutical, agricultural, and food industries.

Cloning, Characterization, Expression Analysis, and Agglutination Studies of Novel Gene Encoding β-d-Galactose, N-Acetyl-d-Glucosamine and Lactose-Binding Lectin from Rice Bean (Vigna umbellata)

Lectins are glycoproteins and known for their peculiar carbohydrate-binding activity and their insect-pest-resistant properties. Earlier we have published our research finding on novel gene encoding Bowman-Birk type protease inhibitor with insecticidal properties from rice bean. This paper presents first report on cloning, sequencing, and expression of RbL ORF of 843bp encoding 280 amino acids long lectin precursor from rice bean (Vigna umbellata) seeds. Blast analysis revealed more than 90% similarity of RbL protein with Vigna aconitifolia and Vigna angularis lectins. Phylogenetic analysis also revealed a close relationship between RbL and other legume lectins. Sequence analysis of genomic DNA revealed intronless nature of RbL gene (GenBank accession No. MT043160). The isolated RbL ORF was expressed in E. coli BL-21(DE3) cells and maximum expression was recorded with 0.5mM IPTG after 4h incubation at 37°C. Western blotting confirmed RbL protein expression in E. coli. Recombinant protein (His6-RbL) of ~ 35kDam.wt was purified using Ni-NTA affinity chromatography to the extent of 0.26mg/ml. In silico analysis characterized RbL protein as acidic, stable, hydrophobic, and secretary protein with one signal peptide cleavage site (A26-A27) and four N-glycosylation sites. Template-based 3D model of RbL was structured using MODELLER tool and validated as good quality model. Structural analysis revealed dominance of β-pleated sheets and β-turns in RbL protein structure. β-D-galactose, N-acetyl-D-glucosamine, and lactose were predicted as putative ligands for RbL protein. Hydrogen bonding and hydrophobic forces were the major interactions between the predicted ligands and RbL protein. Agglutination and agglutination inhibition assays confirmed the binding specificity of RbL protein with the trypsinized rabbit erythrocytes and with the predicted ligands, respectively. Gene ontology analysis functionally annotated RbL protein as a plant defense protein. The novel information generated in the study is not mere pre-experimental findings but could also lay foundation for future research on exploring RbL gene and encoding protein for different biomedical and biotechnological applications.

Ranacyclin-NF, a Novel Bowman-Birk Type Protease Inhibitor from the Skin Secretion of the East Asian Frog, Pelophylax nigromaculatus.

Serine protease inhibitors are found in plants, animals and microorganisms, where they play important roles in many physiological and pathological processes. Inhibitor scaffolds based on natural proteins and peptides have gradually become the focus of current research as they tend to bind to their targets with greater specificity than small molecules. In this report, a novel Bowman–Birk type inhibitor, named ranacyclin-NF (RNF), is described and was identified in the skin secretion of the East Asian frog, Pelophylax nigromaculatus. A synthetic replicate of the peptide was subjected to a series of functional assays. It displayed trypsin inhibitory activity with an inhibitory constant, Ki, of 447 nM and had negligible direct cytotoxicity. No observable direct antimicrobial activity was found but RNF improved the therapeutic potency of Gentamicin against Methicillin-resistant Staphylococcus aureus (MRSA). RNF shared significant sequence similarity to previously reported and related inhibitors from Odorrana grahami (ORB) and Rana esculenta (ranacyclin-T), both of which were found to be multi-functional. Two analogues of RNF, named ranacyclin-NF1 (RNF1) and ranacyclin-NF3L (RNF3L), were designed based on some features of ORB and ranacyclin-T to study structure–activity relationships. Structure–activity studies demonstrated that residues outside of the trypsin inhibitory loop (TIL) may be related to the efficacy of trypsin inhibitory activity.

Molecular insights into the functional role of nitric oxide (NO) as a signal for plant responses in chickpea.

The molecular mechanisms and targets of nitric oxide (NO) are not fully known in plants. Our study reports the first large-scale quantitative proteomic analysis of NO donor responsive proteins in chickpea. Dose response studies carried out using NO donors, sodium nitroprusside (SNP), diethylamine NONOate (DETA) and S-nitrosoglutathione (GSNO) in chickpea genotype ICCV1882, revealed a dose dependent positive impact on seed germination and seedling growth. SNP at 0.1mM concentration proved to be most appropriate following confirmation using four different chickpea genotypes. while SNP treatment enhanced the percentage of germination, chlorophyll and nitrogen contents in chickpea, addition of NO scavenger, cPTIO reverted its impact under abiotic stresses. Proteome profiling revealed 172 downregulated and 76 upregulated proteins, of which majority were involved in metabolic processes (118) by virtue of their catalytic (145) and binding (106) activity. A few crucial proteins such as S-adenosylmethionine synthase, dehydroascorbate reductase, pyruvate kinase fragment, 1-aminocyclopropane-1-carboxylic acid oxidase, 1-pyrroline-5-carboxylate synthetase were less abundant whereas Bowman-Birk type protease inhibitor, non-specific lipid transfer protein, chalcone synthase, ribulose-1-5-bisphosphate carboxylase oxygenase large subunit, PSII D2 protein were highly abundant in SNP treated samples. This study highlights the protein networks for a better understanding of possible NO induced regulatory mechanisms in plants.

Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean.

This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327 bp encoding 109 amino acids was cloned from rice bean seeds using degenerate primer set. BlastP search revealed that the RbTI encoded amino acid of approx 13.0 kDa and shared 99% homology each with BBI from Phaseolus parvulus, Vigna trilobata and Vigna vexilata. Phylogenetic tree also showed close relationship of RbTI with BBI from other members of Leguminaceae family. RbTI gene was further confirmed as intronless (GenBank accession no. KJ159908). The secondary and 3D-structural models for the RbTI were predicted with homology modeling. qRT-PCR studies revealed the highest RbTI expression in the seeds nearing maturity, whereas the low expression of the gene was noticed in young leaves. The isolated RbTI was successfully expressed in Escherichiacoli and the highest expression was recorded after 5.5h of induction. Study on the inhibitory activity of expressed protein against the gut proteases of Hessian fly larvae revealed 87% inhibition. The novel RbTI gene will further broaden the pool of plant defense genes and could be an ideal choice for developing transgenic crops resistant to insect pests with high economic value. In addition, it has the potential to be used as a probe for selection of insect- and pathogen-resistant genotypes.

HV-BBI—A novel amphibian skin Bowman–Birk-like trypsin inhibitor

Here we describe the isolation of a novel C-terminally amidated octadecapeptide—SVIGCWTKSIPPRPCFVK-amide—that contains a disulphide loop between Cys 5 and Cys 15 that is consistent with a Bowman–Birk type protease inhibitor, from the skin secretion of the Chinese Bamboo odorous frog, Huia versabilis. Named HV-BBI, the peptide is encoded by a single precursor of 62 amino acid residues whose primary structure was deduced from cloned skin cDNA. The precursor exhibits the typical organization of that encoding an amphibian skin peptide with a highly-conserved signal peptide, an intervening acidic amino acid residue-rich domain and a single HV-BBI-encoding domain located towards the C-terminus. A synthetic replicate of HV-BBI, with the wild-type K (Lys-8) residue in the presumed P1 position, was found to be a potent inhibitor of trypsin with a K i just slightly less than 19 nM. Substitution at this site with R (Arg) resulted in a significant reduction in potency ( K i 57 nM), whereas replacement of K with F (Phe) resulted in the complete abolition of trypsin inhibitory activity. Thus, HV-BBI is a potent inhibitor of trypsin and the lysyl (K) residue that occupies the P1 position appears to be optimal for potency of action against this protease.

Multigene family for Bowman-Birk type proteinase inhibitors of wild soja and soybean: the presence of two BBI-A genes and pseudogenes.

Genes for Bowman-Birk type protease inhibitors (BBIs) of wild soja (Glycine soja) and soybean (Glycine max) comprise a multigene family. The organization of the genes for wild soja BBIs (wBBIs) was elucidated by an analysis of their cDNAs and the corresponding genomic sequences, and compared with the counterparts in the soybean. The cDNAs encoding three types of wild soja BBIs (wBBI-A, -C, and -D) were cloned. Two subtypes of cDNAs for wBBI-A, designated wBBI-A1 and -A2, were further identified. Similar subtypes (sBBI-A1 and -A2) were also found in the soybean genome. cDNA sequences for wBBIs were highly homologous to those for the respective soybean homologs. Phylogenetic analysis of these cDNAs demonstrated the evolutional proximity between these two leguminae strains.

Processing of soybean hulls to enhance the distribution and extraction of value‐added proteins

AbstractSoybean hulls contain peroxidase (SBP) and Bowman–Birk type protease inhibitor (BBI). SBP is used in the European bread‐baking industry, and BBI isolated from dehulled, defatted soy flour possesses cancer‐preventive and anticarcinogenic properties. Because hulls possess a low percentage of nitrogen, with fibre and other carbohydrates being the major components, our objective was to determine whether air classification of milled hulls could be used to fractionate and enrich these value‐added proteins. Hulls were pin milled three times at 18 000 rpm, followed by air classification to yield fractions with particle size distributions ranging from <15 to >30 µm. The finely ground fractions with particle sizes ranging from <15 to 18 µm were enriched with nitrogen and lipid when compared with the more coarsely ground fractions. Yields of extractable nitrogenous components from aqueous extracts were quantified at a variety of pHs, homogenisation speeds and times. BBI and SBP were confirmed by polyacrylamide gel electrophoresis with known standards. Based on assays for SBP and BBI activities, BBI, though concentrated in the finer fractions, also appeared in the highest‐mass, coarsest fraction, whereas SBP resided mainly in the coarsest fraction. Dry milling and air classification of soybean hulls proved beneficial to distribute SBP into a product stream that facilitates its isolation and purification. Published in 2003 for SCI by John Wiley & Sons, Ltd.

Methionine-induced stabilization of Bowman–Birk protease inhibitor mRNA

Abstract Supplemental methionine in a complete culture medium increases the Bowman-Birk-type protease inhibitor (BBPI) level in cultured developing soybean (Glycine max, L. Merrill) cotyledons. The increase of BBPI level was paralleled by an increase in the mRNA level. Nuclear run-on analysis showed that transcriptional activities of the BBPI genes were not different between cotyledons cultured in media with or without added methionine. Kinetics of changes in BBPI mRNA levels suggested a change in BBPI mRNA stability upon adding or omitting methionine from the medium. Direct measurement of BBPI mRNA by the use of cordycepin revealed that BBPI mRNAs were much more stable in cotyledons cultured in medium supplemented with methionine than in cotyledons cultured in medium without methionine. No major difference or heterogeneity in the length of BBPI mRNA poly(A) tails in cotyledons cultured in medium with or without met was seen.

Crystallographic Refinement of Bowman-Birk Type Protease Inhibitor A-II from Peanut (Arachis hypogaea) at 2·3 Å Resolution

The crystal structure of Bowman-Birk type protease inhibitor A-II from peanut was refined at 2·3 Å resolution using a restrained least-squares method. The crystallographic R-factor is 0·196 for 7697 reflections with F>3σ (F) in the range from 6·0 to 2·3 Å resolution. Two molecules in an asymmetric unit are independently refined and, their structures are compared with each other. The inhibitor molecule has an elongated shape with two reactive sites, one at both ends of the longest dimension. As a secondary structure, a 4-stranded β-sheet-like structure is found, in which two water molecules bind two 2-stranded β-sheets together with six hydrogen bonds. The molecule is constructed by two homologous domains which are related by an intramolecular pseudo 2-fold axis. The structure and atomic B-factors of peptide loops containing a reactive site were compared with that of adzuki bean Bowman-Birk type inhibitor in the complex with bovine β-trypsin. This comparison shows that no significant structural change occurs in the reactive site of inhibitor at the formation of the inhibitor-protease complex, but structural rigidity around the reactive site seems to increase.

The Structure of Bowman-Birk Type Protease Inhibitor A-il from Peanut (Arachis hypogaea) at 3.3 Å Resolution<xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

The structure of Bowman-Birk type protease inhibitor (A-II from peanut) is described at 3.3 A resolution. The molecules form a tetramer with 222 local symmetry in our crystals. Each monomer has an elongated shape with approximate dimensions of 45 X 15 X 15 A and consists of two distinct domains. The three-dimensional structures of the two domains are similar and are related by the intramolecular approximate twofold rotation axis. The two independent protease binding sites protrude from the molecular body on opposite sides. A scheme for the molecular evolution of the double-headed Bowman-Birk type protease inhibitors is proposed, based on the three-dimensional structure.

Structure of the trypsin-binding domain of Bowman-Birk type protease inhibitor and its interaction with trypsin.

The crystal structure of the complex formed by bovine trypsin and Bowman-Birk type protease inhibitor AB-I extracted from azuki beans (Vigna angularis) 'Takara' has been analyzed. The structure was solved by the application of the phase combination of single isomorphous phases and trypsin model phases, followed by phase improvement using the iterative Fourier technique. From the resulting electron density map, a three-dimensional atomic model of the trypsin binding domain of AB-I has been built. The peptide chain at the trypsin reactive site turns back sharply at Pro29 and forms a 9-residue ring (Cys24-Cys32). The 'front side' of this ring, consisting of the reactive site (Cys24-Met28), interacts with trypsin in a similar manner to other families of inhibitors and forms a stable complex, which seems to be maintained by the interactions with the 'back side' of this ring (Pro29-Cys34). The similar spatial arrangements of the 'back side' of this inhibitor and the 'secondary contact region' of the other inhibitors with respect to the reactive site suggest an important common role of these regions in exhibiting inhibitory activity.

Crystallization of Bowman-Birk type protease inhibitor (peanut) and its complex with trypsin.

Crystallization and preliminary crystallographic study of Bowman-Birk type protease inhibitors, A-I, A-II, and B-III from peanut seeds (Arachis hypogaea), and of the A-II + trypsin complex were carried out. A-II, with 70 amino acid residues, crystallizes in a trigonal system, P3(1)21 (or P3(2)21), a = 71.8, c = 65.9 A, Z = 12 or 18. The A-I crystal is isomorphous with that of A-II, indicating that the N-terminal residues are in a disordered state in both crystals. The B-III crystal is monoclinic, C2, a = 119.6, b = 69.6, c = 94.2 A, beta = 115.1 degrees, Z is about 40. The A-II + trypsin complex crystallizes in an orthorhombic system, P2(1)2(1)2(1), a = 55.5, b = 56.0, c = 182.1 A, Z = 4.

Crystallographic studies ofSt. trypsinand Bowman–Birk type protease inhibitor

Crystallographic studies of<i>St. trypsin</i>and Bowman–Birk type protease inhibitor

Preliminary crystallographic study of Bowman-Birk protease inhibitor (adzuki bean) and its complex with trypsin.

Preliminary crystallographic studies of a Bowman-Birk type protease inhibitor, AB-I, from adzuki beans (Phaseolus angularis) 'Takara,' and its complex with trypsin were carried out. AB-I, MW 9100 with 82 amino acid residues, crystallizes in a trigonal space group, P3121 (or P3221), with the following unit cell dimensions: a = 68.7, c = 99.7 A. The asymmetric unit contains two dimer molecules. Structure analysis at 5 A resolution revealed the rough appearance of the dimer. The complex between AB-I and trypsin also could be crystallized in a tetragonal space group, P41212 (or P43212), with the unit cell dimensions, a = 55.4 and c = 181.5 A, and Z = 8. The crystallinity seems to be much better than that of the crystals of the inhibitor alone. The other type of inhibitor from adzuki bean, AB-IIa, was also crystallized.

Amino acid sequence of a trypsin-chymotrypsin inhibitor, B-III, of peanut (Arachis hypogaea).

The amino acid sequence of peanut trypsin-chymotrypsin inhibitor, B-III, was determined by conventional methods. The limited proteolysis of B-III with trypsin indicated the reactive sites of B-III for trypsin to be Arg(10)-Arg(11) and Arg(38)-Ser(39). Comparison of the established sequence of B-III with those of other Bowman-Birk type double-headed protease inhibitors indicated that B-III has four amino acid insertions and one amino acid deletion. It is especially interesting that the amino acid residue in the P1' position (No. 11) of the first reactive site for trypsin is arginine instead of serine, which seems to be conserved at the P1' position of the reactive sites of all Bowman-Birk type protease inhibitors.