Mung Bean Trypsin Inhibitor Research Articles

Reconstructed mung bean trypsin inhibitor targeting cell surface GRP78 induces apoptosis and inhibits tumor growth in colorectal cancer

Glucose regulated protein 78 (GRP78) has been reported to be present on cell membranes of cancer cells but not the normal cells, serving as a potential anti-cancer target. In the present study, a fusion protein containing the GRP78 binding peptide WIFPWIQL and the active fragment of mung bean trypsin inhibitor was constructed, and its targeted anti-tumor effects were investigated both in vitro and in vivo. The results showed that the fusion protein specifically inhibited growth and induced apoptosis in colorectal cancer cells but not in the normal cells. Mechanistically, these anti-tumor effects were attributed to induction of G1 phase arrest and activation of multiple apoptotic pathways. Importantly, the fusion protein could also suppress the growth of xenografted human colorectal carcinoma in vivo. Our study reveals that this fusion protein may be developed as a therapeutic agent for treatment of colon cancer, and holds important implications for developing other anti-cancer peptide drugs.

Mung Bean Trypsin Inhibitor Is Effective in Suppressing the Degradation of Myofibrillar Proteins in the Skeletal Muscle of Blue Scad (Decapterus maruadsi)

Mung bean trypsin inhibitor (MBTI) of the Bowman-Birk family was purified to homogeneity with a molecular mass of approximately 9 kDa on tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 8887.25 Da as determined by matrix-assisted laser desorption/ionization-quadrupole ion trap-time-of-flight mass spectrometry (MALDI-QIT-TOF MS). Using blue scad myofibrillar proteins as targets, it was found that, in the absence of MBTI, proteolysis of myofibrillar proteins, especially myosin heavy chain (MHC), could be identified after incubation at 55 °C for 2 h, while in the presence of MBTI, with a final concentration of 25 ng/mL, proteolysis of these proteins was greatly suppressed even after incubation for 3 h. Although cysteine proteinase inhibitor E-64 was also effective in preventing protein degradation, inhibitors for metallo- and asparatic proteinases did not reveal obvious inhibitory effects. Our present results strongly suggested that the naturally occurring legume bean seed protein MBTI can be used as an effective additive in preventing marine fish blue scad surimi gel softening, which is quite possibly caused by myofibril-bound serine proteinase (MBSP).

Small peptides derived from the Lys active fragment of the mung bean trypsin inhibitor are fully active against trypsin

The Bowman-Birk protease inhibitors have recently attracted attention for their potential as cancer preventive and suppressing agents. They contain two canonical binding loops, both consisting of nine highly conserved residues capable of inhibiting corresponding serine proteases. In this study, we cloned the cDNA of the mung bean trypsin inhibitor, one of the most studied Bowman-Birk protease inhibitors. A modified peptide, Lys33GP, with 33 residues derived from the long chain of the Lys active fragment of mung bean trypsin inhibitor, was successfully expressed in Escherichia coli as a glutathione-S-transferase fusion protein. The recombinant product was obtained with a high yield, and exhibited potent inhibitory activity. Meanwhile, a shorter peptide composed of only 16 residues (the Lys16 peptide), corresponding to the active core of the fragment, was synthesized. Both the recombinant and the synthesized peptides had the same inhibitory activity toward trypsin at a molar ratio of 1 : 1, implying that the Lys16 peptide with two disulfide bonds is possibly the essential structural unit for inhibitory activity. Using site-directed mutagenesis, the P(1) position Lys was replaced by Phe, and the resulting mutant, Lys33K/F, was determined to have potent chymotrypsin inhibitory activity. Both Lys33GP and the Lys33K/F mutant may be potential pharmaceutical agents for the prevention of oncogenesis.

Research retrospect on small molecule protease in-hibitors and peptide toxins

This paper is a retrospect of our research works on small molecular weight protease inhibitors and peptide toxins, all of them are active peptides. The Lysin active fragment of the double headed mung bean trypsin inhibitor has been intensively studied. The approaches of sequence determination, gene expression and mutagenesis as well as peptide synthesis are used to indicate that the essen- tial core of the inhibitor is composed of two conjugated disulfide loops each with nine-residues, which shares high homology with the cyclic peptide sun flower trypsin inhibitor. Another described inhibitor is the Trichosanthes trypsin inhibitor belonging to the squish family, its gene sequence was first reported in this family. The studied peptide toxins include scorpion toxins and conotoxins. Many novel potassium channel toxins were found from the scorpion Buthus martensi Karsch, in- cluding the calcium dependent BK or SK potassium channel toxins and voltage dependent toxins. Some of them have two deferent functions displaying at separated region, their structure-function relationships were elucidated. The expressed recombinant scorpion chloride toxin has a potential in treatment of malignant gliomas. From 16 species of cone snail 50 novel conotoxin peptides were identified, and 134 novel conotoxin genes were cloned. Three unreported superfamilies were characterized, designated as V, Y, K superfamily, respectively; some new disulfide frame and arrangement, unique motif, D-form residue in the toxin primary structures were found; the genomic structure of A superfamily was analysed, in the intro there is a very conserved sequence at the 3’end which could be used as a primer to clone more new conotoxin genes; the biological function of some conotoxins were elucidated.

Purification, characterization and evaluation of insecticidal potential of trypsin inhibitor from mungbean (Vigna radiata L. Wilczek) seeds

Protease inhibitors present in seeds of legumes possess strong inhibitory activity against trypsin and confer resistance against pests. In the present investigation, trypsin inhibitor activity was found in the seed flour extracts of all the eight selected varieties of mungbean under study which was further confirmed by dot blot analysis. All the varieties showed inhibitory activity in vitro against the gut protease of Helicoverpa armigera (HGP). Trypsin inhibitor was purified from mungbean seeds to near homogeneity with 58.1-fold and 22.8% recovery using heat denaturation, NH4(SO4)2 fractionation, ion-exchange chromatography on DEAE-Sephadex A-25 and gel filtration through Sephadex G-75. The molecular mass of the inhibitor was 47 kDa as determined by gel filtration and SDS-PAGE. The inhibitor retained 90% or more activity between pH 4 and 10, however, it was nearly inactive at extreme pH values. The inhibitor was stable up to 80°C but thereafter, the activity decreased gradually retaining nearly 30% of activity when heated at 100°C for 20 min. The inhibitor activity was undetectable at 121°C. Insect bioassay experiment using purified mungbean trypsin inhibitor showed a marked decline in survival (%) of larvae with increase in inhibitor concentration. The larval growth was also extended by the trypsin inhibitor. This study signifies the insecticidal potential of mungbean trypsin inhibitor which might be exploited for raising transgenic plants.

Template‐assisted rational design of peptide inhibitors of furin using the lysine fragment of the mung bean trypsin inhibitor

Highly active, small‐molecule furin inhibitors are attractive drug candidates to fend off bacterial exotoxins and viral infection. Based on the 22‐residue, active Lys fragment of the mung bean trypsin inhibitor, a series of furin inhibitors were designed and synthesized, and their inhibitory activity towards furin and kexin was evaluated using enzyme kinetic analysis. The most potent inhibitor, containing 16 amino acid residues with a K i value of 2.45 × 10−9 m for furin and of 5.60 × 10−7 m for kexin, was designed with three incremental approaches. First, two nonessential Cys residues in the Lys fragment were deleted via a Cys‐to‐Ser mutation to minimize peptide misfolding. Second, residues in the reactive site of the inhibitor were replaced by the consensus substrate recognition sequence of furin, namely, Arg at P1, Lys at P2, Arg at P4 and Arg at P6. In addition, the P7 residue Asp was substituted with Ala to avoid possible electrostatic interference with furin inhibition. Finally, the extra N‐terminal and C‐terminal residues beyond the doubly conjugated disulfide loops were further truncated. However, all resultant synthetic peptides were found to be temporary inhibitors of furin and kexin during a prolonged incubation, with the scissile peptide bond between P1 and P1′ being cleaved to different extents by the enzymes. To enhance proteolytic resistance, the P1′ residue Ser was mutated to d‐Ser or N‐methyl‐Ser. The N‐methyl‐Ser mutant gave rise to a K i value of 4.70 × 10−8 m for furin, and retained over 80% inhibitory activity even after a 3 h incubation with the enzyme. By contrast, the d‐Ser mutant was resistant to cleavage, although its inhibitory activity against furin drastically decreased. Our findings identify a useful template for the design of potent, specific and stable peptide inhibitors of furin, shedding light on the molecular determinants that dictate the inhibition of furin and kexin.

Discovery, Structural Determination, and Putative Processing of the Precursor Protein That Produces the Cyclic Trypsin Inhibitor Sunflower Trypsin Inhibitor 1

Backbone-cyclized proteins are becoming increasingly well known, although the mechanism by which they are processed from linear precursors is poorly understood. In this report the sequence and structure of the linear precursor of a cyclic trypsin inhibitor, sunflower trypsin inhibitor 1 (SFTI-1) from sunflower seeds, is described. The structure indicates that the major elements of the reactive site loop of SFTI-1 are present before processing. This may have importance for a protease-mediated cyclizing reaction as the rigidity of SFTI-1 may drive the equilibrium of the reaction catalyzed by proteolytic enzymes toward the formation of a peptide bond rather than the normal cleavage reaction. The occurrence of residues in the SFTI-1 precursor susceptible to cleavage by asparaginyl proteases strengthens theories that involve this enzyme in the processing of SFTI-1 and further implicates it in the processing of another family of plant cyclic proteins, the cyclotides. The precursor reported here also indicates that despite strong active site sequence homology, SFTI-1 has no other similarities with the Bowman-Birk trypsin inhibitors, presenting interesting evolutionary questions.

녹두(Vigna radiata L.) Trypsin Inhibitor의 정제 및 약물학적 특성

녹두(Vigna radiata L.) Trypsin Inhibitor의 정제 및 약물학적 특성

Studies on an artificial trypsin inhibitor peptide derived from the mung bean trypsin inhibitor: chemical synthesis, refolding, and crystallographic analysis of its complex with trypsin.

The active fragment with Lys at the reactive site of mung bean trypsin inhibitor (MBILF) is composed of two peptide chains, A1 of 26 residues and A2 of 9 residues linked via two disulfide bonds. In the present study, a peptide of 22 residue comprising the sequence of chain A1 from position 3 to 24 was synthesized by the solid-phase method. This synthetic peptide with six Cys residues contains a reactive site at position Lys11I-Ser12I (I denotes an inhibitor residue). Air oxidation and HPLC purification resulted in two antitrypsin active components, SPC1 and SPC2. Neither SPC1 nor SPC2 can stoichiometrically inhibit trypsin. The Ki values of SPC1 and SPC2 are 1.2 x 10(-7) and 4.0 x 10(-8) M, respectively. The complexes of SPC1 and SPC2 with bovine beta-trypsin (BTRY) were crystallized by ammonium sulphate precipitation at pH 6.4 and 6.0, respectively. The two crystals have the same crystal form with space group P2(1)2(1)2(1) and cell dimension of a = 63.2(2) A, b = 63.5(6) A, and c = 69.8(4) A. The crystal structure of one complex, SPC1-BTRY, was determined and refined at 2.2 A resolution to a final R-value of 19.2%. From the resulting electron density map, 9 residues of SPC1, from position 9I to 17I, were identified clearly and three-dimension atomic model of the 9-residue reactive loop formed by a disulfide bridge, Cys9I-Cys17I, was built. No electron density corresponding to the other 13 residues was observed in the present map.(ABSTRACT TRUNCATED AT 250 WORDS)

The crystal structure of the ternary complex of mung bean trypsin inhibitor with two porcine trypsin (PTRY-MBI-PTRY) at 2.8 Å resolution

The crystal structure of the ternary complex of mung bean trypsin inhibitor with two porcine trypsin (PTRY-MBI-PTRY) at 2.8 Å resolution

Isolation and Partial Characterization of a Subtilisin Inhibitor from the Mung Bean (Vigna radiata)

The subtilisin inhibitor (MBSI-A) from the mung bean (Vigna radiata (L.) Wilczek) seed has been purified to homogeneity. MBSI-A consists of a single polypeptide chain of 119 residues, with a high content of glutamic acid/glutamine, aspartic acid/asparagine, valine, threonine, and proline (19, 12, 10, 9, and 8 residue percent, respectively). MBSI-A is a potent inhibitor of subtilisin Carlsberg, but is inactive toward bovine trypsin and alpha-chymotrypsin and the plant cysteinyl proteinase papain. The MBSI is located exclusively in the cytosol of the seed cotyledon cell, unlike the mung bean trypsin inhibitor (MBTI), which is located primarily in the protein bodies. Both MBSI and MBTI accumulate in the seed during the most active period of reserve protein accumulation, 12 to 18 days after flowering. During germination MBSI, like MBTI, is broken down beginning 2 to 3 days after seed imbibition. The disappearance of MBSI-A is accompanied by the transient appearance of a new inhibitor species, MBSI-D. The amino acid composition of MBSI-D suggests that it may be produced by the loss of approximately 20 amino acid residues from MBSI-A.

The proteolysis of trypsin inhibitors in legume seeds.

The seeds of plants often contain large amounts of proteins, which are subjected to extensive proteolytic processing during seed development and subsequent germination. One class of legume seed proteins, the Bowman-Birk-type trypsin inhibitors, has proved especially useful as a subject in studying these events. Sequence studies of the trypsin inhibitors from a number of legume species suggest that many of the inhibitors undergo a limited shortening at the amino terminus during seed development. However, during germination, the inhibitors appear to function as storage proteins. As such, they are subjected to extensive proteolysis, ultimately leading to their destruction. This degradative process has been studied extensively in the mung bean (Vigna radiata [L.] Wilczek). Proteolysis of the mung bean trypsin inhibitor involves, at least initially, an ordered sequence of limited proteolytic cleavages. The two proteases involved in the initial phases of this degradation have been identified and partially characterized.

Crystal structure determination of mung bean trypsin inhibitor Lys fragment-bovine trypsin complex--molecular replacement, electron density map at 3.0 angstron resolution.

The orientation and position of the trypsin molecule in the complex crystal cell mung bean trypsin inhibitor Lys fragment (MBILF)-bovine trypsin (BTRY) have been successfully determined by molecular replacement method with the model of the refined bovine trypsin molecule. Starting from the BTRY coordinates which were oriented and located in the correct azimuth and position in the complex cell according to the result from rotation function and translation function, sim-weighted Fourier map with coefficients 2/Fo/-/Fc/ at 3.0 A resolution was calculated. Besides the electron density which is obviously attributed to itself, in the vicinity of the active site of BTRY the dense contour levels corresponding to the MBILF and and its boundary could be clearly seen in this map. The size of MBILF was approximately estimated at 15 x 15 x 25 A.

Crystal structure determination and molecular model of mung bean trypsin inhibitor lys active fragment–bovine trypsin (MBILF–BTRY) complex at 3.0 Å resolution

Crystal structure determination and molecular model of mung bean trypsin inhibitor lys active fragment–bovine trypsin (MBILF–BTRY) complex at 3.0 Å resolution

Structural analysis of the complex of mung bean trypsin inhibitor with porcine trypsin at 2.8 Å resolution

Structural analysis of the complex of mung bean trypsin inhibitor with porcine trypsin at 2.8 Å resolution

X-ray crystallographic studies on the complex of mung bean trypsin inhibitor with porcine trypsin

X-ray crystallographic studies on the complex of mung bean trypsin inhibitor with porcine trypsin

Characterization of the Proteinase that Initiates the Degradation of the Trypsin Inhibitor in Germinating Mung Beans (Vigna radiata)

The proteinase (proteinase F) responsible for the initial proteolysis of the mung bean (Vigna radiata) trypsin inhibitor (MBTI) during germination has been purified 1400-fold from dry beans. The enzyme acts as an endopeptidase, cleaving the native inhibitor, MBTI-F, to produce the first modified inhibitor form, MBTI-E. The cleavage of the Asp76-Lys77 peptide bond of MBTI-F occurs at a pH optimum of 4.5, with the tetrapeptide Lys-Asp-Asp-Asp being released. Proteinase F exhibited no activity against the modified inhibitor forms MBTI-E and MBTI-C. Vicilin, the major storage protein of the mung bean, does not serve as a substrate for proteinase F between pH 4 and 7. Proteinase F is inhibited by phenylmethylsulfonyl fluoride, chymostatin, p-hydroxymercuribenzoate, and p-chlorophenylsulfonate, but not by iodoacetate and CuCl(2). It is not activated by dithiothreitol, and is stable for extended periods of time (10 months, 4 degrees C, pH 4.0) in the absence of reducing agents. An apparent molecular weight of 65,000 was found for proteinase F by gel filtration. Subcellular fractionation in glycerol suggests that greater than 85% of the proteinase F activity is found in the protein bodies of the ungerminated mung bean. The same studies indicate that at least 56% of the MBTI of the seed is also localized in the protein bodies.

The crystallographic study of the complex of the Lys active fragment of the mung bean trypsin inhibitor with bovine trypsin.

The Lys fragment of mung bean trypsin inhibitor can combine with bovine trypsin to form a complex at an equal molar ratio. The single crystals of the complex were obtained by using the micro-still-setting method and the X-ray diffraction extended to 1.8A resolution. Its space group is P212121 with cell dimensions a = 62.9(1)A, b = 63.4(1)A and c = 69.7 (2)A. There is one complex molecule in a crystallographic asymmetric unit.

Involvement of carboxypeptidase in the degradation of the mung bean (Vigna radiata) trypsin inhibitor during germination and early seedling growth

Examination of the substrate specifity of the carboxypeptidase activity of ungerminated and germinated mung beans (Vigna radiata (L.) Wilczek) reveals the presence of two distinct enzymes. The first of these, carboxypeptidase I, is maximally active against carbobenzyloxy-Ala-Phe. It is present in large amounts in the cotyledons of ungerminated seeds, and declines rapidly during germination. The second, carboxypeptidase II, is most active against carbobenzyloxy-Phe-Ala. This enzyme increases in the cotyledons during germination and seedling growth. The possible involvement of one or both of these enzymes in the degradation of the native mung bean trypsin inhibitor (MBTI - F) and its proteolytically modified forms (MBTI - E, - C, and - E') during germination was also examined. The enzyme catalyzing one step in the conversion of MBTI - E to MBTI - C has been isolated with 557-fold purification from germinated mung beans. The inhibitor-degrading enzyme has a molecular weight of approximately 60,000. It is optimally active against MBTI - E at between pH 4.0 to 4.5. No activity was found with MBTI - F, - E', or - C as substrates. The enzyme has been identified as the carboxypeptidase II on the basis of (1) coelution during chromatography, (2) action on synthetic and natural substrates, (3) sensitivity to enzyme inhibitors, and (4) temporal variation during germination. The MBTI degrading carboxypeptidase removes the two carboxyl-terminal residues from MBTI - E to produce an inhibitor species that co-migrates with MBTI - C on polyacrylamide gel electrophoresis, but has not been subjected to the internal cleavage and proteolysis at the amino-terminus found in MBTI - C.

Amino Acid Sequence of Mung Bean Trypsin Inhibitor and Its Modified Forms Appearing during Germination

The amino acid sequence of the major trypsin inhibitor, F, of ungerminated mung beans (Vigna radiata [L.] Wilczek) was determined by a combination of automatic solid phase and manual sequencing techniques. F is a typical Bowman-Birk-type proteinase inhibitor with 80 amino acid residues and exhibits a high degree of identity with the other sequenced members of the Bowman-Birk family of inhibitors. Thin layer peptide maps of mung bean inhibitors E and C (which appear during germination) indicate that both are derived from inhibitor F by limited specific proteolysis. Loss of the carboxyl-terminal residues 77 to 80 from F produces inhibitor E, while the loss of an additional two carboxyl-terminal residues, the loss of the amino-terminal residues 1 to 8, and an internal cleavage at Ala(35)-Asp(36) produces inhibitor C from E. Another inhibitor species, E', was isolated from ungerminated seeds. It differs from F in the loss of residues 1 to 6. The majority of the proteolytic cleavages noted in the F-E-C-E' system are at peptide bonds involving aspartyl residues.