Amino acid sequence of the protease inhibitor BWI-4a from buckwheat seeds

The complete amino acid sequence of protease inhibitor BWI-4a from buckwheat (Fagopyrum esculentum Moench) seeds, consisting of 67 amino acid residues with a single disulfide bond, has been established by Edman degradation in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Its N terminus is blocked by a pyroglutamic acid residue. Mass spectrometric analysis revealed that inhibitor BWI-4a is present in buckwheat seeds in two isoforms with a single amino acid substitution of Ala40 for Gly40. The reactive site of the inhibitor contains an Arg43-Asp44 bond. Analysis of the amino acid sequence suggests that the buckwheat seed protease inhibitor is a member of the potato proteinase inhibitor I family.     

Cationic Inhibitors of Serine Proteinases from Buckwheat Seeds

Preparations of low molecular weight protein inhibitors of serine proteinases have been obtained from buckwheat (Fagopyrum esculentum) seeds by chromatography of seed extract on trypsin-Sepharose 4B, Mono-Q, and Mono-S ion exchangers (FPLC regime). Their molecular masses, determined by mass spectrometry, were 5203 (BWI-1c), 5347 (BWI-2c), 7760 (BWI-3c), and 6031 daltons (BWI-4c). All of the inhibitors possess high pH- and thermal stability in the pH range 2-12. In addition to trypsin, BWI-3c and BWI-4c inhibited chymotrypsin and subtilisin-like bacterial proteases. The N-terminal sequences of all of the inhibitors were determined: BWI-1c (23 residues), BWI-2c (33 residues), BWI-3c (18 residues), and BWI-4c (20 residues). In their physicochemical properties and N-terminal amino acid sequences, the buckwheat seed trypsin inhibitors BWI-3c and BWI-4c appear to belong to potato proteinase inhibitor I family.     

New protease inhibitors from buckwheat seeds: properties, partial amino acid sequences and possible biological role

Preparations of new low molecular weight protein inhibitors of serine proteinases have been obtained from buckwheat Fagopyrum esculentum seeds by chromatography of seed extracts on trypsin-Sepharose 4B, Mono-Q and Mono-S ion-exchangers. Their molecular masses, determined by mass spectrometry, were equal to 5203 (BWI-1c), 5347 (BWI-2c), 7760 (BWI-3c) and 6031 daltons (BWI-4c). All inhibitors possessed high pH-stability in the pH range 2-12 and thermostability. In addition to trypsin, BWI-3c and BWI-4c inhibitors inhibited chymotrypsin and subtilisin-like proteases. The inhibition constants (Ki) for trypsin, chymotrypsin and subtilisin by the studied inhibitors were determined. The N-terminal sequences of all inhibitors were established: BWI-1c (23 residues), BWI-2c (33 residues), BWI-3c (18 residues) and BWI-4c (20 residues). According to the physicochemical properties and N-terminal amino acid sequences, buckwheat seed protease inhibitors BWI-3c and BWI-4c are suggested to belong to the potato proteinase inhibitor I family.     

The anionic protease inhibitor BWI-1 from buckwheat seeds. Kinetic properties and possible biological role

Kinetic characteristics and effects on the growth of filamentous fungi of one of the main anionic protease inhibitors, BWI-1, isolated from buckwheat seeds, have been studied. The inhibition constants of bovine trypsin, chymotrypsin and cathepsin G from human granulocytes with BWI-1 were found to be 1.1, 67 and 200 n M , respectively. Analysis of the amino acid sequence of BWI-1 in the vicinity of the reactive site revealed its homology to the potato proteinase inhibitor I family. It is suggested that the inability of BWI-1 to bind elastase of human granulocytes is due to the basic nature of the amino acid residue (Arg) at the P_j position in its reactive site. It was demonstrated that BWI-1 was able to suppress the germination of the spores and the growth of the mycelium of two filamentous fungi.     

Buckwheat trypsin inhibitor with helical hairpin structure belongs to a new family of plant defence peptides

A new peptide trypsin inhibitor named BWI-2c was obtained from buckwheat (Fagopyrum esculentum) seeds by sequential affinity, ion exchange and reversed-phase chromatography. The peptide was sequenced and found to contain 41 amino acid residues, with four cysteine residues involved in two intramolecular disulfide bonds. Recombinant BWI-2c identical to the natural peptide was produced in Escherichia coli in a form of a cleavable fusion with thioredoxin. The 3D (three-dimensional) structure of the peptide in solution was determined by NMR spectroscopy, revealing two antiparallel α-helices stapled by disulfide bonds. Together with VhTI, a trypsin inhibitor from veronica (Veronica hederifolia), BWI-2c represents a new family of protease inhibitors with an unusual α-helical hairpin fold. The linker sequence between the helices represents the so-called trypsin inhibitory loop responsible for direct binding to the active site of the enzyme that cleaves BWI-2c at the functionally important residue Arg(19). The inhibition constant was determined for BWI-2c against trypsin (1.7×10(-1)0 M), and the peptide was tested on other enzymes, including those from various insect digestive systems, revealing high selectivity to trypsin-like proteases. Structural similarity shared by BWI-2c, VhTI and several other plant defence peptides leads to the acknowledgement of a new widespread family of plant peptides termed α-hairpinins.     

An antifungal peptide from Fagopyrum tataricum seeds

A major trypsin inhibitor was isolated and characterized from the seeds of the tartary buckwheat (Fagopyrum tataricum) (FtTI) by ammonium sulfate precipitation, ion exchange chromatography and centrifugal ultrafiltration. SDS-PAGE analysis under reducing condition showed that FtTI is a single polypeptide chain with a molecular mass of approximately 14 kDa. The complete amino acid sequence of FtTI was established by automatic Edman degradation and mass spectrometry. It was found that the trypsin inhibitor molecule consists of 86 amino acid residues containing two disulfide bonds which connect Cys⁸ to Cys⁶⁵ and Cys⁴⁹ to Cys⁵⁸. The active site of the inhibitor was found to contain an Asp⁶⁶-Arg⁶⁷ bond. MALDI-TOF analysis showed that FtTI has two isoforms (Mr: 11.487 and 13.838 kDa). Dixon plots revealed a competitive inhibition of trypsin with inhibition constants (Ki) of 1.6 nM. Analysis of the amino acid sequence suggests that FtTI is a member of the protease inhibitor I family. What is more, FtTI exhibited strong inhibitory activity against phytopathogenic fungi.     

Kinetics of interaction of trypsin with an anionic inhibitor of trypsin BWI-1a from buckwheat seeds

The kinetics of binding of bovine trypsin to a proteinaceous inhibitor of trypsin from buckwheat seeds (BWI-1a) has been studied. The association rate constant (k(ass)) was 2.2 x 10(6) M-1 x sec-1 and the dissociation rate constant (k(off)) of the enzyme--inhibitor complex was 3.5 x 10(-3) sec-1; the inhibition constant Ki was 1.5 nM. The inhibitor BWI-1a is of the slow, tightly binding type. The mechanism of the inhibition of bovine trypsin by the trypsin inhibitor BWI-1a was studied. The mechanism of inhibition was found to involve two steps according to the kinetic data.     

Cloning and characterization of a novel trypsin inhibitor (BTIomega1) gene from Fagopyrum esculentum.

Based on the amino acid information of trypsin inhibitor of buckwheat (Fagopyrum Esculentum Moench), degenerated primers were designed and a full-length cDNA sequence named BTIomega1 (Buckwheat Trypsin Inhibitor) was amplified from the leaves RNA by using RT-PCR and rapid amplification of cDNA ends (RACE) methods. Sequence analysis shows that the 392 bp cDNA contained an open reading frame (ORF) of 216 bp, encoding 72 amino acids residues. The deduced amino acid sequence exhibits 96 and 93% homology with BWI-1 and BTI-2, a natural trypsin inhibitor from buckwheat seeds. Southern blotting suggested that three copies of BTIomega1 gene existed in the buckwheat genome. Moreover, a predicted secondary structure and 3D-structural model was constructed by homology modeling. To our knowledge, this is the first all-round report of the gene BTIomega1. The novel BTIomega1 gene has been submitted to the GeneBank under Accession No. DQ289792.     

Cationic Inhibitors of Serine Proteinases from Buckwheat Seeds: Study of Their Interaction with Exogenous Proteinases

The inhibition of exogenous serine proteinases of different origin by cationic protease inhibitors BWI-1c, -2c, -3c, and -4c from buckwheat (Fagopyrum esculentum Moench) seeds has been studied. High efficiency of the inhibitors in binding bovine trypsin and chymotrypsin as well as their broad antiprotease effect, including inhibition of proteinases secreted by fungi and bacteria, has been demonstrated. According to the data obtained, it is proposed that cationic inhibitors from buckwheat seeds may participate in the defense of plants against fungal and bacterial infection.     

荞麦胰蛋白酶抑制剂的原核表达及纯化

根据文献报道的荞麦胰蛋白酶抑制剂的氨基酸序列及本研究室先前已获得的部分基因序列设计引物,经过RT-PCR扩增,获得荞麦胰蛋白酶抑制剂编码区基因全序列.将该基因克隆到原核表达载体pQE-31中,并转化至大肠杆菌M15,经IPTG诱导表达获得可溶性目的蛋白,其表达量约占菌体总蛋白的25%.该目的蛋白经Ni2 -NTA柱亲和纯化,SDS-PAGE分析显示,在大约9kD处出现明显的目的条带,与预计蛋白分子量大小一致.Western blot鉴定证实,目的蛋白N端带有6个组氨酸标签.活性测定表明,目的蛋白具有专一性的胰蛋白酶抑制剂活性,抑制活性约为77U/mg纯化蛋白.本实验为进一步研究荞麦胰蛋白酶抑制剂结构与功能的关系奠定了基础.     

Conformational changes of rBTI from buckwheat upon binding to trypsin: implications for the role of the P(8)' residue in the potato inhibitor I family

BWI-1 (buckwheat trypsin inhibitor), a member of the potato inhibitor I family, suppresses the growth of T-acute lymphoblastic leukemia cells and induces apoptosis in human solid tumor cell lines. Here, we report the crystal structure of rBTI (recombinant buckwheat trypsin inhibitor), a recombinant protein of BWI-1, at 1.84 ? resolution and the structure of rBTI in complex with bovine trypsin at 2.26 ? resolution. A conformational change of Trp53 at the P(8)' position in rBTI was observed upon its binding to trypsin, which is not seen in other members of the potato inhibitor I family reported previously. The role of the P(8)' residue in the potato inhibitor I family was examined by measuring the association and dissociation rates of four rBTI mutants with different substitutions at the P(2) and P(8)' positions when binding to trypsin. One of the mutants, P44T, was found to be a much stronger inhibitor than wild-type rBTI, with a picomolar (pM) dissociation constant. Our results could provide valuable insights for designing a new rBTI-based antitumor drug in the future.     

苦荞和甜荞查尔酮合成酶基因的克隆及序列比较

以苦荞品种‘西农9920’和甜荞品种‘西农9976’为材料,根据其它植物查尔酮合成酶(chalcone synthase,CHS)基因DNA序列的保守区域设计的一对简并引物,进行PCR扩增,从2种荞麦基因组中克隆出了长度均为860 bp的CHS基因片段,对其进行回收、克隆,挑选阳性克隆测序;序列分析表明这2个片段含有CHS基因的N端和C端的结构域,分别为苦荞和甜荞的CHS基因片段,命名为FtCHS和FeCHS。对获得的2种荞麦CHS基因的DNA序列进行比较分析,发现两者间存在多达43处单碱基多态性,这些单碱基多态性可能是苦荞和甜荞种子中类黄酮含量差异的重要原因之一。苦荞和甜荞CHS与其它植物CHS的氨基酸序列的进化分析表明,其与同为蓼科的掌叶大黄和石竹科的满天星的同源性较近。     

The complete amino acid sequence of the major Kunitz trypsin inhibitor from the seeds of Prosopsis juliflora.

The major inhibitor of trypsin in seeds of Prosopsis juliflora was purified by precipitation with ammonium sulphate, ion-exchange column chromatography on DEAE- and CM-Sepharose and preparative reverse phase HPLC on a Vydac C-18 column. The protein inhibited trypsin in the stoichiometric ratio of 1:1, but had only weak activity against chymotrypsin and did not inhibit human salivary or porcine pancreatic alpha-amylases. SDS-PAGE indicated that the inhibitor has a Mr of ca 20,000, and IEF-PAGE showed that the pI is 8.8. The complete amino acid sequence was determined by automatic degradation, and by DABITC/PITC microsequence analysis of peptides obtained from enzyme digestions of the reduced and S-carboxymethylated protein with trypsin, chymotrypsin, elastase, the Glu-specific protease from S. aureus and the Lys-specific protease from Lysobacter enzymogenes. The inhibitor consisted of two polypeptide chains, of 137 residues (alpha chain) and 38 residues (beta chain) linked together by a single disulphide bond. The amino acid sequence of the protein exhibited homology with a number of Kunitz proteinase inhibitors from other legume seeds, the bifunctional subtilisin/alpha-amylase inhibitors from cereals and the taste-modifying protein miraculin.     

The amino acid sequence of a Bowman-Birk type proteinase inhibitor from faba beans (Vicia faba L.).

The amino acid sequence of a Bowman-Birk type proteinase inhibitor (FBI) from seeds of faba bean (Vicia faba L.) was determined by analysis of peptide fragments generated by reduction and S-carboxymethylation of enzymatically modified inhibitors, which were obtained from native FBI by limited proteolysis with TPCK-trypsin or TLCK-chymotrypsin at pH 3.5. The established sequence showed that FBI is highly homologous with Vicia angustifolia inhibitor (VAI0 but lacks the portion corresponding to the C-terminal 9 amino acids of VAI. The trypsin reactive-site peptide bond in FBI was also indicated to be Lys(16)-Ser(17) and the chymotrypsin reactive-site peptide bond to be Tyr(42)-Ser(43) by limited proteolysis with TPCK-trypsin or TLCK-chymotrypsin and by sequence comparison with other Bowman-Birk type inhibitors.     

Isolation of a trypsin inhibitor with deletion of N-terminal pentapeptide from the seeds of Momordica cochinchinensis, the Chinese drug mubiezhi.

A trypsin inhibitor, MCCTI-1, with a molecular weight of 3479 Da as determined by mass spectrometry, was isolated from Momordica cochinchinensis seeds with a procedure involving extraction with 5% acetic acid, ammonium sulfate precipitation, ion exchange chromatography on CM-Sepharose and reverse-phase high performance liquid chromatography. The sequence of its first 13 N-terminal amino acid residues was ILKKCRRDSDCPG which was about 85% identical with the sequence of trypsin inhibitor MCTI-1 from Momordica charantia Linn. When compared with the sequences of most other squash family trypsin inhibitors, the sequence of MCCTI-1 was characterized by the deletion of a pentapeptide from the N-terminus. Trypsin inhibitors also existed in seeds of some hitherto uninvestigated Cucurbitaceae species.     

Molecular genetic analysis of collection of transgenic tobacco plants with buckwheat serine proteases inhibitor gene during long-term subculture

In this paper, the results of long-term screening of independently derived transgenic tobacco plants carrying the synthetic BWI-1a gene of serine proteases inhibitor from buckwheat are presented. For several years periodic spot checks of persistence and expression of the heterologous protective genes in vegetatively cloned collections and seeds of transgenic plants were conducted. The persistence of expression of the target gene after ten years of passage of plants in aseptic culture without selective pressure in their seed progeny for at least three generations and derived callus was shown. Extracts of tissues of all the variants of transgenic plants inhibited the growth of phytopathogenic bacteria and the germination of spores of fungi. The degree of the suppression of a pathogen in this case was hardly reduced. In the second seed generation, the number of defective seeds increased and there was a sharp decline of germination ability of the seeds even in nonselective conditions.     

Complete amino acid sequence of a new murine T-cell growth factor P40

A new murine T-cell growth factor, designated P40, which supports growth of helper T-cells in the absence of interleukin-2, interleukin-4 and antigen has been isolated from helper T-cell lines in sufficient quantities (100 micrograms) to permit its complete amino acid sequence determination. This was achieved by a combination of sensitive peptide mapping using microbore reversed-phase high performance liquid chromatography and automated microsequence analysis. Attempts to obtain N-terminal sequence data on P40 were unsuccessful due to N-terminal blockage of the native molecule. The nature of this N-terminal blocking was established using a combination of amino acid analysis, fast-atom-bombardment mass spectrometry and peptide synthesis. The P40 molecule, a single polypeptide chain comprising 126 amino acid residues, is structurally distinct from other known T-cell growth factors. No similarity was revealed when the amino acid sequence of P40 was compared with other proteins whose biochemical structure is known. The protein sequence data reported here predict four N-linked glycosylation sites in the P40 molecule.     

Purification and characterization of a 24 kDa protein from tartary buckwheat seeds

A 24 kDa protein was isolated from tartary buckwheat seeds by using chromatography of Superdex 75 gel filtration and Resource Q ion-exchange column. SDS-PAGE and Sephacryl S-200 gel filtration were used to provide information about the molecular mass of the protein purified from tartary buckwheat. The protein was composed of 215 amino acid residues and showed strong IgE binding activity in an ELISA test to the sera colleted from two patients allergic to buckwheat. These results suggested that the purified 24 kDa protein from tartary buckwheat seeds was an important functional protein and was relatively specific for buckwheat-allergic patients. It should be a very useful tool in the diagnosis of buckwheat allergy in the future.     

High-resolution structure of a potent, cyclic proteinase inhibitor from sunflower seeds.

Proteinaceous serine proteinase inhibitors are widespread throughout the plant kingdom where they play an important role in protection against pests and pathogens. Here, we describe the isolation and characterisation of a novel 14 amino acid residue cyclic peptide from sunflower seeds, which is a potent inhibitor of trypsin (Ki=100 pM). The crystal structure of this peptide in complex with bovine beta-trypsin shows both sequence and conformational similarity with the trypsin-reactive loop of the Bowman-Birk family of serine proteinase inhibitors. This inhibitor, however, is unique in being monofunctional, cyclic and far shorter (14 amino acid residues) than inhibitors belonging to this family (typically 60-70 amino acid residues). The high potency of this peptide is likely to arise from the considerable structural rigidity achieved through its cyclic nature which is further stabilised by a single internal disulphide bond. This study helps delineate the minimal unit required for effective peptide inhibitors of serine proteinases, and will assist in the further design of inhibitors to this widespread class of enzymes.