Amino acid sequence of the acidic Kunitz-type trypsin inhibitor from winged-bean seed [Psophocarpus tetragonolobus (L.) DC]

The primary sequence of trypsin inhibitor-2 (WBTI-2) fromPsophocarpus tetragonolobus (L.) DC seeds was determined. This inhibitor consists of a single polypeptide chain of 182 amino acids, including four half-cystine residues, and an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2 showed 57% identity to the basic trypsin inhibitor (WBTI-3) and 50% identity to the chymotrypsin inhibitor (WBCI) of winged bean, and 54% identity to the trypsin inhibitor DE-3 fromErythrina latissima seed. The similarity to the soybean Kunitz trypsin inhibitor (40%) and the other Kunitz-type inhibitors fromAdenanthera pavonina (30%) and wheat (26%) was much lower. Sequence comparisons indicate that thePsophocarpus andErythrina inhibitors are more closely related to each other than to other members of the Kunitz inhibitor family.     

Kunitz trypsin inhibitor genes are differentially expressed during the soybean life cycle and in transformed tobacco plants.

We investigated the structure, organization, and developmental regulation of soybean Kunitz trypsin inhibitor genes. The Kunitz trypsin inhibitor gene family contains at least 10 members, many of which are closely linked in tandem pairs. Three Kunitz trypsin inhibitor genes, designated as KTi1, KTi2, and KTi3, do not contain intervening sequences, and are expressed during embryogenesis and in the mature plant. The KTi1 and KTi2 genes have nearly identical nucleotide sequences, are expressed at different levels during embryogenesis, are represented in leaf, root, and stem mRNAs, and probably do not encode proteins with trypsin inhibitor activity. By contrast, the KTi3 gene has diverged 20% from the KTi1 and KTi2 genes, and encodes the prominent Kunitz trypsin inhibitor found in soybean seeds. The KTi3 gene has the highest expression level during embryogenesis, and is also represented in leaf mRNA. All three Kunitz trypsin inhibitor genes are regulated correctly in transformed tobacco plants. Our results suggest that Kunitz trypsin inhibitor genes contain different combinations of cis-control elements that program distinct qualitative and quantitative expression patterns during the soybean life cycle.     

Trypsin inhibitor from the seeds of Acacia confusa.

A trypsin inhibitor (ACTI) was isolated and purified from the seeds of Acacia confusa by gel filtration, and trypsin-Sepharose 4B column affinity chromatography. The molecular weight of ACTI was found to be 21,000 +/- 1,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid composition analysis. ACTI contained four half-cystine and no methionine residues, and was rich in aspartic acid, glutamic acid, glycine, leucine, and lysine residues. The native trypsin inhibitor was composed of two polypeptide chains, and it inhibited trypsin and alpha-chymotrypsin stoichiometrically at the molar ratio of 1:1 and 2:1, respectively. The amino-terminal sequence analysis of the A. confusa trypsin inhibitor A and B chains revealed a more extensive homology with Acacia elata and silk tree trypsin inhibitors, and a less extensive homology with Kunitz soybean trypsin inhibitor.     

胰蛋白酶抑制剂基因siRNA表达体系的构建

以大豆基因组DNA为模板,利用聚合酶链式反应(PCR)技术克隆了大豆胰蛋白酶抑制剂基因KSTI3的全长DNA片段,并将其构建到pMD18-T vector上。核苷酸序列测定结果表明:该基因片段全长654bp,与已发表的KSTI3基因序列同源性达99%。将反义 正义基因片段插入到pBI121 35S启动子下,构建重组质粒pBIKSTI3。通过冻融法将该重组质粒转入农杆菌EHA105中,获得了siRNA表达体系。利用农杆菌介导法将带有pBIKSTI3的菌株转化大豆,从2棵再生植株中得到2 100bp的特异性扩增条带,而未转化的植株中无该片段的产生。     

Sequencing and characterization of the citrus weevil, Diaprepes abbreviatus, trypsin cDNA. Effect of Aedes trypsin modulating oostatic factor on trypsin biosynthesis.

Trypsin mRNA from the citrus weevil, Diaprepes abbreviatus, was reverse transcribed and amplified by PCR. A cDNA species of 513 bp was cloned and sequenced. The 3' and 5' ends of the gene (262 bp and 237 bp, respectively) were amplified by rapid amplification of cDNA ends, cloned and sequenced. The deduced sequence of the trypsin cDNA (860 bp) encodes for 250 amino acids including 11 amino acids of activation and signal peptides and exhibited 16.8% identity to trypsin genes of selected Lepidoptera and Diptera. A three-dimensional model of Diaprepes trypsin contained two domains of beta-barrel sheets as has been found in Drosophila and Neobellieria. The catalytic active site is composed of the canonical triad of His41, Asp92 and Ser185 and a specificity pocket occupied by Asp179 with maximal activity at pH 10.4. Southern blot analysis indicated that at least two copies of the gene are encoded by Diaprepes midgut. Northern blot analysis detected a single RNA band below 1.35 kb at different larval ages (28-100 days old). The message increased with age and was most abundant at 100 days. Trypsin activity, on the other hand, reached a peak at 50 days and fell rapidly afterwards indicating that the trypsin message is probably regulated translationally. Feeding of soybean trypsin inhibitor and Aedes aegypti trypsin modulating oostatic factor affected trypsin activity and trypsin biosynthesis, respectively. These results indicate that Diaprepes regulates trypsin biosynthesis with a trypsin modulating oostatic factor-like signal.     

大豆Kunitz型胰蛋白酶抑制剂新类型Tid的全序列分析

大豆ｋｕｎｉｔｚ型胰蛋白酶抑制剂（ＳＢＴｉＡ２）是一种大量存在于大豆（Ｇｌｙｃｉｎｅｍａｘ）中的种子贮藏蛋白．虽然对它的生化特性及结构已有较多的研究,但它在体内的主要功能仍不很清楚．国际上所发现的３个由显性等位基因Ｔｉａ、Ｔｉｂ、Ｔｉｃ编码的大豆ｋｕｎｉｔｚ胰蛋白酶抑制剂的氨基酸顺序已被明确测定,相互间有一到多个氨基酸残基的不同［１］．Ｔｉｄ是从我国１５０００余份大豆资源中筛选到的唯一一份ｋｕｎｉｔｚ型胰蛋白酶抑制剂位点的新类型,遗传分析证明它是另一个ＳＢＴｉＡ２的显性等位基因［２,３］．严…     

Cotyledon nuclear proteins bind to DNA fragments harboring regulatory elements of phytohemagglutinin genes.

The effects of deleting DNA sequences upstream from the phytohemagglutinin-L gene of Phaseolus vulgaris have been examined with respect to the level of gene product produced in the seeds of transgenic tobacco. Our studies indicate that several upstream regions quantitatively modulate expression. Between -1000 and -675, a negative regulatory element reduces expression approximately threefold relative to shorter deletion mutants that do not contain this region. Positive regulatory elements lie between -550 and -125 and, compared with constructs containing only 125 base pairs of upstream sequences (-125), the presence of these two regions can be correlated with a 25-fold and a 200-fold enhancement of phytohemagglutinin-L levels. These experiments were complemented by gel retardation assays, which demonstrated that two of the three regions bind cotyledon nuclear proteins from mid-mature seeds. One of the binding sites maps near a DNA sequence that is highly homologous to protein binding domains located upstream from the soybean seed lectin and Kunitz trypsin inhibitor genes. Competition experiments demonstrated that the upstream regions of a bean beta-phaseolin gene, the soybean seed lectin gene, and an oligonucleotide from the upstream region of the trypsin inhibitor gene can compete differentially for factor binding. We suggest that these legume genes may be regulated in part by evolutionarily conserved protein/DNA interactions.     

Amino acid sequences of two trypsin inhibitors from winged bean seeds (Psophocarpus tetragonolobus (L)DC.)

The trypsin inhibitor (WTI-1) purified from winged bean seeds is a Kunitz type protease inhibitor having a molecular weight of 19,200. WTI-1 inhibits bovine trypsin stoichiometrically, but not bovine alpha-chymotrypsin. The approximate Ki value for the trypsin-inhibitor complex is 2.5 X 10(-9) M. The complete amino acid sequence of WTI-1 was determined by conventional methods. Comparison of the sequence with that of soybean trypsin inhibitor (STI) indicated that the sequence of WTI-1 had 50% homology with that of STI. WTI-1 was separated into 2 homologous inhibitors, WTI-1A and WTI-1B, by isoelectric focusing. The isoelectric points of WTI-1A and WTI-1B were 8.5 and 9.4, respectively, and their sequences were presumed from their amino acid compositions.     

Structure of soybean Kunitz trypsin inhibitor mRNA determined from cDNA by using oligodeoxynucleotide primers

Oligodeoxynucleotides complementary to the deduced mRNA sequence of soybean Kunitz trypsin inhibitor (KTI) were used to prime the synthesis of cDNA from soybean cotyledon total poly(A) RNA. The primed cDNA was used to select clones from a Glycine max cotyledon cDNA library. Two out of twelve hybridizing clones were shown to contain KTI cDNA. The nucleotide sequence of one clone, pSTI 9-2, was determined and it was found to encompass the complete protein coding region of KTI excet for three C-terminal residues. Trypsin inhibitor is synthesized with a 25 amino acid hydrophobic N-terminal sequence presumed to be a signal peptide. The mature polypeptide encoded by pSTI 9-2 agrees with the published amino acid composition of KTI, but contains two discrepancies at the peptide sequence level.     

Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom

Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibitor from Ophiophagus hannah) was isolated from king cobra venom by three chromatographic steps of gel filtration, trypsin affinity and reverse phase HPLC. OH-TCI is composed of 58 amino acid residues with a molecular mass of 6339Da. Successful expression of OH-TCI was performed as the maltose-binding fusion protein in E. coli DH5alpha. Much different from Oh11-1, the purified native and recombinant OH-TCI both had strong inhibitory activities against trypsin and chymotrypsin although the sequence identity (74.1%) between them is very high. The inhibitor constants (K(i)) of recombinant OH-TCI were 3.91 x 10(-7) and 8.46 x10(-8)M for trypsin and chymotrypsin, respectively. To our knowledge, it was the first report of Kunitz/BPTI serine proteinase inhibitor from snake venom that had equivalent trypsin and chymotrypsin inhibitory activities.     

A novel serine protease inhibitor from Bungarus fasciatus venom

By Sephadex G-50 gel filtration, cation-exchange CM-Sephadex C-25 chromatography and reversed phase high-performance liquid chromatography (HPLC), a novel serine protease inhibitor named bungaruskunin was purified and characterized from venom of Bungarus fasciatus. Its cDNA was also cloned from the cDNA library of B. fasciatus venomous glands. The predicted precursor is composed of 83 amino acid (aa) residues including a 24-aa signal peptide and a 59-aa mature bungaruskunin. Bungaruskunin showed maximal similarity (64%) with the predicted serine protease inhibitor blackelin deduced from the cDNA sequence of the red-bellied black snake Pseudechis porphyriacus. Bungaruskunin is a Kunitz protease inhibitor with a conserved Kunitz domain and could exert inhibitory activity against trypsin, chymotrypsin, and elastase. By screening the cDNA library, two new B chains of beta-bungarotoxin are also identified. The overall structures of bungaruskunin and beta-bungarotoxin B chains are similar; especially they have highly conserved signal peptide sequences. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor.     

Alzheimer's disease amyloid peptide is encoded by two exons and shows similarity to soybean trypsin inhibitor

To better understand the processing of the Alzheimer disease amyloid precursor protein, we have cloned and sequenced that region of the human genome coding for the amyloid peptide. Two exons separated by a 6.2kb intron define this region. Characterization of the A4 peptide amino acid sequence shows similarity to the structure of soybean trypsin inhibitor (Kunitz). Our observation describes a different region of PreA4 than the previously characterized domain of larger amyloid precursor molecules PreA4 751 and 770(2). Moreover, the exon organization, Kunitz domain duplication and transmembrane location of A4 suggest that PreA4 is similar to growth factor precursors and thus may be processed similarly.     

In vivo phosphorylation of a recombinant peptide substrate of CDPK suggests involvement of CDPK in plant stress responses

A phage display library displaying random peptides 15 amino acids in length was screened for peptides that interact with soybean (Glycine max L.) CDPKalpha, an isoform of calcium-dependent protein kinase (EC 2.7.1.37). Interaction of phage displaying the peptide RHPTLTRSPTLRNIQ with CDPKalpha was confirmed in an independent binding assay. A synthetic peptide corresponding to this sequence plus the surrounding amino acids AERHPTLTRSPTLRNIQPPC was synthesized and found to be a substrate of CDPK isoforms alpha, beta, and gamma. A second random peptide phage display library was constructed that displayed the substrate peptide sequence plus an additional 10 random amino acids on its amino-terminal side. Nine new peptides were obtained from the screening, all of which were phosphorylated by CDPKalpha. Sequence VSPRSFWTTWRHPTLTRSPTLRNIQ appeared twice in the screen. Because it agreed well with the consensus phosphorylation site of CDPKs, its coding sequence was cloned and stably transformed into tobacco cells. The substrate peptide expressed in tobacco was phosphorylated by recombinant CDPKalpha in vitro and by endogenous CDPK in vivo. Increased phosphorylation of the peptide substrate in response to hydrogen peroxide treatment was observed in transgenic tobacco cells. These results show that the peptide substrate expressed in tobacco cells can be used as a CDPK activity reporter for in vivo studies.     

Cloning, sequence analysis and crystal structure determination of a miraculin-like protein from Murraya koenigii

Earlier, the purification of a 21.4 kDa protein with trypsin inhibitory activity from seeds of Murraya koenigii has been reported. The present study, based on the amino acid sequence deduced from both cDNA and genomic DNA, establishes it to be a miraculin-like protein and provides crystal structure at 2.9 Å resolution. The mature protein consists of 190 amino acid residues with seven cysteines arranged in three disulfide bridges. The amino acid sequence showed maximum homology and formed a distinct cluster with miraculin-like proteins, a soybean Kunitz super family member, in phylogenetic analyses. The major differences in sequence were observed at primary and secondary specificity sites in the reactive loop when compared to classical Kunitz family members. The crystal structure analysis showed that the protein is made of twelve antiparallel β-strands, loops connecting β-strands and two short helices. Despite similar overall fold, it showed significant differences from classical Kunitz trypsin inhibitors.     

山东产野生大豆胰蛋白酶抑制剂的初步研究

该实验建立了HPLC测定大豆胰蛋白酶抑制剂(STI)活性的方法,并对山东产野生大豆(G.soja)与同地区产的黑豆和黄豆(G.max)的胰蛋白酶抑制活性差异进行了比较.用耦合了胰蛋白酶的亲合色谱柱对野生大豆的STI进行分离纯化,紫外分光光度法比较3种大豆的STI含量;PCR结合TA克隆技术对野生大豆STI中的Kunitz型(KSTI)蛋白基因编码区的氨基酸顺序进行初步测定.结果发现,山东产野生大豆的STI活性和含量均高于同地区产的黑豆和黄豆;山东产野生大豆的KSTI蛋白基因编码区的氨基酸顺序与已知的Tia型基本一致,仅第59位氨基酸由于单核苷酸的置换发生了Ser→Thr的转变,此位置位于活性中心附近.研究表明,山东产野生大豆胰蛋白酶抑制活性较强,且含量高.     

Characterization of a bifunctional wheat inhibitor of endogenous α-amylase and subtilisin

A bifunctional α-amylase/serine protease inhibitor which inhibits germination-specific cereal α-amylases of the Graminae subfamily Festucoideae as well as bacterial subtilisins has been isolated from wheat grains. This protein has M_r ≈20500 and pI ≈7.2. The amino acid composition and N-teminal sequence (45 residues) show that the inhibitor is homologous with cereal and leguminous inhibitors of the soybean trypsin inhibitor (Kunitz) family.     

Dietary mixtures of cysteine and serine proteinase inhibitors exhibit synergistic toxicity toward the red flour beetle,Tribolium castaneum

1. Combinations of a cysteine proteinase inhibitor (CPI) and serine proteinase inhibitors (SPI) in wheat germ diets were toxic to larvae of the red flour beetle, Tribolium castaneum, when tested at levels where individual inhibitors were nontoxic.2. Mixtures of 0.1% (w/w) CPI (E-64) plus 1% of either of three plant SPIs (soybean Kunitz trypsin inhibitor, soybean Bowman-Birk trypsin-chymotrypsin inhibitor, or lima bean trypsin inhibitor) inhibited T. castaneum growth, resulting in 82–97% reduction in larval weight gain 17 days after hatching and 40–60% mortality.3. Supplemention of diet containing 0.1% E-64 plus 1% soybean Kunitz trypsin inhibitor (STI) with a mixture of amino acids at 7% caused a partial reversal of the growth inhibition, with 91% of the larvae surviving.4. Diet containing 0.1% E-64 plus either 5 or 10% STI resulted in 100% mortality of the larvae during the first or second instar.5. Addition of a mixture ofamino acids at 20% to the 0.1% E-64 plus 10% STI diet allowed 89% of the larvae to develop into adults.6. The synergism between different classes of proteinase inhibitors in the insect's diet that enhances growth inhibition and toxicity demonstrates the potential for an insect pest management strategy involving the coordinated manipulation of two or more types of digestive enzyme inhibitor genes in plants.     

Obtaining a Transgenic Upland Cotton Harboring Two Insecticidal Genes

pBinLK carried two insecticidal genes, pea lectin (P-Lec)gene and soybean Kunitz trypsin inhibitor (SKTI) gene, were successfully transferred into 4 upland cotton ( Gossypium hirsutum L. ) cultivars, "Xinluzao-1 ", "Xinluzhong-2", "Jihe-321" and "Liao-9" via Agrobacterium-mediated transformation. Hypocotyl segments from aseptic seedlings were used as receipient. After co-cultivation of hypocotyl segments with A. tumefac/ens (Smith et Townsend) Conn, kanamycin-resistant calli were screened, and somatic embryos and regenerated plants were obtained through various media. Transgenic cotton plants harboring two insecticidal genes were confirmed by NPT-Ⅱ ELISA, PCR and PCR Southern. rllae results of bioassay demonstrated that the transgenic plants showed significant resistance to the larvae of cotton bollwonn (Heliothis armigera Hubner).