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.     

Design of serine proteinase inhibitors by combinatorial chemistry using trypsin inhibitor SFTI-1 as a starting structure.

A small peptide library of monocyclic SFTI-1 trypsin inhibitor from sunflower seeds modified in positions P(1) and P(4)' was synthesized using a portioning-mixing method. The peptide library was deconvoluted by the iterative approach in solution. Two trypsin ([Met(9)]-SFTI-1 and [Arg(5), Abu(9)]-SFTI-1), one chymotrypsin ([Phe(5)]-SFTI-1) and one human elastase ([Leu(5), Trp(9)]-SFTI-1) inhibitors were selected and resynthesized. The values of their association equilibrium constants (K(a)) with target enzymes indicate that they are potent inhibitors. In addition, the last two analoges belong to the most active inhibitors of this size. The results obtained show that the conserved Pro(9) residue in the Bowman-Birk inhibitor (BBI)s is not essential for inhibitory activity.     

A trypsin and chymotrypsin inhibitor from seeds of Bauhenia purpurea

A trypsin and chymotrypsin inhibitor was partially purified from Bauhenia purpurea seeds and separated from a second inhibitor by Ecteola cellulose chromatography. The factor inhibited bovine trypsin and chymotrypsin as well as pronase trypsin and elastase. It formed a complex with trypsin and with chymotrypsin, but a ternary complex could not be detected. Differences were detected in the effect on trypsin and on chymotrypsin, although one enzyme interfered with the inhibition of the other. The results obtained point to two active centers on the inhibitor for the trypsin and chymotrypsin inhibition such that the one cannot complex with the inhibitor after this inhibitor had complexed with the other.     

Three classes of proteinase inhibitor gene have distinct but overlapping patterns of expression in Pisum sativum plants

Genes representative of three gene classes encoding proteinase inhibitor proteins, with distinct spatial expression patterns, were isolated and characterized from Pisum.Under standard plant growth conditions, one class is expressed exclusively in seeds, whereas the other two make minor contributions to seed inhibitor proteins but are also expressed in other organs, predominantly in root endodermal and floral reproductive tissues. Two of the gene classes contain few genes and are genetically linked at the Tri locus, whereas the third class displays complex hybridization patterns to genomic DNA and maps to diverse genetic loci. Expression analysis of this last class suggests that only a small number of these genes are expressed. The quantitative effect of the Tri locus on root and floral inhibitor gene expression was examined in near-isogenic lines of pea. The proteins encoded by the three classes are all members of the same family (Bowman-Birk) of enzyme inhibitors but are distinct in terms of overall sequence, active site sequences and inhibitor function.     

The contribution of key hydrophobic residues in ecotin to enzyme-inhibitor complex stability

The Escherichia coli protease inhibitor ecotin is believed to be implicated in the evasion of host defenses during infection. The protein has also attracted attention as a scaffold for the design of novel, specific protease inhibitors. Ecotin interacts with its targets through two sites. Key hydrophobic residues in both sites (Leu-64, Trp-67, Tyr-69, Met-84, and Met-85) were mutated to alanine and the effects on the inhibition of trypsin, chymotrypsin, and elastase were assessed. Each of these mutant ecotin proteins tested in kinetic assays with these enzymes exerted less inhibitory potency compared to wild-type ecotin. However, these effects were relatively small and not additive.     

Molecular mechanism of enzyme inhibition: prediction of the three-dimensional structure of the dimeric trypsin inhibitor from Leucaena leucocephala by homology modelling

Serine proteinase inhibitors are widely distributed in nature and inhibit the activity of enzymes like trypsin and chymotrypsin. These proteins interfere with the physiological processes such as germination, maturation and form the first line of defense against the attack of seed predator. The most thoroughly examined plant serine proteinase inhibitors are found in the species of the families Leguminosae, Graminae, and Solanaceae. Leucaena leucocephala belongs to the family Leguminosae. It is widely used both as an ornamental tree as well as cattle food. We have constructed a three-dimensional model of a serine proteinase inhibitor from L. leucocephala seeds (LTI) complexed with trypsin. The model was built based on its comparative homology with soybean trypsin inhibitor (STI) using the program, MODELLER6. The quality of the model was assessed stereochemically by PROCHECK. LTI shows structural features characteristic of the Kunitz type trypsin inhibitor and shows 39% residue identity with STI. LTI consists of 172 amino acid residues and is characterized by two disulfide bridges. The protein is a dimer with the two chains being linked by a disulfide bridge. Despite the high similarity in the overall tertiary structure, significant differences exist at the active site between STI and LTI. The present study aims at analyzing these interactions based on the available amino acid sequences and structural data. We have also studied some functional sites such as phosphorylation, myristoylation, which can influence the inhibitory activity or complexation with other molecules. Some of the differences observed at the active site and functional sites can explain the unique features of LTI.     

Structural and mutational analyses of the interaction between the barley alpha-amylase/subtilisin inhibitor and the subtilisin savinase reveal a novel mode of inhibition

Subtilisins represent a large class of microbial serine proteases. To date, there are three-dimensional structures of proteinaceous inhibitors from three families in complex with subtilisins in the Protein Data Bank. All interact with subtilisin via an exposed loop covering six interacting residues. Here we present the crystal structure of the complex between the Bacillus lentus subtilisin Savinase and the barley α-amylase/subtilisin inhibitor (BASI). This is the first reported structure of a cereal Kunitz-P family inhibitor in complex with a subtilisin. Structural analysis revealed that BASI inhibits Savinase in a novel way, as the interacting loop is shorter than loops previously reported. Mutational analysis showed that Thr88 is crucial for the inhibition, as it stabilises the interacting loop through intramolecular interactions with the BASI backbone.     

Characterization of a Kunitz trypsin inhibitor with a single disulfide bridge from seeds of Inga laurina (SW.) Willd

Inga laurina is a tree that belongs to the Mimosoideae sub-family of the Leguminosae. A protein inhibitor of trypsin (ILTI) was isolated from its seeds by ammonium sulphate precipitation, ion-exchange chromatography and rechromatography on an HiTrap Q ion-exchange column. By SDS-PAGE, ILTI yielded a single band with a Mr of 20 kDa with or without reduction. ILTI was found to be a single polypeptide chain containing 180 amino acids, the sequence of which was clearly homologous to the Kunitz family of serine protease plant protein inhibitors, and it also showed significant similarity to the seed storage proteins, sporamin and miraculin. However, ILTI displayed major differences to most other Kunitz inhibitors in that it contained only one disulfide bridge, and did not have two polypeptide chains as for the majority of other Kunitz inhibitors purified from Mimosoideae species. ILTI inhibited bovine trypsin with an equilibrium dissociation constant (K(i)) of 6 x 10(-9)M, but did not inhibit chymotrypsin, papain and alpha-amylase. Its amino acid sequence contained a Lys residue at the putative reactive site (position 64). ILTI was stable over a wide range of temperature and pH and in the presence of DTT.     

Selective Removal of Individual Disulfide Bonds within a Potato Type II Serine Proteinase Inhibitor from Nicotiana alata Reveals Differential Stabilization of the Reactive-Site Loop

The 53-amino-acid trypsin inhibitor 1 from Nicotiana alata (T1) belongs to the potato type II family also known as the PinII family of proteinase inhibitors, one of the major families of canonical proteinase inhibitors. T1 contains four disulfide bonds, two of which (C4-C41 and C8-C37) stabilize the reactive-site loop. To investigate the influence of these two disulfide bonds on the structure and function of potato II inhibitors, we constructed two variants of T1, C4A/C41A-T1 and C8A/C37A-T1, in which these two disulfide bonds were individually removed and replaced by alanine residues. Trypsin inhibition assays show that wild-type T1 has a K_i of < 5 nM, C4A/C41A-T1 has a weaker K_i of ∼ 350 nM, and the potency of the C8A/C37A variant is further decreased to a K_i of ∼ 1.8 μM. To assess the influence of the disulfide bonds on the structure of T1, we determined the structure and dynamics of both disulfide variants by NMR spectroscopy. The structure of C4A/C41A-T1 and the amplitude of intrinsic flexibility in the reactive-site loop resemble that of the wild-type protein closely, despite the lack of the C4-C41 disulfide bond, whereas the timescale of motions is markedly decreased. The rescue of the structure despite loss of a disulfide bond is due to a previously unrecognized network of interactions, which stabilizes the structure of the reactive-site loop in the region of the missing disulfide bond, while allowing intrinsic motions on a fast (picosecond-nanosecond) timescale. In contrast, no comparable interactions are present around the C8-C37 disulfide bond. Consequently, the reactive-site loop becomes disordered and highly flexible in the structure of C8A/C37A-T1, making it unable to bind to trypsin. Thus, the reactive-site loop of T1 is stabilized differently by the C8-C37 and C4-C41 disulfide bonds. The C8-C37 disulfide bond is essential for the inhibitory activity of T1, whereas the C4-C41 disulfide bond is not as critical for maintaining the three-dimensional structure and function of the molecule but is responsible for maintaining flexibility of the reactive-site loop on a microsecond-nanosecond timescale.     

Immunomodulatory activity of a chymotrypsin inhibitor from Momordica cochinchinensis seeds.

Serine protease inhibitors are widely distributed in the plant kingdom. Many of them have been purified and characterized from different species. While the physicochemical properties of these protease inhibitors have been extensively investigated, their biological effects, e.g. immunomodulatory effect, remain relatively unexplored. Recently, we isolated a chymotrypsin-specific inhibitor (MCoCI) from the seeds of Momordica cochinchinensis (Lour) Spreng (Family Cucurbitaceae), the traditional Chinese medicine known as Mubiezhi, which has been used as an antiinflammatory agent. In the present study, the effects of MCoCI on different types of cells of the immune system, including splenocytes, splenic lymphocytes, neutrophils, bone marrow cells and macrophages, were investigated. MCoCI was shown to possess immuno-enhancing and antiinflammatory effects. MCoCI could stimulate the proliferation of different cells of the immune system, e.g. splenocytes, splenic lymphocytes and bone marrow cells, in a manner comparable to that of Concanavalin A. Moreover, MCoCI could also suppress the formation of hydrogen peroxide in neutrophils and macrophages. These immunomodulatory effects may explain some of the therapeutic actions of Mubiezhi.     

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.     

The release of proteinase inhibitors from legume seeds during germination

The seeds of twelve common species of legumes were examined for the release of proteinase inhibitor activity during germination. All species released inhibitory activity against bovine trypsin (EC 3.4.21.4), ranging from 1.0 unit per g dry wt. of seed in 24 hr for soybean (Glycine max), to 0.07 unit per g for broad beans (Vicia faba) and sugar pod peas (Pisum sativum). This release corresponds to approximately 1–13 % of the total trypsin inhibitory activity of the seed, with lentils (Lens culinaris) releasing the greatest percentage, and the scarlet runner bean (Phaseolus coccineus) the least. In most species the amount of inhibitor released increases until 24–48 hr of germination, and then remains roughly the same or decreases slightly by 72 hr of germination. Five species of legumes were also examined for the release of inhibitory activity against bovine chymotrypsin (EC 3.4.21.1). In each case chymotryptic inhibitory activity was released in a manner similar to the trypsin inhibitor.     

Phage display of a double-headed proteinase inhibitor: Analysis of the binding domains of potato proteinase inhibitor II

Potato proteinase inhibitor II (PI2) is a serine proteinase inhibitor composed of two domains that are thought to bind independently to proteinases. To determine the activities of each domain separately, various inactive and active domain combinations were constructed by substituting amino acid residues in the active domains by alanines. These derivatives were expressed as soluble protein inEscherichia coli and exposed on M13 phage as fusions to gene 3 in a phagemid system for monovalent phage display. Inactivation of both active domains by Ala residues reduced binding of phage to trypsin and chymotrypsin by 95%. Ten times more phage were bound to proteinases by domain II compared to domain I, while a point mutation (Leu⁵ Arg) altered the binding specificity of domain I of PI2 phage from chymotrypsin to trypsin. The mutants were used to show that functional PI2 phage mixed with nonfunctional PI2 phage could be enriched 323 000-fold after three rounds of panning. Thus, these results open up the possibility to use phage display for the selection of engineered PI2 derivatives with improved binding characteristics towards digestive proteinases of plants pests.The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number L37519 (p303.51).     

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.     

Proteinase inhibitors in Nauphoeta cinerea midgut.

Proteinase inhibitors were studied in the midgut of Nauphoeta cinerea Oliv. (Blattoptera: Blaberidae) in experimental conditions, excluding their nutritional origin. One trypsin inhibitor (TI) with M(r) 8,000 and two subtilisin inhibitors (SI1 and SI2) with M(r) 13,000 and 8,000 were detected after fractionation of total protein preparation on Sephadex G-50. Ninety-four percent of both types of inhibitors was located in anterior midgut (AM). TI was 120-fold purified by FPLC-chromatography on Mono Q. Its isoelectric point was 4.3. TI lost a large part of activity in acidic and especially in alkaline medium. TI, SI1, and SI2 effectively inhibited activities of endogenous proteinases from posterior midgut (PM) of the cockroach. A search for inhibitor of endogenous unusual SH-dependent proteinase from AM revealed in AM a new inhibitor with M(r) 18,000. It was also inactivated in alkaline medium and was effective against proteinases from PM along with unusual SH-dependent proteinase from AM. A mechanism of regulation of activity of midgut proteinases is proposed based on pH-stability of inhibitors.     

Novel alleles among soybean Bowman-Birk proteinase inhibitor gene families

Trypsin inhibitors have been found in various animals, plants and microorganisms.There were two types of trypsin inhibitors in soybean including Bowman-Birk protease inhibitors(BBI) and Kunitz in-hibitors(KTI).The different BBI genes from wild soybean(G.soja) and cultivated soybean(G.max) formed a multigene family.We constructed a cDNA library of cultivar 'SuiNong 14' seed at the R7 growth stage using the SMART Kit.Seventeen contigs or singletons were highly homologous to soy-bean protease inhibitors.Contigs of 5, 35, 8 and 9 were highly homologous to BBI family members BBI-A1, BBI-A2, BBI-C and BBI-D, respectively.Sequence analyses showed there were novel allelic varia-tions among the 4 BBI members in SuiNong 14.Based on the comparison of soybean seed cDNA li-braries from different developmental stages, it was apparent that the expression of trypsin inhibitors increased during seed development in soybean.Phylogenetic analysis of BBI gene sequences among dicotyledonous and monocotyledonous plants demonstrated that these genes shared a common pro-genitor.     

Characterization of a Kunitz trypsin inhibitor with one disulfide bridge purified from Swartzia pickellii

Swartzia pickellii is a Leguminosae that belongs to the Caesalpinioideae sub-family the Swartzia pickellii Trypsin Inhibitor (SWTI), a serine proteinase inhibitor was isolated from its seeds. SWTI is a single polypeptide chain protein and it's structure has 174 amino acid residues, it homologous to other Kunitz plant inhibitors, however shows some major differences: it contains only one disulfide bridge, instead two which are usually found in plant Kunitz inhibitors, and the SWTI reactive site does not contain the usual Arg or Lys residues at the putative reactive site (position 65). A glycosylation site was detected at Asn38 with 1188 kDa carbohydrate portion. The primary structure micro heterogeneity was found combining the sequence determination and mass spectrometry. Three forms of SWTI were actually defined: two glycosylated forms a 20,204 kDa (Arg 165) and 20,185 kDa (His 165) and one deglycosylated form 19,016 kDa (Arg 165), all of them contain a Met residue at position 130.     

甘薯和花生胰蛋白酶抑制剂的初步研究

许多植物蛋白制成品均含有抑制动物消化的蛋白酶抑制剂。目前已从某些豆类及蔬菜种子中分离出多种对胰蛋白酶具有抑制作用的活性物质。该实验以花生、甘薯等为原料,通过DEAE-Sepharose4BFF阴离子交换柱层析分离胰蛋白酶抑制剂,以N-苯甲酰-L-精氨酸乙酯(BAEE)为底物测定其对胰蛋白酶的抑制活性;将具有抑制活性的组分通过SDS-PAGE测定蛋白质相对分子质量(Mr);以聚丙烯酰胺凝胶等电聚焦电泳测定蛋白质等电点(pI)。结果显示,甘薯中至少有4种胰蛋白酶抑制剂组分,相对分子质量为20～25kD、等电点在pH5.0～6.6之间;花生中至少有三种胰蛋白酶抑制剂组分,相对分子质量为30～70kD、等电点在pH5.0～5.8之间。     

Serine proteinase inhibitors in the Compositae: distribution, polymorphism and properties

Multiple molecular forms of inhibitors of trypsin (TI) and chymotrypsin (CI), which are typical digestive enzymes of insects, mammals and micro-organisms, and subtilisin (SI), a proteinase of many bacteria and phytopathogenic fungi, were identified in seeds and vegetative organs of the majority of 128 wild and cultivated species representing 65 genera of three of the subfamilies of the Compositae. Inhibitors with M(r) ranging from 7450 to 7800 and combining activities towards subtilisin and trypsin and/or chymotrypsin (T/C/SI) had the widest distribution and may be involved in plant defense mechanisms. They were found in many species of the subfamilies Carduoideae (genera Carthamus, Centaurea, Cirsium), Cichorioideae (Lactuca, Taraxacum) and Asteroideae (Helianthus, Cosmos, Bidens). Partial amino acid sequencing showed that the safflower (Carthamus tinctorius) T/C/SI and Cosmos bipinnatus T/C/SI, T/SI and C/SI belonged to the potato I inhibitor family. The most active, variable and heterogeneous inhibitors were found in species of the tribe Heliantheae, which is placed in the evolutionary advanced subfamily Asteroideae. Seeds of Helianthus species, Eclipta prostrata, Gailardia aristata, Zinnia elegans and Silphium perfoliatum contained various TI with M(r) ranging from 1500 to 14,750, with some also containing SI. H. annuus seeds contain a unique cyclic TI of M(r) 1514 and similar TI were also present in other Helianthus spp. and the related species Tithonia diversifolia. Zinnia elegans contained a TI with M(r) 11,350 which appeared to represent a novel type of inhibitor distantly related to the cereal subgroup of Bowman-Birk inhibitors. TI and T/SI varied widely in H. annuus lines and wild Helianthus species in their presence or absence and composition. Similar T/SI components were found in the cultivated diploid H. annuus and annual diploid species with the B genome but not in perennials with the A genome. Some T/SI, SI and TI were detected in vegetative organs of sunflower and other Compositae. Studies of the polymorphism and distribution of proteinase inhibitors are relevant to the evolution of protective protein systems and the mechanisms of resistance to pathogenic organisms in the Compositae and other plants.