Comparative structure analysis of proteinase inhibitors from the desert locust, Schistocerca gregaria.

The solution structure of three small serine proteinase inhibitors, two natural and one engineered protein, SGCI (Schistocerca gregaria chymotrypsin inhibitor), SGCI[L30R, K31M] and SGTI (Schistocerca gregaria trypsin inhibitor), were determined by homonuclear NMR-spectroscopy. The molecules exhibit different specificities towards target proteinases, where SGCI is a good chymotrypsin inhibitor, its mutant is a potent trypsin inhibitor, and SGTI inhibits both proteinases weakly. Interestingly, SGTI is a much better inhibitor of insect proteinases than of the mammalian ones used in common assays. All three molecules have a similar fold composed from three antiparallel beta-pleated sheets with three disulfide bridges. The proteinase binding loop has a somewhat distinct geometry in all three peptides. Moreover, the stabilization of the structure is different in SGCI and SGTI. Proton-deuterium exchange experiments are indicative of a highly rigid core in SGTI but not in SGCI. We suggest that the observed structural properties play a significant role in the specificity of these inhibitors.     

Monoclonal Antibodies Against Soybean Bowman-Birk Inhibitor Recognize the Protease-Reactive Loops

Monoclonal antibodies against soybean Bowman-Birk protease inhibitor (BBI) have been generated and used to detect and quantify BBI in foods, soybean germplasm, and animal tissues and fluids. The purpose of this study was to determine the recognition sites of two monoclonal antibodies to BBI (mAb 238 and mAb 217) in relation to the protease-inhibitory sites of BBI. The results showed that (1) the binding of mAb 238 can be blocked by trypsin and that of mAb 217 by chymotrypsin; (2) the trypsin or chymotrypsin inhibitory activities of BBI are blocked by mAb 238 or mAb 217, respectively; and (3) mAb 238 failed to recognize a tryptic loop mutant BBI variant and mAb 217 was unable to bind a chymotryptic loop mutant BBI variant. These findings demonstrate that the epitopes recognized by mAb 238 and mAb 217 reside, at least in part, in the tryptic and chymotryptic loops of BBI, respectively.     

The Bowman-Birk inhibitor. Trypsin- and chymotrypsin-inhibitor from soybeans

Four decades of studies on the isolation, characterization, properties, structure, function and possible uses of the Bowman-Birk trypsin- and chymotrypsin-inhibitor from soybeans are reviewed. Starting from Bowman's Acetone Insoluble factor, designated Ai, AA and SBTIAA, the Bowman-Birk inhibitor (BBI) was found to be a protein molecule consisting of a chain of 71 amino acids cross linked by 7 disulfide bonds, with a tendency to self-associate. BBI possesses two independent sites of inhibition, one at Lys 16-Ser 17 against trypsin and the other at Leu 43-Ser 44 against chymotrypsin. It forms a 1:1 complex with either trypsin or chymotrypsin and a ternary complex with both enzymes. Ingestion of BBI by rats, chicks or quails affects the size and protein biosynthesis of the pancreas. Establishment of the full covalent structure of BBI revealed a high homology in the sequences around the two inhibitory sites, suggesting evolutionary gene duplication from a single-headed ancestral inhibitor. Scission of BBI by CNBr followed by pepsin results in two active fragments, one that inhibits trypsin and the other, chymotrypsin. Replacements and substitutions in the reactive sites result in changes in inhibitory activity and in specificity of inhibition. Conformation studies, labeling of BBI with a photoreactive reagent, chemical synthesis of cyclic peptides that include inhibitory sites, in vitro synthesis of BBI, and species specificity regarding the inhibited enzymes are described. The significance of BBI as a prototype of a family of inhibitors present in all legume seeds is discussed.     

Studies on simultaneous inhibition of trypsin and chymotrypsin by horsegram Bowman-Birk inhibitor

Bowman-Birk inhibitors (BBI) isolated from plant seeds are small proteins active against trypsin and/or chymotrypsin. These inhibitors have been extensively studied in terms of their structure, interactions, function and evolution. Examination of the known three-dimensional structures of BBIs revealed similarities and subtle differences. The hydrophobic core, deduced from surface accessibility and hydrophobicity plots, corresponding to the two tandem structural domains of the double headed BBI are related by an almost exact two-fold, in contrast to the reactive site loops which depart appreciably from the two-fold symmetry. Also, the orientations of inhibitory loops in soybean and peanut inhibitors were different with respect to the rigid core. Based on the structure of Adzuki bean BBI-trypsin complex, models of trypsin and chymotryspin bound to the monomeric soybean BBI (SBI) were constructed. There were minor short contacts between the two enzymes bound to the inhibitor suggesting near independence of binding. Binding studies revealed that the inhibition of one enzyme in the presence of the other is associated with a minor negative cooperativity. In order to assess the functional significance of the reported oligomeric forms of BBI, binding of proteases to the crystallographic and non-crystallographic dimers as found in the crystal structure of peanut inhibitor were examined. It was found that all the active sites in these oligomers cannot simultaneously participate in inhibition.     

Identification of chymotrypsin inhibitors from a second-generation template assisted combinatorial peptide library.

In an earlier study (McBride JD, Freeman N, Domingo GJ, Leatherbarrow RJ. Selection of chymotrypsin inhibitors from a conformationally-constrained combinatorial peptide library. J. Mol. Biol. 1996; 259: 819-827) we described a resin-bound cyclic peptide library, constructed based on the sequence of the anti-tryptic reactive site loop of Bowman Birk Inhibitor (BBI), a proteinase inhibitor protein. This library was used to identify re-directed chymotrypsin inhibitors with Ki values as low as 17 nM. We have now extended this work by constructing an enhanced library in which a further position, at the P4 site of the inhibitor, has been randomized. This new library has variation at three target locations (P4, P1 and P2) within the inhibitory loop region, producing 8,000 variants. Screening this library allowed selection of new inhibitor sequences with Ki values as low as 3.4 nM. The success of this approach is reflected by the fact that the inhibition constant given by the selected peptide sequence is slightly lower than that reported against chymotrypsin for the most studied full length BBI protein, Soybean BBI 2-IV.     

Interaction between duodenase, a proteinase with dual specificity, and soybean inhibitors of Bowman-Birk and Kunitz type

The interaction between duodenase, which belongs to a group of Janus-faced proteinases, and classical Bowman--Birk (BBI) and Kunitz (STI) type inhibitors from soybean was investigated. Duodenase was shown to interact only with the antichymotrypsin site (Leu-Ser) of BBI, whereas the antitrypsin site (Lys-Ser) of the inhibitor appeared to be vacant and capable of interaction with trypsin. The inhibition constants of duodenase by BBI, the BBI--trypsin complex, and STI were 4, 400, and 40 nM, respectively.     

Atomic resolution crystal structure of HV‐BBI protease inhibitor from amphibian skin in complex with bovine trypsin

Protease inhibitors of the Bowman‐Birk (BBI) family are commonly found in plants and animals where they play a protective role against invading pathogens. Here, we report an atomic resolution (1Å) crystal structure of a peptide inhibitor isolated from a skin secretion of a Chinese bamboo odorous frog Huia versabilis (HV‐BBI) in complex with trypsin. HV‐BBI shares significant similarities in sequence with a previously described inhibitor from a diskless‐fingered odorous frog Odorrana graham (ORB). However, the latter is characterized by more than a 16,000 fold higher K_i against trypsin than HV‐BBI. Comparative analysis of trypsin cocrystal structures of HV‐BBI and ORB and additionally that of Sunflower Trypsin Inhibitor (SFTI‐1) together with accessory information on the affinities of inhibitor variants allowed us to pinpoint the inhibitor moiety responsible for the observed large difference in activity and also to define the extent of modifications permissible within the common protease‐binding loop scaffold of BBI inhibitors. We suggest that modifications outside of the inhibitory loop permit the evolution of specificity toward different enzymes characterized by trypsin‐like specificity. Proteins 2015; 83:582–589. © 2014 Wiley Periodicals, Inc.     

Toxicity to the pea aphid Acyrthosiphon pisum of anti-chymotrypsin isoforms and fragments of Bowman-Birk protease inhibitors from pea seeds

Aphids feed on a protein-poor diet and are insensitive to several serine protease inhibitors. However, among the Bowman-Birk family of plant trypsin inhibitors (BBI), some members display significant toxicity to the pea aphid Acyrthosiphon pisum. A BBI isoform purified from pea seeds (PsTI-2) displays an IC50 of 41 microM and a LC50 of 48 microM at 7 days. Our data show that the chymotrypsin-directed active site from these bifunctional inhibitors is responsible for this activity, and that artificial cyclic peptides bearing the Bowman-Birk anti-chymotrypsin head induce much greater toxicity and growth inhibition than their anti-trypsin counterparts. The toxic syndrome included a rapid behavioural response of aphids on diets containing the toxic peptides, with induced restlessness after only 1 h of exposure to the chymotrypsin inhibitor. Nevertheless, chymotrypsin activity was not detected in aphid guts, using two chromogenic chymotrypsin substrates, and the physiological target of the chymotrypsin inhibitor remains unknown. These data show for the first time that plant chymotrypsin inhibitors, still widely unexplored, may act as paradoxical toxicants to aphids and serve as defensive metabolites for phloem-feeding insects.     

The effect of variation within inhibitory domains on the activity of pea protease inhibitors from the Bowman-Birk class

We have investigated the properties of variant pea seed protease inhibitors, homologous to the anti-carcinogenic Bowman-Birk inhibitor (BBI) from soybean but differing most significantly in amino acid sequences at the two independent sites of protease inhibition. The pea protease inhibitors were expressed, using Aspergillus niger, with yields of up to 23 mg secreted recombinant protein per litre of media. The recombinant proteins showed protease inhibitory activity and were deduced to be disulphide-bonded correctly; limited post-translational processing had occurred at the amino-terminal ends of all proteins. Differences in trypsin and chymotrypsin specific inhibitory activities, and in inhibition constants, were observed in studies of the two recombinant variants and BBI.     

Differential inhibition of Helicoverpa armigera gut proteinases by proteinase inhibitors of pigeonpea (Cajanus cajan) and its wild relatives

The seeds of 36 pigeonpea [Cajanus cajan (L) Millsp.] cultivars, resistant and susceptible to pests and pathogens and 17 of its wild relatives were analysed for inhibitors of trypsin, chymotrypsin, and insect gut proteinases to identify potential inhibitors of insect (Helicoverpa armigera) gut enzymes. Proteinase inhibitors (PIs) of pigeonpea cultivars showed total inhibition of trypsin and chymotrypsin, and moderate inhibition potential towards H. armigera proteinases (HGP). PIs of wild relatives exhibited stronger inhibition of HGP, which was up to 87% by Rhynchosia PIs. Electrophoretic detection of HGPI proteins and inhibition of HGP isoforms by few pigeonpea wild relative PIs supported our enzyme inhibitor assay results. Present results indicate that PIs exhibit wide range of genetic diversity in the wild relatives of pigeonpea whereas pigeonpea cultivars (resistant as well as susceptible to pests and pathogens) are homogeneous. The potent HGPIs identified in this study need further exploration for their use in strengthening pigeonpea defence against H. armigera.     

Serpins of oat (Avena sativa) grain with distinct reactive centres and inhibitory specificity

Most proteinase inhibitors from plant seeds are assumed to contribute to broad-spectrum protection against pests and pathogens. In oat (Avena sativa L.) grain the main serine proteinase inhibitors were found to be serpins, which utilize a unique mechanism of irreversible inhibition. Four distinct inhibitors of the serpin superfamily were detected by native PAGE as major seed albumins and purified by thiophilic adsorption and anion exchange chromatography. The four serpins OSZa-d are the first proteinase inhibitors characterized from this cereal. An amino acid sequence close to the blocked N-terminus, a reactive centre loop sequence, and the second order association rate constant (ka') for irreversible complex formation with pancreas serine proteinases at 24 degrees C were determined for each inhibitor. OSZa and OSZb, both with the reactive centre scissile bond P1-P1' Thr downward arrow Ser, were efficient inhibitors of pancreas elastase (ka' > 105M-1 s-1). Only OSZb was also an inhibitor of chymotrypsin at the same site (ka' = 0.9 x 105M-1 s-1). OSZc was a fast inhibitor of trypsin at P1-P1' Arg downward arrow Ser (ka' = 4 x 106M-1 s-1); however, the OSZc-trypsin complex was short-lived with a first order dissociation rate constant kd = 1.4 x 10-4 s-1. OSZc was also an inhibitor of chymotrypsin (ka' > 106M-1 s-1), presumably at the overlapping site P2-P1 Ala downward arrow Arg, but > 90% of the serpin was cleaved as substrate. OSZd was cleaved by chymotrypsin at the putative reactive centre bond P1-P1' Tyr downward arrow Ser, and no inhibition was detected. Together the oat grain serpins have a broader inhibitory specificity against digestive serine proteinases than represented by the major serpins of wheat, rye or barley grain. Presumably the serpins compensate for the low content of reversible inhibitors of serine proteinases in oats in protection of the grain against pests or pathogens.     

Differential inhibition of serine proteinases by rabbit alpha 1-proteinase inhibitors F and S

Inhibition of six serine proteinases (bovine trypsin and chymotrypsin, equine leucocyte proteinases type 1 and 2A, porcine pancreatic elastase type III and rabbit plasmin) by rabbit alpha 1-proteinase inhibitors F and S was studied. In each case examined, the F form reacted more rapidly. The number of moles of an enzyme inhibited by one mole of alpha 1-proteinase inhibitor in a complete reaction (molar inhibitory capacity) ranged from 0.26 (leucocyte proteinase type 1) to 1.01 (trypsin). More significantly, however, the molar inhibitory capacities of both alpha 1-proteinase inhibitors differed for the same enzymes. The highest F/S inhibitory ratio was recorded with chymotrypsin (1.88), and the lowest with elastase (0.69). These differences in molar inhibitory capacities are likely to reflect the dual nature of the reaction between the inhibitor and a proteinase, that is, either complex formation or inactivation of alpha 1-proteinase inhibitor without enzyme inhibition. No evidence was obtained to suggest that differential reactivity and differential inhibitory capacity are interdependent. The observations are consistent with the view that rabbit alpha 1-proteinase inhibitors F and S are closely related yet functionally distinct proteins.     

The Detection of Inhibitors of the Sclerotinia sclerotiorum (Lib.) de Bary Extracellular Proteinases in Sunflower

Abstract: Water-soluble protein fractions from leaves, seeds and heads of sunflower were shown to contain inhibitors of trypsin, chymotrypsin and extracellular proteinases from Sclerotinia sclerotiorum , a pathogen of sunflower, and Colletotrichum lindemuthianum. These included bifunctional inhibitors of trypsin and subtilisin. Comparison with the patterns of inhibition of standard proteinases indicated that the major extracellular proteinases of S. sclerotiorum are subtilisin-like. It is speculated that the sunflower inhibitors play a role in conferring resistance to fungal infection.     

Characterization of midgut proteinase activities of white grubs: Lepidiota noxia,Lepidiota negatoria,and Antitrogus consanguineus (scarabaeidae,melolonthini)

The proteinases in the midguts of three scarab white grub species, Lepidiota noxia, L. negatoria, and Antitrogus consanguineus, were investigated to classify the proteinases present and to determine the most effective proteinase inhibitor for potential use as an insect control agent. pH activity profiles indicated the presence of serine proteinases and the absence of cysteine proteinases. This was confirmed by the lack of inhibition by specific cysteine proteinase inhibitors. Trypsin, chymotrypsin, elastase, and leucine aminopeptidase activities were detected by using specific synthetic substrates. A screen of 32 proteinase inhibitors produced 9 inhibitors of trypsin, chymotrypsin, and elastase which reduced proteolytic activity by greater than 75%. © 1995 Wiley-Liss, Inc.     

Overlapping binding sites for trypsin and papain on a Kunitz-type proteinase inhibitor from Prosopis juliflora

Proteinase inhibitors are among the most promising candidates for expression by transgenic plants and consequent protection against insect predation. However, some insects can respond to the threat of the proteinase inhibitor by the production of enzymes insensitive to inhibition. Inhibitors combining more than one favorable activity are therefore strongly favored. Recently, a known small Kunitz trypsin inhibitor from Prosopis juliflora (PTPKI) has been shown to possess unexpected potent cysteine proteinase inhibitory activity. Here we show, by enzyme assay and gel filtration, that, unlike other Kunitz inhibitors with dual activities, this inhibitor is incapable of simultaneous inhibition of trypsin and papain. These data are most readily interpreted by proposing overlapping binding sites for the two enzymes. Molecular modeling and docking experiments favor an interaction mode in which the same inhibitor loop that interacts in a canonical fashion with trypsin can also bind into the papain catalytic site cleft. Unusual residue substitutions at the proposed interface can explain the relative rarity of twin trypsin/papain inhibition. Other changes seem responsible for the relative low affinity of PTPKI for trypsin. The predicted coincidence of trypsin and papain binding sites, once confirmed, would facilitate the search, by phage display for example, for mutants highly active against both proteinases.     

Structure-function relationship of bromelain isoinhibitors from pineapple stem

Bromelain isoinhibitors from pineapple stem (BIs) are unique double-chain inhibitors and inhibit the cysteine proteinase bromelain competitively. The three-dimensional structure was shown to be composed of two distinct domains, each of which is formed by a three-stranded anti-parallel beta-sheet. Unexpectedly, BIs were found to share similar folding and disulfide-bond connectivities not with the cystatin superfamily, but with Bowman-Birk trypsin/chymotrypsin inhibitor (BBI). The structural similarity between them suggests that BIs and BBI have evolved from a common ancestor and differentiated in function during the course of molecular evolution.     

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 conserved cis peptide bond is necessary for the activity of Bowman-Birk inhibitor protein

The Bowman-Birk inhibitor (BBI) family of protease inhibitors has an inhibitory region comprising a disulfide-linked nine-residue loop that adopts the characteristic canonical motif found in many serine protease inhibitors. A unique feature of the BBI loop is the presence of a cis peptide bond at the edge of the inhibitory loop. BBI-related protein fragments that encapsulate this loop retain the structure and inhibitory activity of the parent protein. The most common BBI loop sequence has a proline-proline element with a cis-trans geometry at P3'-P4'. We have examined this element by analysis of the inhibitory activity and structure for a series of synthetic fragments where each of these proline residues has been systematically replaced with alanine. The results show that only when a proline is present at P3' are potent inhibition and a cis peptide bond at that position in the solution structure observed, suggesting that this conformation is required for biological activity. Though a P4' proline is not essential for activity, it effectively stabilizes the cis conformation at P3' by suppressing alternative conformations. This is most evident from the Pro-Ala variant, which comprises a 1:1 mixture of slowly exchanging and structurally different cis and trans isomers. Monitoring the action of trypsin on this mixture by NMR shows that this protease interacts selectively with the cis P3' structure, providing direct evidence for the link between activity and the nativelike structure of the cis isomer. This is, to the best of our knowledge, the first example where cis isomer selectivity can be demonstrated for a proteinase.     

Preparation of photoreactive derivatives of trypsin-chymotrypsin inhibitors from soybeans and chick peas by selective modification of lysine residues

Photoreactive derivatives of the Bowman-Birk trypsin-chymotrypsin inhibitor (BBI) from soybeans and of CI, the trypsin-chymotrypsin inhibitor from chick peas, were prepared by selective modification of the epsilon-amino groups of lysine residues with 2-nitro-4(5)-azidophenylsulfenyl chlorides (2,4(5)-NAPS-C1). The ultraviolet absorption spectra of the photolabeled inhibitors indicated that three out of the five lysines of BBI and one of the seven lysines of CI were modified. The inhibitory activity of the modified inhibitors towards trypsin and chymotrypsin was not reduced even after photolysis. The specific lysine residues that constitute the trypsin-inhibitory sites of BBI and CI did not react with the photoreactive reagents. Further modification of the photoreactive derivatives of BBI and CI with maleic anhydride, directed towards the trypsin-reactive sites, resulted in almost complete loss of the trypsin-inhibiting activity without reducing the ability to inhibit chymotrypsin. A pronounced potentiation effect (approximately 2x) of the chymotrypsin inhibiting activity was noted for 2,5-NAPS-CI and it was retained even after maleylation followed by photolysis, raising the possibility of exposure of an additional chymotrypsin inhibitory site in CI.     

Genome organization of Bowman–Birk inhibitor in common bean (Phaseolus vulgaris L.)

A BAC library from common bean has been used in order to isolate the entire multigene Bowman–Birk serine protease inhibitor family and to study its genome organization. Using a previously isolated trypsin/chymotrypsin inhibitor nucleotide sequence as probe, two positive BAC clones were identified. The P2B8 BAC clone, of about 135 kbp and containing the complete BBI family, was chosen and partially sequenced. Our results confirm that a small multigene family codes for three double-headed inhibitors named: tc-BBI-1, tc-BBI-2 and et-BBI. They contain the binding loop trypsin/chymotrypsin (tc-BBI-1 and tc-BBI-2) and the elastase/trypsin one (et-BBI), respectively. Genes coding for tc-BBI-1 and et-BBI, were found to be very close to each other and arranged in a head to head fashion. Southern blot hybridisation on genomic DNA digested with PstI enzyme suggests that all three genes are present in a fragment of 19 kbp. Northern blot analyses on RNA isolated from various common bean organs showed that the expression of tc-BBI-1 and et-BBI was restricted to the developing cotyledons. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.