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.     

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.     

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.     

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.     

Bowman–Birk inhibitors in Lens: identification and characterization of two paralogous gene classes in cultivated lentil and wild relatives

In order to investigate the genetic structure of lentil Bowman–Birk inhibitors (BBIs), primers were designed on pea BBI sequences. The sequences obtained from lentil DNA, using these primers, indicate that lentil possesses at least two paralogous genes. Protein sequences translated in silico from lentil DNA sequences suggest that the two coded proteins are highly similar to Pisum trypsin inhibitor TI1 and TI6 BBIs, respectively. In fact, both are double-headed inhibitors, one class showing the presence of a trypsin- and a chymotrypsin-reactive site, the other showing two trypsin-inhibition sites, similar to pea TI1 and TI6, respectively. The same primers were used to amplify sequences from the DNA of other Lens species. The results strongly support that all species of Lens possess the same classes of BBI coding genes, orthologous to those identified in the cultivated lentil. Lens nigricans showed the most diverging sequences both at the nucleotide and the amino acid level. The similarity of the two gene classes identified in the genus Lens to those of Pisum and the observations that the patterns of expression of the Lens genes are equivalent to those of pea orthologous genes, possibly imply that BBIs in Lens are coded by gene classes with similar genome organization and function to those of pea. Finally, a phyletic analysis, based on the comparison of sequences obtained from other species belonging to the Vicieae tribe of the Fabaceae family, strongly suggests that all Vicieae could have a similar genome organization and function for BBI genes, and that this could be a general rule in all the Fabaceae family.Publication of the Institute of Plant Genetics N. 50     

Participation of S-S loops in inhibitory activity of peanut protease inhibitor B-III

A peanut Bowman-Birk (BBI) type protease inhibitors B-III has two regions, 1 and 2, homologous with each other. Each region contains three S-S loops and a reactive site in its outermost loop. The inhibitor was used to investigate the contribution of the S-S loops of BBI-type inhibitors to their inhibitory activity. Two steps of Edman degradation of the native inhibitor cleaved loop III (the innermost S-S loop) of region 1 of B-III, and the antichymotryptic activity of the first reactive site decreased to about 1/4 of that of native B-III. A third step of Edman degradation split loop II and the inhibitory activity at that site became extremely low (about 1/200 of the original value). These results suggest that protease inhibitor B-III maintains its active conformation by means of the three S-S loops and that the conformation is markedly changed by the splitting of loop II.     

A simple method to determine trypsin and chymotrypsin inhibitory activity

A colorimetric method for serine protease inhibition was modified using N-Acetyl-DL-Phenylalanine beta-Naphthylester (APNE) as the substrate and o-Dianisidine tetrazotized (oD) as the dye. The reaction generated a single peak absorbing at 530 nm for both trypsin and chymotrypsin. Standard curves with increasing enzyme concentrations showed strong linearity. A standard curve for the serine protease inhibitor, Bowman-Birk Inhibitor (BBI), has been made using this modified method. The IC50 for 3 U of trypsin was found to be 33 ng and the IC50 obtained for 3 mU of chymotrypsin was 53 ng. A recombinant BBI (rBBI) gene was constructed, cloned and expressed in the yeast Pichia pastoris. Evaluating samples of rBBI for protease inhibitory activity by the gel activity method failed to quantify the inhibitor amounts, due to high sensitivity for trypsin inhibition and low sensitivity for chymotrypsin inhibition. After development, the results could not be quantified, even to the extent that 1 microl of rBBI could not be detected with chymotrypsin inhibition. Therefore, a modified method for trypsin and chymotrypsin inhibition was used to evaluate the level of rBBI-expression for these same samples. The level of rBBI expression was calculated to be 50-56 ng/microl of media. These amounts fit into the range of values previously obtained by Western blot analysis. This modified method allows us to combine the sensitivity of the gel activity method with the quantification attributes of a Western blot. Thus, the modified method represents a significant improvement in speed, sensitivity and reproducibility over the gel activity method.     

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 (1)H-NMR solution structure of the antitryptic core peptide of Bowman-Birk inhibitor proteins: a minimal canonical loop

Bowman-Birk inhibitor (BBI) proteins contain an inhibitory motif comprising a disulfide-bonded sequence that interacts with serine proteinases. Recently, a small 14-residue peptide from sunflowers (SFTI-1), which has potent anti-trypsin activity, has been found to have the same motif. However, this peptide also has an unusual head-to-tail cyclisation. To address the role of the core inhibitory sequence itself, we have solved the (1)H-NMR solution structure of an antitryptic 11-residue cyclic peptide that corresponds to the core reactive site loops of both SFTI-1 and Bowman-Birk inhibitor proteins. A comparison is made between the secondary chemical shifts found in this family and the canonical regions of several other inhibitors, giving some insight into relative flexibility and hydrogen bonding patterns in these inhibitors. The solution structure of the core peptide in isolation is found to retain essentially the same three-dimensional arrangement of both backbone and side chains as observed in larger antitryptic BBI and SFTI-1 fragments as well as in the complete proteins. The retention of the canonical conformation in the core peptide explains the peptids inhibitory potency. It therefore represents a minimization of both the BBI and SFTI-1 sequences. We conclude that the core peptide is a conformationally defined, canonical scaffold, which can serve as a minimal platform for the engineering of biological activity.     

Eliminating Anti-Nutritional Plant Food Proteins: The Case of Seed Protease Inhibitors in Pea

Several classes of seed proteins limit the utilisation of plant proteins in human and farm animal diets, while plant foods have much to offer to the sustainable intensification of food/feed production and to human health. Reduction or removal of these proteins could greatly enhance seed protein quality and various strategies have been used to try to achieve this with limited success. We investigated whether seed protease inhibitor mutations could be exploited to enhance seed quality, availing of induced mutant and natural Pisum germplasm collections to identify mutants, whilst acquiring an understanding of the impact of mutations on activity. A mutant (TILLING) resource developed in Pisum sativum L. (pea) and a large germplasm collection representing Pisum diversity were investigated as sources of mutations that reduce or abolish the activity of the major protease inhibitor (Bowman-Birk) class of seed protein. Of three missense mutations, predicted to affect activity of the mature trypsin / chymotrypsin inhibitor TI1 protein, a C77Y substitution in the mature mutant inhibitor abolished inhibitor activity, consistent with an absolute requirement for the disulphide bond C77-C92 for function in the native inhibitor. Two further classes of mutation (S85F, E109K) resulted in less dramatic changes to isoform or overall inhibitory activity. The alternative strategy to reduce anti-nutrients, by targeted screening of Pisum germplasm, successfully identified a single accession (Pisum elatius) as a double null mutant for the two closely linked genes encoding the TI1 and TI2 seed protease inhibitors. The P. elatius mutant has extremely low seed protease inhibitory activity and introgression of the mutation into cultivated germplasm has been achieved. The study provides new insights into structure-function relationships for protease inhibitors which impact on pea seed quality. The induced and natural germplasm variants identified provide immediate potential for either halving or abolishing the corresponding inhibitory activity, along with associated molecular markers for breeding programmes. The potential for making large changes to plant protein profiles for improved and sustainable food production through diversity is illustrated. The strategy employed here to reduce anti-nutritional proteins in seeds may be extended to allergens and other seed proteins with negative nutritional effects. Additionally, the novel variants described for pea will assist future studies of the biological role and health-related properties of so-called anti-nutrients.     

A Low Dose of Fermented Soy Germ Alleviates Gut Barrier Injury,Hyperalgesia and Faecal Protease Activity in a Rat Model of Inflammatory Bowel Disease

Pro-inflammatory cytokines like macrophage migration inhibitory factor (MIF), IL-1β and TNF-α predominate in inflammatory bowel diseases (IBD) and TNBS colitis. Increased levels of serine proteases activating protease-activated receptor 2 (PAR-2) are found in the lumen and colonic tissue of IBD patients. PAR-2 activity and pro-inflammatory cytokines impair epithelial barrier, facilitating the uptake of luminal aggressors that perpetuate inflammation and visceral pain. Soy extracts contain phytoestrogens (isoflavones) and serine protease inhibitors namely Bowman-Birk Inhibitors (BBI). Since estrogens exhibit anti-inflammatory and epithelial barrier enhancing properties, and that a BBI concentrate improves ulcerative colitis, we aimed to evaluate if a fermented soy germ extract (FSG) with standardized isoflavone profile and stable BBI content exert cumulative or synergistic protection based on protease inhibition and estrogen receptor (ER)-ligand activity in colitic rats. Female rats received orally for 15 d either vehicle or FSG with or without an ER antagonist ICI 182.780 before TNBS intracolonic instillation. Macroscopic and microscopic damages, myeloperoxidase activity, cytokine levels, intestinal paracellular permeability, visceral sensitivity, faecal proteolytic activity and PAR-2 expression were assessed 24 h, 3 d and 5 d post-TNBS. FSG treatment improved the severity of colitis, by decreasing the TNBS-induced rise in gut permeability, visceral sensitivity, faecal proteolytic activity and PAR-2 expression at all post-TNBS points. All FSG effects were reversed by the ICI 182.780 except the decrease in faecal proteolytic activity and PAR-2 expression. In conclusion, the anti-inflammatory properties of FSG treatment result from two distinct but synergic pathways i.e an ER-ligand and a PAR-2 mediated pathway, providing rationale for potential use as adjuvant therapy in IBD.     

Functional comparison of homologous members of three groups of Kunitz-type enzyme inhibitors from potato tubers ( Solanum tuberosum L.)

For functional studies, nine cDNAs encoding Kunitz-type enzyme inhibitors from potato tubers were expressed as GST (glutathione S transferase)-tagged fusion proteins in the fission yeast Schizosaccharomyces pombe. The inhibitors represented the three major homology groups A, B and C found in tubers. Members of the same homology group were at least 90% identical in sequence. The purified GST fusion proteins were tested for their ability to inhibit the proteases trypsin, alpha-chymotrypsin, subtilisin, papain and aspergillopepsin I, and for inhibition of the growth of fungi. Fusion proteins belonging to the same and different homology groups were found to exhibit distinct protease inhibition profiles. Removal of the GST tag by cleavage with enterokinase did not change the inhibition profile but increased the inhibitory activity. Group A and B inhibitors affected the proteases to different extents, whereas group C inhibitors showed only weak or no protease inhibition. One fusion protein completely inhibited aspergillopepsin I. One fusion protein each of groups A and B strongly inhibited mycelial growth of the fungus Fusarium moniliforme. The results suggest functional polymorphism among closely related members of the Kunitz-type inhibitor family.     

Specific inhibitors prevent proteolytic degradation of recombinant proteins expressed in High Five cells

The insect cell line BTI-TN-5B1-4 (High Five) is frequently used to express recombinant proteins in large amounts using the baculovirus expression system. However, extensive proteolytic degradation of recombinant proteins is often encountered. Furthermore, we have observed that recombinant proteins migrate in SDS-PAGE in agreement with poly-ubiquitinated forms of the protein, suggesting a ubiquitin/proteasome degradation pathway. Here, we describe a systematic study unraveling the effect of adding proteasome inhibitors or specific protease inhibitors to the growth medium of High Five insect cells infected with recombinant baculovirus. Furthermore, protease inhibitors were added to the lysis buffer to establish the most efficient way to inhibit proteolytic activity after lysis of baculovirus-infected cells expressing recombinant proteins. We conclude that a combination of adding protease inhibitors to the growth medium and to the lysis buffer minimizes the proteolytic activity in High Five cells. The most efficient protease inhibitors were E-64 in the growth medium together with Leupeptin in the lysis buffer at concentrations higher than with available cocktails of inhibitors. The optimal treatment of High Five cells is different from the optimal treatment of Sf9 cells. For proteins susceptible to ubiquitinylation, a treatment of insect cell cultures with the proteasome inhibitor MG132 (LLL) leads to a considerable reduction of the yield of production of recombinant protein.     

Bowman-Birk protease inhibitor from the seeds of Vigna unguiculata forms a highly stable dimeric structure

Different protease inhibitors including Bowman-Birk type (BBI) have been reported from the seeds of Vigna unguiculata. Protease isoinhibitors of double-headed Bowman-Birk type from the seeds of Vigna unguiculata have been purified and characterized. The BBI from Vigna unguiculata (Vu-BBI) has been found to undergo self-association to form very stable dimers and more complex oligomers, by size-exclusion chromatography and SDS-PAGE in the presence of urea. Many BBIs have been reported to undergo self-association to form homodimers or more complex oligomers in solution. Only one dimeric crystal structure of a BBI (pea-BBI) is reported to date. We report the three-dimensional structure of a Vu-BBI determined at 2.5 A resolution. Although, the inhibitor has a monomer fold similar to that found in other known structures of Bowman-Birk protease inhibitors, its quaternary structure is different from that commonly observed in this family. The structural elements responsible for the stability of monomer molecule and dimeric association are discussed. The Vu-BBI may use dimeric or higher quaternary association to maintain the physiological state and to execute its biological function.     

Crystal structure of the Bowman-Birk inhibitor from barley seeds in ternary complex with porcine trypsin

The structure and function of Bowman-Birk inhibitors (BBIs) from dicotyledonous plants such as soybean have been studied extensively. In contrast, relatively little is known about the BBIs from monocotyledonous plants such as barley, which differ from dicot BBIs in size and tertiary structure. The BBI from barley seeds (BBBI) consists of 125 amino acid residues with two separate inhibitory loops. Previously we determined the high-resolution structure of a 16 kDa BBBI in the free state. The BBBI folds into two compact domains (N and C domain) with tertiary structures that are similar to that of the 8 kDa BBI from dicots. Here we report the structure of a 1:2 complex between BBBI and porcine pancreatic trypsin (PPT) at 2.2 A resolution. This structure confirms that several regions, including the inhibitory loops in the free BBBI structure, show exceptionally low temperature factors and a distorted conformation due to crystalline packing in the lattice. Extensive analysis of the interaction between BBBI and trypsin, and comparison with other known canonical inhibitor-protease complexes, reveals that the mode of interaction between BBBI and PPT is similar to that of known serine protease inhibitors, as expected; however, several unique features are also identified in the primary binding sites near the inhibitory loops as well as in additional binding sites. The carboxy-terminal tail of the inhibitor extends into the interface between the two trypsin molecules and interacts with both of them simultaneously. The longest distance between the two P1 residues (Arg17 and Arg76) in the complex structure is approximately 34 A, which is shorter than in the free inhibitor, but it is still possible for BBBI to bind and inhibit two trypsin molecules simultaneously and independently.     

Kinetics of the inhibition of calcium/calmodulin-dependent protein kinase II by pea protein-derived peptides

Calcium/calmodulin-dependent protein kinase II (CaMKII) catalyzes the phosphorylation of various cellular proteins and excessive activities have been implicated in the pathogenesis of various chronic diseases. We hypothesized that positively charged peptides can be produced through enzymatic hydrolysis of pea proteins; such peptides could then bind to negatively charged calmodulin (CaM) at a physiological pH level and inhibit CaMKII activity. Pea protein isolate was hydrolyzed with an alkaline protease (alcalase) and filtered through a 1000-mol wt cutoff membrane. The permeate, which contained low-molecular weight peptides, was used to isolate cationic peptides on an SP-Sepharose column by ion exchange chromatography. Separation of the permeate on the SP-Sepharose column yielded two fractions with net positive charges that were subsequently used for enzyme inhibition studies. Fraction I eluted earlier from the column and contained lower contents of lysine and arginine than Fraction II, which eluted later. Results show that both peptide fractions inhibited CaMKII activity mostly in a competitive manner, although kinetic data suggested that inhibition by Fraction II may be of the mixed type. Kinetic analysis (K(m) and K(i)) showed that affinity of peptides in Fraction II for CaM was more than that in Fraction I, which was directly correlated with the higher inhibitory properties of Fraction II against CaMKII. The results suggest that it may be possible to use pea protein-derived cationic peptides to modulate CaMKII activities.     

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.