Isolation and characterization of isoinhibitors of the potato protease inhibitor I family from the latex of the rubber trees, Hevea brasiliensis

Three isoinhibitors have been isolated to homogeneity from the C-serum of the latex of the rubber tree, Hevea brasiliensis clone RRIM 600, and named HPI-1, HPI-2a and HPI-2b. The three inhibitors share the same amino acid sequence (69 residues) but the masses of the three forms were determined to be 14,893+/-10, 7757+/-5, and 7565+/-5, respectively, indicating that post-translational modifications of the protein have occurred during latex collection. One adduct could be removed by reducing agents, and was determined to be glutathione, while the other adduct could not be removed by reducing agents and has not been identified. The N-termini of the inhibitor proteins were blocked by an acetylated Ala, but the complete amino acid sequence analysis of the deblocked inhibitors by Edman degradation of fragments from endopeptidase C digestion and mass spectrometry confirmed that the three isoinhibitors were derived from a single protein. The amino acid sequence of the protein differed at two positions from the sequence deduced from a cDNA reported in GenBank. The gene coding for the inhibitor is wound-inducible and is a member of the potato inhibitor I family of protease inhibitors. The inhibitor strongly inhibited subtilisin A, weakly inhibited trypsin, and did not inhibit chymotrypsin. The amino acid residues at the reactive site P(1) and P(1)(') were determined to be Gln45 and Asp46, respectively, residues rarely reported at the reactive site in potato inhibitor I family members. Comparison of amino acid sequences revealed that the HPI isoinhibitors shared from 33% to 55% identity (50-74% similarity) to inhibitors of the potato inhibitor I family. The properties of the isoinhibitors suggest that they may play a defensive role in the latex against pathogens and/or herbivores.     

Characterization of low-molecular-mass trypsin isoinhibitors from oil-rape (Brassica napus var. oleifera) seed.

A new low-molecular-mass (6767.8 Da) serine proteinase isoinhibitor has been isolated from oil-rape (Brassica napus var. oleifera) seed, designated 5-oxoPro1-Gly62-RTI-III. The 5-oxoPro1-Gly62-RTI-III isoinhibitor is longer than the Asp2-Pro61-RTI-III and the Ser3-Pro61-RTI-III forms, all the other amino acid residues being identical. In RTI-III isoinhibitors, the P1-P1' reactive site bond (where residues forming the reactive site have been identified as PnellipsisP1 and P1'ellipsisPn', where P1-P1' is the inhibitor scissile bond) has been identified at position Arg21-Ile22. The inhibitor disulphide bridges pattern has been determined as Cys5-Cys27, Cys18-Cys31, Cys42-Cys52 and Cys54-Cys57. The disulphide bridge arrangement observed in the RTI-III isoinhibitors is reminiscent of that found in a number of toxins (e.g. erabutoxin b). Moreover, the organization of the three disulphide bridges subset Cys5-Cys27, Cys18-Cys31 and Cys42-Cys52 is reminiscent of that found in epidermal growth factor domains. Preliminary 1H-NMR data indicates the presence of alphaalphaNOEs and 3JalphaNH coupling constants, typical of the beta-structure(s). These data suggest that the three-dimensional structure of the RTI-III isoinhibitors may be reminiscent of that of toxins and epidermal growth factor domains, consisting of three-finger shaped loops extending from the crossover region. Values of the apparent association equilibrium constant for RTI-III isoinhibitors binding to bovine beta-trypsin and bovine alpha-chymotrypsin are 3.3 x 109 m-1 and 2.4 x 106 m-1, respectively, at pH 8.0 and 21.0 degrees C. The serine proteinase : inhibitor complex formation is a pH-dependent entropy-driven process. RTI-III isoinhibitors do not show any similarity to other serine proteinase inhibitors except the low molecular mass white mustard trypsin isoinhibitor, isolated from Sinapis alba L. seed (MTI-2). Therefore, RTI-III and MTI-2 isoinhibitors could be members of a new class of plant serine proteinase inhibitors.     

Isolation and characterization of a wound-induced trypsin inhibitor from alfalfa leaves

A trypsin inhibitor from leaves of field-grown alfalfa plants has been purified and shown to be the same trypsin inhibitor that is wound induced in leaves of young growth chamber grown plants. This inhibitor accounts for the major trypsin inhibitory activity found in both field-grown and wound-induced plants. The inhibitor exhibits a molecular weight of about 7500 and is specific for trypsin with a Ki of 1 X 10(-10) M. Analysis of the purified inhibitor by cation-exchange high-performance liquid chromatography revealed the presence of four isoinhibitor species that have identical immunological and inhibitory properties. The amino acid analysis of the four species indicates small but significant differences. Immunological double diffusion comparisons of the alfalfa inhibitor with the Bowman-Birk and Kunitz soybean inhibitors did not reveal any cross-reactivity although the amino acid content of the alfalfa inhibitor resembles those of Bowman-Birk family members.     

The isoinhibitors of chymotrypsin/elastase from Ascaris lumbricoides: the reactive site

Five isoinhibitors of chymotrypsin/elastase present in aqueous extracts of Ascaris were isolated. The reactive site in each isoinhibitor, the peptide bond that during encounter is positioned over the catalytic site in chymotrypsin, is Leu-Met. This bond was hydrolyzed by incubating intact isoinhibitors with 5-25 mol% chymotrypsin at pH 3.2 for 4-6 days (isoinhibitor 1) or 2.5-5 weeks (isoinhibitors 2-5). The reaction under these conditions did not proceed beyond 60% modified isoinhibitor (peptide bond hydrolyzed) and 40% intact inhibitor. The Leu-Met bond, hydrolyzed in modified isoinhibitor, can be resynthesized at pH 7.6 by incubating modified inhibitor with a stoichiometric amount of chymotrypsin bound to Sepharose CL-4B and then dissociating the complex in a kinetically controlled fashion with 5% trichloroacetic acid. The product, intact inhibitor, was obtained in greater than 80% yield. The site in the isoinhibitor that is positioned over the catalytic site in elastase during encounter is the same as for encounter with chymotrypsin. The Leu-Met bond hydrolyzed during encounter with elastase can be resynthesized by chymotrypsin. Chymotrypsin and elastase bind to the inhibitor at the same site.     

The isoinhibitors of chymotrypsin/elastase from Ascaris lumbricoides: the primary structure

The complete primary structure of five chymotrypsin/elastase isoinhibitors isolated from Ascaris lumbricoides was determined by conventional methods. These structures represent the first sequence set for the Ascaris inhibitor family. All five isoinhibitors are single-chain polypeptides crosslinked by five disulfide bridges. Isoinhibitor 1 consists of 63 amino acid residues and has glycine at the N-terminal and histidine at the C-terminal. Isoinhibitors 2-5 all have arginine at the N-terminal, differ at positions 25 and 40, and have different C-terminal regions. Isoinhibitors 2 and 4 have asparagine at positions 25 and serine at position 40, whereas isoinhibitors 3 and 5 have lysine and threonine at these positions, respectively. The different C-terminal regions of isoinhibitors 2-5 account for their varying lengths. Isoinhibitor 1 has no sequence heterogeneity. Frequent repetitions of various dipeptides and one tripeptide are evident along the peptide chain of isoinhibitors 2-5. None of the isoinhibitors contains the aromatic amino acids phenylalanine or tyrosine. Comparison of the amino acid sequence of isoinhibitor 1 with the sequence of isoinhibitors 2-5 shows that they differ at a minimum of 16 positions. The primary structures of isoinhibitors 1-5 from Ascaris do not demonstrate a great degree of homology when compared with the sequence of presently known proteinase inhibitors. However, these isoinhibitors share with a very large number of inhibitor families the presence of half-cystine in the P3 position.     

Ascaris suum: biosynthesis and isoinhibitor profile of chymotrypsin/elastase isoinhibitors

Chymotrypsin/elastase isoinhibitors were radiolabeled when live, adult Ascaris suum were incubated in tissue culture medium (NCTC-135) supplemented with L-[35S]cysteine. This is the first demonstration that the synthesis of these proteins occurs in A. suum; the isoinhibitors are not host products utilized by the parasite against its host. Of five chymotrypsin/elastase isoinhibitors demonstrable in A. suum, only isoinhibitors 1, 4, and 5 were found in each worm. The amino acid sequences of these three isoinhibitors indicate that they are gene products and are not obtained by modification after translation. The two inhibitors that are not observed could arise by limited proteolysis. The same chymotrypsin/elastase isoinhibitor profile present in each nematode eliminates any speculation that the multiple forms arise from an adaptation between A. suum and its host. A new chymotrypsin/elastase isoinhibitor nomenclature is proposed, so that isoinhibitor 1 is now Isoinhibitor A, and isoinhibitors 4 and 5 are now Isoinhibitors B and C, respectively.     

Trichuris suis: a secretory chymotrypsin/elastase inhibitor with potential as an immunomodulator

A serine protease inhibitor, termed TsCEI, was purified from adult-stage Trichuris suis by acid precipitation, affinity chromatography (elastase-agarose), and reverse-phase HPLC. The molecular weight of TsCEI was estimated at 6.437 kDa by laser desorption mass spectrometry. TsCEI potently inhibited both chymotrypsin (K(i) = 33.4 pM) and pancreatic elastase (K(i) = 8.32 nM). Neutrophil elastase, chymase (mouse mast cell protease-1, mMCP-1), and cathepsin G were also inhibited by TsCEI, whereas trypsin, thrombin, and factor Xa were not. The cDNA-derived amino acid sequence of the mature TsCEI consisted of 58 residues including 9 cysteine residues with a molecular mass of 6.196 kDa. TsCEI displayed 48% sequence identity to a previously characterized trypsin/chymotrypsin inhibitor of T. suis, TsTCI. TsCEI showed 36% sequence identity to a protease inhibitor from the hemolymph of the honeybee Apis mellifera. Sequence similarity was also detected with the trypsin/thrombin inhibitor of the European frog Bombina bombina, the elastase isoinhibitors of the nematode Anisakis simplex, and the chymotrypsin/elastase and trypsin inhibitors of the nematode Ascaris suum. The inhibitors of T. suis, an intestinal parasite of swine, may function as components of a parasite defense mechanism by modulating intestinal mucosal mast cell-associated, protease-mediated, host immune responses.     

Nature of the tryptic/chymotryptic inhibitor from horsegram (Dolichos biflorus).

A protease inhibitor specific to trypsin and chymotrypsin was purified from horsegram (Dolichos biflorus) with the inhibition index 0.24 micrograms/micrograms for trypsin and 0.36 micrograms/micrograms for chymotrypsin. In SDS-PAGE, the inhibitor protein was seen as a single band with apparent molecular mass Mr = 15,500. However, on fast protein liquid chromatography (FPLC) or non-denaturating PAGE, the inhibitor resolved into four components revealing the existence of isoinhibitors. Data on amino acid analysis indicate that the isoinhibitors are closely related. The major amino acids in the inhibitor are half cystine (18.9 mole %), aspartic acid (12.7 mole %) and serine (14.3 mole %). The inhibitor was partially stable to 0.1% sodium dodecyl sulphate, 8M urea or 6M guanidine hydrochloride. The inhibitory activity was lost on reduction or carboxamidomethylation or acetylation. Modification of the arginine groups or CNBr cleavage of the protein did not result in significant loss of either tryptic or chymotryptic inhibitory activities. The isoinhibitors separated by FPLC reacted with polyclonal antibody raised in rabbits and had pI values ranging from 4.8-5.1. The horsegram inhibitor thus resembles other Bowman-Birk protease inhibitors.     

Proteinase isoinhibitors from bovine spleen: primary structure of an intermediate in the processing of the precursor

The complete amino acid sequence of the proteinase inhibitor III from bovine spleen is reported. It consists of 62 amino acid residues and is identical to that of spleen inhibitor II (an isoinhibitor of the bovine pancreatic trypsin inhibitor, which shares with the latter 89% of sequence identity), except for four extra residues at the C-terminal side. Inhibitor III appears to be an intermediate in the processing of the putative 100-residue primary expression product, which leads to the mature inhibitor II. These results and those previously obtained for another intermediate, isoinhibitor I, are indicative of the following order for the last steps of the precursor processing inhibitor I----inhibitor III----inhibitor II. The mature protein and the two intermediates isolated have a very similar antiproteolytic activity. However, their in vivo target enzyme(s) are not yet known, as also the target enzyme of the bovine pancreatic trypsin inhibitor is not known. Thus, the available data would indicate that either the three isoinhibitors have a distinct functional role, by inhibiting different target enzymes, or inhibitors I and III are obligatory intermediates for directing the final targeting of the mature, functionally relevant inhibitor II.     

Isolation and characterization of a proteinaceous inhibitor of microbial proteinases induced during the hypersensitive reaction of tobacco to tobacco mosaic virus

A proteinase inhibitor is strongly induced in tobacco leaves reacting hypersensitively to tobacco mosaic virus. The tobacco inhibitor is highly active against four different serine endoproteinases of fungal and bacterial origin (EC 3.4.21.14) but inhibits poorly two serine endoproteinases of animal origin, trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1). The inhibitor has been purified to homogeneity by successive steps of conventional and high-performance liquid chromatography. When electrophoresed under denaturing conditions, it behaves as a small polypeptide with a molecular weight of about 6,000. From its amino acid composition and NH2-terminal amino acid sequence analysis, it appears that the inhibitor belongs to the potato inhibitor I family. A polyclonal antiserum was raised against the purified tobacco inhibitor and was used in immunoblotting experiments to follow inhibitor accumulation during the hypersensitive reaction of tobacco to tobacco mosaic virus. The inhibitor is highly efficient and might represent a potent fungicide and/or bactericide to be used in plant biotechnology.     

Purification and characterization of proteinase inhibitors from wild soja (Glycine soja) seeds

Nine proteinase inhibitors, I-VIIa, VIIb, and VIII, were isolated from wild soja seeds by ammonium sulfate fractionation and successive chromatographies on SP-Toyopearl 650M, Sephacryl S-200SF, and DEAE-Toyopearl 650S columns. Reverse-phase HPLC finally gave pure inhibitors. All of the inhibitors inhibited trypsin with dissociation constants of 3.2-6.2 x 10(-9) M. Some of the inhibitors inhibited chymotrypsin and elastase as well. Two inhibitors (VIIb and VIII) with a molecular weight of 20,000 were classified as a soybean Kunitz inhibitor family. Others (I-VIla) had a molecular weight of about 8,000, and were stable to heat and extreme pH, suggesting that these belonged to the Bowman-Birk inhibitor family. Partial amino acid sequences of four inhibitors were also analyzed. The complete sequence of inhibitor IV was ascertained from the nucleotide sequences of cDNA clones encoding isoinhibitors homologous to soybean C-II.     

Isolation of acidic acrosin isoinhibitors (BUSI I A, BUSI IB1 and BUSI I B2) from bull seminal plasma.

Three natural proteinase isoinhibitors with low isoelectric points BUSI I A (pI = 3.9), BUSI I B1 (pI = 3.4 and BUSI I B2 (pI = 3.7) were isolated from bull seminal plasma by gel filtration on Sephadex G-50 and ion exchange chromatography on DEAE-Sephadex and SE-Sephadex. Isoinhibitors Bl and B2 have identical amino acid composition. Isoinhibitor A contains six amino acid residues less than isoinhibitors B1 and B2. Since sugars have been detected in the isoinhibitors, heterogeneity may also be due to the sugar component. The isoinhibitors show the same inhibitory properties; all of them inhibit acrosin, trypsin and chymotrypsin. Glandular kallikrein is also inhibited, but to a very low extent only. The molecular weight (Mr approximately 8 900) was determined by gel filtration.     

Characterization of genomic sequence coding for bromelain inhibitors in pineapple and expression of its recombinant isoform

Bromelain inhibitor (BI) is a cysteine proteinase inhibitor isolated from pineapple stem (Reddy, M. N., Keim, P. S., Heinrikson, R. L., and Kézdy, F. J. (1975) J. Biol. Chem. 250, 1741-1750). It consists of eight isoinhibitors, and each isoinhibitor has a two-chain structure. In this study, the genomic DNA has been cloned and found to encode a precursor protein with 246 amino acids (M(r) = approximately 27,500) containing three isoinhibitor domains (BI-III, -VI, and -VII) that are 93% identical to one another in amino acid sequences. The gene structure indicated that these isoinhibitors are produced by removal of the N-terminal pre-peptide (19 residues), 3 interchain peptides (each 5 residues), 2 interdomain peptides (each 19 residues), and the C-terminal pro-peptide (18 residues). Moreover, all the amino acid sequences of bromelain isoinhibitors could be explained by removal of one or two amino acids from BI-III, -VI, and -VII with exopeptidases. A recombinant single-chain BI-VI with and without the interchain peptide showed the same and no bromelain inhibitory activity as compared with the native BI-VI, respectively. These results indicate that the interchain peptide plays an important role of the folding process of the mature isoinhibitors.     

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.     

Identification of a novel four-domain member of the proteinase inhibitor II family from the stigmas of Nicotiana alata

Proteinase inhibitors (PIs) of the potato type II family have been identified in a number of solanaceous species. Most family members have two PI domains which are specific for either chymotrypsin or trypsin. More recently family members have been described with three or six repeated PI domains. Here we describe a novel four-domain family member produced in the stigmas and leaves of the ornamental tobacco, Nicotiana alata, which has high sequence identity with a six-domain member from the same species. Both proteins are produced as precursors that enter the secretory pathway and are subsequently processed into a series of 6 kDa PIs. The four- and six-domain precursor proteins were isolated from immature stigmas and characterised by mass spectrometry which revealed that both proteins had been trimmed at the N-terminus, at a position corresponding to the predicted signal peptide cleavage site. Furthermore, no post-translational modifications were apparent.     

Isolation and characterization of a novel, developmentally regulated proteinase inhibitor I protein and cDNA from the fruit of a wild species of tomato

A novel member of the proteinase Inhibitor I family having a trypsin inhibitor specificity was isolated from the fruit of the wild tomato species Lycopersicon peruvianum (L.) Mill. (LA 107) and characterized. The protein is among the isoinhibitors of Inhibitor I that comprise 50% of the soluble proteins in the fruit of this wild species of tomato. A cDNA corresponding to the inhibitor protein and mRNA was isolated and characterized. The Inhibitor I mRNA represented 0.06% of the poly(A) RNA and gene copy number reconstruction experiments gave an estimate of two to four genes/haploid genome. The open reading frame of the cDNA codes for a protein of 111 amino acids having a 42-amino acid prepropolypeptide. The NH2-terminal sequence of the first 21 amino acids of the purified Inhibitor I protein confirmed that the cDNA was identical to the protein. The amino acid sequence of the L. peruvianum fruit Inhibitor I exhibits 74% identity with the wound-inducible Inhibitor I from tomato leaves. Whereas all previously identified members of the Inhibitor I family have either Met, Leu, or Asp at the P1 site and can inhibit enzymes such as chymotrypsin, subtilisin, and elastase, the fruit Inhibitor I possesses Lys at the P1 position. Thus, this is the first member of the extensive Inhibitor I family from plants and animals that exhibits trypsin inhibitory specificity. The presence of this inhibitor in wild tomato fruit may reflect a functional role to protect the tissues against herbivory.     

Modification of the isoinhibitors of human serum alpha 1-proteinase inhibitor (alpha 1-antitrypsin) by pancreatic proteases

Due to the action of a serum protease, the two most cathodal isoinhibitors of the alpha 1-proteinase inhibitor (alpha 1-PI) are cleaved at the Gly5-Asp6 bond and lack two negative charges. In spite of this, these can bind trypsin and chymotrypsin, showing that the N-terminal pentapeptide is not indispensable for inhibition function. Pancreatic proteases also cleave a bond near the N-terminus in alpha 1-PI, resulting in a loss of two negative charges and a corresponding cathodal shift in the electrofocusing behavior of the isoinhibitors. Trypsin cleaves isoinhibitors near the N-terminus at a large inhibitor excess and unless an additional cleavage takes place, at least two of the new isoinhibitors remain active. An additional cleavage(s), most likely at a distance of 30-40 residues from the C-terminus results in a corresponding decrease of the molecular mass and a loss of inhibition function. Although the C-terminal cleavage peptide does separate from the protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it remains associated with it under conditions of polyacrylamide gel isoelectric focusing. Chymotrypsin also cleaved alpha 1-PI near the N-terminus but this could be observed only at protease excess and the modified isoinhibitors did not form complexes with chymotrypsin. The molecular polymorphism of alpha 1-PI is partly explained by the absence of the N-terminal pentapeptide from some of the isoinhibitors.     

Purification, characterization, and cDNA cloning of a Bowman-Birk type trypsin inhibitor from Apios americana Medikus tubers

An Apios americana trypsin inhibitor, AATI, was purified from Apios tubers by chromatography on DEAE Cellulofine A-500 and Sephadex G-50. The molecular mass of AATI was determined to be 6,437 Da by matrix-assisted laser desorption and ionization time-of-flight mass spectrometer (MALDI-TOF-MS). It showed strong inhibitory activity toward serine proteases, and the inhibition constants toward trypsin and chymotrypsin were 3.0 x 10(-9) M and 1.0 x 10(-6) M respectively. The inhibitory activity was not affected by heating at 80 degrees C for 2 h or by incubation at a wide range of pH values, suggesting that AATI has remarkable heat-stability and pH-stability. AATI cDNA consists of 552 nucleotides, and includes an open reading frame encoding a protein of 116 amino acids. The results of N-terminal amino acid sequencing of AATI and MALDI-TOF-MS analysis suggested that the deduced amino acid sequence had 50 and seven extra amino acids at the N- and C-termini respectively. Thus the mature AATI protein consists of 59 amino acid residues. Comparison of the amino acid sequence with those of the trypsin inhibitors from plants suggests that AATI belongs to the Bowman-Birk family and that it contains two possible reactive sites toward trypsin at Lys62 and Arg88.     

Amino acid sequence of thePhaseolus vulgaris var. “Fogo na Serra” inhibitor and interactive surface modeling for the enzyme-inhibitor complex

A trypsin and chymotrypsin inhibitor from seeds ofPhaseolus vulgaris var. “Fogo na Serra” (PFSI) was purified and its complete amino acid sequence was determined using Edman degradation methods. The inhibitor was found to belong to the Bowman-Birk family of enzymatic inhibitors; it has 82 amino acid residues and a 8.985-kDa molecular mass. The PFSI/α-chymotrypsin binary complex has been modeled using the Turkey ovomucoid inhibitor third domain (OMTKY3) bound toα-chymotrypsin [Fujinagaet al. (1987),J. Mol. Biol.,195, 397–418. template. The model allowed identification of the binding surface.     

Purification, characterization and primary structure of a chymotrypsin inhibitor from Naja atra venom

A chymotrypsin inhibitor, designated NA-CI, was isolated from the venom of the Chinese cobra Naja atra by three-step chromatography. It inhibited bovine alpha-chymotrypsin with a Ki of 25 nM. The molecular mass of NA-CI was determined to be 6403.8 Da by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) analysis. The complete amino acid sequence was determined after digestion of S-carboxymethylated inhibitor with Staphylococcus aureus V8 protease and porcine trypsin. NA-CI was a single polypeptide chain composed of 57 amino acid residues. The main contact site with the protease (P1) has a Phe, showing the specificity of the inhibitor. NA-CI shared great similarity with the chymotrypsin inhibitor from Naja naja venom (identities=89.5%) and other snake venom protease inhibitors.