Amino-acid sequence of a trypsin/chymotrypsin inhibitor from giant taro (Alocasiamacrorrhiza)

Giant taro (Alocasiamacrorrhiza) contains a protein which inhibits both trypsin and chymotrypsin. This trypsin/chymotrypsin inhibitor exists as a dimer of two identical monomers each with slight polymorphism and is an attractive candidate for conferring insect resistance in transgenic plants. The 184 amino-acid sequence (molecular mass of 19774 Da for the Met-24, Glu-50 form) has been determined and is compared with those of other Kunitz-type trypsin, chymotrypsin and subtilisin inhibitors. There appears to be greater ‘homology’ between the giant taro inhibitor and those inhibitors from other monocotyledons than inhibitors from dicotyledons. The P₁ loop region is different from that of other Kunitz-type inhibitors and contains a sequence Leu-Ala-Phe-Phe-Pro at residues 56–60. This section of sequence differs only by a Leu/Ile replacement to a tight binding inhibitor of neutrophil elastase, Recently produced by genetic engineering. The most likely candidate for the P₁ residue in the giant taro trypsin/chymotrypsin inhibitor is Leu-56.     

Isolation and characterization of four serine proteinase inhibitors (serpins) from hemolymph of Manduca sexta

Four serine proteinase inhibitors have been isolated from hemolymph of fifth instar larvae of Manduca sexta. One of these, an inhibitor specific for elastase, has been previously shown to be a member of the serpin family of serine proteinase inhibitors. Of the three remaining inhibitors, two are specific for chymotrypsin and one for trypsin. The four inhibitors have molecular weights of approx. 47,000 and isoelectric points between 4.4 and 4.8. The four proteins have very similar amino acid compositions, and NH₂-terminal sequence analysis suggests that they represent members of a gene family.     

Proteinase inhibitors in rat serum. Purification and partial characterization of three functionally distinct trypsin inhibitors.

Three different serine proteinase inhibitors were isolated from rat serum and purified to apparent homogeneity. One of the inhibitors appears to be homologous to alpha 1-proteinase inhibitor isolated from man and other species, but the other two, designated rat proteinase inhibitor I and rat proteinase inhibitor II, seem to have no human counterpart. alpha 1-Proteinase inhibitor (Mr 55000) inhibits trypsin, chymotrypsin and elastase, the three serine proteinases tested. Rat proteinase inhibitor I (Mr 66000) is active towards trypsin and chymotrypsin, but is inactive towards elastase. Rat proteinase inhibitor II (Mr 65000) is an effective inhibitor of trypsin only. Their contributions to the trypsin-inhibitory capacity of rat serum are about 68, 14 and 18% for alpha 1-proteinase inhibitor, rat proteinase inhibitor I and rat proteinase inhibitor II respectively.     

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.     

Isolation and characterization from potato tubers of two polypeptide inhibitors of serine proteinases

Two polypeptides, isolated to electrophoretic homogeneity from Russet Burbank potato tubers, are powerful inhibitors of pancreatic serine proteinases. One of the inhibitors, called polypeptide trypsin inhibitor, PTI, has a molecular weight of 5100, and inhibits bovine trypsin. The inhibitor is devoid of methionine, histidine, and tryptophan and contains eight half-cystine residues as four disulfide bridges. The second inhibitor, polypeptide chymotrypsin inhibitor II, PCI-II, has a molecular weight of 5700 and powerfully inhibits chymotrypsin. This inhibitor is also devoid of methionine and tryptophan but it contains only six of half-cystines as three disulflde bonds. Both polypeptides strongly inhibit pancreatic elastase. In immunological double diffusion assays, polypeptide trypsin inhibitor and polypeptide chymotrypsin inhibitor II exhibit a high degree of immunological identity (a) with each other, (b) with a polypeptide chymotrypsin inhibitor (PCI-I, M_r 5400) previously isolated from potato tubers, and (c) with inhibitor II, a larger (monomer M_r ~ 12,000) inhibitor of both trypsin and chymotrypsin which has also been previously isolated from potato tubers. The four polypeptide proteinase inhibitors now isolated from Russet Burbank potato tubers cumulatively inhibit all five major intestinal digestive endo- and exoproteinases of animals. The inhibitors are thought to be antinutrients that are present as part of the natural chemical defense mechanisms of potato tubers against attacking pests.     

Purification and some properties of two proteinase inhibitors (DE-1 and DE-3) from Erythrina latissima (broad-leaved erythrina) seed

Two proteinase inhibitors, DE-1 and DE-3, were purified from Erythrina latissima seeds. Whereas DE-1 inhibits bovine chymotrypsin and not bovine trypsin, DE-3 inhibits trypsin but not chymotrypsin. The molecular weights and the amino acid compositions of the two inhibitors resemble the corresponding properties of the Kunitz-type proteinase inhibitors. The N-terminal primary structure of DE-3 showed homology with soybean trypsin inhibitor (Kunitz) and also with the proteinase inhibitors (A-II and B-II) from Albizzia julibrissin seed.     

Three-dimensional Structure of a Kunitz-type Inhibitor in Complex with an Elastase-like Enzyme

Elastase-like enzymes are involved in important diseases such as acute pancreatitis, chronic inflammatory lung diseases, and cancer. Structural insights into their interaction with specific inhibitors will contribute to the development of novel anti-elastase compounds that resist rapid oxidation and proteolysis. Proteinaceous Kunitz-type inhibitors homologous to the bovine pancreatic trypsin inhibitor (BPTI) provide a suitable scaffold, but the structural aspects of their interaction with elastase-like enzymes have not been elucidated. Here, we increased the selectivity of ShPI-1, a versatile serine protease inhibitor from the sea anemone Stichodactyla helianthus with high biomedical and biotechnological potential, toward elastase-like enzymes by substitution of the P1 residue (Lys¹³) with leucine. The variant (rShPI-1/K13L) exhibits a novel anti-porcine pancreatic elastase (PPE) activity together with a significantly improved inhibition of human neuthrophil elastase and chymotrypsin. The crystal structure of the PPE·rShPI-1/K13L complex determined at 2.0 Å resolution provided the first details of the canonical interaction between a BPTI-Kunitz-type domain and elastase-like enzymes. In addition to the essential impact of the variant P1 residue for complex stability, the interface is improved by increased contributions of the primary and secondary binding loop as compared with similar trypsin and chymotrypsin complexes. A comparison of the interaction network with elastase complexes of canonical inhibitors from the chelonian in family supports a key role of the P3 site in ShPI-1 in directing its selectivity against pancreatic and neutrophil elastases. Our results provide the structural basis for site-specific mutagenesis to further improve the binding affinity and/or direct the selectivity of BPTI-Kunitz-type inhibitors toward elastase-like enzymes.     

Production and properties of recombinant glutenin-hydrolyzing proteinases from Eurygaster integriceps Put.

cDNAs coding for a mature form of glutenin-hydrolyzing trypsin-like proteinase (referred to as glutenin-hydrolyzing proteinase 3 or GHP3) from the insect pest Eurygaster integriceps Put. and a zymogen of this proteinase containing a signal peptide required for protein secretion were cloned into vectors pPIC9 and pPIC3.5, respectively. The constructs were used for protein expression in cells of the methylotrophic yeast Pichia pastoris. The recombinant protein corresponding to the mature form of the proteinase was secreted into the culture medium and possessed proteolytic activity, while the zymogen acquired activity after trypsin treatment. Both recombinant enzymes hydrolyzed high-molecular weight glutenin subunits from wheat of the variety Ege-88 and a range of other soft and durum wheat varieties. Chymotrypsin inhibitor I from potatoes and related inhibitors from seeds of plants of the subclass Asteridae, the Kunitz-type trypsin inhibitor from soybeans, and bovine aprotinin had a weak inhibitory effect on the recombinant proteinases, while the Bowman-Birk trypsin and chymotrypsin inhibitor from soybeans did not interact with these enzymes.     

Heterologous expression, purification, and properties of a chymotrypsin inhibitor isolated from potatoes

The PKPIJ-B gene encoding a chymotrypsin inhibitor from a subfamily of potato Kunitz-type proteinase inhibitors (PKPI) in potatoes (Solanum tuberosum L. cv. Yubilei Zhukova) was cloned into a pET23a vector and then expressed in Escherichia coli. The recombinant PKPIJ-B protein obtained in the inclusion bodies was denatured, purified by high-performance liquid chromatography (HPLC) on Mono Q under denaturing conditions, and renaturated. The renaturated protein was additionally purified using HPLC on DEAE-ToyoPearl. The PKPIJ-B protein efficiently suppressed chymotrypsin activity, had a weaker effect on trypsin, and inhibited the growth and development of phytopathogenic microorganisms affecting potato plants.     

Cysteine proteinases substitute for serine proteinases in the midgut glands of Crangon crangon and Crangon allmani (Decapoda: Caridea)

The utilization of dietary proteins in crustaceans is facilitated by a set of peptide hydrolases which are often dominated by “trypsin-like” serine proteinases. As expected, the North Sea shrimps Crangon crangon and Crangon allmani showed in their midgut glands high proteolytic activities. However, the majority of animals lacked trypsin and chymotrypsin. Conversely, a minority of about 10% of the animals had elevated trypsin activities. The appearance of trypsin was neither related to the mode of feeding nor to the nutritive state of the animals. When present, trypsin was expressed in both species as a single isoform of apparently 20 kDa. The lack of serine proteinases was also confirmed by inhibitor assays. AEBSF, a serine proteinase inhibitor, slightly reduced total proteinase activity by less than 10%. In contrast E 64, a cysteine proteinase inhibitor, caused a reduction of more than 70% of total proteinase activity, indicating that a substantial share of proteolytic activity is caused by cysteine proteinases. Cathepsin L-like proteinases were identified as major cysteine proteinases.A comparison with the eucarid crustaceans Pandalus montagui, Pagurus bernhardus, Cancer pagurus and Euphausia superba showed a similar high level of total proteinase activity in all species. Trypsin, however, varied significantly between species showing lowest activities in Caridea and the highest activity in E. superba. E 64 suppressed total proteinase activity by more than 70% in Crangon species but not in C. pagurus and E. superba. In contrast, the serine proteinase inhibitor AEBSF had only little effect in Caridea but was most effective in P. bernhardus, C. pagurus and E. superba. The results may indicate different traits of food utilization strategies in some eucarid crustaceans. Caridea may express predominantly cysteine proteinase, while in Anomura, Brachyura and Euphausiacea, serine proteinases may prevail.     

Epidermal growth factor-urogastrone receptor: selective alteration in simian virus 40 transformed mouse fibroblasts

Comparative data on the properties of four thiol proteinase inhibitors, and of four serine proteinase inhibitors (two subtilisin and two trypsin inhibitors) isolated from seeds of Vigna are presented. They were similar in their molecular weights (5000–15,000) and dissociation constants (10^?8–10^?9m). The range of isoelectric points of the thiol proteinase inhibitors was 6.5 to 10.6, and of the serine proteinase inhibitors was 5.0 to 5.9. The amino acid compositions of one papain isoinhibitor, one of subtilisin, and one of trypsin are presented. Papain inhibitor A1 and subtilisin inhibitor 2a were low in cystine. All of the inhibitors were stable upon heating to 80 °C for 5 min at low pH. The subtilisin inhibitor did not bind to catalytically inactive subtilisin derivatives, whereas the papain inhibitor was stoichiometrically bound to the Hg or thioacetamide derivatives of papain. Incubation of the subtilisin inhibitor with catalytic amounts of subtilisin led to the formation of a modified form with the same inhibitor activity as the native inhibitor but with a different electrophoretic mobility. There was no indication of a similar modification of the papain inhibitor by papain. Separate sites are present on the trypsin-chymotrypsin inhibitors for trypsin and chymotrypsin. The papain inhibitors have the same binding sites for papain and ficin.     

Purification and Characterization of a Proteinase Inhibitor from Field Bean, Dolichos lablab perpureus L.

A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS–PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an M _r of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI–trypsin/chymotrypsin complexes by difference spectral studies. Apparent K _a values of complexes of inhibitor with trypsin and chymotrypsin were 2.1 × 10⁷ M^?1 and 3.1 × 10⁷ M^?1, respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively.     

Purification and biochemical characterization of a serine proteinase inhibitor from Derris trifoliata Lour. seeds: Insight into structural and antimalarial features

A potent serine proteinase inhibitor was isolated and characterized from the seeds of the tropical legume liana, Derris trifoliata (DtTCI) by ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography. SDS-PAGE as well as MALDI-TOF analysis showed that DtTCI is a single polypeptide chain with a molecular mass of ∼20 kDa. DtTCI has three isoinhibitors (pI: 4.55, 5.34 and 5.72) and, inhibited both trypsin and chymotrypsin in a 1:1 molar ratio. Both Dixon plots and Lineweaver-Burk double reciprocal plots revealed a competitive inhibition of trypsin and chymotrypsin activity, with inhibition constants (K_i) of 1.7 × 10⁻¹⁰ and 1.25 × 10⁻¹⁰ M, respectively. N-terminal sequence of DtTCI showed over 50% similarity with numerous Kunitz-type inhibitors of the Papilionoideae subfamily. High pH amplitude and broad temperature optima were noted for DtTCI, and time course experiments indicated a gradual loss in inhibitory potency on treatment with dithiothreitol (DTT). Circular Dichroism (CD) spectrum of native DtTCI revealed an unordered structure whereas exposure to thermal-pH extremes, DTT and guanidine hydrochloride (Gdn HCl) suggested that an abundance of β-sheets along with intramolecular disulfide bonds provide conformational stability to the active site of DtTCI, and that severity of denaturants cause structural modifications promoting inhibitory inactivity. Antimalarial studies of DtTCI indicate it to be a potent antiparasitic agent.     

Proteinase inhibitors from Erythrina corallodendron and Erythrina cristagalli seeds

Eight and five proteinase inhibitors were purified from Erythrina corallodendron and E. cristagalli seeds, respectively, by gel filtration followed by ion exchange chromatography on DEAE-cellulose and DEAE-sepharose. Each inhibitor consists of 161–163 amino acids (M_r 18 000) including four half-cystine residues and resembles the Kunitz-type proteinase inhibitors. The N-terminal amino acid sequence of trypsin inhibitor DE-7 from E. corallodendron seed resembles those of other Erythrina species. For the other inhibitors no free N-terminal amino acid was found. DE-1,-2,-3,-4 and -5 from the seed of E. corallodendron contain potent inhibitors for α-chymotrypsin and they have practically no action on trypsin. From the same seed, inhibitors DE-6, -7 and -8 strongly inhibit trypsin and also inhibit α-chymotrypsin to varying degrees. From the seeds of E. cristagalli, inhibitors DE-1 and -8 inhibit trypsin strongly and DE-2, -3 and -4 are strongly inhibitory for α-chymotrypsin. On summarizing the inhibitor characteristics of the Kunitz-type proteinase inhibitors from the seeds of eight different species of Erythrina, it was obvious that there is a relationship between the alanine content of the inhibitors and their activities. A high alanine content is associated with potent α-chymotrypsin activities and low alanine content with strong trypsin activities.     

Rhipicephalus sanguineus trypsin inhibitors present in the tick larvae: isolation,characterization, and partial primary structure determination

Blood sucking animals are a rich source of proteinase inhibitors mainly those that interfere in their host hemostatic systems. The tick Rhipicephalus sanguineus is an ectoparasite of dogs and other animals. The aims of this work were the purification and characterization of serine proteinase inhibitors present in R. sanguineus larvae (RsTI). The inhibitors (RsTI) were isolated by affinity chromatography on trypsin-Sepharose and ion exchange chromatographies in Resource Q and Mono S columns. These RsTIs were separated in around 12 different protein peaks, when they showed molecular masses between 8 and 18 kDa, by SDS-PAGE. Purified RsTIs presented differences in the specificity for different serine proteinases. RsTIQ2 was, better inhibitor than RsTIQ7 and RsTIS5 for neutrophil elastase, plasmin, and HuPK with dissociation constants (K(i)) of 1.3, 3.2, and 22 nM, respectively. Other inhibitors such as RsTIQ7, RsTIS3, and RsTIS5 also affected neutrophil elastase and plasmin with K(i) in the nM range. The RsTIQ2, RsTIQ7, and RsTIS5 amino acid sequence data allowed classifying them as members of the Kunitz-type serine proteinase inhibitor family, even though the RsTI role is still unknown. Our present results showed that serine proteinase inhibitors from R. sanguineus are similar to inhibitors from Boophilus microplus other hard tick species, suggesting a similar role of these inhibitors in hard tick species and also as a potential tool to generate or improve vaccine against different ectoparasites with an unique antigen.     

Kinetic mechanism of chicken liver xanthine dehydrogenase.

Human inter-alpha-trypsin inhibitor (I alpha I) is a plasma proteinase inhibitor active against cathepsin G, leucocyte elastase, trypsin and chymotrypsin. It owes its broad inhibitory specificity to tandem Kunitz-type inhibitory domains within an N-terminal region. Sequence studies suggest that the reactive-centre residues critical for inhibition are methionine and arginine. Reaction of I alpha I with the arginine-modifying reagent butane-2,3-dione afforded partial loss of inhibitory activity against both cathepsin G and elastase but complete loss of activity against trypsin and chymotrypsin. Reaction of I alpha I with the methionine-modifying reagent cis-dichlorodiammineplatinum(II) resulted in partial loss of activity against cathepsin G and elastase but did not affect inhibition of either trypsin or chymotrypsin. Employment of both reagents eliminated inhibition of cathepsin G and elastase. These findings suggest that both cathepsin G and elastase are inhibited at either of the reactive centres of I alpha I. Trypsin and chymotrypsin, however, appear to be inhibited exclusively at the arginine reactive centre.     

The reactive sites of proteinase inhibitors from Erythrina seeds

Although the Kunitz-type proteinase inhibitors from the seeds of various Erythrina species have similar molecular weights (approximately 20,000), and share many other chemical characteristics, they could nevertheless be divided into three groups on the basis of their relative abilities to inhibit chymotrypsin, trypsin and tissue plasminogen activator. Group a inhibitors were relatively specific for chymotrypsin; they were poor inhibitors of trypsin and had no apparent effect upon tissue plasminogen activator. Group b proteins inhibited trypsin strongly and chymotrypsin slightly less effectively. They had no effect upon t-PA. Group c inhibitors inhibited trypsin, chymotrypsin and t-PA. Analysis of the amino acid composition of the three groups of inhibitors revealed major differences in alanine content. Minor differences in the content of most other amino acids were also noticed. Group b and group c inhibitors had, in most cases, the same reactive sites (Arg-Ser). The sequences neighbouring the reactive sites showed a significant degree of homology. Chemical modification of arginine in proteinase inhibitors from the seeds of E. latissima and soybeans using 1-2-cyclohexanedione confirmed the presence or absence of arginine in the reactive sites.     

Heterologous expression, purification, and properties of a potato protein inhibitor of serine proteinases

The gene PKPI-B10 [AF536175] encoding in potato (Solanum tuberosum L., cv. Istrinskii) a Kunitz-type protein inhibitor of proteinases (PKPI) has been cloned into the pET23a vector and then expressed in Escherichia coli. The recombinant protein PKPI-B10 obtained as inclusion bodies was denatured, separated from admixtures by ion-exchange fast protein liquid chromatography (FPLC) on MonoQ under denaturing conditions, and renatured. The native protein was additionally purified by ion-exchange FPLC on DEAE-Toyopearl. The PKPI-B10 protein effectively inhibits the activity of trypsin, significantly weaker suppresses the activity of chymotrypsin, and has no effect on other serine proteinases: human leukocyte elastase, subtilisin Carlsberg, and proteinase K, and also the plant cysteine proteinase papain.     

Two Kazal-type protease inhibitors from Macrobrachium nipponense and Eriocheir sinensis: comparative analysis of structure and activities

Kazal-type inhibitors (KPIs) play important roles in many biological and physiological processes, such as blood clotting, the immune response and reproduction. In the present study, two male reproductive tract KPIs, termed Man-KPI and Ers-KPI, were identified in Macrobrachium nipponense and Eriocheir sinensis, respectively. The inhibitory activities of recombinant Man-KPI and Ers-KPI against chymotrypsin, elastase, trypsin and thrombin were determined. The results showed that both of them strongly inhibit chymotrypsin and elastase. Kinetic studies were performed to elucidate their inhibition mechanism. Furthermore, individual domains were also expressed to learn further which domain contributes to the inhibitory activities of intact KPIs. Only Man-KPI_domain3 is active in the inhibition of chymotrypsin and elastase. Meanwhile, Ers-KPI_domain2 and 3 are responsible for inhibition of chymotrypsin, and Ers-KPI_domains2, 3 and 4 are responsible for the inhibition of elastase. Meanwhile, the inhibitory activities of these two KPIs toward Macrobrachium rosenbergii, M. nipponense and E. sinensis sperm were compared with that of the Kazal-type peptidase inhibitor (MRPINK) characterized from the M. rosenbergii reproductive tract in a previous study. The results demonstrated that KPIs can completely inhibit the gelatinolytic activities of sperm proteases from their own species, while different levels of cross-inhibition were observed between KPI and proteases from different species. These results may provide new perspective to further clarify the mechanism of KPI-proteases interaction in the male reproductive system.     

Primary structure of potato kunitz-type serine proteinase inhibitor

The serine proteinase inhibitor (PSPI-21) isolated from potato tubers (Solanum tuberosum L.) comprises two protein species with pI 5.2 and 6.3, denoted as PSPI-21-5.2 and PSPI-21-6.3, respectively. They were separated by anion exchange chromatography on a Mono Q FPLC column. Both species tightly inhibit human leukocyte elastase, whereas their interaction with trypsin and chymotrypsin is substantially weaker. The sequences of both PSPI-21-5.2 and PSPI-21-6.3 were determined by analysis of overlapping peptides obtained from the oxidized or reduced and S-pyridylethylated proteins after digestion with trypsin or pepsin. Both species of PSPI-21 are composed of two chains, named chains A and B, which are linked by a disulfide bridge between Cys(146) and Cys(157). The other disulfide bridge is located within the A chains between Cys(48) and Cys(97). The amino acid sequences of the large A chains of the two forms, consisting of 150 amino acids residues each, differ in a single residue at position 52. The small chains B, containing 37 and 36 residues in PSPI-21-6.3 and PSPI-21-5.2, respectively, have nine different residues. The entire amino acid sequences of the two inhibitors show a high degree of homology to the other Kunitz-type proteinase inhibitors from plants.