Types Of Protease Inhibitors Research Articles

Towards a comprehensive view of the primary structure of venom proteins from the parasitoid wasp Pimpla hypochondriaca

Venom from the parasitoid wasp Pimpla hypochondriaca has potent in vivo activity against insect haemocytes and disrupts host immune responses. Using hybridisation techniques, and more recently random sequence analysis, we had previously identified cDNAs encoding 10 venom proteins from this wasp and deduced their primary structures. We have now extended the random sequence analysis and discovered a further nine cDNAs encoding proteins with predicted signal sequences. The mature proteins were calculated to have masses of between 4 and 22 kDa. Post-signal sequence residues predicted from the cDNAs matched those derived by Edman degradation from venom proteins separated using gel filtration and reverse phase chromatography, confirming that the cloned cDNAs encode proteins which are secreted into the venom sac. Proteins containing at least six cysteine residues were abundant and seven of these cysteine-rich venom proteins, cvp1–7, were identified. The sequences of some of these proteins were similar, or contained similar cysteine arrangements, to Kunitz type protease inhibitors, pacifastin, the trypsin inhibitor domain protein family, atracotoxin and omega-conotoxin, respectively, which occur in a diverse range of animals including spiders, molluscs, humans and grasshoppers. Two small venom proteins, svp1 and svp2, as well as cvp7 did not have similar sequences to proteins in the GenBank protein database suggesting they may be highly specialised venom components. The random sequencing approach has provided a rapid means of determining the primary structure of the majority of Pimpla hypochondriaca venom proteins.

Towards a comprehensive view of the primary structure of venom proteins from the parasitoid wasp Pimpla hypochondriaca

Venom from the parasitoid wasp Pimpla hypochondriaca has potent in vivo activity against insect haemocytes and disrupts host immune responses. Using hybridisation techniques, and more recently random sequence analysis, we had previously identified cDNAs encoding 10 venom proteins from this wasp and deduced their primary structures. We have now extended the random sequence analysis and discovered a further nine cDNAs encoding proteins with predicted signal sequences. The mature proteins were calculated to have masses of between 4 and 22 kDa. Post-signal sequence residues predicted from the cDNAs matched those derived by Edman degradation from venom proteins separated using gel filtration and reverse phase chromatography, confirming that the cloned cDNAs encode proteins which are secreted into the venom sac. Proteins containing at least six cysteine residues were abundant and seven of these cysteine-rich venom proteins, cvp1–7, were identified. The sequences of some of these proteins were similar, or contained similar cysteine arrangements, to Kunitz type protease inhibitors, pacifastin, the trypsin inhibitor domain protein family, atracotoxin and omega-conotoxin, respectively, which occur in a diverse range of animals including spiders, molluscs, humans and grasshoppers. Two small venom proteins, svp1 and svp2, as well as cvp7 did not have similar sequences to proteins in the GenBank protein database suggesting they may be highly specialised venom components. The random sequencing approach has provided a rapid means of determining the primary structure of the majority of Pimpla hypochondriaca venom proteins.

Multigene family for Bowman-Birk type proteinase inhibitors of wild soja and soybean: the presence of two BBI-A genes and pseudogenes.

Genes for Bowman-Birk type protease inhibitors (BBIs) of wild soja (Glycine soja) and soybean (Glycine max) comprise a multigene family. The organization of the genes for wild soja BBIs (wBBIs) was elucidated by an analysis of their cDNAs and the corresponding genomic sequences, and compared with the counterparts in the soybean. The cDNAs encoding three types of wild soja BBIs (wBBI-A, -C, and -D) were cloned. Two subtypes of cDNAs for wBBI-A, designated wBBI-A1 and -A2, were further identified. Similar subtypes (sBBI-A1 and -A2) were also found in the soybean genome. cDNA sequences for wBBIs were highly homologous to those for the respective soybean homologs. Phylogenetic analysis of these cDNAs demonstrated the evolutional proximity between these two leguminae strains.

Processing of soybean hulls to enhance the distribution and extraction of value‐added proteins

AbstractSoybean hulls contain peroxidase (SBP) and Bowman–Birk type protease inhibitor (BBI). SBP is used in the European bread‐baking industry, and BBI isolated from dehulled, defatted soy flour possesses cancer‐preventive and anticarcinogenic properties. Because hulls possess a low percentage of nitrogen, with fibre and other carbohydrates being the major components, our objective was to determine whether air classification of milled hulls could be used to fractionate and enrich these value‐added proteins. Hulls were pin milled three times at 18 000 rpm, followed by air classification to yield fractions with particle size distributions ranging from <15 to >30 µm. The finely ground fractions with particle sizes ranging from <15 to 18 µm were enriched with nitrogen and lipid when compared with the more coarsely ground fractions. Yields of extractable nitrogenous components from aqueous extracts were quantified at a variety of pHs, homogenisation speeds and times. BBI and SBP were confirmed by polyacrylamide gel electrophoresis with known standards. Based on assays for SBP and BBI activities, BBI, though concentrated in the finer fractions, also appeared in the highest‐mass, coarsest fraction, whereas SBP resided mainly in the coarsest fraction. Dry milling and air classification of soybean hulls proved beneficial to distribute SBP into a product stream that facilitates its isolation and purification. Published in 2003 for SCI by John Wiley & Sons, Ltd.

Structure–activity and structure–metabolism relationships of HIV protease inhibitors containing the 3-hydroxy-2-methylbenzoyl-allophenylnorstatine structure

A series of peptidomimetic human immunodeficiency virus (HIV) protease inhibitors containing substituted allophenylnorstatine [Apns: (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] were designed and synthesized. From the structure–metabolism relationship of this type of HIV protease inhibitors, the compounds having para substitution of the phenyl ring of Apns and/or 2,6-disubstitution of the P2′ benzylamine were found to be able to avoid the P2 phenol glucuronidation that occurs with SM-319777 (formerly named JE-2147, KNI-764); one of the main metabolic pathways of SM-319777. These new analogues, such as SM-322377, had more desirable pharmacokinetic profiles and more potent antiviral activity against not only wild type HIV-1 but also the multi-drug-resistant HIV-1 than SM-319777.

Characterization of Cl-HCO3 exchange in basolateral membrane of rat distal colon.

Sodium-independent Cl movement (i.e., Cl-anion exchange) has not previously been identified in the basolateral membranes of rat colonic epithelial cells. The present study demonstrates Cl-HCO3 exchange as the mechanism for 36Cl uptake in basolateral membrane vesicles (BLMV) prepared in the presence of a protease inhibitor cocktail from rat distal colon. Studies of 36Cl uptake performed with BLMV prepared with different types of protease inhibitors indicate that preventing the cleavage of the COOH-terminal end of AE2 protein by serine-type proteases was responsible for the demonstration of Cl-HCO3 exchange. In the absence of voltage clamping, both outward OH gradient (pHout/pHin: 7.5/5.5) and outward HCO3 gradient stimulated transient 36Cl uptake accumulation. However, voltage clamping with K-ionophore, valinomycin, almost completely (87%) inhibited the OH gradient-driven 36Cl uptake, whereas HCO3 gradient-driven 36Cl uptake was only partially inhibited (38%). Both electroneutral HCO3 and OH gradient-driven 36Cl uptake were 1) completely inhibited by DIDS, an anion exchange inhibitor, with a half-maximal inhibitory constant (Ki) of approximately 26.9 and 30.6 microM, respectively, 2) not inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid(NPPB), a Cl channel blocker, 3) saturated by increasing extravesicular Cl concentration with a Km for Cl of approximately 12.6 and 14.2 mM, respectively, and 4) present in both surface and crypt cells. Intracellular pH (pHi) was also determined with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetomethylester (BCECF-AM) in an isolated superfused crypt preparation. Removal of Cl resulted in a DIDS-inhibitable increase in pHi both in HCO3-buffered and in the nominally HCO3-free buffered solutions (0.28 +/- 0.02 and 0.11 +/- 0.02 pH units, respectively). We conclude that a carrier-mediated electroneutral Cl-HCO3 exchange is present in basolateral membranes and that, in the absence of HCO3, Cl-HCO3 exchange can function as a Cl-OH exchange and regulate pHi across basolateral membranes of rat distal colon.

Structural Basis of Inhibition Revealed by a 1:2 Complex of the Two-headed Tomato Inhibitor-II and Subtilisin Carlsberg

Multidomain proteinase inhibitors play critical roles in the defense of plants against predation by a wide range of pests. Despite a wealth of structural information on proteinase-single domain inhibitor interactions, the structural basis of inhibition by multidomain proteinase inhibitors remains poorly understood. Here we report the 2.5-A resolution crystal structure of the two-headed tomato inhibitor-II (TI-II) in complex with two molecules of subtilisin Carlsberg; it reveals how a multidomain inhibitor from the Potato II family of proteinase inhibitors can bind to and simultaneously inhibit two enzyme molecules within a single ternary complex. The N terminus of TI-II initiates the folding of Domain I (Lys-1 to Cys-15 and Pro-84 to Met-123) and then completes Domain II (Ile-26 to Pro-74) before coming back to complete the rest of Domain I (Pro-84 to Met-123). The two domains of TI-II adopt a similar fold and are arranged in an extended configuration that presents two reactive site loops at the opposite ends of the inhibitor molecule. Each subtilisin molecule interacts with a reactive site loop of TI-II through the standard, canonical binding mode. Remarkably, a significant distortion of the active site of subtilisin is induced by the presence of phenylalanine in the P1 position of reactive site loop II of TI-II. The structure of the TI-II.(subtilisin)2 complex provides a molecular framework for understanding how multiple inhibitory domains in a single Potato II type proteinase inhibitor molecule from the Potato II family act to inhibit proteolytic enzymes.

Acyl sulfonamides as potent protease inhibitors of the hepatitis C virus full-Length NS3 (Protease-Helicase/NTPase): A comparative study of different C-terminals

Synthesis and inhibitory potencies of three types of protease inhibitors of the hepatitis C virus (HCV) full-length NS3 (protease-helicase/NTPase) are reported: (i) inhibitors comprising electrophilic serine traps (pentafluoroethyl ketones, α-keto acids, and α-ketotetrazoles), (ii) product-based inhibitors comprising a C-terminal carboxylate group, and (iii) previously unexplored inhibitors comprising C-terminal carboxylic acid bioisosteres (tetrazoles and acyl sulfonamides). Bioisosteric replacement with the tetrazole group provided inhibitors equally potent to the corresponding carboxylates, and substitution with the phenyl acyl sulfonamide group yielded more potent inhibitors. The hexapeptide inhibitors Suc-Asp- d-Glu-Leu-Ile-Cha-Nva-NHSO 2Ph and Suc-Asp- d-Glu-Leu-Ile-Cha-ACPC-NHSO 2Ph with K i values of 13.6 and 3.8 nM, respectively, were approximately 20 times more potent than the corresponding inhibitors with a C-terminal carboxylate and were comparable to the carboxylate-based inhibitor containing the native cysteine, Suc-Asp- d-Glu-Leu-Ile-Cha-Cys-OH ( K i=28 nM). The acyl sulfonamide group constitutes a very promising C-terminal functionality that allows for prime site optimization.

Possible identity of IL-8 converting enzyme in human fibroblasts as a cysteine protease

A converting activity was characterized in human diploid fibroblasts, which secrete 72IL-8 and 77IL-8 in treatment with IFN-β and poly I: poly C. 77IL-8 was significantly converted to 72IL-8 by a partially purified fraction of the culture supernatant of human diploid fibroblasts. The converting activity, which was temperature-dependent and optimal at pH 6, was completely inhibited by cysteine protease inhibitors, antipain dihydrochloride and E-64, but not by other types of protease inhibitors. These data clearly show that human diploid fibroblasts are capable of processing IL-8 to produce a mature IL-8 and that the putative converting enzyme appears to be a cysteine protease.

Molecular evolution of Bowman–Birk type proteinase inhibitors in flowering plants

The Bowman–Birk family (BBI) of proteinase inhibitors is probably the most studied family of plant inhibitors. We describe the primary structure and the gene expression profile of 14 putative BBIs from the sugarcane expressed sequence tag database and show how we used these newly discovered sequences together with 87 previously described BBI sequences from the GenBank database to construct phylogenetic trees for the BBI family. Phylogenetic analysis revealed that BBI-type inhibitors from monocotyledonous and dicotyledonous plants could be clearly separated into different groups, while the overall topology of the BBI tree suggests a different pattern of evolution for BBI families in flowering plants. We also found that BBI proteinase inhibitors from dicotyledonous plants were well conserved, accumulating only slight differences during their evolution. In addition, we found that BBIs from monocotyledonous plants were highly variable, indicating an interesting process of evolution based on internal gene duplications and mutation events.

Mechanisms of early virologic failure in antiretroviral-naive patients starting protease inhibitor-containing regimens: the APROVIR Study.

The virologic and pharmacologic mechanisms of virologic failure (VF) were studied in 243 antiretroviral-naive patients starting first-line protease inhibitor (PI)-containing therapy (nelfinavir in 66% and indinavir in 19%). Among the 220 patients with follow-up data, VF occurred in 35 (16%) during the first year of follow-up. A higher baseline virus load and poorer adherence to therapy were associated with VF. At the time of VF, key PI-resistance mutations were detected in 11 (48%) of 23 patients who started on nelfinavir but were absent in 6 patients with indinavir treatment failure. PI plasma levels were more often below the range of active concentrations in VF with wild-type viruses (74%) than in VF with PI-resistant viruses (25%; P=.02). The mechanisms of early VF and of selection of PI-resistant viruses differed by type of PI and were dependent on PI plasma levels.

Gene expression in haemocytes of kuruma prawn, Penaeus japonicus, in response to infection with WSSV by EST approach

Gene expression in haemocytes of the kuruma prawn (Penaeus japonicus) was investigated using an expressed sequence tag (EST) approach. Partial nucleotide sequences of cDNA library clones constructed from normal and white spot syndrome virus (WSSV)—infected P. japonicus haemocytes were determined. Of 635 clones obtained from the normal library, 284 (44·7%) significantly matched sequences in GenBank, and of 370 clones obtained from WSSV-infected library, 174 (47·0%) significantly matched sequences in the database. One hundred fifty-two deduced proteins were newly identified. Of these, 28 types were involved in biodefence. For the prophenoloxidase system, there are prophenoloxidase, coagulation factor G-β chain precursor, factor D, Masquarade-like protease, transglutaminase (TGase), clottable protein and eight types of protease inhibitors (two types of antileukoproteinase, alpha-2-macroglobulin, chelonianin, elastase inhibitor, two types of Kazal inhibitor and Kunitz-type inhibitor). For antibacterial peptides, there are bactinecin 11, penaeidin-2 precursor and lysozyme c type. The others defence-related proteins are basophil leukocyte interleukin-3-regulated protein, natural killer enhancing factor (NK-EF), integral membrane protein (CD34+), ESM-1, Notch homologue and Drac homologue. For the adhesion proteins, there are β-integrin, cell adhesion molecule (CAM) and three types of collagens. All ESTs representing protease inhibitors and tumour-related proteins were found only in the WSSV-infected library. Those encoding for apoptotic peptides were expressed at high levels in infected library. The putative defence proteins accounted for 2·7% of total ESTs in a normal shrimp library and 15·7% of the total ESTs in an infected library.

Factors related to lipodystrophy and metabolic alterations in patients with human immunodeficiency virus infection receiving highly active antiretroviral therapy.

Morphologic and metabolic changes associated with protease inhibitor (PI) therapy have been reported since the introduction of PIs for treatment of human immunodeficiency virus infection. These changes were measured 12-20 months after initiation of PI therapy in a cross-sectional study involving 614 patients from the Antiprotéases Cohorte (APROCO) Study (Agence Nationale de Recherches sur le Sida-EP11). The prevalence was 21% for isolated peripheral atrophy, 17% for isolated fat accumulation, 24% for mixed syndrome, 23% for glucose metabolism alterations, 28% for hypertriglyceridemia (triglyceride level, > or =2.2 mM), and 57% for hypercholesterolemia (cholesterol level, > or =5.5 mM). Age was significantly associated with different phenotypes of lipodystrophy and metabolic alterations, but body-mass index, CD4(+) cell count, and type of nucleoside reverse-transcriptase inhibitor or PI received were not constantly associated with these changes. Furthermore, in all models tested, exposure to stavudine was associated with lipoatrophy and exposure of ritonavir was associated with hypertriglyceridemia. Detection and management of these disorders should be implemented to prevent further complications.

Molecular characterization of four alpha-1-antitrypsin variant alleles found in a Japanese population: a mutation hot spot at the codon for amino acid 362

In this study alpha-1-antitrypsin (AAT) phenotypes at the protease inhibitor (PI) locus were determined by isoelectric focusing of native and desialylated serum samples from 236 Japanese subjects living in the western part of Japan. The shifts in relative mobility between some PI types were observed before and after desialylation. This technique was useful in distinguishing between some PI M subtypes and variants. The molecular basis of four variant alleles, including two new alleles found in this study, was characterized: PI E tokyo [Lys 335( AAG)→ Glu( GAG)] and PI N nagato [Leu 276(C TG)→Pro(C CG)] arose from PI M1(Val 213) and PI M2, respectively. A new PI P yonago [Asp 19(G AT)→Ala(G CT)] originated from PI M1(Val 213). A new PI M5 gunma [Pro 362( CCC)→Ser( TCC)], arising from PI M3, was the sixth allele involving a mutation at codon 362, which is suggested to be a mutation hot spot. PI M5 gunma was likely to show normal AAT levels and function although the mutations occurred near codon 358 for Met 358. The molecular basis of PI variant alleles found in Japanese was different from that reported in previous studies.

A proteolytically truncated form of free CD18, the common chain of leukocyte integrins, as a novel marker of activated myeloid cells

An unusual CD18 monoclonal antibody (mAb) MEM-148 binds, in contrast to standard CD18 mAbs, specifically to peripheral blood monocytes and neutrophils activated by various stimuli such as phorbol myristate acetate, opsonized zymosan, heat-aggregated immunoglobulin, and (after priming with lipopolysaccharide, tumor necrosis factor, or granulocyte-macrophage colony-stimulating factor) also by formyl-methionyl-leucyl-phenylalanine. In addition, in vivo activated neutrophils obtained from urine of patients following recent prostatectomy were also strongly positive for MEM-148. On the activated myeloid cells the mAb recognized a 65- to 70-kd protein identified immunochemically and by mass spectrometric peptide sequencing as a membrane-anchored fragment of CD18 (the common chain of leukocyte integrins) produced by proteolytic cleavage. The CD18 fragment originated mainly from integrin molecules stored intracellularly in resting cells, it was unassociated with CD11 chains, and its formation was inhibited by several types of protease inhibitors. Thus, the 65- to 70-kd CD18 fragment represents a novel abundant activation marker of myeloid cells of so far unknown function but possibly involved in conformational changes in leukocyte integrin molecules resulting in increased affinity to their ligands.

Proteolysis of the urokinase-type plasminogen activator receptor by metalloproteinase-12: implication for angiogenesis in fibrin matrices

AbstractPericellular proteolysis plays an important role in cell migration and the formation of new capillary structures. The plasminogen activator/plasmin and matrix degrading metalloproteinase (MMP) cascades act together in the remodeling of matrix and cell-matrix contacts. Previously we have shown that the formation of capillary structures by human foreskin microvascular endothelial cells (hMVECs) in a 3-dimensional fibrin matrix requires a functional urokinase-type plasminogen activator receptor (u-PAR). Here we report on the unexpected finding that inhibition of hMVEC-derived MMP activity by BB94 (batimastat) increased the outgrowth of capillary structures in a fibrin matrix. BB94 prevented the release of the u-PA binding domain D1 of u-PAR and thereby increased the number of functional u-PARs on hMVECs without affecting the u-PAR messenger RNA levels. Comparison of various types of protease inhibitors pointed to the prime involvement of MMP activity. Using recombinant MMPs it was shown that MMP-12 activity was able to release the D1 domain of cellularly expressed u-PAR. In addition, the expression of MMP-12 in control and basic fibroblast growth factor/tumor necrosis factor-α–stimulated hMVECs was shown by reverse transcriptase–polymerase chain reaction, suggesting that endothelial cell–derived MMP-12 may be involved in angiogenesis-related u-PAR shedding. This new mechanism of u-PAR cleavage provides new insights into the mutual interactions between the MMP and u-PA/plasmin systems. Moreover, it may be helpful in the interpretation of recent data on the use of specific MMP inhibitors in the treatment of several types of cancer.

Genomic Structure of the Immune-related Serpin Gene in the Fall Webworm, Hyphantria cunea

We previously identified a novel immune-related serine type protease inhibitor cDNA from Hyphantria cunea (Drury) (Shin et al., 1998) that suggested a specific inhibitor directed at the protease involved in the activation of prophenoloxidase (proPO) (Park et al., 2000). Herein, we report complete nucleotide sequence and the exon-intron structure of the gene. The Hyphantria serpin gene spans 18 kb and is composed of eight exons and seven introns coding for an 1.8-kb mRNA. In the 5′ flanking sequence, we found several putative regulatory motifs including an enhancer, two NF-IL6 motifs, a TNF-α responsive element, an interferon-γ-responsive element, and a GATA motif element that have been found in the promoters of the acute phase protein genes of mammals and insects. In addition, a 9-bp inverted repeat sequence located from −270 to −221 region was also found. A reactive site loop sequence was located in exon 8 flanked by 3′ untranslation sequence. Phylogenetic analysis showed that this Hyphantria serpin did not cluster with either insect or mammalian serpins that are known, or suspected, to be associated with immune function. Southern blot analysis showed that the Hyphantria serpin gene is present as a single copy in the genome.

Linkage Analysis of Alpha 1-Antitrypsin Deficiency: Lessons for Complex Diseases

Objectives: Severe alpha 1-antitrypsin (A1AT) deficiency is the one proven genetic risk factor for chronic obstructive pulmonary disease (COPD). Familial aggregation has been demonstrated for COPD among individuals who do not have A1AT deficiency, but linkage analysis of COPD has not been reported. To investigate the optimal phenotype definitions and analytical methods for the linkage analysis of COPD, we examined a set of 28 A1AT- deficient families containing 155 individuals. We have used the protease inhibitor (PI) type as a genetic marker rather than a disease gene, and we have performed linkage analysis between PI type and serum A1AT level and spirometry-related phenotypes. Methods: Linkage analysis was performed on the quantitative phenotypes forced expiratory volume at 1 s (FEV₁ as % predicted), the ratio of FEV₁ to forced vital capacity (FEV₁/FVC as % predicted), and serum A1AT level using the variance component approach in SOLAR, the generalized estimating equation approach in RELPAL, and the model-based classical lod score method in LINKAGE. Linkage analysis with qualitative A1AT and spirometry phenotypes was performed using a model-based method (LINKAGE) and a model-free method (GENEHUNTER). Adjustments for smoking effects were investigated under each method. Results: All of the methods demonstrated linkage of PI type to serum A1AT level. Interestingly, however, the other quantitative phenotypes provided only weak evidence for linkage of PI type to lung disease. Better evidence for linkage of lung disease to PI type was found using a moderate or a mild threshold for the definition of airflow obstruction. Conclusions: For linkage analysis of spirometry phenotypes in A1AT deficiency, qualitative phenotypes provided stronger evidence for linkage than quantitative phenotypes. Possible contributors to the stronger evidence for linkage to qualitative spirometry phenotypes include the ascertainment scheme and the nonnormality of the pulmonary function data in PI Z subjects. This study provides guidelines for studies of the genetics of COPD unrelated to A1AT deficiency.

Protein engineering of novel proteinase inhibitors and their effects on the growth of Spodoptera exigua larvae.

Novel types of proteinase inhibitors with multi-inhibitory activities were generated by replacement of phytocystatin domains in sunflower multi-cystatin (SMC) by the serine proteinase inhibitor BGIT from bitter gourd seeds. Two chimeric inhibitors SMC-T3 and SMC-T23, in which the third domain in SMC and the second and third domains in SMC were replaced by BGIT, acquired trypsin inhibitory activity (Ki: 1.46 x 10(-7) M and 1.75 x 10(-7) M), retaining inhibitory activity toward papain (Ki: 4.5 x 10(-8) M and 1.52 x 10(-7) M), respectively. We compared the chimeric inhibitors and the recombinant SMC (r-SMC) in relation to their effects on the growth of larval Spodoptera exigua. When the second instar larvae were reared on a diet containing rSMC, SMC-T3, or SMC-T23 for ten days, a significant reduction in weight gain was observed. Mean weights for rSMC, SMC-T3, and SMC-T23 were 43 mg, 32 mg, and 43 mg, respectively, as compared with that (60 mg) for the absence of the inhibitor. In contrast, BGIT had little effect on the growth of the S. exigua larvae. This result indicated that the chimeric inhibitor SMC-T3 with two phytocystatin domains and one serine proteinase inhibitor domain is an efficient inhibitor of proteinases in the S. exigua larvae. Therefore, this novel type of proteinase inhibitor with multi-inhibitory activities may represent a promising protein for successful application to a transgenic plant with insect resistance.

Proteinase inhibitors from the medicinal leech Hirudo medicinalis.

The medicinal leech Hirudo medicinalis produces various types of proteinase inhibitors: bdellins (inhibitors of trypsin, plasmin, and acrosin), hirustasin (inhibitor of tissue kallikrein, trypsin, alpha-chymotrypsin, and granulocyte cathepsin G), tryptase inhibitor, eglins (inhibitors of alpha-chymotrypsin, subtilisin, and chymasin and the granulocyte proteinases elastase and cathepsin G), inhibitor of factor Xa, hirudin (thrombin inhibitor), inhibitor of carboxypeptidase, and inhibitor of complement component C1s. This review summarizes data on their primary and tertiary structures, action mechanisms, and biological activities.