Isolation and characterization of inhibitors of neutral proteinases from spleen

Two different types of neutral proteinase inhibitors were isolated from postmicrosomal supernatant of bovine spleen. Inhibitor A with molecular weight 40,000 inhibits elastases and chymotrypsin-like neutral proteinases from bovine spleen, whereas inhibitor B with estimated molecular weight 20 000 inhibits only chymotrypsin-like neutral proteinase.     

Identity of kininogenase and plasminogen activators in human granulocytes

It was shown that the plasminogen activator inhibitor, ZGlyGlyArgCH2Cl, inactivates the kininogenase and plasminogen activator activities in the whole human granulocyte lysate and human granulocyte proteinase fractions isolated by isoelectrofocusing from the granulocyte lysate (pH 3-10). The kinetics of irreversible inhibition of the ZGlyGlyArgpNA-amidase activity in granulocyte proteinase fractions (pI 10.75, 8.9 and 8.3) by ZGlyGlyArgCH2Cl was measured. These data confirm the earlier obtained results on the trypsin-like nature of the human granulocyte plasminogen activator and its identity to this cell kininogenase.     

Inhibition of human granulocyte elastase and kininogenase

Using an inhibitory analysis, the different enzymatic nature of kininogenase and elastase activities in serine proteinase fractions (pI 8.3-10.75) isolated from human granulocyte lysates by isoelectrofocusing was demonstrated. The thermo- and acid-stable serine proteinase inhibitor from rabbit serum was shown to completely inhibit the kininogenase activity in these fractions but to have no inhibiting action on the elastase activity. On the contrast, the specific granulocyte elastase inhibitor, N-3-carbomethoxypropanoyl-L-alanyl-L-alanyl-L-prolyl-L-valyl-chloromethylketone , inhibits granulocyte elastase and does not inhibit the kininogenase activity in lysate fractions. The efficiency of granulocyte elastase inhibition by this chloromethylketone is evaluated by the kinetic parameters k3, Ki. The values of k3/Ki for granulocyte elastase forms with pI of 10.75, 8.9 and 8.0 are 1430, 670 and 360 M-1 S-1, respectively and show effective inhibition of the three forms by this inhibitor. Based on the different degree of inhibition of the three elastase forms by chloromethylketone inhibitor the existence of the family of elastaselike enzymes in human granulocytes is postulated.     

Identity of human granulocyte kininogenase and plasminogen activator

Preparative isoelectrofocusing used for fractionating the whole human granulocyte lysate serine proteinases revealed multiple forms of elastase, cathepsin G, kininogenase, human granulocytes plasminogen activator (pI 6.2-10.75). Kinetic characteristics of their substrate specificity were also obtained. It is shown that serine kininogenase of human granulocytes is not identical with elastase as it had been supposed before, it is of trypsin-like nature and is identical with plasminogen activator of these cells. The results obtained reveal new aspects in comprehension of the role of the granulocyte plasminogen activator in development of the inflammatory reaction. It is found that acid-stable proteinase inhibitors formed from blood plasma inter-alpha-inhibitor of trypsin, have an inhibitory effect on the granulocyte plasminogen activator, that supports an assumption on the anti-inflammatory function of these inhibitors.     

Gene expression patterns of Helicoverpa armigera gut proteases

Relative quantification of reported gut proteinase cDNAs from Helicoverpa armigera larvae fed on various host plants (cotton, chickpea, pigeonpea, tomato and okra), non-host plant PIs (winged bean, bitter gourd, ground nut, and capsicum) and during larval development has been carried out using semi-quantitative RT-PCR. Five trypsin-like and three chymotrypsin-like proteinases were categorized as insensitive or sensitive to most of the proteinase inhibitors (PIs) and insensitive/sensitive to specific PIs based on their expression analysis. These results were supported by amino acid sequence analysis, where diverged amino acids were observed in the regions, which are reported to be involved in typical trypsin-trypsin inhibitor interactions and critical for proteinase inhibitor resistance. Among exopeptidases (five aminopeptidase and three carboxypeptidase), HaAmi4 and HaAmi5 of aminopeptidase and HaCar1 of carboxypeptidase exhibited considerable differential expression. Elastase and cathepsin B-like proteinases were expressed at negligible levels. The proteases identified in the study would be ideal candidates for further interactions studies with protease inhibitors to understand the structural reasons of protease inhibitor insensitivity.     

Effects of proteinase inhibitors on preimplantation embryos in the rat

Proteinase inhibitors of microbial origin were injected into the uterine horns of mated rats at 14:00 h on Day 5 of pregnancy (spermatozoa in vaginal smear = Day 1), and 5 or 6 h later the embryos were flushed from the horns and examined. Chymostatin and alpha-MAPI, inhibitors of chymotrypsin-like serine proteinase and thiol proteinases, as well as thiolstatin, an inhibitor of thiol proteinases, significantly inhibited embryo growth. The inhibitory activity of alpha-MAPI on embryonic growth was distinctly greater than that of thiolstatin, although the ID50 values of the two inhibitors to papain are similar. Antipain and leupeptin which are inhibitors of trypsin-like and thiol proteinases, and talopeptin, an inhibitor of metal proteinases, significantly interrupted the removal of the zona pellucida from expanding blastocysts. These results suggest that (1) a chymotrypsin-like proteinase seems to be important to the growth of the embryo, (2) a thiol proteinase may participate in embryonic growth, and (3) a trypsin-like proteinase and a metal proteinase are likely to participate in zonalysis.     

Purification and identification of two serine class proteinases from dog mast biochemically and immunologically similar to human proteinases tryptase and chymase

Serine class proteinases with trypsin-like and chymotrypsin-like specificity were purified from dog mastocytoma tissue. An antiserum was produced against the chymotrypsin-like proteinase. The antiserum reacted with mast cells in skin sections prepared from normal dogs consistent with the proteinase being a mast cell constituent. The antiserum also cross-reacted with the major chymotrypsin-like proteinase isolated from normal dog skin and partially cross-reacted with human skin chymase. No cross-reaction was detected with rat chymase. The trypsin-like proteinase from dog mastocytoma tissue was similar to tryptase isolated from human skin. It had a similar subunit structure, was not inhibited by many protein proteolytic enzyme inhibitors, bound to heparin, and reacted strongly with antiserum against human tryptase. Antiserum against human tryptase also reacted with mast cells in skin sections prepared from normal dog skin. No immunocytochemical labeling of rat skin mast cells was observed with anti-human tryptase. These studies establish the presence of a trypsin-like and chymotrypsin-like proteinase in dog skin mast cells and provide immunological evidence which suggests that both proteinases are more closely related to human than rat mast cell proteinases. These immunological and biochemical relationships are important when comparing the roles of these proteinases in different animals.     

Human skin chymotryptic proteinase. Isolation and relation to cathepsin g and rat mast cell proteinase I

A chymotrypsin-like proteinase was purified 2400-fold from human skin. The procedure involves extraction of the proteinase from skin in 2 M KCl, precipitation with protamine chloride, fractionation by gel filtration chromatography, and fractionation by chromatography using a CH-Sepharose-D-tryptophan methyl ester affinity column. The properties of this proteinase were compared to the rat mast cell proteinase I and human cathepsin G. Differences were observed in the rates at which the proteinases were inhibited by diisopropyl fluorophosphate, the sensitivity of the proteinases to protein proteolytic inhibitors, the relative hydrolytic rates of the proteinases for a series of substrates, and the kinetic constants of the proteinases for synthetic substrates. The human skin proteinase did not react with antiserum to the rat skin proteinase and did not elute in the same position as the rat skin proteinase on gel filtration columns. These data demonstrate that the human skin proteinase is distinct from the other proteinases. Extracts of involved skin from patients with cutaneous mastocytosis had 15-fold higher levels of chymotryptic activity than extracts of uninvolved skin or skin from normal controls. The enzymatic properties of the material extracted from the biopsied skin were similar to those of the proteinase from normal skin, suggesting that the human skin chymotrypsin-like proteinase is a mast cell constituent.     

Characterization of neutral proteinases from human spleen]

Several proteinases hydrolyzing histone and caseine in neutral media were obtained by Sephadex G-100 fractionation of water and salt (1 M KCl) extracts of human spleen. The level of the activity of proteinases in the extracts was very low as a result of the presence of an inhibitor. Neutral proteinases were found in two protein fractions. The "high-molecular-weight-" proteinases were inhibited by DFP and therefore they were attributed to a group of serine proteinases. The "low-molecular-weight" fraction contained neutral SH-dependent proteinase(s) and DFP-inhibited enzymes. In this fraction, the kininogenase activity and the hydrolysis of Boc-1-ananine p-nitrophenyl ester, N-benzoyl-L-tryosine ethyl ester and N-benzoyl-DL-arginine p-nitroanilide were observed.     

Digestive proteinases in Lasioderma serricorne (Coleoptera: Anobiidae)

The cigarette beetle, Lasioderma serricorne (Fabricius), is a common pest of stored foods. A study of digestive proteinases in L. serricorne was performed to identify potential targets for proteinaceous biopesticides, such as proteinase inhibitors. Optimal casein hydrolysis by luminal proteinases of L. serricorne was in pH 8.5-9.0 buffers, although the pH of luminal contents was slightly acidic. Results from substrate and inhibitor analyses indicated that the primary digestive proteinases were serine proteinases. The most effective inhibitors of caseinolytic hydrolysis were from soybean (both Bowman Birk and Kunitz), with some inhibition by chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone, and leupeptin. Casein zymogram analysis identified at least eight proteolytic activities. Activity blot analyses indicated one major proteinase activity that hydrolysed the trypsin substrate N-alpha-benzoyl-L-arginine rho-nitroanilide, and three major proteinase activities that hydrolysed the chymotrypsin substrate N-succinyl ala-ala-pro-phe rho-nitroanilide. The absence of cysteine, aspartic, and metallo proteinases in L. serricorne digestion was evidenced by the lack of activation by thiol reagents, alkaline pH optima, and the results from class-specific proteinase inhibitors. The data suggest that protein digestion in L. serricorne is primarily dependent on trypsin- and chymotrypsin-like proteinases.     

Effect of cysteine proteinase inhibitors on murine B16 melanoma cell invasion in vitro

Various types of proteinases are implicated in the malignant progression of human and animal tumors. Proteinase inhibitors may therefore be useful as therapeutic agents in anti-invasive and anti-metastatic treatment. The aims of this study were (1) to estimate the relative importance of proteinases in B16 cell invasion in vitro using synthetic, class-specific proteinase inhibitors and (2) to assess the inhibitory effect of some naturally occurring cysteine proteinase inhibitors. Serine proteinase inhibitor reduced invasiveness by up to 24%, whereas inhibition of aspartic proteinases reduced invasion by 11%. Synthetic inhibitors of cysteine proteinases markedly impaired invasion: cathepsin B inhibitors, particularly Ca-074Me, inhibited invasion from 20-40%, whereas cathepsin L inhibitor Clik 148 reduced invasion by 11%. The potato cysteine proteinase inhibitor PCPI 8.7 inhibited invasion by 21%, whereas another potato inhibitor, PCPI 6.6, and the mushroom cysteine proteinase inhibitor clitocypin had no effects. As the inhibitors that inhibited cathepsin B were in general more efficient at impairing the invasiveness, we conclude that of the two cysteine proteinases, cathepsin B plays a more important role than cathepsin L in murine melanoma cell invasion.     

Intracellular distribution of neutral proteinases and inhibitors in pig leucocytes. Isolation of two inhibitors of neutral proteinases.

Granule and post-granular-supernatant fractions were obtained from pig leucocyte cells by differential centrifugation in 0.34 M sucrose. Granule extract possesses proteinase activity at acid and at neutral pH. Three groups of neutral and a group of acid proteinases were isolated from granule extracts by chromatography on DEAE-cellulose. In the first group are present elastase-like and plasminogen-activator proteinases, that are inhibited by diisopropylphosphorofluoridate, alpha1-antitrypsin, intracellular leucocyte inhibitor and partly with p-aminomethylbenzoic acid and Trasylol. The second group of neutral proteinases is unstable under the conditions of isolation used the third group of neutral proteinases comprises collagenases that are inhibited by ethylenediamine tetraacetic acid disodium salt, alpha1-antitrypsin and leucocyte inhibitor. The acid proteinases are inhibited only with pepstatin, up to 90%. In the post-granular supernatant was found the acid proteinase activity towards hemoglobin and casein, and non-stable neutral proteolytic activity towards bovine serum albumin and serum gamma globulin. In the post-granular supernatant also the inhibitors of neutral proteinases were found. By gel filtration on Sephadex G-100 and ion-exchange chromatography on CM-cellulose two inhibitors of neutral proteinases were isolated. The majority of the inhibitor capacity (about 80%) of post-granular supernatant was eluted together with ovalbumin (Mr 43000) and the remainder with cytochrome c (12300). These inhibitors inhibit the granule neutral proteinases, acting on all substrates used, but do not inhibit granule acid proteinase. Inhibition effects of post-granular-supernatant inhibitors on trypsin and chymotrypsin were obtained only when bovine serum albumin was used as substrate. Inhibitors of post-granular supernatant are stable at pH 6-8, but unstable in the pH rnage 2-5 and are thermolabile.     

Biosynthesis, purification, and characterization of the human coronavirus 229E 3C-like proteinase.

Coronavirus gene expression involves proteolytic processing of the gene 1-encoded polyprotein(s), and a key enzyme in this process is the viral 3C-like proteinase. In this report, we describe the biosynthesis of the human coronavirus 229E 3C-like proteinase in Escherichia coli and the enzymatic properties, inhibitor profile, and substrate specificity of the purified protein. Furthermore, we have introduced single amino acid substitutions and carboxyl-terminal deletions into the recombinant protein and determined the ability of these mutant 3C-like proteinases to catalyze the cleavage of a peptide substrate. Using this approach, we have identified the residues Cys-3109 and His-3006 as being indispensable for catalytic activity. Our results also support the involvement of His-3127 in substrate recognition, and they confirm the requirement of the carboxyl-terminal extension found in coronavirus 3C-like proteinases for enzymatic activity. These data provide experimental evidence for the relationship of coronavirus 3C-like proteinases to other viral chymotrypsin-like enzymes, but they also show that the coronavirus proteinase has additional, unique properties.     

Identification of a cathepsin G-like proteinase in the MCTC type of human mast cell

Human mast cells can be divided into two subsets based on serine proteinase composition: a subset that contains the serine proteinases tryptase and chymase (MCTC), and a subset that contains only tryptase (MCT). In this study we examined both types of mast cells for two additional proteinases, cathepsin G and elastase, which are the major serine proteinases of neutrophils. Because human mast cell chymase and cathepsin G are both chymotrypsin-like proteinases, the properties of these enzymes were further defined to confirm their distinctiveness. Comparison of their N-terminal sequences showed 30% nonidentity over the first 35 amino acids, and comparison of their amino acid compositions demonstrated a marked difference in their Arg/Lys ratios, which was approximately 1 for chymase and 10 for cathepsin G. Endoglycosidase F treatment increased the electrophoretic mobility of chymase on SDS gels, indicating significant N-linked carbohydrate on chymase; no effect was observed on cathepsin G. Immunoprecipitation and immunoblotting with specific antisera to each proteinase revealed little, if any, detectable cross-reactivity. Immunocytochemical studies showed selective labelling of MCTC type mast cells by cathepsin G antiserum in sections of human skin, lung, and bowel. No labeling of mast cells by elastase antiserum was detected in the same tissues, or in dispersed mast cells from lung and skin. A protein cross-reactive with cathepsin G was identified in extracts of human skin mast cells by immunoblot analysis. This protein had a slightly higher Mr (30,000) than the predominant form of neutrophil cathepsin G (Mr 28,000), and could not be separated from chymase (Mr 30,000) by SDS gel electrophoresis because of the size similarity. Using casein, a protein substrate hydrolyzed at comparable rates by chymase and cathepsin G, it was shown that about 30% of the caseinolytic activity in mast cell extracts was sensitive to inhibitors of cathepsin G that had no effect on chymase. Hydrolytic activity characteristic of elastase was not detected in these extracts. These studies indicate that human MCTC mast cells may contain two different chymotrypsin-like proteinases: chymase and a proteinase more closely related to cathepsin G, both of which are undetectable in MCT mast cells. Neutrophil elastase, on the other hand, was not detected in human mast cells by our procedures.     

3,4-dichloroisocoumarin-induced activation of the degradation of beta-casein by the bovine pituitary multicatalytic proteinase complex.

The breakdown of beta-casein (caseinolytic activity) by the bovine pituitary multicatalytic proteinase complex (MPC) is initiated by a fourth active site different from the previously described chymotrypsin-like activity (cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide, where Cbz is benzyloxycarbonyl), trypsin-like activity (cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide), and peptidylglutamyl peptide bond-hydrolyzing (PGP) activity (cleavage of Cbz-Leu-Leu-Glu-2-naphthylamide) (Yu, B., Pereira, M. E., and Wilk, S. (1991) J. Biol. Chem. 266, 17396-17400). 3,4-Dichloroisocoumarin, a serine proteinase inhibitor, stimulated the caseinolytic activity of bovine pituitary or lens MPC, 3-18-fold under conditions under which the other three catalytic activities were inactivated. Addition of hydroxylamine to the modified enzyme did not reverse the effects of the inhibitor. A form of the proteinase exhibiting only 2-4% of control chymotrypsin-like, trypsin-like, and PGP activities degraded beta-casein with no accumulation of intermediate peptides. 3,4-Dichloroisocoumarin, by reacting with the chymotrypsin-like, trypsin-like, and/or PGP-active sites, may promote a conformational change of MPC, rendering the caseinolytic active site accessible to the substrate. Once bound to the active site, beta-casein is rapidly degraded either by the caseinolytic component itself or by a cooperative interaction with catalytic centers that are not affected by the serine proteinase inhibitor. These results imply that the caseinolytic component does not belong to the class of serine proteinases. Other proteins tested were not degraded by the 3,4-dichloroisocoumarin-treated enzyme, suggesting that the conformation of beta-casein may be more adequate for degradation by the caseinolytic component.     

The interaction of a trypsin-dependent neutral protease and its inhibitor found in tumour cells. Analysis of complex kinetic data involved in a thiol-disulphide exchange mechanism

Ehrlich ascites tumour cells contain a granule-derived zymogen which on trypsin activation yields a collegenolytic neutral protease. The preparation of the granule fraction by subcellular fractionation procedure results in the preparation of a second fraction referred to as the post-granule supernatant fraction. The post-granule supernatant fraction contains a latent form of the granule-derived neutral protease and an excess of cytoplasmic inhibitor for this enzyme. The inhibitor of neutral protease is also capable of inhibiting trypsin and in each case the chemical mechanism of enzyme.inhibitor complex formation has been shown to be a reversible thiol-disulphide exchange. The post-granule supernatant fraction exhibited complex kinetic data when the interactions between the inhibitor, the latent enzymes and trypsin were examined simultaneously by incremental analysis. The data were interpreted and quantitatively analysed by computer analysis. It was demonstrated that the conventional types of analysis could not have provided meaningful interpretations of the experimental data provided by these complex-interacting systems.     

Biochemical characterization of midgut digestive proteases from Mamestra brassicae (cabbage moth; Lepidoptera: Noctuidae) and effect of soybean Kunitz inhibitor (SKTI) in feeding assays

Proteolytic activities in soluble protein extracts from Mamestra brassicae (cabbage moth) larval midgut were analysed using specific peptide substrates and proteinase inhibitors. Serine proteinases were the major activities detected, with chymotrypsin-like and trypsin-like activities being responsible for approximately 62% and 19% of the total proteolytic activity towards a non-specific protein substrate. Only small amounts of elastase-like activities could be detected. The serine proteinases were active across the pH range 7-12.5, with both trypsin-like and chymotrypsin-like activities maximal at pH 11.5. The digestive proteinases were stable to the alkaline environment of the lepidopteran gut over the timescale of passage of food through the gut, with 50% of trypsin and 40% of chymotrypsin activity remaining after 6h at pH 12, 37 degrees C. Soybean Kunitz trypsin inhibitor (SKTI) ingestion by the larvae had a growth-inhibitory effect, and induced inhibitor-insensitive trypsin-like activity. Qualitative and quantitative changes in proteinase activity bands after gel electrophoresis of gut extracts were evident in SKTI-fed larvae when compared with controls, with increases in levels of most bands, appearance of new bands, and a decrease in the major proteinase band present in extracts from control insects.     

Cysteine proteinases from Schistosoma haematobium adult worms.

To identify and characterize cysteine proteinases from Schistosoma haematobium, lyophilized adult worms were homogenized, and enzymes were isolated and purified. From extracts prepared in acidic buffer, 3 putative cysteine proteinases were identified either directly or indirectly. The first proteinase (ShCP1) was identified by labeling with a radioiodinated inhibitor, Z-Tyr-Ala-CHN2, as a 35-kDa protein. However, it could not be detected by silver staining, amino acid sequencing, or by a monoclonal antibody specific for a similar molecule from Schistosoma mansoni. A second cysteine proteinase, ShCP2, was purified by gel filtration and dialysis. This 32-kDa molecule was thiol-dependent and was labeled with Z-Tyr-Ala-CHN2. The amino terminal amino acid sequence of ShCP2 showed remarkable similarity (up to 77%) to that of S. mansoni cathepsin B (SmCP2) as well as to mammalian cysteine proteinases. Both ShCP1 and ShCP2 reacted with polyclonal antibodies against S. mansoni, suggesting the existence of shared antigenic epitopes. A third activity, ShCP3, was identified as possibly a distinct proteinase based on its similarities to a 28-kDa cysteine proteinase from S. mansoni. This preliminary investigation demonstrates that the overall profile of cysteine proteinases in S. haematobium is very similar to that of S. mansoni.     

A serine proteinase inhibitor produced by an HTLV I virus-transformed human T lymphocyte line

A previous report from our laboratory indicated that a proteinase inhibitor is produced by rabbit T lymphocytes. We now report that a human T cell line, C91/PL, produces a proteinase inhibitor which inhibits the enzymatic activity of trypsin and kallikrein. This newly identified proteinase inhibitor (LPI 1) did not inhibit the enzymatic activity of four other serine proteinases (thrombin, plasmin, chymotrypsin, or pancreatic elastase), a thiol proteinase (papain), or a carboxyl proteinase (pepsin). Active synthesis of LPI 1 by the C91/PL cell line was shown by the appearance of similar levels of inhibitory activity in sequential cell supernatants, lack of appearance of inhibitor in supernatants of cells killed by heat or sodium azide or of viable cells in the presence of cyclohexamide, and incorporation of a radiolabeled amino acid into newly synthesized inhibitor. Although both the inhibitor of rabbit origin and of human origin are proteins produced by T cells and have similar inhibitory specificity, important differences were observed: LPI 1 is sensitive to boiling and the two inhibitors migrate differently upon electrophoresis in substrate-containing polyacrylamide gel. Furthermore, LPI 1 was produced by a cell line of the T4 phenotype which had been established by in vitro viral transformation of human cord blood lymphocytes with HTLV 1 whereas the inhibitor of rabbit origin was produced by normal splenic T cells. Three other human T cell lines of the T4 phenotype, MOLT-13, KE-37, and HPB-ALL, from patients with acute lymphoblastic leukemia did not produce a proteinase inhibitor. Thus, the production of proteinase inhibitors does not appear to be a general characteristic of human T cell lines nor of the T4 subset. Proteinase inhibitors produced by T cells may have an immunoregulatory role in proteinase-mediated physiological processes.