Physiological adaptation explains the insensitivity of Baris coerulescens to transgenic oilseed rape expressing oryzacystatin I

Larvae of Baris coerulescens Scop. (Coleoptera: Curculionidæ) exhibit a complex array of gut proteinase activities comprising cysteine and serine proteinases. The major cysteine proteinase activity, showing an optimum at pH 6.0, corresponds to at least 4 different proteinases. On the contrary, the minor serine proteinase activity, with an optimum at pH 9.0, seems to be due essentially to a single proteinase. The cysteine proteinase inhibitor oryzacystatin I (OC-I) inhibits completely the cysteine proteinase activity in vitro. However, larval growth and survival were not significantly different on control and transgenic oilseed rape plants expressing high levels of active OC-I. In larvae grown on transgenic plants, cysteine proteinase activity was dramatically decreased, whereas serine proteinase activity was increased by more than 2-fold, when compared to larvae raised on control plants. For both activities, no new proteinase was detected in insects fed plants expressing OC-I. These results suggest that partial compensation of the inhibition of cysteine proteinase activity by the increase in serine proteinase activity allowed the larvae to overcome the effects of OC-I consumption. This case illustrates problems that could arise when trying to achieve high levels of protection for plants against Coleopteran pests possessing a complex digestive proteinase pool.     

Complexity in specificities and expression of Helicoverpa armigera gut proteinases explains polyphagous nature of the insect pest

Helicoverpa armigera is a devastating pest of cotton and other important crop plants all over the world. A detailed biochemical investigation of H. armigera gut proteinases is essential for planning effective proteinase inhibitor (PI)-based strategies to counter the insect infestation. In this study, we report the complexity of gut proteinase composition of H. armigera fed on four different host plants, viz. chickpea, pigeonpea, cotton and okra, and during larval development. H. armigera fed on chickpea showed more than 2.5- to 3-fold proteinase activity than those fed on the other host plants. H. armigera gut proteinase composition revealed the predominance of serine proteinase activity; however, the larvae fed on pigeonpea revealed the presence of metalloproteases and low levels of aspartic and cysteine proteases as well. Gut proteinase activity increased during larval development with the highest activity seen in the fifth instar larvae which, however, declined sharply in the sixth instar. Over 90% of the gut proteinase activity of the fifth instar larvae was of the serine proteinase type, however, the second instar larvae showed the presence of proteinases of other mechanistic classes like metalloproteases, aspartic and cysteine proteases along with serine proteinase activity as evident by inhibition studies. Analysis of fecal matter of larvae showed significant increase in proteinase activity when fed on an artificial diet with or without non-host PIs than larvae fed on a natural diet. The diversity in the proteinase activity observed in H. armigera gut and the flexibility in their expression during developmental stages and depending upon the diet provides a base for selection of proper PIs for insect resistance in transgenic crop plants.     

Processing of the lactococcal extracellular serine proteinase.

Activity of the lactococcal cell envelope-located serine proteinase depends on the presence of membrane-associated lipoprotein PrtM. To differentiate between the action of the proteinase and the action of PrtM in the process of proteinase maturation, an inactive form of the lactococcal proteinase was constructed. This was done by mutating one of the three amino acids thought to constitute the active site of the enzyme. The secreted form of this inactivated proteinase was the same size as the inactive secreted form of the proteinase produced in the absence of PrtM. Both inactive proteinases are larger than the active proteinase. Isolation of proteinase by washing lactococcal cells carrying the complete proteinase gene in a Ca(2+)-free buffer was prevented by the absence of prtM or the absence of a functional active site. We propose that PrtM, during or after membrane translocation of the proteinase, effects the autoproteolytic removal of the N-terminal pro region of the proteinase. Subsequent C-terminal autodigestion results in the release of the enzyme from the lactococcal cells.     

Detection and evaluation of serine proteinase by affinity chromatography on immobilized-aprotinin inRicinus communia

Neutral proteinase was found in the leaves ofRicinus communie as assayed with α-casein and H-D-Val-Leu-Lys-pNA as substrates. The enzyme is maximally active at pH around 7.4. A selective adsorbent for serine proteinase was prepared by attaching aprotinin to aminoalkyl-porous glass. When partially purified leaf proteinase was passed through a column containing this adsorbent, the proteinase activity present was bound to the porous glass. The proteinase eluted at IM NaCl was inhibited by aprotinin, leupeptin, DFP, phenylmethylsulfonyl fluoride (PMSF) and serine proteinase inhibitor fromR, communis leaves, whereas pepstatin, EDTA, EGTA, and DTT had no effect on the enzyme. This inhibition profile suggests the leaf proteinase is a neutral proteinase, such as a serine proteinase.     

Processing of the lactococcal extracellular serine proteinase

Activity of the lactococcal cell envelope-located serine proteinase depends on the presence of membrane-associated lipoprotein PrtM. To differentiate between the action of the proteinase and the action of PrtM in the process of proteinase maturation, an inactive form of the lactococcal proteinase was constructed. This was done by mutating one of the three amino acids thought to constitute the active site of the enzyme. The secreted form of this inactivated proteinase was the same size as the inactive secreted form of the proteinase produced in the absence of PrtM. Both inactive proteinases are larger than the active proteinase. Isolation of proteinase by washing lactococcal cells carrying the complete proteinase gene in a Ca(2+)-free buffer was prevented by the absence of prtM or the absence of a functional active site. We propose that PrtM, during or after membrane translocation of the proteinase, effects the autoproteolytic removal of the N-terminal pro region of the proteinase. Subsequent C-terminal autodigestion results in the release of the enzyme from the lactococcal cells.     

Purification of a neutral proteinase, associated with the actomyosin complex, from uterine myometrium.

We have purified and characterized a neutral proteinase activity from pig uterine myometrium. The proteinase co-purified with the actomyosin complex and could only be separated from it by a high concentration of a chaotropic ion, 3M-NaBr. The proteinase was further purified by gel filtration and affinity chromatography. The purified protein showed a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis corresponding to an Mr of 28 000. Gel filtration on Sephadex G-100 in a buffer containing 3M-NaBr gave an Mr of 27 500. Without the addition of the chaotropic Br- ion, the proteinase aggregates to high-Mr forms of more than 10(6)Da. The proteinase has optimum hydrolytic activity with casein as substrate at pH 7.5-8.0. The thiol-group-blocking reagents p-chloromercuribenzoate, p-chloromercuribenzenesulphonate and Hg2+, as well as soya-bean trypsin inhibitor and 4-aminobenzamidine, inhibited the proteinase. Other bivalent cations, chelating agents and the serine-specific reagents 7-amino-1-chloro-3-tosylamido-L-heptan-2-one and phenylmethanesulphonyl fluoride were without any effect on proteinase activity. The proteinase degraded myosin very rapidly at a molar ratio of proteinase to myosin of 1:50, concomitant with the rate of loss of the ATPase activity. Compared with myosin, actin was only a poor substrate and was degraded at a much lower rate, even at a high molar ratio of the proteinase to actin.     

Evidence indicating that the multicatalytic proteinase of rabbit reticulocytes is not incorporated as a core enzyme into a 26 S proteinase complex.

We have reinvestigated the recent proposal that the multicatalytic proteinase, together with other components of reticulocyte lysate, may become incorporated into a very large, "26 S" proteinase complex via an ATP-dependent process. Different from these published results, we consistently isolate the multicatalytic proteinase as a 650,000 Da "20 S" multisubunit proteinase. Analysis on nondenaturing polyacrylamide gels of reticulocyte fractions containing the putative complexed form of the multicatalytic proteinase reveal that activity against succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin is associated with two groups of protein of different molecular mass. One migrates like multicatalytic proteinase purified to homogeneity, displays, on sodium dodecyl sulfate gels, a set of protein species in the range of 23,000-32,000 Da, characteristic of the multicatalytic proteinase, and is recognized by a monospecific antibody to the enzyme. In contrast, the activity associated with the higher molecular mass (26 S) proteinase complex lacks the typical multicatalytic proteinase subunits and is devoid of antigenic material, when tested with the antibody. These results confirm and extend our recent findings in mouse liver by showing that the multicatalytic proteinase is not a constituent of a 26 S proteinase complex.     

Degradations of human immunoglobulins and hemoglobin by a 60 kDa cysteine proteinase of Trichomonas vaginalis

The present study was undertaken to investigate the role of cysteine proteinase of Trichomonas vaginalis in escaping from host defense mechanism. A cysteine proteinase of T. vaginalis was purified by affinity chromatography and gel filtration. Optimum pH for the purified proteinase activity was 6.0. The proteinase was inhibited by cysteine and serine proteinase inhibitors such as E-64, NEM, IAA, leupeptin, TPCK and TLCK, and also by Hg²⁺, but not affected by serine-, metallo-, and aspartic proteinase inhibitors such as PMSF, EDTA and pepstatin A. However, it was activated by the cysteine proteinase activator, DTT. The molecular weight of a purified proteinase was 62 kDa on gel filtration and 60 kDa on SDS-PAGE. Interestingly, the purified proteinase was able to degrade serum IgA, secretory IgA, and serum IgG in time- and dose-dependent manners. In addition, the enzyme also degraded hemoglobin in a dose-dependent manner. These results suggest that the acidic cysteine proteinase of T. vaginalis may play a dual role for parasite survival in conferring escape from host humoral defense by degradation of immunoglobulins, and in supplying nutrients to parasites by degradation of hemoglobin.     

Regulation of proteinase synthesis in Lactococcus lactis

The synthesis of extracellular serine proteinase of Lactococcus lactis was studied during the growth in a batch and a continuous culture on chemically defined media. In a batch culture the proteinase synthesis started during the exponential phase of growth and the highest proteinase concentrations were found at the end of the exponential and beginning of the stationary phase of growth. During the growth in a lactose-limited chemostat with amino acids as the sole source of nitrogen, the specific rate of proteinase synthesis was maximal at a μof 0.23 h^?1. At higher growth rates the proteinase productin declined. The proteinase synthesis was dependent on the amino acid sources in the medium. In batch cultures of L. lactis grown on a chemically defined medium with amino acids, the proteinase production was increased four-fold compared to media containing casein or a tryptic digest of casein as the sole source of nitrogen. The inhibition of the rate of proteinase synthesis by casein and peptides was also observed during the growth in a chemostat. The addition of the dipeptide leucylproline (final concentration of 100 μM) to a lactose-limited continuous culture during the steady state (D = 0.23 h^?1) resulted in a transient inhibition of the rate of proteinase synthesis. This suggested that exogenously supplied peptides control the regulation of proteinase synthesis of L. lactis.     

Purification and properties of a novel latent proteinase showing myosin heavy chain-degrading activity from threadfin-bream muscle

A novel latent proteinase of which activity was induced by heating in the presence of NaCl was purified to homogeneity from threadfin-bream muscle by a combination of DEAE-cellulose, Con A-Sepharose, Arg-Sepharose, and Shim-pack HAC chromatographies. This proteinase was a glycoprotein having a monomeric subunit structure; Mr was estimated to be 77,000 on SDS-PAGE analysis. The proteinase hydrolyzed Boc-Leu-Thr-Arg-MCA as well as myosin heavy chain in the presence of 2-4% NaCl at pH 7.0 and at 60 degrees C, optimally. The proteinase was classified as serine proteinase based on the effects of soybean trypsin inhibitor, leupeptin, and antipain.     

Mechanism of staphylococcal serine proteinase inactivation by lymphocytes and granulocytes

Stricking differences were observed in the mechanism of interaction between staphylococcal serine proteinase and surface of human granulocytes or lymphocytes despite the fact that incubation of this enzyme with both types of cells leads to analogical decrease of proteinase activity. Interaction of proteinase with lymphocytes releases peptides smaller than these released spontaneously by non-treated lymphocytes or lymphocytes treated with DFP-proteinase. However, in supernatants of lymphocytes neither complex of proteinase with cell derived molecules nor changes of electrophoretic mobility of proteinase was found. Products of proteinase—lymphocyte reaction have a proliferative effect on intact lymphocytes, which is greater that the one of active proteinase. On the other hand granulocytes are resistant to proteinase and bind active proteinase as well as the DFP-proteinase in the receptor mediated way, followed by endocytosis with the affinity similar to the one in monocytes.     

Isolation and characterization of the gene encoding a novel, thermostable serine proteinase from the mould Tritirachium album Limber

A number of proteinases are induced and secreted into the culture medium of Tritirachium album Limber when the nitrogen source is limited to exogenous proteins. We have constructed a cDNA library using the polyadenylated RNA isolated during the nutritional induction with bovine serum albumin. A full-length clone of a gene for a new proteinase (named proteinase R) was identified from this library. This clone contained sequences coding for the 108-amino-acid prepro-leader as well as for the 279-amino-acid mature proteinase. Proteinase R apparently belongs to the subtilisin group of serine proteases that contains disulphide bonds. Homology between proteinase R and proteinase K was found to be about 87% at the nucleotide as well as at the amino acid level. The Brookhaven Protein Data Base co-ordinate file of proteinase K was used as a template to study the proteinase R substitutions in three-dimensional space. The majority of the substitutions of proteinase R with respect to proteinase K were found to be on the exterior of the protein model.     

Effect of Culture Medium Composition and pH on the Production of M Protein and Proteinase by Group A Streptococci

The effects of pH, yeast extract, and neopeptone on the production of extracellular proteinase and M protein by group A streptococci were studied with a type 1 strain capable of producing both M protein and proteinase. The strain DS 2036-66 grew moderately well in a semisynthetic broth. M protein was produced without adding peptides to the medium. When added to a medium with 1% glucose, yeast extract (0.1%) was found to stimulate both growth and proteinase formation. Limiting the glucose to 0.25% prevented a drop in pH below 6.7 and prevented proteinase formation. Although less growth occurred with limited glucose, M protein of high specific activity was produced with an actual increase in acid-extractable M protein during the stationary phase of growth. When the medium was buffered at pH 7.85 with tris(hydroxymethyl)aminomethane buffer, 0.5% neopeptone prevented proteinase formation. This was true even in the presence of 1% glucose and 0.1% yeast extract, which resulted in a fall in pH to about 4.8 by 48 hr. Growth was greater than in Todd Hewitt broth, but the specific activity of M protein was considerably less than that found in the medium with glucose limited to 0.25%. Neopeptone was found to have little direct action on crude streptococcal proteinase. Instead, the evidence suggested that neopeptone somehow prevents proteinase elaboration. Yeast extract, on the other hand, appears to stimulate proteinase elaboration. To prevent proteinase formation, neopeptone must be added early, during the logarithmic phase of growth or at the start. In contrast, when yeast extract was added as late as 24 hr, it resulted in the elaboration of extracellular proteinase and in the decline of M protein. When 38 M nontypable strains from the diagnostic laboratory were tested for proteinase activity under conditions similar to those used in the diagnostic laboratory, only six produced much proteinase.     

26S multicatalytic proteinase complexes decrease during the differentiation of murine erythroleukemia cells.

Changes in multicatalytic proteinase activity during differentiation were investigated using Me2SO-induced differentiation of murine erythroleukemia cells as a model. The apparent ATP-dependent multicatalytic proteinase activity decreased in the Me2SO-treated cells with ATP-dependent incorporation of [3H]diisopropyl fluorophosphate decreasing notably after Me2SO-treatment. This decrease in activity does not seem to arise from a cessation of cell-proliferation, because no significant changes in proteinase activity were observed under different culture conditions. Hydroxyapatite column chromatography was employed to analyze the form of multicatalytic proteinase. It was clearly demonstrated that the 26S form of the proteinase decrease in the differentiated cells relative to normal cells. Multicatalytic proteinase-associated proteins that bind to the proteinase in an ATP-dependent manner were purified on an anti-multicatalytic proteinase IgG conjugated column. Only a small amount of protein was recovered from the differentiated cells. These results suggest that the decrease in multicatalytic proteinase-associated proteins that occurs upon cell-differentiation abolishes the ATP-dependent activity of the proteinase.     

Compensatory proteolytic responses to dietary proteinase inhibitors in the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae)

Increasing levels of inhibitors that target cysteine and/or serine proteinases were fed to Tribolium castaneum larvae, and the properties of digestive proteinases were compared in vitro. Cysteine proteinases were the major digestive proteinase class in control larvae, and serine proteinase activity was minor. Dietary serine proteinase inhibitors had minimal effects on either the developmental time or proteolytic activity of T. castaneum larvae. However, when larvae ingested cysteine proteinase inhibitors, there was a dramatic shift from primarily cysteine proteinases to serine proteinases in the proteinase profile of the midgut. Moreover, a combination of cysteine and serine proteinase inhibitors in the diet prevented this shift from cysteine proteinase-based digestion to serine proteinase-based digestion, and there was a corresponding substantial retardation in growth. These data suggest that the synergistic inhibitory effect of a combination of cysteine and serine proteinase inhibitors in the diet of T. castaneum larvae on midgut proteolytic activity and beetle developmental time is achieved through the prevention of the adaptive proteolytic response to overcome the activity of either type of inhibitor.     

The multicatalytic proteinase of mammalian cells

A high-molecular-weight nonlysosomal proteinase has recently been discovered in mammalian cells. It is a widely distributed and abundant enzyme which has attracted attention because of its complex multisubunit structure and its unusual catalytic properties. The 700-kDa proteinase is composed of many different types of low-molecular-weight subunits (Mr 21,000-34,000) arranged in a hollow cylindrical structure. This 20 S complex is very similar, if not identical, to the 19-20 S cylindrical particles, ring-type particles, or prosomes which have been isolated from several different types of eukaryotic cells. The proteinase appears to have at least two distinct catalytic sites and can cleave bonds on the carboxyl side of basic, hydrophobic, or acidic amino acid residues. Inhibition of proteinase activity by thiol reagents supports the suggestion that the enzyme is a cysteine proteinase but there is some evidence that it may be a serine proteinase and the catalytic mechanism is at present unknown. ATP has little effect on proteinase activity in most purified preparations but recently the proteinase has been implicated in ATP-dependent pathways of protein degradation. Ther is a second type of high-molecular-weight complex multisubunit proteinase, a 26 S particle, which catalyzes the ATP-dependent degradation of ubiquitin-protein conjugates. The precise function of these two complex proteinases in intracellular proteolysis remains to be elucidated.     

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.     

Interaction between proteolytic strains of Lactococcus lactis influenced by different types of proteinase during growth in milk.

The influence of the type of cell envelope-located proteinase (PI versus PIII) on the associative growth of Lactococcus lactis in milk was studied. Two genetically engineered strains, differing only by the type of proteinase, were first used as a model study. An interaction occurred during the second exponential growth phase of the mixed culture and resulted in a decrease in growth rate of the PI-type proteinase strain, whereas that of the PIII-type proteinase strain remained unaffected. The reduction in proteolytic activity of the PI-type proteinase strain (presumably resulting from an inhibition of the synthesis of the enzyme) due to the peptides released by the PIII-type proteinase was found to be partly responsible for this interaction. Extension of the study to wild-type proteinase-positive L. lactis strains showed a systematic imbalance of the mixture of the two strains in favor of the PIII-type proteinase strain.     

Inhibition of platelet aggregation by a fibrinogenase from Naja nigricollis venom is independent of fibrinogen degradation

Fibrinogenases, proteinases which release peptides from the carboxy-terminal end of fibrinogen, are classified as alpha-fibrinogenases or beta-fibrinogenases, based on their ability to preferentially attack the A alpha or B beta chain, respectively, of fibrinogen. alpha-Fibrinogenases have been shown to inhibit platelet aggregation whereas beta-fibrinogenases do not. We have studied the inhibition of platelet aggregation by proteinase F1, an alpha-fibrinogenase from Naja nigricollis venom. This proteinase inhibits whole blood aggregation in a dose-dependent manner, with an IC50 value of 145 micrograms. However, the proteinase fails to inhibit aggregation in washed platelet suspensions. Thus, proteinase F1 appears to require a plasma factor to cause inhibition. Since fibrinogen acts as an adhesive protein which links platelets during aggregation, and since proteinase F1 cleaves fibrinogen, we investigated the role of fibrinogen in the inhibition of platelet aggregation by proteinase F1. The degradation products of fibrinogen formed by the proteinase did not cause significant inhibition. Thus, the inhibition of platelet aggregation appears to be independent of the formation of fibrinogen degradation products. We also studied the effect of proteinase F1 on aggregation of platelets that were reconstituted with defibrinogenated plasma. The proteinase inhibited aggregation of platelets even in the absence of plasma fibrinogen. Proteinase F1 was about 4-fold more potent in inhibiting platelet aggregation in defibrinogenated blood. From these results, we conclude that the inhibition of platelet aggregation by proteinase F1 from N. nigricollis venom is independent of its action on fibrinogen.