Proteases and Their Involvement in the Infection and Immobilization of Nematodes by the Nematophagous Fungus Arthrobotrys oligospora

The nematophagous fungus Arthrobotrys oligospora produced extracellular proteases when grown in a liquid culture, as revealed by measuring the hydrolysis of the chromogenic substrate Azocoll. The extracellular protease activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and other serine protease inhibitors and partly inhibited by the aspartate protease inhibitor pepstatin and by a cysteine protease inhibitor [l-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane, or E-64]. Substrate gel electrophoresis showed that the fungus produced several different proteases, including multiple serine proteases. The function of proteases in the infection of nematodes was examined by treating the fungus with various protease inhibitors. None of the inhibitors tested affected the adhesion of nematodes to the traps, but incubating trap-bearing mycelium with a serine protease inhibitor, PMSF, antipain, or chymostatin, or the metalloprotease inhibitor phenanthroline significantly decreased the immobilization of nematodes captured by the fungus. Inhibitors of cysteine or aspartic proteases did not affect the immobilization of captured nematodes. The effects of PMSF on the immobilization of nematodes were probably due to serine proteases produced by the fungus, since the effects were observed when unbound inhibitor was washed away from the fungus before the nematodes were added to the system. No effects were observed when the nematodes only were pretreated with PMSF.     

Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.     

Degradation of human hemoglobin by Prevotella intermedia

In this study, the ability of Prevotella intermedia, an obligate anaerobic rod, to degrade human hemoglobin was determined by SDS-PAGE and the degradation was quantified by scanning densitometry. Both bacterial cells and culture supernatants degraded hemoglobin. The hemoglobin degradation by P. intermedia was time-dependent, heat sensitive, pH related and was not influenced by iron restriction. Inhibition studies demonstrated that a cysteine protease might be involved in hemoglobin degradation and this protease might require metal ions for its activity and it might be thiol-requiring and trypsin-inducible. The results indicate that P. intermedia is capable to release heme from hemoglobin, hence provide a source of iron for its proliferation.     

Investigation of sequential behavior of carboxyl protease and cysteine protease activities in virus-infected Sf-9 insect cell culture by inhibition assay

Proteases produced during the culture of Spodoptera frugiperda Sf-9 cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV) were assayed with various protease inhibitors. This inhibitory analysis revealed that: (1) carboxyl and cysteine proteases were predominantly produced by the insect cells infected with recombinant AcNPV, the gene of which encoded a variant of green fluorescent protein in a portion of the polyhedrin gene of the baculovirus, and (2) the protease activity was almost completely blocked by pepstatin A (carboxyl protease inhibitor) and E64 (cysteine protease inhibitor) in an additive manner in the presence of EDTA. Utilizing the additive property of the inhibitors, the inhibition-based protease assay discriminated between the two protease activities and elucidated the sequential behavior of the carboxyl and cysteine proteases produced in the virus-infected Sf-9 cell culture. The carboxyl protease(s) existed in the virus-infected cells all the time and their level in the medium continuously increased. Uninfected cells also contained a carboxyl protease activity, the level of which was similar to that of the virus-infected cells. At a certain time after virus infection, the cysteine protease activity was largely increased in the virus-infected cells and a significant amount of the protease(s) was released into the medium, due to the cell membranes losing their integrity. The behavior of intracellular and extracellular cysteine protease activities coincided with that of a recombinant protein whose expression was under the control of the viral polyhedrin promoter. Similar examinations with wt-AcNPV-infected and uninfected insect cells showed that the inhibition-based protease assay was useful for analyzing the carboxyl protease and cysteine protease activities emerging in the insect cell (Sf-9)/baculovirus expression system.     

Proteolysis of the calcium-dependent protease inhibitor by myocardial calcium-dependent protease

Bovine heart peak II calcium-dependent protease was capable of hydrolyzing its specific inhibitor protein at high molar ratios of protease to inhibitor. The proteolysis was inhibited by leupeptin and required millimolar calcium. Thus, it appeared to be attributable to the calcium-dependent protease and not to possible contaminating proteases in the purified preparations of inhibitor or calcium-dependent protease. Incubation of the purified inhibitor with the calcium-dependent protease produced a discrete pattern of inhibitor fragments on Western blots developed with an inhibitor-specific monoclonal antibody. Traces of similar or identical lower molecular weight immunoreactive material could be observed in Western blots of bovine heart extracts, and the immunoreactivity present as these lower molecular weight forms could be increased by incubation of the extracts with calcium ion. These results suggest that the inhibitor can be proteolyzed to low molecular weight forms which can be detected in cardiac tissue extracts, and that calcium-dependent protease(s) may be responsible for this phenomenon.     

HIV-1 protease inhibitory activity of L-694,746, a novel metabolite of L-689,502.

L-689,502 is a potent inhibitor of HIV-1 protease activity in vitro. Microbial biotransformations of L-689,502 by cultures belonging to the genus Streptomyces sp. were performed. Extracts of culture broths were examined for the production of metabolites of L-689,502 that could inhibit HIV-1 protease activity. One culture, MA 6804 (Streptomyces lavendulae, ATCC 55095), produced L-694,746 that, while being structurally related to L-689,502, is a novel metabolite and a potent inhibitor of HIV-1 protease.     

A novel protease obtained from FBS-containing culture supernatant,that processes single chain form hepatocyte growth factor to two chain form in serum-free culture

The recombinant human hepatocyte growth factor (r-hHGF) produced by Chinese hamster ovary cells transfected with hHGF cDNA (CHO BD-24 cells) was the two chain form in fetal bovine serum (FBS) containing culture. However, in serum-free culture the non-processed r-hHGF, single chain form, was detected with two chain form r-hHGF. We purified the protease that proteolytically processed single chain r-hHGF to two chain form r-hHGF. A protease was purified to give a single peak from the culture supernatant by use of several column chromatographies. When this protease was added to serum-free culture of CHO BD-24 cells, the proteolytic processing of single chain r-hHGF to two chain form r-hHGF was completely achieved. This protease was found to be composed of two peptide chains with molecular mass of 38 kDa under non-reducing condition by SDS-PAGE. The results of N-terminal amino acid sequence analysis and inhibitor selectivity suggested that this protease was a novel serine protease originating from fetal bovine serum.     

Presence of the acute phase protein, bikunin, in the endometrium of gilts during estrous cycle and early pregnancy

Noninvasive, epitheliochorial placental attachment in the pig is regulated through endometrial production of protease inhibitors. The objective of the present study was to determine if the light-chain serine protease inhibitor of the inter-alpha-trypsin inhibitor family, bikunin, is produced by the porcine endometrium during the estrous cycle and early pregnancy. Western blot analysis revealed the presence of bikunin in uterine flushings of gilts collected during the luteal phase of the estrous cycle and early pregnancy (Days 12-18). However, bikunin unbound to the inter-alpha-trypsin heavy chains was detected only in endometrial explant culture medium obtained from estrus and pregnant (Days 12, 15, and 18) gilts. Endometrial bikunin gene expression was lowest on Day 10 of the estrous cycle and pregnancy, followed by a 30- to 77-fold increase on Day 15 of the estrous cycle and pregnancy. Bikunin gene expression decreased on Day 18 of the estrous cycle, whereas endometrial bikunin gene expression continued to increase in pregnant gilts. Bikunin mRNA was localized to the uterine glands between Days 15 and 18 of the estrous cycle and pregnancy. In addition to its role as a protease inhibitor, bikunin functions in stabilization of the extracellular matrix, which suggests that bikunin could be involved with facilitating placental attachment to the uterine epithelial surface in the pig.     

Suppression of the degradation of recombinant human apolipoprotein E by a protease inhibitor obtained from fetal bovine serum in serum-free culture

The recombinant human apolipoprotein E (Apo-E) produced by Chinese hamster ovary cells (CHO-322 cells) in serum free culture was degraded to 24K and 23K fragments that contained N-terminal amino acid. The degradation site of Apo-E to 24K fragment was between Arg180 and Leu181 and the C-terminal amino acid of 23K fragment was Gly169. In fetal bovine serum (FBS)-containing culture, the degradation was inhibited. However, in calf serum (CS) the inhibitory activity was not detected. Thus, we attempted the purification of the factor with this inhibitory activity from FBS. A protease inhibitor was purified to give a single peak from FBS by ammonium sulfate precipitation and combination of several column chromatographies. When this FBS-derived protease inhibitor (FBS-d-PI) was added to serum-free culture of CHO-322 cells, degradation of recombinant Apo-E to the 24K and 23K fragments was dose-dependently suppressed and accumulation of intact Apo-E in culture supernatant was observed. FBS-d-PI was found to be a glycoprotein with relative molecular size of 75K daltons under reducing condition, and 85K daltons under nonreducing condition by SDS-PAGE. A complex of FBS-d-PI and a cellular protease was also detected in culture supernatant by western blot analysis using mouse monoclonal antibodies against FBS-d-PI.     

Cysteine protease is a major exotoxin of pathogenic luminous Vibrio harveyi in the tiger prawn, Penaeus monodon

The role of an extracellular cysteine protease, produced by pathogenic luminous Vibrio harveyi strain 820514 originally isolated from diseased tiger prawn (Penaeus monodon), in the disease process in the prawns was studied. The protease was lethal to P. monodon with an LD50 value of 0.3 microgram protein g-1 prawn. The lethal toxicity of the extracellular products (ECP) of the bacterium was neutralized by pre-incubation of the ECP with rabbit antiserum to the cysteine protease. Pre-incubation of ECP with CuCl2 (an inhibitor of cysteine protease) also inhibited toxicity. This suggests that cysteine protease is the major toxin produced by the bacterium. The present protease is the first toxic cysteine protease to be found in Vibrio species.     

Characterization of an extracellular protease inhibitor of Bacillus brevis HPD31 and nucleotide sequence of the corresponding gene.

A novel proteinaceous protease inhibitor was isolated from the culture supernatant of Bacillus brevis HPD31. The protease inhibitor of B. brevis (designated BbrPI) was produced extracellularly in multiple forms having at least three different molecular weights. One of them, BbrPI-a, was purified to near homogeneity and only showed inhibitory activity toward serine proteases, such as trypsin, chymotrypsin, and subtilisin. BbrPI was presumed to form a trypsin-inhibitor complex in a molar ratio of 1:1. The inhibitor was found to be heat resistant at neutral and acidic pHs. The gene coding for BbrPI was cloned into Escherichia coli, and its nucleotide sequence was determined. The sequence suggested that BbrPI is produced with a signal peptide of 24 amino acid residues. The amino acid sequence of the protein deduced from the DNA sequence contained the amino acid sequences of amino termini of the inhibitors, a, b, and c, and their putative precursor determined chemically. The molecular weight of the precursor was about 33,000, and the molecular weights of inhibitors a, b, and c were about 22,000, 23,500, and 24,000, respectively. It is presumed that the secreted precursor protein, which is probably inactive, is cleaved by protease into several active protease inhibitor molecules. BbrPI shows no significant homology to the protease inhibitors described previously and is unique in not having any cysteine residues in its molecule.     

Characterization of an extracellular protease inhibitor of Bacillus brevis HPD31 and nucleotide sequence of the corresponding gene.

A novel proteinaceous protease inhibitor was isolated from the culture supernatant of Bacillus brevis HPD31. The protease inhibitor of B. brevis (designated BbrPI) was produced extracellularly in multiple forms having at least three different molecular weights. One of them, BbrPI-a, was purified to near homogeneity and only showed inhibitory activity toward serine proteases, such as trypsin, chymotrypsin, and subtilisin. BbrPI was presumed to form a trypsin-inhibitor complex in a molar ratio of 1:1. The inhibitor was found to be heat resistant at neutral and acidic pHs. The gene coding for BbrPI was cloned into Escherichia coli, and its nucleotide sequence was determined. The sequence suggested that BbrPI is produced with a signal peptide of 24 amino acid residues. The amino acid sequence of the protein deduced from the DNA sequence contained the amino acid sequences of amino termini of the inhibitors, a, b, and c, and their putative precursor determined chemically. The molecular weight of the precursor was about 33,000, and the molecular weights of inhibitors a, b, and c were about 22,000, 23,500, and 24,000, respectively. It is presumed that the secreted precursor protein, which is probably inactive, is cleaved by protease into several active protease inhibitor molecules. BbrPI shows no significant homology to the protease inhibitors described previously and is unique in not having any cysteine residues in its molecule.     

A protease inhibitor produced by Streptomyces lividans 66 exhibits inhibitory activities toward both subtilisin BPN' and trypsin.

A proteinaceous protease inhibitor was isolated from the culture broth of Streptomyces lividans 66 by a series of purification steps (salting out by ammonium sulfate, ion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on Phenyl-Sepharose, and gel-filtration on Sephacryl S-200), and was named S. lividans protease inhibitor (SLPI). The purified SLPI existed in a dimeric form consisting of two identical subunits, each of which was composed of 107 amino acids. SLPI exhibited strong inhibitory activity toward subtilisin BPN'. These features were similar to those of protein protease inhibitors produced by other Streptomyces (SSI family inhibitor). In addition, SLPI was capable of inhibiting trypsin with an inhibitor constant (Ki) of about 10(-9) M. The primary structure of SLPI and location of two disulfide bridges were homologous to those of the other serine protease inhibitors of Streptomyces. The reactive site of SLPI was found to be Arg67-Glu68 from the sequence analysis of cleaved SLPI which was produced by acidification of subtilisin-SLPI complex. An Arg residue at the P1 site was consistent with the trypsin-inhibitory property of SLPI. Sequence comparison with other members of the SSI family revealed that amino acid replacements in SLPI were mainly localized on the surface of the SLPI molecule, and many of the amino acid residues in beta-sheets and hydrophobic core were well conserved.     

Purification and Properties of an Extracellular Protease Produced by the Entomopathogenic Fungus Beauveria bassiana

Beauveria bassiana GK2016 grown in a medium with gelatin as the sole carbon and nitrogen source produced an extracellular protease. The protease production was highest when the fungus was grown on a semiliquid medium and was purified about 18-fold, with a recovery of 21%. The protease molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be about 35,000. It had an optimum activity at pH 8.5 and 37 degrees C and was rapidly inactivated at 50 degrees C. Its enzymatic activity was that of an endopeptidase which hydrolyzed elastin, casein, and gelatin but was much less active on bovine serum albumin and collagen. No trypsinlike activity was detected on N-alpha-benzoyl-dl-arginine-p-nitroanilide. It was, however, inhibited by phenylmethylsulfonyl fluoride, indicating that a serine residue is present in the active site. The protease was unaffected by metal-chelating agents, sulfhydryl reagents, trypsin inhibitor, and chymotrypsin inhibitor.     

Effect of environmental haemin upon the physiology and biochemistry of Prevotella intermedia R78

The effect of environmental haemin on the physiology and biochemistry of Prevotella intermedia R78 grown in batch culture was assessed. Extent and rate of growth increased as the environmental haemin concentration was raised. In addition, cell morphology was predominantly cocco-bacillary when cultured in high haemin environments, while bacillary forms were prevalent in low haemin conditions (< 2.5 mumol l-1). Cells harvested from low haemin environments produced greater numbers of extracellular vesicles and greater amounts of peptidolytic activity, haemagglutinating potential and haemin binding activity when compared with cells harvested from high haemin conditions. The results of the present study indicate that aspects of the biochemistry and physiology of P. intermedia are influenced by changes in environmental haemin levels.     

Production of specific monoclonal antibodies against the active sites of human pancreatic secretory trypsin inhibitor variants by in vitro immunization with synthetic peptides

Specific monoclonal antibodies against the active sites of two genetically engineered pancreatic secretory trypsin inhibitor (PSTI) variants (PSTI 0 and PSTI 4) were produced. The protease inhibitors PSTI 0 and PSTI 4 differ only by three amino acid substitution at their active sites. PSTI 0 inhibits trypsin, whereas PSTI 4 inhibits human granulocyte elastase and chymotrypsin. Immunization was performed in vitro with a synthetic heptapeptide that covers the mutated region of the protein. For this purpose in vitro culture conditions for the production of specific monoclonal antibodies against synthetic peptides were improved. The monoclonal antibodies obtained react specifically with the corresponding protease inhibitor variant. Competition experiments with trypsin and human elastase demonstrate that the protease displace the monoclonal antibody from the active site of PSTI 0 and PSTI 4 respectively.     

Extracellular serine protease synthesis by mosquito-pathogenic strains of Bacillus sphaericus

Nine strains of Bacillus sphaericus toxic to mosquito larvae produced haloes of hydrolysis when cultured on casein-nutrient agar. In batch culture, protease synthesis by B. sphaericus BSE 18 occurred during exponential growth and was repressed by high concentrations of peptone. Extracellular protease from this bacterium showed optimal activity at about pH 10.2, was inhibited by phenylmethylsulphonyl chloride and chymostatin but not soybean trypsin inhibitor or EDTA. Hydrolysis of N -CBZ-glycine-nitrophenyl ester was consistent with the major enzyme being a serine protease.     

Trypsin action on the growth of Sendai virus in tissue culture cells. V. An activating enzyme for Sendai virus in the chorioallantoic fluid of the embryonated chicken egg

A trypsin-like protease which is responsible for activation of Sendai virus was found in the chorioallantoic fluid (CAF) of embryonated chicken eggs. Treatment of the inactive form of Sendai virus, grown in LLC-MK2 cells, with CAF enhanced both hemolytic activity and infectivity for the cells. Soybean trypsin inhibitor restrained the enhancing activity of CAF. These results indicate that CAF contains a trypsin-like protease which activates the inactive form of Sendai virus. The activation was strongly inhibited by phenylmethylsulfonylfluoride, ethylenediaminetetraacetate, antipain, and leupeptin but not by tosyllysylchloromethylketone, suggesting that the activating enzyme in CAF is a protease similar to but not identical with trypsin. The inactive form of the virion was produced in ovo when the seed virus was inoculated along with antipain or leupeptin. In deembryonated chicken eggs in which CAF was substituted for a culture medium, multiple cycle growth occurred, but not when soybean trypsin inhibitor was present. These observations indicate that some activating enzyme, possibly the same one as found in CAF, was secreted from the chorioallantoic membrane.     

Coaggregation of Porphyromonas gingivalis and Prevotella intermedia.

Porphyromonas gingivalis cells coaggregated with Prevotella intermedia cells. The coaggregation was inhibited with L-arginine, L-lysine, Nalpha-p-tosyl-L-lysine chloromethyl ketone, trypsin inhibitor, and leupeptin. Heat- and proteinase K-treated P. gingivalis cells showed no coaggregation with P. intermedia cells, whereas heat and proteinase K treatments of P. intermedia cells did not affect the coaggregation. The vesicles from P. gingivalis culture supernatant aggregated with P. intermedia cells, and this aggregation was also inhibited by addition of L-arginine or L-lysine and by heat treatment of the vesicles. The rgpA rgpB, rgpA kgp, rgpA rgpB kgp, and rgpA kgp hagA mutants of P. gingivalis did not coaggregate with P. intermedia. On the other hand, the fimA mutant lacking the FimA fimbriae showed coaggregation with P. intermedia as well as the wild type parent. These results strongly imply that a heat-labile and proteinous factor on the cell surface of P gingivalis, most likely the gingipain-adhesin complex, is involved in coaggregation of P. gingivalis and P. intermedia.     

Effect of a Microbial Acid Protease Inhibitor (S-PI) on the Production of Fungal Acid Protease

Cladosporium sp. No. 45–2, an acid protease-producing microorganism, was cultured in medium containing a microbial acid protease inhibitor (S–PI). By the addition of S–PI, the amount of acid protease in the culture broth showed an increase of 50~80% over those of normal culture (S–PI-free). Acid protease was purified from the S–PI-added culture filtrate, and its enzymatic and physicochemical properties were compared with those of acid protease obtained from normal culture. It was determined that the acid protease obtained from S–PI-added culture was the same as that of normal culture, but that the productivity was increased by the addition of S–PI.

The increase in acid protease productivity is assumed to be due to a change in metabolism by the addition of S–PI.