Analysis of the effects of Perkinsus marinus proteases on plasma proteins of the Eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas).

We employed two in vitro buffer systems to determine the potential pathogenic effects of Perkinsus marinus serine proteases on the plasma proteins of the eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas). Specifically, this study characterized the oyster plasma protein targets of P. marinus proteases. Additionally, protease-specific inhibitory activity was revealed upon comparison of artificial (PBS) and endogenous (plasma-based) diluents employed during protease digestions. It was found that a C. virginica plasma protein of approximately 35 kDa was eliminated when a standard buffer (PBS) was used as a diluent; however, this protein was preserved when a low-molecular-weight, plasma-based, diluent was used. The results strongly indicate that low-molecular-weight inhibitors of P. marinus proteases are present in oyster plasma. A control (nonparasitic) serine protease, alpha-chymotrypsin, was employed to ascertain the specificity of the protease inhibitors. Although alpha-chymotrypsin possesses ample proteolytic activity for C. virginica plasma proteins, the anti-proteases could specifically inhibit only P. marinus proteases. Such specificity of anti-protease activity is not uncommon among low-molecular-weight serine proteases. The hemolymph target protein was isolated by 2D electrophoresis and isoelectrically isolated for further characterization by N-terminal amino acid sequencing.     

A serine alkaline protease from the fungusConidiobolus coronatus with a distinctly different structure than the serine protease subtilisin Carlsberg

In view of the functional similarities between subtilisin Carlsberg and the alkaline protease fromConidiobolus coronatus, the biochemical and structural properties of the two enzymes were compared. In spite of their similar biochemical properties, e.g., pH optima, heat stability, molecular mass, pI, esterase activity, and inhibition by diisopropyl fluorophosphate and phenylmethlysulfonylfluoride, the proteases were structurally dissimilar as revealed by (1) their amino acid compositions, (2) their inhibition by subtilisin inhibitor, (3) their immunological response to specific anti-Conidiobolus protease antibody, and (4) their tryptic peptide maps. Our results demonstrate that although they are functionally analogous, theConidiobolus protease is structurally distinct from subtilisin Carlsberg. TheConidiobolus protease was also different from other bacterial and animal proteases (e.g. pronase, protease K, trypsin, and chymotrypsin) as evidenced by their lack of response to anti-Conidiobolus protease antibody in double diffusion and in neutralization assays. TheConidiobolus serine protease fails to obey the general rule that proteins with similar functions have similar primary sequences and, thus, are evolutionarily related. Our results strengthen the concept of convergent evolution for serine proteases and provide basis for research in evolutionary relationships among fungal, bacterial, and animal proteases.     

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 broad spectrum Kunitz type serine protease inhibitor secreted by the hookworm Ancylostoma ceylanicum

Although blood-feeding hookworms infect over a billion people worldwide, little is known about the molecular mechanisms through which these parasitic nematodes cause gastrointestinal hemorrhage and iron deficiency anemia. A cDNA corresponding to a secreted Kunitz type serine protease inhibitor has been cloned from adult Ancylostoma ceylanicum hookworm RNA. The translated sequence of the A. ceylanicum Kunitz type inhibitor 1 (AceKI-1) cDNA predicts a 16-amino acid secretory signal sequence, followed by a 68-amino acid mature protein with a molecular mass of 7889 daltons. Recombinant protein (rAceKI-1) was purified from induced lysates of Escherichia coli transformed with the rAceKI-1/pET 28a plasmid, and in vitro studies demonstrate that rAceKI-1 is a tight binding inhibitor of the serine proteases chymotrypsin, pancreatic elastase, neutrophil elastase, and trypsin. AceKI-1 inhibitory activity is present in soluble protein extracts and excretory/secretory products of adult hookworms but not the infective third stage larvae. The native AceKI-1 inhibitor has been purified to homogeneity from soluble extracts of adult A. ceylanicum using size exclusion and reverse-phase high pressure liquid chromatography. As a potent inhibitor of mammalian intestinal proteases, AceKI-1 may play a role in parasite survival and the pathogenesis of hookworm anemia.     

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.     

Peptidyl boronates inhibit Salmonella enterica serovar Typhimurium Lon protease by a competitive ATP-dependent mechanism

Lon is a homo-oligomeric ATP-dependent serine protease that functions in the degradation of damaged and certain regulatory proteins. This enzyme has emerged as a novel target in the development of antibiotics because of its importance in conferring bacterial virulence. In this study, we explored the mechanism by which the proteasome inhibitor MG262, a peptidyl boronate, inhibits the peptide hydrolysis activity of Salmonella enterica serovar Typhimurium Lon. In addition, we synthesized a fluorescent peptidyl boronate inhibitor based upon the amino acid sequence of a product of peptide hydrolysis by the enzyme. Using steady-state kinetic techniques, we have shown that two peptidyl boronate variants are competitive inhibitors of the peptide hydrolysis activity of Lon and follow the same two-step, time-dependent inhibition mechanism. The first step is rapid and involves binding of the inhibitor and formation of a covalent adduct with the active site serine. This is followed by a second slow step in which Lon undergoes a conformational change or isomerization to increase the interaction of the inhibitor with the proteolytic active site to yield an overall inhibition constant of 5-20 nM. Although inhibition of serine and threonine proteases by peptidyl boronates has been detected previously, Lon is the first protease that has required the binding of ATP in order to observe inhibition.     

Molecular characterization of a gene encoding extracellular serine protease isolated from a subtilisin inhibitor-deficient mutant of Streptomyces albogriseolus S-3253.

An extracellular serine protease produced by a mutant, M1, derived from Streptomyces albogriseolus S-3253 that no longer produces a protease inhibitor (Streptomyces subtilisin inhibitor [SSI]) was isolated. A 20-kDa protein was purified by its affinity for SSI and designated SAM-P20. The amino acid sequence of the amino-terminal region of SAM-P20 revealed high homology with the sequences of Streptomyces griseus proteases A and B, and the gene sequence confirmed the relationships. The sequence also revealed a putative amino acid signal sequence for SAM-P20 that apparently functioned to allow secretion of SAM-P20 from Escherichia coli carrying the recombinant gene. SAM-P20 produced by E. coli cells was shown to be sensitive to SSI inhibition.     

A novel locust (Schistocerca gregaria) serine protease inhibitor with a high affinity for neutrophil elastase

We have purified to homogeneity two forms of a new serine protease inhibitor specific for elastase/chymotrypsin from the ovary gland of the desert locust Schistocerca gregaria. This protein, greglin, has 83 amino acid residues and bears putative phosphorylation sites. Amino acid sequence alignments revealed no homology with pacifastin insect inhibitors and only a distant relationship with Kazal-type inhibitors. This was confirmed by computer-based structural studies. The most closely related homologue is a putative gene product from Ciona intestinalis with which it shares 38% sequence homology. Greglin is a fast-acting and tight binding inhibitor of human neutrophil elastase (k(ass)=1.2x10(7) M(-1) x s(-1), K(i)=3.6 nM) and subtilisin. It also binds neutrophil cathepsin G, pancreatic elastase and chymotrypsin with a lower affinity (26 nM< or =K(i)< or =153 nM), but does not inhibit neutrophil protease 3 or pancreatic trypsin. The capacity of greglin to inhibit neutrophil elastase was not significantly affected by exposure to acetonitrile, high temperature (90 degrees C), low or high pH (2.5-11.0), N-chlorosuccinimide-mediated oxidation or the proteolytic enzymes trypsin, papain and pseudolysin from Pseudomonas aeruginosa. Greglin efficiently inhibits the neutrophil elastase activity of sputum supernatants from cystic fibrosis patients. Its biological function in the locust ovary gland is currently unknown, but its physicochemical properties suggest that it can be used as a template to design a new generation of highly resistant elastase inhibitors for treating inflammatory diseases.     

Recombinant chymotrypsin inhibitor 2: expression, kinetic analysis of inhibition with alpha-chymotrypsin and wild-type and mutant subtilisin BPN', and protein engineering to investigate inhibitory specificity and mechanism

The serine protease inhibitor chymotrypsin inhibitor 2 (CI2 or BSPI2) has been expressed in Escherichia coli with the pINIIIompA3 expression vector to produce 20-40 mg/L of culture. Recombinant CI2 purified from this system has been characterized and found to be identical with CI2 from barley. Slow-binding kinetics were observed for the interaction between CI2 and subtilisin BPN', with Ki = 2.9 x 10(-12) M. Analysis of slow-binding data indicates that binding of the inhibitor follows the simplest model of E + I = EI with no kinetically detectable intermediate steps or proteolytic cleavage of the reactive site bond in CI2 (Met-59-Glu-60). This, in agreement with crystallographic data, indicates that the enzyme-inhibitor adduct is the Michaelis complex, which is not chemically processed by the enzyme. Three mutant CI2 molecules with new P1 residues have also been examined with a range of serine proteases, including a mutant subtilisin. In agreement with earlier studies, we find the P1 amino acid an important determinant of specificity. CI2 Met----Lys-59 was found to be a temporary inhibitor of subtilisin BPN' but an effective inhibitor of subtilisin Carlsberg and subtilisin BPN'(Glu----Ser-156). The structural reasons for this are discussed in relation to mechanisms of inhibition of serine proteases.     

A Second Kazal-like protease inhibitor from Phytophthora infestans inhibits and interacts with the apoplastic pathogenesis-related protease P69B of tomato

The plant apoplast forms a protease-rich environment in which proteases are integral components of the plant defense response. Plant pathogenic oomycetes, such as the potato (Solanum tuberosum) and tomato (Lycopersicon esculentum) pathogen Phytophthora infestans, secrete a diverse family of serine protease inhibitors of the Kazal family. Among these, the two-domain EPI1 protein was shown to inhibit and interact with the pathogenesis-related protein P69B subtilase of tomato and was implicated in counter-defense. Here, we describe and functionally characterize a second extracellular protease inhibitor, EPI10, from P. infestans. EPI10 contains three Kazal-like domains, one of which was predicted to be an efficient inhibitor of subtilisin A by an additivity-based sequence to reactivity algorithm (Laskowski algorithm). The epi10 gene was up-regulated during infection of tomato, suggesting a potential role during pathogenesis. Recombinant EPI10 specifically inhibited subtilisin A among the major serine proteases, and inhibited and interacted with P69B subtilase of tomato. The finding that P. infestans evolved two distinct and structurally divergent protease inhibitors to target the same plant protease suggests that inhibition of P69B could be an important infection mechanism for this pathogen.     

Purification and characterization of an acidic trypsin/subtilisin inhibitor from tortoise egg white

Egg whites of three species of tortoise and turtle have been compared by gel chromatography for inhibitory activity against proteases. The egg white of Geomda trijuga trijuga Schariggar contains trypsin/subtilisin inhibitor while the egg white of Caretta caretta Linn. contains both trypsin and chymotrypsin inhibitors. No protease inhibitory activity has been detected in the egg white of Trionyx gangeticus Cuvier. An acidic trypsin/subtilisin inhibitor has been purified to homogeneity from the egg white of tortoise (G. trijuga trijuga). It is a single polypeptide chain of 100 amino acid residues, having a molecular weight of 11 700. It contains six disulphide bonds and is devoid of methionine and carbohydrate moiety. Its isoelectric point is at pH 5.95 and is stable at 100°C for 4 h at neutral pH. The inhibitor inhibits both trypsin and subtilisin by forming enzyme-inhibitor complexes at a molar ratio close to unity. Their dissociation contants are 7.2·10^?9 M for bovine trypsin adn 5.5·10^?7 M for subtilisin. Chemical modification of amino groups with trinitrobenzene sulfonate has reduced its inhibitory activities against both trypsin and subtilisin, but the loss of its trypsin inhibitory activity is faster than of its subtilisin inhibitory activity. It has independent binding sites for inhibition of trypsin and subtilisin.     

Purification of a cysteine protease inhibitor from larval hemolymph of the tobacco hornworm (Manduca sexta) and functional expression of the recombinant protein

A cysteine protease inhibitor (CPI) with an apparent molecular mass of 11.5kDa was purified from larval hemolymph of the tobacco hornworm (Manduca sexta) by gel filtration on Sephadex G-50 followed by hydrophobic and ion-exchange column chromatographies. The purified cysteine proteinase inhibitor, denoted as MsCPI, strongly inhibited the plant cysteine protease, papain, with a K(i) value of 5.5 x 10(-9)M. Nucleotide sequence analysis of a partial cDNA encoding MsCPI indicated that MsCPI consists of 105 amino acid residues in a sequence that is similar to sarcocystatin A from Sarcophaga peregrina. However, northern blotting and PCR analyses using the specific primers of MsCPI suggested that the mRNA encoding MsCPI had a size of more than 12 kilobases, which included at least six tandemly repeated MsCPI segments. MsCPI was expressed in Escherichia coli and the recombinant protein effectively inhibited cysteine proteases from plants as well as from animals such as cathepsins B (K(i), 6.8 nM), H (3.0 nM), and L (0.87 nM). There was no inhibition exhibited toward trypsin, chymotrypsin, subtilisin, pepsin or themolysin.     

Effect of inhibitory activity of mutation at reaction site P4 of the Streptomyces subtilisin inhibitor, SSI

The protein Streptomyces subtilisin inhibitor, SSI, efficiently inhibits a bacterial serine protease, subtilisin BPN'. We recently demonstrated that functional change in SSI was possible simply by replacing the amino acid residue at the reactive P1 site (methionine 73) of SSI. The present paper reports the additional effect of replacing methionine 70 at the P4 site of SSI (Lys73) on inhibitory activity toward two types of serine proteases, trypsin (or lysyl endopeptidase) and subtilisin BPN'. Conversion of methionine 70 at the P4 site of SSI(Lys73) to glycine or alanine resulted in increased inhibitory activity toward trypsin and lysyl endopeptidase, while replacement with phenylalanine weakened the inhibitory activity toward trypsin. This suggests that steric hindrance at the P4 site of SSI(Lys73) is an obstacle for its binding with trypsin. In contrast, the same P4 replacements had hardly any effect on inhibitory activity toward subtilisin BPN'. Thus the subsite structure of subtilisin BPN' is tolerant to these replacements. This contrast in the effect of P4 substitution might be due to the differences in the S4 subsite structures between the trypsin-like and the subtilisin-like proteases. These findings demonstrate the importance of considering structural complementarity, not only at the main reactive site but also at subsites of a protease, when designing stronger inhibitors.     

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.     

Interactions of Streptomyces serine-protease inhibitors with Streptomyces griseus metalloendopeptidase II.

Streptomyces griseus metalloendopeptidase II (SGMPII) was shown to form tight complexes with several Streptomyces protein inhibitors which had been believed to be specific to serine proteases, such as Streptomyces subtilisin inhibitor (SSI), plasminostreptin (PS), and alkaline protease inhibitor-2c' (API-2c'), as well as with Streptomyces metalloprotease inhibitor (SMPI). The dissociation constants of complexes between SGMPII and these inhibitors were successfully determined by using a novel fluorogenic bimane-peptide substrate. The values ranged from nM to pM. The results of studies by gel chromatographic and enzymatic analyses indicated that SGMPII is liberated from the complex with SSI by the addition of subtilisin BPN'. SGMPII and subtilisin BPN' proved, therefore, to interact with SSI in a competitive manner, despite the difference in the chemical nature of their active sites.     

Alpha-2-macroglobulin-like protease inhibitor from the egg white of cuban crocodile (Crocodylus rhombifer)

A high molecular weight protease inhibitor was purified from the egg white of Cuban crocodile (Crocodylus rhombifer). It inhibited the casein hydrolyzing activity of trypsin, subtilisin and papain. Its native molecular weight was 730,000 and it consisted of four subunits of equal molecular weight, each pair of which were disulfide bonded. The amino acid composition, circular dichroic spectrum and electron micrographs of this protein are also presented. Upon incubation with trypsin this protein yielded a fragment of Mr = 80,000, similar in size to the one known to originate from alpha 2-macroglobulin under the same conditions. The molecular parameters of this protein and the broad inhibitory activity towards thiol and serine proteases with different substrate specificities suggest that it is a protein closely related to alpha 2-macroglobulin in mammalian serum. From its native molecular weight and amino acid composition we believe that this protein is also a reptilian counterpart of the avian ovomacroglobulin described by Miller and Feeney (3).     

Macromolecular chelation as an improved mechanism of protease inhibition: structure of the ecotin-trypsin complex.

The 2.4 A crystal structure (R = 0.180) of the serine protease inhibitor ecotin was determined in a complex with trypsin. Ecotin's dimer structure provides a second discrete and distal binding site for trypsin and, as shown by modelling experiments, other serine proteases. The second site is approximately 45 A from the reactive/active site of the complex and features 13 hydrogen bonds, including six that involve carbonyl oxygen atoms and four bridged by water molecules. Contacts ecotin makes with trypsin's active site are similar to, though more extensive than, those found between trypsin and basic pancreatic trypsin inhibitor. The side chain of ecotin Met84 is found in the substrate binding pocket of trypsin where it makes few contacts, but also does not disrupt the solvent structure or cause misalignment of the scissile bond. This first case of protein dimerization being used to augment binding energy and allow chelation of a target protein provides a new model for protein-protein interactions and for protease inhibition.     

Purification and characterisation of a protease inhibitor from Streptomyces chromofuscus 34-1 with an antiviral activity

The aim of this study was to purify and characterise a novel protease inhibitor (PISC-2002) isolated from culture supernatants of Streptomyces chromofuscus. PISC-2002 was purified by anion-exchange chromatography, and RP-HPLC analysis. PISC-2002 had a molecular mass of 11.2 kDa and a high content of hydrophobic amino acids and proline. N-terminal sequence gave two sequences differing by one residue. The main sequence is ASLPAVSALVLTV and the shorter sequence is SLPAVSALVLTV. This shows its homology to Streptomyces subtilisin inhibitor family. Besides its large spectrum of powerful inhibitory activities against various serine proteases, PISC-2002 displayed significant antiviral effect against influenza virus A/Rostock/34 (H7N7).