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.     

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.     

A comparison of three heparin-binding serine proteinase inhibitors.

The purpose of this study was to compare three heparin-binding plasma proteinase inhibitors in order to identify common and unique features of heparin binding and heparin-enhanced proteinase inhibition. Experiments with antithrombin, heparin cofactor, and protein C inhibitor were performed under identical conditions in order to facilitate comparisons. Synthetic peptides corresponding to the putative heparin binding regions of antithrombin, heparin cofactor, and protein C inhibitor bound to heparin directly and interfered in heparin-enhanced proteinase inhibition assays. All three inhibitors obeyed a ternary complex mechanism for heparin-enhanced thrombin inhibition, and the optimum heparin concentration was related to the apparent heparin affinity of the inhibitor. The maximum inhibition rate and rate enhancement due to heparin appeared to be unique properties of each inhibitor. In assays with heparin oligosaccharides of known size, only the antithrombin-thrombin reaction exhibited a sharp threshold for rate enhancement at 14-16 saccharide units. Acceleration of antithrombin inhibition of factor Xa, heparin cofactor inhibition of thrombin, and protein C inhibitor inhibition of thrombin, activated protein C, and factor Xa did not require a minimum saccharide size. The differences in heparin size dependence and rate enhancement of proteinase inhibition by these inhibitors might reflect differences in the importance of the ternary complex mechanism and other mechanisms, alterations in inhibitor reactivity, and orientation effects in heparin-enhanced proteinase inhibition.     

Surface interaction of human granulocytes and lymphocytes with staphylococcal extracellular serine proteinase

Enzymatic activity of purified staphylococcal extracellular serine proteinase decreases as a result of incubation with granulocytes as well as with lymphocytes taken from peripheral blood of healthy donors. However, specific proteinase binding was observed only in the case of granulocytes but not in peripheral lymphocytes.     

Human seminal proteinase and prostate-specific antigen are the same protein

Human seminal proteinase and prostate-specific antigen (PSA) were each isolated from human seminal fluid and compared. Both are glycoproteins of 32-34 kDa with protease activities. Based on some physicochemical,enzymatic and immunological properties,it is concluded that these proteins are in fact identical.The protein exhibits properties similar to kallikrein-like serine protease, trypsin,chymotrypsin and thiol acid protease.Tests of the activity of the enzyme against some potential natural and synthetic substrates showed that bovine serum albumin was more readily hydrolysed than casein.The results of this study should be useful in purifying and assaying this protein.Based on published studies and the present results,the broad proteolytic specificity of human seminal proteinase suggests a role for this protein in several physiological functions.     

Purification and characterization of an extracellular thiol-containing serine proteinase from Thermomyces lanuginosus.

An extracellular protease produced by the filamentous fungus Thermomyces lanuginosus has been purified and characterized. The results indicate that the enzyme, which we have called humicolin, is a thiol-containing serine protease with a molecular mass of 38,000 kilodaltons. Secretion of humicolin, which is glycosylated, is tightly regulated by protein substrates. Kinetic characterization has revealed that humicolin activity is highly dependent upon the deprotonation of a group with a pKa of 6.6 and that the enzyme has a specificity for phenylalanine in the P1 position of the substrate.     

Dietary regulation of serine proteinases that are resistant to serine proteinase inhibitors

Ingestion of Kunitz soybean trypsin inhibitor (STI) by larval Helicoverpa zea, Agrotis ipsilon, and Trichoplusia ni extended the retention time of food in the digestive tract and increased the level of activity of proteolytic enzymes that were not susceptible to inhibition by STI. The level of enhancement of activity of STI-resistant (STI-R) enzyme(s) was directly influenced by the dosage and timing of exposure to STI. However, not all proteinase inhibitors (PIs) enhanced the level of proteinase inhibitor resistant (PI-R) enzymes, even if those PIs inhibited a significant proportion of enzyme activity. These findings suggest that a complex system may be responsible for the regulation of proteolytic enzymes in the midgut of larval Lepidoptera, and one hypothesis for this regulation is proposed.     

Caldolase, a chelator-insensitive extracellular serine proteinase from a Thermus spp.

An extracellular alkaline serine proteinase from Thermus strain ToK3 was isolated and purified to homogeneity by (NH4)2SO4 precipitation followed by ion-exchange chromatography on DEAE-cellulose and QAE-Sephadex, affinity chromatography on N alpha-benzyloxycarbonyl-D-phenylalanyl-triethylenetetraminyl-Sepha rose 4B and gel-filtration chromatography on Sephadex G-75. The purified enzyme had a pI of 8.9 and an Mr determined by gel-permeation chromatography of 25,000. The specific activity was about 37,700 proteolytic units/mg with casein as substrate, and the pH optimum was 9.5. Proteolytic activity was inhibited by low concentrations of di-isopropyl phosphorofluoridate and phenylmethanesulphonyl fluoride, but was unaffected by EDTA, EGTA, o-phenanthroline, N-ethyl-5-phenylisoxazolium-3'-sulphonate, N alpha-p-tosyl-L-phenylalanylchloromethane, N alpha-p-tosyl-L-lysylchloromethane, trypsin inhibitors and pepstatin A. The enzyme contained approx. 10% carbohydrate and four disulphide bonds. No Ca2+, Zn2+ or free thiol groups were detected. It hydrolysed several native and dye-linked proteins and synthetic chromogenic peptides and esters. The enzyme was very thermostable (half-life values were 840 min at 80 degrees C, 45 min at 90 degrees C and 5 min at 100 degrees C). The enzyme was unstable at low ionic strength: after 60 min at 75 degrees C in 0.1 M-Tris/acetate buffer, pH 8, only 20% activity remained, compared with no loss in 0.1 M-Tris/acetate buffer, pH 8, containing 0.4 M-NaCl.     

Purification and characterization of an extracellular serine proteinase from Acanthamoeba castellanii

An extracellular proteinase of Acanthamoeba castellanii was purified and its biochemical and pathological properties were characterized. The molecular mass of the purified enzyme was approximately 42 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephacryl S-200 HR gel-filtration chromatography. Therefore, its structure seemed to be monomeric with a single polypeptide. Its activity was inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride. Its activity was optimum at 30 to 50 degrees C with a maximum at 50 degrees C; optimal pH was 8.0. As much as 70% of the enzyme activity was maintained at 50 degrees C for at least 12 h but was rapidly inactivated thereafter. The purified enzyme degraded collagen and rabbit corneal extract. Furthermore, it exhibited strong cytopathic effects on human corneal epithelial cells and fibroblast cells. These suggest the possible role of this enzyme in the pathogenesis of Acanthamoeba.     

Detection of serine proteinase inhibitors in human cornified cells

A screening test for serine proteinase inhibitors revealed trypsin and urokinase inhibitors in the extract of human cornified cells. No inhibition for α-chymotrypsin, thrombin or plasmin was detected. Characterization of the inhibitors separated with a Sephacryl S-200 gel column demonstrated that: 1) trypsin inhibitor with a molecular weight of 45,000 was labile to heat, acid and alkali and showed temporary inhibition, and 2) urokinase inhibitor with a molecular weight of 35,000 was found relatively stable and exhibited time dependent inhibition. Both were distinct from a known thiol proteinase inhibitor which showed high stability and immediate inhibition. Regulatory roles of serine proteinase inhibitors are postulated.     

Granzyme activity in the inflamed lung is not controlled by endogenous serine proteinase inhibitors

Numerous lung diseases, such as hypersensitivity pneumonitis (HP), are characterized by the presence of activated alveolar CTL and NK cells. Since these cells produce granzymes, granzyme A and B levels in bronchoalveolar lavage (BAL) fluids from 14 normal subjects and 12 patients with HP were measured by ELISA. Median (range) BAL granzyme A and B levels were 4 (0-37) and 0 (0-6) pg/ml in normal subjects. BAL granzyme levels were significantly higher in HP patients, being at 74 (0-1,889) and 10 (0-78) pg/ml for granzymes A and B, respectively. In vitro, neither of the three main serine protease inhibitors of the lung, namely alpha1-antitrypsin, secretory leukocyte protease inhibitor, and elafin, showed any effect on granzyme A or B activity. In addition, granzyme A was shown to be fully active in BAL fluids. Hence, these data show that granzyme activity may be poorly controlled by protease inhibitors in inflamed tissues. Thus, granzymes could contribute to tissue remodeling and inflammation characterizing HP.     

Localization and accessibility of antigenic sites of the extracellular serine proteinase of Lactococcus lactis.

Lactococcus lactis strains produce an extracellular subtilisin-related serine proteinase in which immunologically different components can be distinguished. Monoclonal antibodies specific for the different proteinase components have been raised and their epitopes were identified. By Western-blot analysis it was found that all monoclonal antibodies recognize all denatured proteinase components. The distinction between the different components could be made under native conditions only, indicating that binding regions are masked in the native molecule. In a L. lactis proteinase which was inactivated by the substitution Asp30----Asn under native conditions, only one epitope could be detected. This demonstrates that autoproteolytic activity is required to make specific binding regions accessible for (monoclonal) antibodies.     

A serine proteinase inhibitor from frog eggs with bacteriostatic activity

By Sephadex G-50 gel filtration, Resource Q anionic exchange and C4 reversed phase liquid high performance liquid chromatography, a proteinase inhibitor protein (Ranaserpin) was identified and purified from the eggs of the odour frog, Rana grahami. The protein displayed a single band adjacent to the molecular weight marker of 14.4 kDa analyzed by SDS-PAGE. The inhibitor protein homogeneity and its molecular weight were confirmed again by MALDI-TOF mass spectrometry analysis. The MALDI-TOF mass spectrum analysis gave this inhibitor protein an m/z of 14422.26 that was matched well with the result from SDS-PAGE. This protein is a serine proteinase inhibitor targeting multiple proteinases including trypsin, elastase, and subtilisin. Ranaserpin inhibited the proteolytic activities of trypsin, elastase, and subtilisin. It has an inhibitory constant (K(i)) of 6.2 x 10(-8) M, 2.7 x 10(-7) M and 2.2 x 10(-8) M for trypsin, elastase, and subtilisin, respectively. This serine proteinase inhibitor exhibited bacteriostatic effect on Gram-positive bacteria Bacillus subtilis (ATCC 6633). It was suggested that ranaserpin might act as a defensive role in resistance to invasion of pests or pathogens. This is the first report of serine proteinase inhibitor and its direct defensive role from amphibian eggs.     

Identification of a gene required for maturation of an extracellular lactococcal serine proteinase.

Directly upstream of the Lactococcus lactis subsp. cremoris Wg2 proteinase gene is an oppositely directed open reading frame (ORF1). The complete nucleotide sequence of ORF1, encoding a 33-kilodalton protein, was determined. A protein of approximately 32 kilodaltons was synthesized when ORF1 was expressed in Escherichia coli by using a T7 RNA polymerase-specific promoter. L. lactis subsp. lactis MG1363 transformants carrying the proteinase gene but lacking ORF1 were phenotypically proteinase deficient, unlike transformants carrying both the proteinase gene and ORF1. Synthesis and secretion of proteinase antigen by L. lactis could be detected with proteinase-directed monoclonal antibodies regardless of whether ORF1 was present. The requirement of ORF1 for proteinase activation was reflected in a reduction in the molecular weight of the secreted proteinase. Furthermore, deletion of the 130 C-terminal amino acids of the Wg2 proteinase prevented attachment of the enzyme to lactococcal cells.     

Molecular recognition. Conformational analysis of limited proteolytic sites and serine proteinase protein inhibitors

The conformations of known tryptic limited proteolytic sites have been analysed and compared to the structures of the binding regions of serine proteinase inhibitors, as they are found when complexed to a serine proteinase. Conformational parameters studied include main-chain torsion angles, root-mean-square fits, accessibility, mobility and protrusion indices. As observed before, the inhibitors share a common main-chain conformation at the binding loop from P3-P'3 (Schechter & Berger notation), which is maintained throughout all the serine proteinase inhibitor families for which X-ray data is available, despite lack of similarity in the rest of the protein. This canonical structure is not found amongst the limited proteolytic sites (or nicksites), which differ markedly from the inhibitor binding loop conformation, and also amongst themselves. The experimentally determined nicksites are in general both accessible and protruding; as are the inhibitor binding loops, as well as being typically flexible regions of structure, as denoted by elevated temperature factors from crystallographic determinations. For cleavage by serine proteinases these loops must radically alter their local conformations and a large motion of the loop relative to the structure, in some cases, would be required to orientate these sites for cleavage.     

The structural puzzle of how serpin serine proteinase inhibitors work

Serine proteinase cleavage of proteins is essential to a wide variety of biological processes and is primarily regulated by protein inhibitors. Many inhibitors are conformationally rigid simulations of optimal serine proteinase substrates, which makes them highly efficient competitive inhibitors of target proteinases. In contrast, members of the serpin family of serine proteinase inhibitors display extensive flexibility and polymorphism, particularly in their reactive site segments and in β-sheet secondary structure, which can take up and expel strands. Reactive site and β-sheet polymorphism appear to be coupled in the serpins and may account for the extreme stability of serpinproteinase complexes through the insertion of the reactive site strand into a β-sheet. These unusual properties may have opened an adaptive pathway of proteinase regulation that was unavailable to the conformationally rigid proteinase inhibitors.     

The role of scaffolding in standard mechanism serine proteinase inhibitors

In single domain, "standard mechanism" protein inhibitors of serine proteinases, about a dozen residues make contact with the cognate enzyme. The remainder of the molecule, the scaffolding, holds the reactive site region of the inhibitor in a canonical conformation, improves the binding by about six orders of magnitude and protects it from proteolysis. However, the stability and global structure of the scaffolding is irrelevant to inhibition, provided that inhibition is measured much below the melting temperature, Tm.