Pericellular proteolysis by neutrophils in the presence of proteinase inhibitors: effects of substrate opsonization

Inflammatory cells are capable of degrading extracellular matrix macromolecules in vivo in the presence of proteinase inhibitors. We and others have hypothesized that such proteolysis is permitted in large part by mechanisms operative in the immediate pericellular environment, especially at zones of contact between inflammatory cells and insoluble matrix components. To further test this hypothesis in vitro, we have used a model system in which viable polymorphonuclear neutrophils (PMN) are allowed to contact a surface coated with proteinase-sensitive substrate, and in which PMN interaction with the surface can be modulated. We have evaluated proteolysis of the surface-bound protein in the presence and absence of proteinase inhibitors. Our results were: (a) In the presence (but not in the absence) of proteinase inhibitors, proteolysis was confined to sharply marginated zones subjacent to the cells; (b) opsonization of the surface enhanced spreading of the PMN, (c) opsonization diminished the effectiveness of alpha-1-proteinase inhibitor (alpha-1-PI) and alpha-2-macroglobulin as inhibitors of proteolysis of surface-bound protein; (d) anti-oxidants did not alter the effectiveness of alpha-1-PI in inhibiting proteolysis of opsonized substrate by PMN; and (e) PMN could restrict entry of alpha-1-PI into zones of contact with opsonized surfaces. We conclude that: (a) In the presence of proteinase inhibitors, PMN can express sharply marginated and exclusively pericellular proteolytic activity; (b) locally high proteinase concentrations and/or exclusion of proteinase inhibitors from pericellular microenvironments may be important mechanisms for pericellular matrix degradation by PMN; and (c) these observations may have general relevance to extracellular matrix remodeling by a variety of inflammatory and other cell types.     

A 25-kDa Serine Peptidase with Keratinolytic Activity Secreted by <Emphasis Type="Italic">Coccidioides immitis</Emphasis>

Coccidioides immitis is the causative agent of coccidioidomycosis, a systemic mycosis that attacks humans and a wide variety of animals. In the present study, we showed that the C. immitis mycelial form is able to release proteolytic enzyme into the extracellular environment. Under chemically defined growth conditions, mycelia secreted seven distinct polypeptides ranging from 15 to 65 kDa and an extracellular peptidase of 25 kDa. This enzyme had its activity fully inhibited by phenylmethylsulphonyl fluoride, a serine peptidase inhibitor. Conversely, metallo, cysteine, and aspartyl peptidase inhibitors did not alter the 25-kDa enzyme behavior. This extracellular serine peptidase was able to degrade keratin, a fibrous protein that composes human epidermis. Additionally, this peptidase cleaved different protein substrates, including gelatin, casein, hemoglobin, and albumin. Curiously, an 18-kDa serine peptidase activity was evidenced solely when casein was used as the co-polymerized protein substrate into the gel. The existence of different secreted peptidases could be advantageous for the adaptation of C. immitis to distinct environments during its complex life cycle.     

Heterogeneous production of metallo-type peptidases in parasites belonging to the family Trypanosomatidae

Proteolytic enzymes play a central role in the physiology of all living organisms, participating in several metabolic pathways and in different phases of parasite-host interactions. We have identified cell-associated peptidase activities in 33 distinct flagellates, including representatives of almost all known trypanosomatid genera parasitizing insects (Herpetomonas, Crithidia, Leishmania, Trypanosoma, Leptomonas, Phytomonas, Blastocrithidia and Endotrypanum) as well as the biflagellate kinetoplastid Bodo, by using SDS-PAGE containing gelatin as co-polymerized substrate and proteolytic inhibitors. Under the alkaline pH (9.0) conditions employed, all the flagellates presented at least one peptidase, with the exception of Crithidia acanthocephali and Phytomonas serpens, which did not display any detectable proteolytic enzyme activity. All the proteolytic activities were completely inhibited by 1,10-phenanthroline, a zinc-chelating agent, putatively identifying these activities as metallo-type peptidases. EDTA and EGTA, two other metallopeptidase inhibitors, E-64 (a cysteine peptidase inhibitor), pepstatin A (an aspartyl peptidase inhibitor) and PMSF (a serine peptidase inhibitor) did not interfere with the metallopeptidase activities detected in the studied trypanosomatids. Conversely, Bodo-derived peptidases were resistant to 1,10-phenanthroline and only partially inhibited by EDTA, showing a distinct inhibition profile. Together, our data demonstrated great heterogeneity of expression of metallopeptidases in a wide range of parasites belonging to the family Trypanosomatidae.     

Modulation of bovine microvascular endothelial cell proteolytic properties by inhibitors of angiogenesis

A tightly controlled increase in extracellular proteolysis, restricted both in time and space, is an important component of the angiogenic process, while anti-proteolysis is effective in inhibiting angiogenesis. By focussing on the plasminogen activator (PA)-plasmin system, the objective of the present studies was to assess whether previously described inhibitors of angiogenesis modify bovine microvascular endothelial cell proteolytic properties. We demonstrate that although synthetic angiostatic steroids (U-24067 and U-42129), heparin, suramin, interferon alpha-2a, and retinoic acid are all inhibitors of in vitro angiogenesis, each of these agents has distinct effects on the plasminogen-dependent proteolytic system. Specifically, angiostatic steroids and interferon alpha-2a reduce urokinase-type PA (u-PA) and PA inhibitor-1 activity, while heparin and retinoic acid increase u-PA activity. Suramin reduces cell-associated u-PA activity and greatly increases PAI-1 production at doses which induce monolayer disruption. These findings demonstrate that a spectrum of alterations in extracellular proteolysis is associated with anti-angiogenesis, and that anti-angiogenesis and anti-proteolysis are not necessarily correlated. A reduction in extracellular proteolysis would be expected to reduce invasion, whereas an increase in proteolysis might modulate the activity of inhibitory cytokines, which in turn could reduce endothelial cell proliferation and migration and inhibit angiogenesis. The spectrum of effects on different elements of the PA system observed in response to the agents assessed suggests that the role of modulations in extracellular proteolytic activity in anti-angiogenesis is likely to be varied and complex. © 1994 Wiley-Liss, Inc.     

Engineered resistance against proteinases

Exogenous proteinase inhibitors are valuable and economically interesting protective biotechnological tools. We examined whether small proteinase inhibitors when fused to a selected target protein can protect the target from proteolytic degradation without simultaneously affecting the function and activity of the target domain. Two proteinase inhibitors were studied: a Kazal-type silk proteinase inhibitor (SPI2) from Galleria mellonella, and the Cucurbita maxima trypsin inhibitor I (CMTI I). Both inhibitors target serine proteinases, are small proteins with a compact structure stabilized by a network of disulfide bridges, and are expressed as free polypeptides in their natural surroundings. Four constructs were prepared: the gene for either of the inhibitors was ligated to the 5' end of the DNA encoding one or the other of two selected target proteins, the coat protein (CP) of Potato potyvirus Y or the Escherichia coli beta-glucuronidase (GUS). CMTI I fused to the target proteins strongly hampered their functions. Moreover, the inhibitory activity of CMTI I was retained only when it was fused to the CP. In contrast, when fused to SPI2, specific features and functions of both target proteins were retained and the inhibitory activity of SPI2 was fully preserved. Measuring proteolysis in the presence or absence of either inhibitor, we demonstrated that proteinase inhibitors can protect target proteins used either free or as a fusion domain. Interestingly, their inhibitory efficiency was superior to that of a commercial inhibitor of serine proteinases, AEBSF.     

Extracellular Peptidase in the Fungal Pathogen Pseudallescheria boydii

Pseudallescheria boydii is a ubiquitous filamentous fungus capable of causing invasive disease in humans. In the present study, using sodium dodecyl sulfate–polyacrylamide gels containing bovine serum albumin as co-polymerized substrate, we identified a 28-kDa proteolytic activity released to the extracellular environment by mycelia of P. boydii. This peptidase was detected during the growth of P. boydii in Sabouraud-dextrose medium for 13 days and reached its maximal production on day 7. The 28-kDa peptidase was active in acidic pH (5.5) and had its activity completely blocked by 1,10-phenanthroline, a potent zinc-metallopeptidase inhibitor. Two other metallopeptidase inhibitors, EDTA and EGTA, were also tested and no alterations were observed in the activity of the 28-kDa extracellular peptidase. Likewise, E-64 (a cysteine peptidase inhibitor), phenylmethylsulphonyl fluoride (a serine peptidase inhibitor), and pepstatin A (an aspartyl peptidase inhibitor) did not significantly alter the enzymatic behavior. Collectively, we described for the first time the expression of an extracellular metallopeptidase in the human opportunistic fungal pathogen P. boydii.     

Recombinant Lactococcus starters as a potential source of additional peptidolytic activity in cheese ripening

AIMS: The aim of this study was to modulate the lactococcal proteolytic system for enhancement of the cheese ripening process. METHODS AND RESULTS: The genes encoding PepN, PepC, PepX and PepI peptidases of a highly proteolytic Lactobacillus helveticus strain were transferred into Lactococcus lactis in a food-grade cloning system. A comparison of the relative peptidase activities from the transformants with those from the untransformed host, determined in the conditions of maturing cheese, showed that an increase in peptidase activity could be achieved by introducing a selected peptidase gene from Lact. helveticus into L. lactis. CONCLUSIONS: Recombinant L. lactis starter strains, carrying a peptidase gene from Lact. helveticus, may have an important contribution to the proteolysis of maturing cheese by producing an additional peptidolytic enzyme activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will be of importance in shortening the ripening period and production of special cheeses (e.g. reduced-fat cheeses) with improved characteristics.     

Evolution of peptidase diversity

A wide variety of peptidases associate with vital biological pathways, but the origin and evolution of their tremendous diversity are poorly defined. Application of the MEROPS classification to a comprehensive set of genomes yields a simple pattern of peptidase distribution and provides insight into the organization of proteolysis in all forms of life. Unexpectedly, a near ubiquitous core set of peptidases is shown to contain more types than those unique to higher multicellular organisms. From this core group, an array of eukaryote-specific peptidases evolved to yield well known intracellular and extracellular processes. The paucity of peptidase families unique to higher metazoa suggests gains in proteolytic network complexity required a limited number of biochemical inventions. These findings provide a framework for deeper investigation into the evolutionary forces that shaped each peptidase family and a roadmap to develop a timeline for their expansion as an interconnected system.     

Protease activities of rumen protozoa

Intact, metabolically active rumen protozoa prepared by gravity sedimentation and washing in a mineral solution at 10 to 15 degrees C had comparatively low proteolytic activity on azocasein and low endogenous proteolytic activity. Protozoa washed in 0.1 M potassium phosphate buffer (pH 6.8) at 4 degrees C and stored on ice autolysed when they were warmed to 39 degrees C. They also exhibited low proteolytic activity on azocasein, but they had a high endogenous proteolytic activity with a pH optimum of 5.8. The endogenous proteolytic activity was inhibited by cysteine proteinase inhibitors, for example, iodoacetate (63.1%) and the aspartic proteinase inhibitor, pepstatin (43.9%). Inhibitors specific for serine proteinases and metalloproteinases were without effect. The serine and cysteine proteinase inhibitors of microbial origin, including antipain, chymostatin, and leupeptin, caused up to 67% inhibition of endogenous proteolysis. Hydrolysis of casein by protozoa autolysates was also inhibited by cysteine proteinase inhibitors. Some of the inhibitors decreased endogenous deamination, in particular, phosphoramidon, which had little inhibitory effect on proteolysis. Protozoal and bacterial preparations exhibited low hydrolytic activities on synthetic proteinase and carboxypeptidase substrates, although the protozoa had 10 to 78 times greater hydrolytic activity (per milligram of protein) than bacteria on the synthetic aminopeptidase substrates L-leucine-p-nitroanilide, L-leucine-beta-naphthylamide, and L-leucinamide. The aminopeptidase activity was partially inhibited by bestatin. It was concluded that cysteine proteinases and, to a lesser extent, aspartic proteinases are primarily responsible for proteolysis in autolysates of rumen protozoa. The protozoal autolysates had high aminopeptidase activity; low deaminase activity was observed on endogenous amino acids.     

Protease activities of rumen protozoa.

Intact, metabolically active rumen protozoa prepared by gravity sedimentation and washing in a mineral solution at 10 to 15 degrees C had comparatively low proteolytic activity on azocasein and low endogenous proteolytic activity. Protozoa washed in 0.1 M potassium phosphate buffer (pH 6.8) at 4 degrees C and stored on ice autolysed when they were warmed to 39 degrees C. They also exhibited low proteolytic activity on azocasein, but they had a high endogenous proteolytic activity with a pH optimum of 5.8. The endogenous proteolytic activity was inhibited by cysteine proteinase inhibitors, for example, iodoacetate (63.1%) and the aspartic proteinase inhibitor, pepstatin (43.9%). Inhibitors specific for serine proteinases and metalloproteinases were without effect. The serine and cysteine proteinase inhibitors of microbial origin, including antipain, chymostatin, and leupeptin, caused up to 67% inhibition of endogenous proteolysis. Hydrolysis of casein by protozoa autolysates was also inhibited by cysteine proteinase inhibitors. Some of the inhibitors decreased endogenous deamination, in particular, phosphoramidon, which had little inhibitory effect on proteolysis. Protozoal and bacterial preparations exhibited low hydrolytic activities on synthetic proteinase and carboxypeptidase substrates, although the protozoa had 10 to 78 times greater hydrolytic activity (per milligram of protein) than bacteria on the synthetic aminopeptidase substrates L-leucine-p-nitroanilide, L-leucine-beta-naphthylamide, and L-leucinamide. The aminopeptidase activity was partially inhibited by bestatin. It was concluded that cysteine proteinases and, to a lesser extent, aspartic proteinases are primarily responsible for proteolysis in autolysates of rumen protozoa. The protozoal autolysates had high aminopeptidase activity; low deaminase activity was observed on endogenous amino acids.     

The structure of chagasin in complex with a cysteine protease clarifies the binding mode and evolution of an inhibitor family

Protein inhibitors of proteolytic enzymes regulate proteolysis and prevent the pathological effects of excess endogenous or exogenous proteases. Cysteine proteases are a large family of enzymes found throughout the plant and animal kingdoms. Disturbance of the equilibrium between cysteine proteases and natural inhibitors is a key event in the pathogenesis of cancer, rheumatoid arthritis, osteoporosis, and emphysema. A family (I42) of cysteine protease inhibitors (http://merops.sanger.ac.uk) was discovered in protozoan parasites and recently found widely distributed in prokaryotes and eukaryotes. We report the 2.2 A crystal structure of the signature member of the I42 family, chagasin, in complex with a cysteine protease. Chagasin has a unique variant of the immunoglobulin fold with homology to human CD8alpha. Interactions of chagasin with a target protease are reminiscent of the cystatin family inhibitors. Protein inhibitors of cysteine proteases may have evolved more than once on nonhomologous scaffolds.     

A role for type I signal peptidase in Staphylococcus aureus quorum sensing

The Staphylococcus aureus Agr quorum-sensing system modulates the expression of extracellular virulence factors. The Agr system is controlled by an autoinducing peptide (AIP) molecule that is secreted during growth. In the AIP biosynthetic pathway, two proteolytic events are required to remove the leader and tail segments of AgrD, the peptide precursor of AIP. The only protein known to be involved in this pathway is AgrB, a membrane endopeptidase that removes the AgrD carboxy-tail. We designed a synthetic peptide substrate and developed an assay to detect peptidases that can remove the N-terminal leader of AIP. Several peptidase activities were detected in S. aureus extracts and these activities were present in both wild-type and agr mutant strains. Only one of these peptidases cleaved in the correct position and all properties of this enzyme were consistent with type I signal peptidase. Subsequent cloning and purification of the two known S. aureus signal peptidases, SpsA and SpsB, demonstrated that only SpsB catalysed this activity in vitro. To investigate the role of SpsB in AIP biosynthesis, SpsB peptide inhibitors were designed and characterized. The most effective inhibitor blocked SpsB activity in vitro and showed antibacterial activity against S. aureus. Importantly, the inhibitor reduced expression of an Agr-dependent reporter and inhibited AIP production in S. aureus, indicating a role for SpsB in quorum sensing.     

Modes of inhibition of cysteine proteases

Cysteine proteases are involved in many physiological processes and their hyperactivity may lead to severe diseases. Nature has developed various strategies to protect cells and whole organisms against undesired proteolysis. One of them is the control of proteolytic activity by inhibition. This paper presents the mechanisms underlying the action of proteinaceous inhibitors of cysteine proteinases and covers propeptides binding backwards relative to the substrate or distorting the protease catalytic centre similarly to serpins, the p35 protein binding covalently to the enzyme, and cystatins that are exosite binding inhibitors. The paper also discusses tyropins and chagasins that, although unrelated to cystatins, inhibit cysteine proteinases by a similar mechanism, as well as inhibitors of the apoptosis protein family that bind in a direction opposite to that of the substrate, similarly to profragments. Special attention is given to staphostatins, a novel family of inhibitors acting in an unusual manner.     

How to use the MEROPS database and website to help understand peptidase specificity

The MEROPS website ( https://www.ebi.ac.uk/merops ) and database was established in 1996 to present the classification and nomenclature of proteolytic enzymes. This was expanded to include a classification of protein inhibitors of proteolytic enzymes in 2004. Each peptidase or inhibitor is assigned to a distinct identifier, based on its biochemical and biological properties, and homologous sequences are assembled into a family. Families in which the proteins share similar tertiary structures are assembled into a clan. The MEROPS classification is thus a hierarchy with at least three levels (protein‐species, family, and clan) showing the evolutionary relationship. Several other data collections have been assembled, which are accessed from all levels in the hierarchy. These include, sequence homologs, selective bibliographies, substrate cleavage sites, peptidase–inhibitor interactions, alignments, and phylogenetic trees. The substrate cleavage collection has been assembled from the literature and includes physiological, pathological, and nonphysiological cleavages in proteins, peptides, and synthetic substrates. In this article, we make recommendations about how best to analyze these data and show analyses to indicate peptidase binding site preferences and exclusions. We also identify peptidases where co‐operative binding occurs between adjacent binding sites.     

Proteolytic activity of rumen microorganisms and effects of proteinase inhibitors.

Proteolytic activity of the bovine rumen microflora was studied with azocasein as the substrate. Approximately 25% of the proteolytic activity of rumen contents was recovered in the strained rumen fluid fraction, and the balance of the activity was associated with the particulate fraction. The proportion of proteinase activity associated with particulate material decreased when the quantity of particulate material in rumen contents was reduced. The specific activity of the proteinase from the bacterial fraction was 6 to 10 times higher than that from the protozoal fraction. Proteinase inhibitors of synthetic, plant, and microbial origin were tested on proteolytic activity of the separated bacteria. Synthetic proteinase inhibitors that caused significant inhibition of proteolysis included phenylmethylsulfonyl fluoride, N-tosyl-1-lysine chloromethyl ketone, N-tosylphenylalanine chloromethyl ketone, EDTA, cysteine, dithiothreitol, iodoacetate, and Merthiolate. Plant proteinase inhibitors that had an inhibitory effect included soybean trypsin inhibitors types I-S and II-S and the lima bean trypsin inhibitor. Proteinase inhibitors of microbial origin that showed an inhibitory effect included antipain, leupeptin, and chymostatin; phosphoramidon and pepstatin had little effect. We tentatively concluded that rumen bacteria possess, primarily, serine, cysteine, and metalloproteinases.     

Proteolytic activity of rumen microorganisms and effects of proteinase inhibitors

Proteolytic activity of the bovine rumen microflora was studied with azocasein as the substrate. Approximately 25% of the proteolytic activity of rumen contents was recovered in the strained rumen fluid fraction, and the balance of the activity was associated with the particulate fraction. The proportion of proteinase activity associated with particulate material decreased when the quantity of particulate material in rumen contents was reduced. The specific activity of the proteinase from the bacterial fraction was 6 to 10 times higher than that from the protozoal fraction. Proteinase inhibitors of synthetic, plant, and microbial origin were tested on proteolytic activity of the separated bacteria. Synthetic proteinase inhibitors that caused significant inhibition of proteolysis included phenylmethylsulfonyl fluoride, N-tosyl-1-lysine chloromethyl ketone, N-tosylphenylalanine chloromethyl ketone, EDTA, cysteine, dithiothreitol, iodoacetate, and Merthiolate. Plant proteinase inhibitors that had an inhibitory effect included soybean trypsin inhibitors types I-S and II-S and the lima bean trypsin inhibitor. Proteinase inhibitors of microbial origin that showed an inhibitory effect included antipain, leupeptin, and chymostatin; phosphoramidon and pepstatin had little effect. We tentatively concluded that rumen bacteria possess, primarily, serine, cysteine, and metalloproteinases.     

Prolyl tripeptidyl peptidase from Porphyromonas gingivalis. A novel enzyme with possible pathological implications for the development of periodontitis

Porphyromonas gingivalis possesses a complex proteolytic system, which is essential for both its growth and evasion of host defense mechanisms. In this report we characterized, both at a protein and genomic level, a novel peptidase of this system with prolyl tripeptidyl peptidase activity. The enzyme was purified to homogeneity, and its enzymatic activity and biochemical properties were investigated. The amino acid sequence at the amino terminus and of internal peptide fragments enabled identification of the gene encoding this enzyme, which we refer to as PtpA for prolyl tripeptidyl peptidase A. The gene encodes an 82-kDa protein, which contains a GWSYGG motif, characteristic for members of the S9 prolyl oligopeptidase family of serine proteases. However, it does not share any structural similarity to other tripeptidyl peptidases, which belong to the subtilisin family. The production of prolyl tripeptidyl peptidase may contribute to the pathogenesis of periodontal tissue destruction through the mutual interaction of this enzyme, host and bacterial collagenases, and dipeptidyl peptidases in the degradation of collagen during the course of infection.     

Signal peptide peptidase and gamma-secretase share equivalent inhibitor binding pharmacology

The enzyme gamma-secretase has long been considered a potential pharmaceutical target for Alzheimer disease. Presenilin (the catalytic subunit of gamma-secretase) and signal peptide peptidase (SPP) are related transmembrane aspartyl proteases that cleave transmembrane substrates. SPP and gamma-secretase are pharmacologically similar in that they are targeted by many of the same small molecules, including transition state analogs, non-transition state inhibitors, and amyloid beta-peptide modulators. One difference between presenilin and SPP is that the proteolytic activity of presenilin functions only within a multisubunit complex, whereas SPP requires no additional protein cofactors for activity. In this study, gamma-secretase inhibitor radioligands were used to evaluate SPP and gamma-secretase inhibitor binding pharmacology. We found that the SPP enzyme exhibited distinct binding sites for transition state analogs, non-transition state inhibitors, and the nonsteroidal anti-inflammatory drug sulindac sulfide, analogous to those reported previously for gamma-secretase. In the course of this study, cultured cells were found to contain an abundance of SPP binding activity, most likely contributed by several of the SPP family proteins. The number of SPP binding sites was in excess of gamma-secretase binding sites, making it essential to use selective radioligands for evaluation of gamma-secretase binding under these conditions. This study provides further support for the idea that SPP is a useful model of inhibitory mechanisms and structure in the SPP/presenilin protein family.     

Recombinant interferon-γ secreted by chinese hamster ovary-320 cells cultivated in suspension in protein-free media is protected against extracellular proteolysis by the expression of natural protease inhibitors and by the addition of plant protein hydrolysates to the culture medium

Summary Biosafety requirements increasingly restrict the cultivation of mammalian cells producing therapeutic glycoproteins to conditions that are devoid of any compound of animal origin. On cultivation in serum-free media, the proteases inhibitors, usually found in serum, cannot protect secreted recombinant proteins against unwanted endogenous proteolysis. Chinese hamster ovary (CHO) cells, secreting recombinant human interferon-γ (CHO-320 cell line) and cultivated in suspension in an original protein-free medium, expressed at least two members of the matrix metalloproteinases (MMP), either at the cell surface (proMMP-14 and MMP-14) or secreted (proMMP-9). In addition, tissue- and urinary-type plasminogen activators were also secreted in such culture conditions. At the cell surface, dipeptidyl peptidase IV and tripeptidyl peptidase II (TPPII) activities were also detected, and their activities decreased during time course of batch cultures. The proteolytic activities of these proteins were counterbalanced by (1) their expression as zymogens (proMMP-9, proMMP-14), (2) the expression of their natural inhibitors, tissue inhibitors of metalloproteinases-1 and-2 and plasminogen activator inhibitor-1 (PAI-1), or (3) the addition of plant protein hydrolysates to the culture medium, acting as a nonspecific source of TPPII inhibitors. This study points out that, even in protein-free media, recombinant proteins secreted by CHO cells are actively protected against physiological and unwanted extracellular proteolysis either by endogenous or by exogenous inhibitors.