The sulfate groups of chondroitin sulfate- and heparan sulfate-containing proteoglycans in neutrophil plasma membranes are novel binding sites for human leukocyte elastase and cathepsin G

Human leukocyte elastase (HLE) and cathepsin G (CG) are expressed at high levels on the surface of activated human neutrophils (PMN) in catalytically active but inhibitor-resistant forms having the potential to contribute to tissue injury. Herein we have investigated the mechanisms by which HLE and CG bind to PMN plasma membranes. (125)I-Labeled HLE and CG bind to PMN at 0 degrees C in a saturable and reversible manner (K(D) = 5.38 and 4.36 x 10(-7) m and 11.5 and 8.1 x 10(6) binding sites/cell, respectively). Incubation of PMN with radiolabeled HLE and CG in the presence of a 200-fold molar excess of unlabeled HLE, CG, myeloperoxidase, lactoferrin, proteinase 3, phenylmethylsulfonyl fluoride (PMSF)-inactivated HLE, or PMSF-inactivated CG inhibited binding of radiolabeled ligands. This indicates that these PMN granule proteins share binding sites on PMN and that functional active sites of HLE and CG are not required for their binding to PMN. The sulfate groups of heparan sulfate- and chondroitin sulfate-containing proteoglycans are the PMN binding sites for HLE and CG since binding of HLE and CG to PMN was inhibited by incubating PMN with 1) trypsin, chondroitinase ABC, and heparitinases, but not other glycanases, and 2) purified chondroitin sulfates, heparan sulfate, and other sulfated molecules, but not with non-sulfated glycans. Thus, heparan sulfate- and chondroitin sulfate-containing proteoglycans are low affinity, high volume PMN surface binding sites for HLE and CG, which are well suited to bind high concentrations of active serine proteinases released from degranulating PMN.     

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.     

Proteinase Activity during Tracheary Element Differentiation in Zinnia Mesophyll Cultures

The zinnia (Zinnia elegans) mesophyll cell culture tracheary element (TE) system was used to study proteinases active during developmentally programmed cell death. Substrate-impregnated gels and single-cell assays revealed high levels of proteinase activity in differentiating TEs compared with undifferentiated cultured cells and expanding leaves. Three proteinases (145, 28, and 24 kD) were exclusive to differentiating TEs. A fourth proteinase (59 kD), although detected in extracts from all tissues examined, was most active in differentiating TEs. The 28- and 24-kD proteinases were inhibited by thiol proteinase inhibitors, leupeptin, and N-[N-(L-3-trans-carboxirane-2-carbonyl)-L-leucyl]-agmatine (E-64). The 145- and 59-kD proteinases were inhibited by the serine proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF). Extracts from the TE cultures contained sodium dodecyl sulfate-stimulated proteolytic activity not detected in control cultures. Sodium dodecyl sulfate-stimulated proteolysis was inhibited by leupeptin or E-64, but not by PMSF. Other tissues, sucrose-starved cells and cotyledons, that contain high levels of proteolytic activity did not contain TE-specific proteinases, but did contain higher levels of E-64-sensitive activities migrating as 36- to 31-kD enzymes and as a PMSF-sensitive 66-kD proteinase.     

Bioactive proteinase 3 on the cell surface of human neutrophils: quantification, catalytic activity, and susceptibility to inhibition

Although proteinase 3 (PR3) is known to have the potential to promote inflammation and injure tissues, the biologic forms and function of PR3 in polymorphonuclear neutrophils (PMN) from healthy donors have received little attention. In this paper, we show that PMN contain 3.24 +/- SD 0.24 pg of PR3 per cell, and that the mean concentration of PR3 in azurophil granules of PMN is 13.4 mM. Low levels of PR3 are detectable on the cell surface of unstimulated PMN. Exposure of PMN to cytokines or chemoattractants alone induces modest (1.5- to 2.5-fold) increases in cell surface-bound PR3. In contrast, brief priming of PMN with cytokines, followed by activation with a chemoattractant, induces rapid and persistent, 5- to 6-fold increases in cell surface expression of PR3, while causing minimal free release of PR3. Membrane-bound PR3 on PMN is catalytically active against Boc-Alanine-Alanine-Norvaline-thiobenzyl ester and fibronectin, but in marked contrast to soluble PR3, membrane-bound PR3 is resistant to inhibition by physiologic proteinase inhibitors. PR3 appears to bind to the cell surface of PMN via a charge-dependent mechanism because exposure of fixed, activated PMN to solutions having increasing ionic strength results in elution of PR3, HLE, and CG, and there is a direct relationship between their order of elution and their isoelectric points. These data indicate that rapidly inducible PR3 expressed on the cell surface of PMN is an important bioactive form of the proteinase. If PR3 expression on the cell surface of PMN is dysregulated, it is well equipped to amplify tissue injury directly, and also indirectly via the generation of autoantibodies.     

Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases

Serine proteinases of human polymorphonuclear neutrophils play an important role in neutrophil-mediated proteolytic events; however, the non-oxidative mechanisms by which the cells can degrade extracellular matrix in the presence of proteinase inhibitors have not been elucidated. Herein, we provide the first report that human neutrophils express persistently active cell surface-bound human leukocyte elastase and cathepsin G on their cell surface. Unstimulated neutrophils have minimal cell surface expression of these enzymes; however, phorbol ester induces a 30-fold increase. While exposure of neutrophils to chemoattractants (fMLP and C5a) stimulates modest (two- to threefold) increases in cell surface expression of serine proteinases, priming with concentrations of lipopolysaccharide as low as 100 fg/ml leads to striking (up to 10-fold) increase in chemoattractant-induced cell surface expression, even in the presence of serum proteins. LPS-primed and fMLP-stimulated neutrophils have approximately 100 ng of cell surface human leukocyte elastase activity per 10(6) cells. Cell surface- bound human leukocyte elastase is catalytically active, yet is remarkably resistant to inhibition by naturally occurring proteinase inhibitors. These data indicate that binding of serine proteinases to the cell surface focuses and preserves their catalytic activity, even in the presence of proteinase inhibitors. Upregulated expression of persistently active cell surface-bound serine proteinases on activated neutrophils provides a novel mechanism to facilitate their egress from the vasculature, penetration of tissue barriers, and recruitment into sites of inflammation. Dysregulation of the cell surface expression of these enzymes has the potential to cause tissue destruction during inflammation.     

Leukocyte cell surface proteinases: regulation of expression, functions, and mechanisms of surface localization

A number of proteinases are expressed on the surface of leukocytes including members of the serine, metallo-, and cysteine proteinase superfamilies. Some proteinases are anchored to the plasma membrane of leukocytes by a transmembrane domain or a glycosyl phosphatidyl inositol (GPI) anchor. Other proteinases bind with high affinity to classical receptors, or with lower affinity to integrins, proteoglycans, or other leukocyte surface molecules. Leukocyte surface levels of proteinases are regulated by: (1) cytokines, chemokines, bacterial products, and growth factors which stimulate synthesis and/or release of proteinases by cells; (2) the availability of surface binding sites for proteinases; and/or (3) internalization or shedding of surface-bound proteinases. The binding of proteinases to leukocyte surfaces serves many functions including: (1) concentrating the activity of proteinases to the immediate pericellular environment; (2) facilitating pro-enzyme activation; (3) increasing proteinase stability and retention in the extracellular space; (4) regulating leukocyte function by proteinases signaling through cell surface binding sites or other surface proteins; and (5) protecting proteinases from inhibition by extracellular proteinase inhibitors. There is strong evidence that membrane-associated proteinases on leukocytes play critical roles in wound healing, inflammation, extracellular matrix remodeling, fibrinolysis, and coagulation. This review will outline the biology of membrane-associated proteinases expressed by leukocytes and their roles in physiologic and pathologic processes.     

Cytokines and proteases in invasive processes: molecular similarities between inflammation and cancer.

Tumor-derived serine proteinases and metalloproteinases have been associated with invasion and metastasis of cancer cells. Leukocytes, particularly monocytes/macrophages and neutrophils, actively synthesize and store these proteolytic enzymes. The production by tumor cells of chemotactic factors that attract white blood cells raises questions that are important for the basic researcher as well as the clinical scientist. Are the proteinases, which have the capacity to dissolve the extracellular matrix and by this solubilization promote cell migration, the same in tumor cells as in normal cells? Is the production of chemotactic factors by tumor cells a coincident epiphenomenon of the malignant state or a selective way to parasitize the host? Does the early attraction of leukocytes to the tumor site contribute to early host defense against cancer? Does our knowledge about mechanisms of action of cytokines have implications for therapy of the cancer patient? Recent experimental data give hints to the answers to these questions and make it possible to deduce a fundamental model of cytokine mediated proteolysis in tissue remodelling.     

Proteases produced by activated neutrophils are able to release soluble CD23 fragments endowed with proinflammatory effects.

Polymorphonuclear neutrophils (PMNs) are the major source of proteolytic activities involved mainly in tissue injuries observed in chronic inflammatory disorders. High levels of soluble forms of CD23 (the low-affinity receptor for IgE) were found in biological fluids from these patients, and recent reports focused on a CD23-mediated regulation of inflammatory response. In this context, we show here that co-culture of activated PMN with CD23+ B cells resulted in a drastic release of soluble CD23 fragments from the cell surface. This cleavage was inhibited by serine proteases inhibitors, including a1-antitrypsin. We next demonstrated that purified human leukocyte elastase or cathepsin G efficiently cleaved membrane CD23 on B cells with a high specificity. Soluble fragments released by serine proteases-mediated CD23 proteolysis stimulated resting monocytes to produce oxidative burst and proinflammatory cytokine without any co-stimulatory signal. This work strongly supports the idea that the capacity of PMN-derived proteases to release soluble forms of CD23 participates in the inflammatory process mediated by these cells.     

Elastase and cathepsin G of human monocytes: heterogeneity and subcellular localization to peroxidase-positive granules

We used antibodies to human leukocyte ("neutrophil") elastase and cathepsin G to localize the corresponding antigens in human neutrophils, monocytes, and alveolar macrophages by immunohistochemistry. Furthermore, we combined immunogold localization with enzyme histochemistry to localize proteinase antigens and endogenous peroxidase activity in the same sections. As expected, all neutrophils contained both elastase and cathepsin G, and the proteinases localized to granules with peroxidase activity. In contrast, marked heterogeneity in monocyte staining for elastase, cathepsin G, and endogenous peroxidase was found. Sixty percent or more were unstained, while the remainder varied greatly in staining intensity. The elastase and cathepsin G in monocytes were localized by immunoelectron microscopy, combined with histochemistry, to cytoplasmic granules which had peroxidase activity. Alveolar macrophages were unstained. Therefore, a subpopulation of peripheral blood monocytes contains leukocyte elastase and cathepsin G in a cell compartment from which these enzymes may potentially be released into the extracellular space. The occurrence of peroxidase and neutral proteinases in the same granules in monocytes could permit the H2O2-myeloperoxidase-halide system and the neutral proteinases to act in concert in such functions as microbe killing and extracellular proteolysis.     

Neutrophil serine proteinases activate human nonepithelial cells to produce inflammatory cytokines through protease-activated receptor 2

Protease-activated receptors (PARs) compose a family of G protein-coupled receptors activated by proteolysis with exposure of their tethered ligand. Recently, we reported that a neutrophil-derived serine proteinase, proteinase 3 (PR3), activated human oral epithelial cells through PAR-2. The present study examined whether other neutrophil serine proteinases, human leukocyte elastase (HLE), and cathepsin G (Cat G) activate nonepithelial cells, human gingival fibroblasts (HGF). HLE and Cat G as well as PR3 activated HGF to produce IL-8 and monocyte chemoattractant protein 1. Human oral epithelial cells but not HGF express mRNA and protein of secretory leukocyte protease inhibitor, an inhibitor of HLE and Cat G, and recombinant secretory leukocyte protease inhibitor clearly inhibited the activation of HGF induced by HLE and Cat G but not by PR3. HGF express PAR-1 and PAR-2 mRNA in the cells and the proteins on the cell surface. HLE and Cat G cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. Treatment with trypsin, an agonist for PAR-2, and a synthetic PAR-2 agonist peptide induced intracellular Ca(2+) mobilization and rendered cells refractory to subsequent stimulation with HLE and Cat G. The production of cytokine induced by HLE and Cat G and the PAR-2 agonist peptide was completely abolished by inhibition of phospholipase C. These findings suggest that neutrophil serine proteinases have equal ability to activate human nonepithelial cells through PAR-2 to produce inflammatory cytokines and may control a number of inflammatory processes such as periodontitis.     

Purification of properoxinectin, a myeloperoxidase homologue and its activation to a cell adhesion molecule

Peroxidases are important mediators of innate immune reactions throughout the animal kingdom. In many arthropods a myeloperoxidase homologue, peroxinectin, is known to function as a cell adhesion factor and an opsonin. Here, we report in the freshwater crayfish Pacifastacus leniusculus the isolation of properoxinectin, inactive in cell adhesion, and we also show that properoxinectin is produced in the mature blood cells whereas the hematopoietic tissue contains very little of this protein. Both properoxinectin and peroxinectin are catalytically active as peroxidases, at least when using low molecular weight substrates. The extracellular processing of properoxinectin into an active cell adhesion protein was found to involve proteolytic steps shared with the prophenoloxidase activating system to yield catalytically active phenoloxidase. Thus, the regulation of activities by two ancient metalloproteins, both potentially producing highly toxic substances aimed at pathogens, is carried out by limited proteolysis. The proteolytic processing is triggered in the presence of microbial compounds such as beta-glucans or lipopolysaccharide after the release of properoxinectin and prophenoloxidase activating serine proteinases from the blood cells.     

Activation of progelatinase A (MMP-2) by neutrophil elastase, cathepsin G, and proteinase-3: a role for inflammatory cells in tumor invasion and angiogenesis

Gelatinase A (MMP-2), a matrix metalloproteinase (MMP) involved in tumor invasion and angiogenesis, is secreted as an inactive zymogen (proMMP-2) and activated by proteolytic cleavage. Here we report that polymorphonuclear neutrophil (PMN)-derived elastase, cathepsin G, and proteinase-3 activate proMMP-2 through a mechanism that requires membrane-type 1 matrix metalloproteinase (MT1-MMP) expression. Immunoprecipitation of human PMN-conditioned medium with a mixture of antibodies to elastase, cathepsin G, and proteinase-3 abolished proMMP-2 activation, whereas individual antibodies were ineffective. Incubation of HT1080 cells with either purified PMN elastase or cathepsin G or proteinase-3 resulted in dose-and time-dependent proMMP-2 activation. Addition of PMN-conditioned medium to MT1-MMP expressing cells resulted in increased proMMP-2 activation and in vitro invasion of extracellular matrix (ECM), but had no effect with cells that express no MT1-MMP. MMP-2 activation by PMN-conditioned medium or purified elastase was blocked by the elastase inhibitor alpha(1)-antitrypsin but not by Batimastat, an MMP inhibitor, showing that elastase activation of MMP-2 is not mediated by MMP activities. The PMN-conditioned medium-induced increase in cell invasion was blocked by Batimastat as well as by alpha(1)-antitrypsin, showing that PMN serine proteinases trigger a proteinase cascade that entails proMMP-2 activation: this gelatinase is the downstream effector of the proinvasive activity of PMN proteinases. These findings indicate a novel role for PMN-mediated inflammation in a variety of tissue remodeling processes including tumor invasion and angiogenesis.     

Neutrophil serine proteinases cleave bacterial flagellin, abrogating its host response-inducing activity

After bacterial infection, neutrophils dominate the cellular infiltrate. Their main function is assumed to be killing invading pathogens and resolving the inflammation they cause. Activated neutrophils are also known to release a variety of molecules, including the neutrophil serine proteinases, extracellularly. The release of these proteinases during inflammation creates a proteolytic environment where degradation of different molecules modulates the inflammatory response. Flagellin, the structural component of flagella on many bacterial species, is a virulence factor with a strong proinflammatory activity on epithelial cells and other cell types. In this study we show that both human and mouse neutrophil serine proteinases cleave flagellin from Pseudomonas aeruginosa and other bacterial species. More important, cleavage of P. aeruginosa flagellin by the neutrophil serine proteinases neutrophil elastase and cathepsin G resulted in loss of the biological activity of this virulence factor, as evidenced by the lack of innate host defense gene expression in human epithelial cells. The finding that flagellin is susceptible to cleavage by neutrophil serine proteinases suggests a novel role for these enzymes in the inflammatory response to infection. Not only can these enzymes kill bacteria, but they also degrade their virulence factors to halt the inflammatory response they trigger.     

Exogenous leukocyte and endogenous elastases can mediate mitogenic activity in pulmonary artery smooth muscle cells by release of extracellular matrix-bound basic fibroblast growth factor

There is increasing evidence that extracellular matrix (ECM)-degrading proteinases contribute to the process of medial hypertrophy and neointimal proliferation in pulmonary vascular diseases. However, little is known about how proteinases, specifically elastases, induce vascular smooth muscle cell (SMC) hyperplasia. Our objective was to determine whether exogenous human leukocyte elastase (HLE), as well as endogenous vascular elastase, could release basic fibroblast growth factor (bFGF), a potent mitogen stored in the ECM surrounding SMCs. Cultured ovine and porcine pulmonary artery SMC were pre-incubated with [¹²⁵I]-bFGF. After removal of unbound [¹²⁵I]-bFGF, administration of HLE (0–1.0 μg/ml, 1 h) resulted in a concentration-dependent accumulation of [¹²⁵I]-bFGF in the conditioned medium, mirrored by depletion from the ECM. The serine elastase inhibitor elafin blocked this HLE-mediated action. Assessment by Western immunoblotting further demonstrated that HLE evoked the release of ECM-bound endogenous bFGF. When incubated with serum-starved SMC, conditioned medium from HLE-treated cells stimulated [³H]-thymidine incorporation, a feature neutralized by bFGF antibodies. In addition, SMC exposed to serum treated elastin (STE), previously shown to stimulate endogenous vascular elastase, liberated bioavailable bFGF from ECM stores, as determined by autoradiography, Western immunoblotting, and stimulation of DNA synthesis and SMC proliferation. Chondroitin sulfate, an inhibitor of STE-induced elastase activity, attenuated the release of bFGF. Our studies demonstrate that HLE, secreted by inflammatory cells, and endogenous vascular elastase release matrix-bound bFGF, suggesting a mechanism whereby elastases, through degradation of ECM, induce SMC proliferation associated with progressive vascular disease. © 1996 Wiley-Liss, Inc.     

Proteinases and their inhibitors in cells and tissues

A large body of evidence has been assembled to indicate the substantial importance of proteolytic processes in various physiological functions. It has recently become clear too that endo-acting peptide bond hydrolases provisionally characterized and classified at present as serine, cysteine, aspartic and metallo together with unknown catalytic mechanism proteinases sometimes act in cascades. They are controlled by natural proteinase inhibitors present in cells and body fluids. In the first part of the present monograph the author was concerned to present an overview on the morphological and physiological approach to localization, surveying reaction principles and methods suitable for visualization of proteolytic enzymes and their natural and synthetic inhibitors. In the second part the roles played by proteinases have been summarized from the point of view of cell biology. The selection of earlier and recent data reviewed on the involvement of proteolysis in the behavior of individual cells reveals that enzymes, whether they be exogeneous or intrinsic, can be effective and sensitive modulators of cellular growth and morphology. There exists a close correlation between malignant growth and degradation of cells. It appears likely that as yet unknown or at least so far inadequately characterized factors that influence the survival or the death of cells may turn out to be proteinases. The causal role of extracellular proteolysis in cancer cell metastases, in stopping cancer cell growth and in cytolysis remains for further investigated. Ovulation, fertilization and implantation are basic biological functions in which proteolytic enzymes play a key role. The emergence of new approaches in reproductive biology and a growing factual basis will inevitably necessitate a reevaluation of present knowledge of proteolytic processes involved. The molecular aspects of intracellular protein catabolism have been discussed in terms of the inhibition of lysosomal and/or non-lysosomal protein breakdown. Peptide and protein hormone biosynthesis and inactivation are still at the centre of interest in cell biology, and a number of proteinases have been implicated in both processes. A number of conjectures partly based on the author's own work have been discussed which suggest the possibility of the involvement of proteolysis in exocytosis and endocytosis. The author's optimistic conclusion is that through the common action of biochemists, cell biologists, cytochemists, and pharmacologists the mystery of cellular proteolysis is beginning to be solved.     

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

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

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 action of neutrophil serine proteases on elastin and its precursor

This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass spectrometric techniques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three proteases in tropoelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P₁, which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr. CG shows a strong preference for the charged amino acid Lys at P₁ in tropoelastin, whereas Lys was not identified at P₁ in CG digests of elastin due to extensive cross-linking at Lys residues in mature elastin. All three serine proteases showed a clear preference for Pro at P₂ and P₄′. With respect to the liberation of potentially bioactive peptides from elastin, the study revealed that all three serine proteases have a similar ability to release bioactive sequences, with CG producing the highest number of these peptides. In bioactivity studies, potentially bioactive peptides that have not been investigated on their bioactivity to date, were tested. Three new bioactive GxxPG motifs were identified; GVYPG, GFGPG and GVLPG.     

An unusual specificity in the activation of neutrophil serine proteinase zymogens

The majority of proteinases exist as zymogens whose activation usually results from a single proteolytic event. Two notable exceptions to this generalization are the serine proteinases neutrophil elastase (HNE) and cathepsin G (cat G), proteolytic enzymes of human neutrophils that are apparently fully active in their storage granules. On the basis of amino acid sequences inferred from the gene and cDNAs encoding these enzymes, it is likely that both are synthesized as precursors containing unusual C-terminal and N-terminal peptide extensions absent from the mature proteins. We have used biosynthetic radiolabeling and radiosequencing techniques to identify the kinetics of activation of both proteinases in the promonocyte-like cell line U937. We find that both N- and C-terminal extensions are removed about 90 min after the onset of synthesis, resulting in the activation of the proteinases. HNE and cat G are, therefore, transiently present as zymogens, presumably to protect the biosynthetic machinery of the cell from adventitious proteolysis. Activation results from cleavage following a glutamic acid residue to give an activation specificity opposite to those of almost all other serine proteinase zymogens, but shared, possibly, by the "granzyme" group of related serine proteinases present in the killer granules of cytotoxic T-lymphocytes and rat mast cell proteinase II.