Studies on cytokine activation of listericidal activity in murine macrophages

The ability of a variety of soluble factors, alone or in combination, to endow murine resident peritoneal macrophages with listericidal activity was assessed. Inhibition of growth and (or) killing of Listeria in infected macrophages was determined by the uptake of [3H]uracil following lysis of the infected macrophage monolayers. Interferon-gamma was shown to induce modest listericidal activity in murine resident macrophages as compared with untreated monolayers. Treatment with tumour necrosis factor alpha also induced significant listericidal activity in this system. Among other cytokines tested, IL-4 induced an ability to inhibit growth of Listeria in resident macrophages. The ability of cytokines tested, IL-4 induced an ability to inhibit growth of Listeria in resident macrophages. The ability of cytokines to act in an additive or synergistic fashion with IFN-gamma was also investigated. Combinations of IFN-gamma and IL-4 and IFN-gamma and IL-2 induced listericidal activity not greater than that seen with IFN-gamma alone. IFN-gamma and TNF-alpha were shown to increase bactericidal activity in an additive fashion. However, elicited macrophages were shown to spontaneously exert a significant listericidal activity that was not enhanced by cytokine treatment. Collectively, these findings show that cytokine treatment induced rather modest enhancement in listericidal activity in murine resident peritoneal macrophages and no enhancement whatsoever in elicited macrophages. Thus, in in vivo situations where Listeria organisms are completely cleared from the infected organs, mechanisms other than lymphokine-induced listericidal activity of resident macrophages would seem to be operating.     

Methionine sulfoxide and proteolytic cleavage contribute to the inactivation of cathepsin G by hypochlorous acid: an oxidative mechanism for regulation of serine proteinases by myeloperoxidase

Using myeloperoxidase and hydrogen peroxide, activated neutrophils produce high local concentrations of hypochlorous acid (HOCl). They also secrete cathepsin G, a serine protease implicated in cytokine release, receptor activation, and degradation of tissue proteins. Isolated cathepsin G was inactivated by HOCl but not by hydrogen peroxide in vitro. We found that activated neutrophils lost cathepsin G activity by a pathway requiring myeloperoxidase, suggesting that oxidants generated by myeloperoxidase might regulate cathepsin G activity in vivo. Tandem mass spectrometric analysis of oxidized cathepsin G revealed that loss of a peptide containing Asp108, which lies in the active site, associated quantitatively with loss of enzymatic activity. Catalytic domain peptides containing Asp108 were lost from the oxidized protein in concert with the conversion of Met110 to the sulfoxide. Release of this peptide was blocked by pretreating cathepsin G with phenylmethylsulfonyl fluoride, strongly implying that oxidation introduced proteolytic cleavage sites into cathepsin G. Model system studies demonstrated that methionine oxidation can direct the regiospecific proteolysis of peptides by cathepsin G. Thus, oxidation of Met110 may contribute to cathepsin G inactivation by at least two distinct mechanisms. One involves direct oxidation of the thioether residue adjacent to the aspartic acid in the catalytic domain. The other involves the generation of new sites that are susceptible to proteolysis by cathepsin G. These observations raise the possibility that oxidants derived from neutrophils restrain pericellular proteolysis by inactivating cathepsin G. They also suggest that methionine oxidation could render cathepsin G susceptible to autolytic cleavage. Myeloperoxidase may thus play a previously unsuspected role in regulating tissue injury by serine proteases during inflammation.     

Additive enhancement of neutrophil collagenase activity by HOCl and cathepsin G.

Using preparations of latent collagenase derived from neutrophil specific granule extracts, the relative effects of cathepsin G and HOCl on activation of neutrophil collagenase were determined using a quantitative collagenase assay. Enhancement of collagenase activity by cathepsin G and physiologically relevant concentrations of HOCl were comparable, but HOCl mediated collagenase activation was reversible in the presence of HOCl scavenger. Collagenase activity in preparations treated with cathepsin G and HOCL simultaneously or sequentially was significantly greater than activity in preparations treated with HOCl or cathepsin G alone. The results indicated additive, yet independent enhancing effects of HOCl and cathepsin G on activity of neutrophil collagenase.     

Cathepsin G is a strong platelet agonist released by neutrophils.

The present studies were undertaken to characterize a serine protease released by N-formyl-L-Met-L-Leu-L-Phe (fMet-Leu-Phe)-stimulated neutrophils that rapidly induces platelet calcium mobilization, secretion and aggregation. The biological activity associated with this protease was unaffected by leupeptin, was only weakly diminished by N-p-tosyl-L-Lys-chloromethane, but was strongly inhibited by alpha 1-antitrypsin, soyabean trypsin inhibitor, N-tosyl-L-Phe-chloromethane and benzoyloxycarbonyl-Gly-Leu-Phe-chloromethane (Z-Gly-Leu-PheCH2Cl). These observations indicated that the biological activity of neutrophil supernatants could be attributed to a chymotrypsin-like enzyme such as cathepsin G. Furthermore, platelet aggregation and 5-hydroxytryptamine release induced by cell-free supernatants from fMet-Leu-Phe-stimulated neutrophils were found to be blocked by antiserum to cathepsin G in a concentration-dependent manner but were unaffected by antiserum to elastase. The biological activity present in neutrophil supernatants co-purified with enzymic activity for cathepsin G during sequential Aprotinin-Sepharose affinity chromatography and carboxymethyl-Sephadex chromatography. SDS/polyacrylamide-gel electrophoresis of the reduced, purified protein, demonstrated three polypeptides with apparent Mr values of 31,500, 29,000 and 28,000 and four polypeptides were resolved on acid-gel electrophoresis. Purified cathepsin G from neutrophils cross-reacted with anti-(cathepsin G) serum in a double immunodiffusion assay and elicited platelet calcium mobilization, 5-hydroxytryptamine secretion and aggregation. Calcium mobilization and secretion induced by low concentrations of cathepsin G were partially dependent on arachidonic acid metabolites and ADP, while stimulation by higher enzyme concentrations was independent of amplification pathways, indicating that cathepsin G is a strong platelet agonist. These results suggest that pathological processes which stimulate neutrophils and release cathepsin G can in turn result in the recruitment and activation of platelets.     

Cleavage of the protein III and major iron-regulated protein of Neisseria gonorrhoeae by lysosomal cathepsin G

Incubation of either 125I-labelled or unlabelled Neisseria gonorrhoeae with enzymically active preparations of human polymorphonuclear leucocyte lysosomal cathepsin G revealed that surface-exposed outer-membrane proteins were susceptible to proteolytic modification. Electroimmunoblotting experiments confirmed that outer-membrane protein III (PIII) and the major iron-regulated protein (MIRP), two conserved gonococcal proteins, were cleaved by cathepsin G. A direct relationship was observed between susceptibility to the antibacterial properties of cathepsin G and cleavage of PIII among isogenic strains differing in their level of resistance to the bactericidal activity of cathepsin G. Although the antibacterial property of cathepsin G is known to be independent of serine-esterase activity, the data suggest that gonococcal outer-membrane proteins are involved in the binding of cathepsin G, and that variation in the level of resistance reflects the degree to which target outer-membrane proteins such as PIII are exposed.     

Cathepsin G-Dependent Modulation of Platelet Thrombus Formation In Vivo by Blood Neutrophils

Neutrophils are consistently associated with arterial thrombotic morbidity in human clinical studies but the causal basis for this association is unclear. We tested the hypothesis that neutrophils modulate platelet activation and thrombus formation in vivo in a cathepsin G-dependent manner. Neutrophils enhanced aggregation of human platelets in vitro in dose-dependent fashion and this effect was diminished by pharmacologic inhibition of cathepsin G activity and knockdown of cathepsin G expression. Tail bleeding time in the mouse was prolonged by a cathepsin G inhibitor and in cathepsin G knockout mice, and formation of neutrophil-platelet conjugates in blood that was shed from transected tails was reduced in the absence of cathepsin G. Bleeding time was highly correlated with blood neutrophil count in wildtype but not cathepsin G deficient mice. In the presence of elevated blood neutrophil counts, the anti-thrombotic effect of cathepsin G inhibition was greater than that of aspirin and additive to it when administered in combination. Both pharmacologic inhibition of cathepsin G and its congenital absence prolonged the time for platelet thrombus to form in ferric chloride-injured mouse mesenteric arterioles. In a vaso-occlusive model of ischemic stroke, inhibition of cathepsin G and its congenital absence improved cerebral blood flow, reduced histologic brain injury, and improved neurobehavioral outcome. These experiments demonstrate that neutrophil cathepsin G is a physiologic modulator of platelet thrombus formation in vivo and has potential as a target for novel anti-thrombotic therapies.     

Interaction of heparin cofactor II with neutrophil elastase and cathepsin G

We investigated the interaction of the human plasma proteinase inhibitor heparin cofactor II (HC) with human neutrophil elastase and cathepsin G in order to examine 1) proteinase inhibition by HC, 2) inactivation of HC, and 3) the effect of glycosaminoglycans on inhibition and inactivation. We found that HC inhibited cathepsin G, but not elastase, with a rate constant of 6.0 x 10(6) M-1 min-1. Inhibition was stable, with a dissociation rate constant of 1.0 x 10(-3) min-1. Heparin and dermatan sulfate diminished inhibition slightly. Both neutrophil elastase and cathepsin G at catalytic concentrations destroyed the thrombin inhibition activity of HC. Inactivation was accompanied by a dramatic increase in heat stability, as occurs with other serine proteinase inhibitors. Proteolysis of HC (Mr 66,000) produced a species (Mr 58,000) that retained thrombin inhibition activity, and an inactive species of Mr 48,000. Amino acid sequence analysis led to the conclusion that both neutrophil elastase and cathepsin G cleave HC at Ile66, which does not affect HC activity, and at Val439, near the reactive site Leu444, which inactivates HC. Since cathepsin G is inhibited by HC and also inactivates HC, we conclude that cathepsin G participates in both reactions simultaneously so that small amounts of cathepsin G can inactivate a molar excess of HC. High concentrations of heparin and dermatan sulfate accelerated inactivation of HC by neutrophil proteinases, with heparin having a greater effect. Heparin and dermatan sulfate appeared to alter the pattern, and not just the rate, of proteolysis of HC. We conclude that while HC is an effective inhibitor of cathepsin G, it can be proteolyzed by neutrophil proteinases to generate first an active inhibitor and then an inactive molecule. This two-step mechanism might be important in the generation of chemotactic activity from the amino-terminal region of HC.     

Acquired cellular immunity: extracellular killing of Listeria monocytogenes by a product of immunologically activated macrophages

A listericidal factor was released by monolayers of peritoneal cells from BCG-immune guinea pigs following incubation with PPD in cell culture. Significantly less listericidal factor was released by monolayers of BCG-immune cells incubated without PPD, or by monolayers of nonimmune cells incubated in the presence or absence of antigen. Culture supernatants containing listericidal factor were active at a dilution of 1:10, but supernatants diluted 1:100 were not listericidal. Dialysis of supernatants against fresh tissue culture medium did not affect their ability to kill Listeria, although heating at 60 °C for 30 min destroyed all activity. Supernatants from cultures of guinea pig fibroblasts or ascites-form hepatoma were not listericidal.     

Identification of the primary antimicrobial domains in human neutrophil cathepsin G

Lysosomal cathepsin G from human neutrophils is a chymotrypsin-like protease which also possesses antimicrobial activity. The antimicrobial activity, however, is independent of protease activity, because treatment of this enzyme with the irreversible serine protease inhibitor diisopropylfluorophosphate has no effect on its antimicrobial action. In this study, we found that digestion of cathepsin G with clostripain caused a loss of proteolytic activity in this neutrophil proteinase. However, bactericidal activity in in vitro assays against Staphylococcus aureus and Neisseria gonorrhoeae was retained. Fractionation of the clostripain-digested cathepsin G mixture yielded two distinct antimicrobial peptides. The sequences of these peptides were IIGGR and HPQYNQR (residues 1-5 and 77-83 in cathepsin G, respectively). Synthetic peptides corresponding to these sequences were also prepared and found to exert broad-spectrum antimicrobial activity in vitro, displaying conditions of temperature- and pH-dependent optima for antimicrobial action resembling that of the full-length enzyme. Depending on the target bacterial strain, these peptides exhibited antimicrobial activity between 5.0 x 10(-5) and 4.0 x 10(-4) M. Significantly, replacement of certain residues within these peptides with either alanine or valine significantly reduced their antibacterial capacities. Our studies suggest that cathepsin G has two antimicrobial sequences, either or both of which may contribute to its bactericidal activity.     

How immune peptidases change specificity: cathepsin G gained tryptic function but lost efficiency during primate evolution

Cathepsin G is a major secreted serine peptidase of neutrophils and mast cells. Studies in Ctsg-null mice suggest that cathepsin G supports antimicrobial defenses but can injure host tissues. The human enzyme has an unusual "Janus-faced" ability to cleave peptides at basic (tryptic) as well as aromatic (chymotryptic) sites. Tryptic activity has been attributed to acidic Glu(226) in the primary specificity pocket and underlies proposed important functions, such as activation of prourokinase. However, most mammals, including mice, substitute Ala(226) for Glu(226), suggesting that human tryptic activity may be anomalous. To test this hypothesis, human cathepsin G was compared with mouse wild-type and humanized active site mutants, revealing that mouse primary specificity is markedly narrower than that of human cathepsin G, with much greater Tyr activity and selectivity and near absence of tryptic activity. It also differs from human in resisting tryptic peptidase inhibitors (e.g., aprotinin), while favoring angiotensin destruction at Tyr(4) over activation at Phe(8). Ala(226)Glu mutants of mouse cathepsin G acquire tryptic activity and human ability to activate prourokinase. Phylogenetic analysis reveals that the Ala(226)Glu missense mutation appearing in primates 31-43 million years ago represented an apparently unprecedented way to create tryptic activity in a serine peptidase. We propose that tryptic activity is not an attribute of ancestral mammalian cathepsin G, which was primarily chymotryptic, and that primate-selective broadening of specificity opposed the general trend of increased specialization by immune peptidases and allowed acquisition of new functions.     

Identification of a cathepsin G-like proteinase in the MCTC type of human mast cell

Human mast cells can be divided into two subsets based on serine proteinase composition: a subset that contains the serine proteinases tryptase and chymase (MCTC), and a subset that contains only tryptase (MCT). In this study we examined both types of mast cells for two additional proteinases, cathepsin G and elastase, which are the major serine proteinases of neutrophils. Because human mast cell chymase and cathepsin G are both chymotrypsin-like proteinases, the properties of these enzymes were further defined to confirm their distinctiveness. Comparison of their N-terminal sequences showed 30% nonidentity over the first 35 amino acids, and comparison of their amino acid compositions demonstrated a marked difference in their Arg/Lys ratios, which was approximately 1 for chymase and 10 for cathepsin G. Endoglycosidase F treatment increased the electrophoretic mobility of chymase on SDS gels, indicating significant N-linked carbohydrate on chymase; no effect was observed on cathepsin G. Immunoprecipitation and immunoblotting with specific antisera to each proteinase revealed little, if any, detectable cross-reactivity. Immunocytochemical studies showed selective labelling of MCTC type mast cells by cathepsin G antiserum in sections of human skin, lung, and bowel. No labeling of mast cells by elastase antiserum was detected in the same tissues, or in dispersed mast cells from lung and skin. A protein cross-reactive with cathepsin G was identified in extracts of human skin mast cells by immunoblot analysis. This protein had a slightly higher Mr (30,000) than the predominant form of neutrophil cathepsin G (Mr 28,000), and could not be separated from chymase (Mr 30,000) by SDS gel electrophoresis because of the size similarity. Using casein, a protein substrate hydrolyzed at comparable rates by chymase and cathepsin G, it was shown that about 30% of the caseinolytic activity in mast cell extracts was sensitive to inhibitors of cathepsin G that had no effect on chymase. Hydrolytic activity characteristic of elastase was not detected in these extracts. These studies indicate that human MCTC mast cells may contain two different chymotrypsin-like proteinases: chymase and a proteinase more closely related to cathepsin G, both of which are undetectable in MCT mast cells. Neutrophil elastase, on the other hand, was not detected in human mast cells by our procedures.     

Differential effects of concanavalin A and phytohemagglutinin on murine immunity. Suppression and enhancement of cell-mediated immunity.

To assess the effects of the selective T-cell mitogens concanavalin A (Con A) and phytohemagglutinin P (PHA) on cell-mediated immunity (CMI), the mitogens were injected before, with, or after intravenous (iv) challenge of mice with Listeria monocytogenes. Mitogenic treatment differentially influenced the CMI response to Listeria. Con A enhanced listericidal activity when given before or with Listeria challenge, but Con A suppressed the CMI response when given after infection with Listeria. In contrast, PHA enhanced listericidal activity at all intervals. Since Con A, but not PHA, affected the growth of Listeria in the spleens of mice 24 and 48 hr after infection, Con A was shown to have an immediate effect on the development of listericidal activity and PHA was shown to have a delayed effect. In addition, Con A induction of immediate nonspecific listericidal activity was short-lived, while PHA induced a longer-lasting effect on resistance to Listeria. The mitogen-induced effects in the CMI response to Listeria were shown to be dependent upon the activities of activated T cells. The enhancement and suppression of listericidal activity appears to result from the activation of different T-cell subpopulations, known to be stimulated preferentially by Con A or PHA.     

Cathepsin G binding to human platelets. Evidence for a specific receptor.

We have shown previously that purified human neutrophil cathepsin G is a strong platelet agonist. We now demonstrate that cathepsin G exhibits saturable, reversible binding to human platelets which is characteristic of binding to a specific receptor. At room temperature, cathepsin G displayed apparent positive co-operativity of binding, as indicated by sigmoidal binding curves and a Hill coefficient greater than unity. By contrast, binding curves conducted with native enzyme at 0.5 degrees C displayed a much smaller degree of sigmoidicity, and binding studies performed with phenylmethanesulphonyl fluoride-treated enzyme at 22-25 degrees C exhibited hyperbolic binding curves. The concentrations of cathepsin G required to give half-saturation (S0.5) with inhibitor-treated enzyme or with native enzyme at either room temperature or 0.5 degrees C were all similar, suggesting that sigmoidal binding curves did not result from an alteration in the affinity of the binding sites for cathepsin G. However, platelets bound approximately twice as many molecules of native enzyme as molecules of phenylmethanesulphonyl fluoride-treated cathepsin G per cell. From these observations it can be inferred that the apparent positive co-operativity may in part reflect the exposure of binding sites due to the proteolytic activity of cathepsin G. However, this conclusion is not supported by experiments conducted with subsaturating cathepsin G concentrations, which demonstrated that ligand binding did not show an expected increase at longer time intervals. Measurement of Ca2+ mobilization and cathepsin G binding in the same platelet suspensions demonstrated that elevations in cytosolic free Ca2+ concentration had achieved near-maximal levels in the presence of 15 micrograms of cathepsin G/ml, whereas maximal binding was observed at approx. 35 micrograms/ml, indicating that only a fraction of the total binding sites need be occupied to elicit platelet activation. Pretreatment of platelets with forskolin or phorbol 12-myristate 13-acetate (PMA) decreased cathepsin G binding by approx. 60% and 40% respectively, indicating that the receptor may be desensitized or down-regulated by phosphorylation due to protein kinases. Since forskolin and PMA could diminish receptor availability by activating negative feedback mechanisms, inhibition of negative signal-transduction pathways could conversely play a role in the up-regulation of cathepsin G binding. In any event, these results show that cathepsin G is an agonist that must bind to platelets to initiate processes associated with cell activation, and suggest a role for cathepsin G in platelet function.     

Isolation, characterization, and amino-terminal amino acid sequence analysis of human neutrophil cathepsin G from normal donors

Human neutrophil cathepsin G from normal donors has been purified 82-fold using an isolation procedure which included sequential sodium chloride extraction, Aprotonin-Sepharose affinity chromatography, CM-cellulose ion-exchange chromatography, and AcA44 gel filtration chromatography. The inclusion of this last purification step was crucial for separating inactive lower molecular weight species from the active forms of neutrophil cathepsin G and resulted in a higher specific activity of the final preparation. SDS polyacrylamide gradient gel electrophoresis of the purified reduced protein demonstrated three discrete polypeptides of Mr 31,000, 30,000, and 29,500. Peptide analysis of tryptic digests indicated that these three polypeptides are structurally related to each other and represent microheterogeneity of the purified protein. The cathepsin G peptide maps were distinctly different from the peptide maps of neutrophil elastase. The apparent isoelectric points of these forms as determined by two-dimensional electrophoresis was approximately 8.0. Utilizing microsequencing techniques, the first 25 residues of normal neutrophil cathepsin G have been determined and shown to be identical (except for residue 11) with the sequence of 21 residues of cathepsin G isolated from leukemic myeloid cells. A high degree of homology was found when the amino-terminal regions of neutrophil cathepsin G, rat mast cell protease II (65%) and two human serine proteinases, factor D (52%) and neutrophil elastase (48%), were compared. A precipitating monospecific antiserum to cathepsin G was produced by repeated immunizations of guinea pigs. This antiserum has been used in immunoblotting experiments to demonstrate that the intracellular form(s) of this enzyme is the same approximate Mr as the purified enzyme, and to develop a solid-phase radioimmunoassay for measuring neutrophil cathepsin G in the range 5-50 ng/ml.     

Kinetic mechanism of chicken liver xanthine dehydrogenase.

Human inter-alpha-trypsin inhibitor (I alpha I) is a plasma proteinase inhibitor active against cathepsin G, leucocyte elastase, trypsin and chymotrypsin. It owes its broad inhibitory specificity to tandem Kunitz-type inhibitory domains within an N-terminal region. Sequence studies suggest that the reactive-centre residues critical for inhibition are methionine and arginine. Reaction of I alpha I with the arginine-modifying reagent butane-2,3-dione afforded partial loss of inhibitory activity against both cathepsin G and elastase but complete loss of activity against trypsin and chymotrypsin. Reaction of I alpha I with the methionine-modifying reagent cis-dichlorodiammineplatinum(II) resulted in partial loss of activity against cathepsin G and elastase but did not affect inhibition of either trypsin or chymotrypsin. Employment of both reagents eliminated inhibition of cathepsin G and elastase. These findings suggest that both cathepsin G and elastase are inhibited at either of the reactive centres of I alpha I. Trypsin and chymotrypsin, however, appear to be inhibited exclusively at the arginine reactive centre.     

Human-derived vascular smooth muscle cells produce angiotensin II by changing to the synthetic phenotype

We investigated whether vascular smooth muscle cells (VSMC)-derived from human produce angiotensin (Ang) II upon change from the contractile phenotype to the synthetic phenotype by incubation with fibronectin (FN). Expression of alpha-smooth muscle (SM) actin, apparent in the contractile phenotype, was decreased by FN. Expressions of matrix Gla and osteopontin, apparent in the synthetic phenotype, were increased by FN. Ang II measured by radioimmunoassay (RIA) was significantly increased in human VSMC by FN. Expression of mRNAs for Ang II-generating proteases cathepsin D, cathepsin G, ACE, and chymase was increased by FN. Expressions of cathepsin D and cathepsin G proteins were also increased by FN. Ang I-generating activity, which was inhibited by an aspartyl protease inhibitor pepstatin A, was readily detected in the conditioned medium from human VSMC. Antisense oligodeoxynucleotides (ODNs) that hybridize with cathepsin D and cathepsin G significantly inhibited FN-increased Ang II in conditioned medium and cell extracts. In VSMC conditioned medium, FN-induced elevation of Ang II was significantly inhibited by temocapril but not by chymostatin. Ang II type 1 receptor antagonist CV11974 completely, and antisense cathepsin D and cathepsin G ODNs partially inhibited the FN-stimulated growth of human VSMC. These results indicate that the change of homogeneous cultures of human VSMC from the contractile to the synthetic phenotype sequentially increases expression of proteases cathepsin D, cathepsin G, and ACE, production of Ang II and productions of growth factors, culminating in VSMC proliferation. These findings implicate a new mechanism for the pathogenesis of human vascular proliferative diseases.     

Human cathepsin G. Catalytic and immunological properties.

1. The specificity of cathepsin G, a neutral proteinase from human spleen, was examined by use of low-molecular-weight substrates. The enzyme was found to hydrolyse several synthetic substrates also hydrolysed by chymotrypsin, but with different kinetic constants. 2. Maximal activity against benzoyl-DL-phenylalanine 2-naphthol ester and azo-casein was in the range pH 7.5-8.0. 3. The sensitivity of cathepsin G to the action of potential inhibitors was determined, and compared with those of bovine chymotrypsin and subtilisin. Cathepsin G showed the characteristics of a serine proteinase, but was less affected by the chloromethyl ketone of tosylphenylalanine than was chymotrypsin. 4. A rabbit anti-(human cathepsin G) serum was raised, and precipitin lines formed in agarose gel were stained for activity of the enzyme. 5. Cathepsin G was shown to be immunologically identical with the chymotrypsin-like enzyme of the azurophil granules of the neutrophil granulocytes.     

New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops.

Cathepsin G has both trypsin- and chymotrypsin-like activity, but studies on its enzymatic properties have been limited by a lack of sensitive synthetic substrates. Cathepsin G activity is physiologically controlled by the fast acting serpin inhibitors alpha1-antichymotrypsin and alpha1-proteinase inhibitor, in which the reactive site loops are cleaved during interaction with their target enzymes. We therefore synthesized a series of intramolecularly quenched fluorogenic peptides based on the sequence of various serpin loops. Those peptides were assayed as substrates for cathepsin G and other chymotrypsin-like enzymes including chymotrypsin and chymase. Peptide substrates derived from the alpha1-antichymotrypsin loop were the most sensitive for cathepsin G with kcat/Km values of 5-20 mM-1 s-1. Substitutions were introduced at positions P1 and P2 in alpha1-antichymotrypsin-derived substrates to tentatively improve their sensitivity. Replacement of Leu-Leu in ortho-aminobenzoyl (Abz)-Thr-Leu-Leu-Ser-Ala-Leu-Gln-N-(2, 4-dinitrophenyl)ethylenediamine (EDDnp) by Pro-Phe in Abz-Thr-Pro-Phe-Ser-Ala-Leu-Gln-EDDnp produced the most sensitive substrate of cathepsin G ever reported. It was cleaved with a specificity constant kcat/Km of 150 mM-1 s-1. Analysis by molecular modeling of a peptide substrate bound into the cathepsin G active site revealed that, in addition to the protease S1 subsite, subsites S1' and S2' significantly contribute to the definition of the substrate specificity of cathepsin G.     

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.