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.     

The effects of heparin cofactor II-derived chemotaxins on neutrophil actin conformation and cyclic AMP levels

The serine proteinase inhibitor heparin cofactor II (HC) can be cleaved by polymorphonuclear leukocyte (PMN) elastase (LE) to yield potent chemotactic activity for PMN and monocytes. In contrast to the bacterially-derived chemotaxin formyl-Met-Leu-Phe (fMLP), the HC-derived chemotaxin does not stimulate PMN degranulation or oxidative burst activity. We compared the effects of HC-derived chemotaxins to the effects of fMLP on PMN actin conformation and on the cAMP levels. Both the HC chemotaxins and fMLP rapidly induced an increase in F-actin which was similar in magnitude and time-course. However, in contrast to fMLP, HC-derived chemotaxins did not elevate cAMP levels. HC-derived chemotaxins may be useful probes of chemotactic responses, since they do not have the mixed biological activities of fMLP.     

Human leukocyte elastase hydrolysis of peptides derived from human elastin exon 24

In normal and pathological tissues, polymorphonuclear leukocyte proteases (elastase, cathepsin G and proteinase 3) may generate soluble peptides through limited proteolysis of elastin, the main component of mature elastic fibres. Elastin-derived peptides display diverse biological activities including cell migration, differentiation, proliferation, chemotaxis, tumor progression and up-regulation of metalloproteinases. To be biologically active, their structures must adopt a beta-turn conformation which accommodates to the cell surface-located elastin binding protein. In this study, we established that human elastin exon 24-derived peptides are hydrolysed by leukocyte elastase, when the active site is fully occupied (from S(5) to S'(3)). As shown by mass spectrometry analyses, a major cleavage site was demonstrated at a Val-Ala bond and a minor one at Gly-Val bond. For longer peptides, the hydrolysed fragments could themselves be re-hydrolysed. If the shortest fragments do not contain the GxxPG sequence known to stimulate cellular effects, some of the intermediates together with hydrolysis fragments generated by other proteases such as proteinase 3, may possess this motif.     

Role of calpains and kininogens in inflammation.

Neutrophil chemotactic activity was found in the autodigest of calcium dependent cysteine proteinase (calpain) I purified from human erythrocytes, an active peptide was isolated, and its structure was determined. It was an N-acetyl nonapeptide with the sequence: N-acetyl Ser-Glu-Glu-Ile-Ile-Thr-Pro-Val-Tyr. This peptide was identical with the N-terminal amino acid sequence of the large subunit of calpain I deduced from cDNA sequence, except that the peptide was lacking a methionine residue and was acetylated at the N-terminus. A number of N-acetyl peptides with N-terminal amino acid sequences of large and small subunits of calpains I and II were synthesized and their chemotactic activity was estimated. In addition to the N-acetyl nonapeptide from calpain I large subunit, several peptides of different lengths from the small subunit showed dose-dependent migrations of neutrophils. They include N-acetyl tetra, hepta, octa, nona and larger size peptides. Further, it was also revealed that when calpain was incubated with high molecular weight (HMW) or low molecular weight (LMW) kininogen, kinin liberation occurred with simultaneous inhibition of calpains by kininogens. These data suggest that chemical mediators generated from the calpain-kininogen system may participate in migration and accumulation of neutrophils to the inflammatory locus.     

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.     

Proteolysis of macrophage inflammatory protein-1alpha isoforms LD78beta and LD78alpha by neutrophil-derived serine proteases

Macrophage inflammatory protein-1alpha (MIP-1alpha) is a chemokine that leads to leukocyte recruitment and activation at sites of infection. Controlling chemokine activity at sites of infection is important, since excess accumulation of leukocytes may contribute to localized tissue damage. Neutrophil-derived serine proteases modulate the bioactivity of chemokine and cytokine networks through proteolytic cleavage. Because MIP-1alpha is temporally expressed with neutrophils at sites of infection, we examined proteolysis of MIP-1alpha in vitro by the neutrophil-derived serine proteases: cathepsin G, elastase, and proteinase 3. Recombinant human MIP-1alpha isoforms LD78beta and LD78alpha were expressed and purified, and the protease cleavage sites were analyzed by mass spectrometry and peptide sequencing. Chemotactic activities of parent and cleavage molecules were also compared. Both LD78beta and LD78alpha were cleaved by neutrophil lysates at Thr16-Ser17, Phe24-Ile25, Tyr28-Phe29, and Thr31-Ser32. This degradation was inhibited by serine protease inhibitors phenylmethylsulfonyl fluoride and 4-(2-aminoethyl)-benzenesulfonyl fluoride. Incubation of the substrates with individual proteases revealed that cathepsin G preferentially cleaved at Phe24-Ile25 and Tyr28-Phe29, whereas elastase and proteinase 3 cleaved at Thr16-Ser17 and Thr31-Ser32. Proteolysis of LD78beta resulted in loss of chemotactic activity. The role of these proteases in LD78beta and LD78alpha degradation was confirmed by incubation with neutrophil lysates from Papillon-Lefevre syndrome patients, demonstrating that the cell lysates containing inactivated serine proteases could not degrade LD78beta and LD78alpha. These findings suggest that severe periodontal tissue destruction in Papillon-Lefevre syndrome may be related to excess accumulation of LD78beta and LD78alpha and dysregulation of the microbial-induced inflammatory response in the periodontium.     

Divergent effects of alpha1-antitrypsin on neutrophil activation, in vitro

alpha1-Antitrypsin (AAT) is a major circulating serine proteinase inhibitor in humans. The anti-proteinase activity of AAT is inhibited by chemical modification. These include inter- or intramolecular polymerisation, oxidation, complex formation with target proteinases (e.g., neutrophil elastase), and/or cleavage by multi-specific proteinases. In vivo, several modified forms of AAT have been identified which stimulate biological activity in vitro unrelated to inhibition of serine proteinases. In this study we have examined the effects of native and polymerised AAT and C-36 peptide, a proteolytic cleavage product of AAT, on human neutrophil activation, in vitro. We show that the C-36 peptide displays striking concentration-dependent pro-inflammatory effects on human neutrophils, including induction of neutrophil chemotaxis, adhesion, degranulation, and superoxide generation. In contrast to C-36 peptide, native and polymerised AAT at similar and higher concentrations showed no effects on neutrophil activation. These results suggest that cleavage of AAT may not only abolish its proteinase inhibitor activity, but can also generate a powerful pro-inflammatory activator for human neutrophils.     

Chemotactic peptide uptake in acute pancreatitis: correlation with tissue accumulation of leukocytes.

Chemotactic peptides bind specifically to receptors on leukocyte membranes. This property makes them prospective vehicles to evaluate inflammation and infection. We used two well-established models of acute pancreatitis to quantitate the binding of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine-lysine (fMLFK) to leukocytes and its correlation to degree of organ inflammation. Uptake of the (99m)Tc-labeled nicotinyl hydrazine-derivatized chemotactic peptide analog fMLFK-HYNIC was measured in blood, pancreas, lung, and muscle specimens in rats with edematous or necrotizing pancreatitis and was compared with neutrophil sequestration assessed by myeloperoxidase activity and histology. Chemotactic peptide uptake in the pancreas was increased in mild and severe pancreatitis compared with controls, with higher levels in severe than in mild disease, and correlated with tissue myeloperoxidase activity (r = 0.7395, P < 0.001). Increased pulmonary uptake only in severe pancreatitis reflected pancreatitis-induced neutrophil sequestration in the lungs. Muscle uptake was unchanged compared with controls. Edema formation did not affect chemotactic peptide uptake. The data suggest that uptake of chemotactic peptides can contribute to quantitative assessment of neutrophils in localized inflammatory processes and is independent of associated edema formation or microcirculatory compromise.     

Neutrophil elastase cleaves the murine hemidesmosomal protein BP180/type XVII collagen and generates degradation products that modulate experimental bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease associated with autoantibodies against the hemidesmosomal proteins BP180 and BP230. In the IgG passive transfer model of BP, blister formation is triggered by anti-BP180 IgG and depends on complement activation, mast cell degranulation, and neutrophil recruitment. Mice lacking neutrophil elastase (NE) do not develop experimental BP. Here, we demonstrated that NE degrades recombinant mouse BP180 within the immunodominant extracellular domain at amino acid positions 506 and 561, generating peptide p561 and peptide p506. Peptide p561 is chemotactic for neutrophils both in vitro and in vivo. Local injection of NE into B6 mice recruits neutrophils to the skin, and neutrophil infiltration is completely blocked by co-injection with the NE inhibitor α1-proteinase inhibitor. More importantly, NE directly cleaves BP180 in mouse and human skin, as well as the native human BP180 trimer molecule. These results demonstrate that (i) NE directly damages the extracellular matrix and (ii) NE degradation of mouse BP180 generates neutrophil chemotactic peptides that amplify disease severity at the early stage of the disease.     

Proteinase 3 hydrolysis of peptides derived from human elastin exon 24

Summary. In normal and pathological tissues, elastin-derived peptides proceed of elastin degradation by polymorphonuclear leukocyte proteases: elastase, cathepsin G and proteinase 3. They were demonstrated to have a chemotactic activity, to promote cell proliferation and protease release, . . .. To be biologically active, their structures, which reflect elastase specificity, must adopt a β-turn conformation which accommodate to the cell surface-located elastin binding protein. In this study, we establish that human elastin exon 24-derived peptides containing at least two repeated VGVAPG sequences are hydrolyzed by the proteinase 3 (Pr3). As shown by mass spectrometry analyses, the demonstrated cleavage sites are in agreement with previously reported Pr3 substrate specificity and its lengthy substrate binding site. The characterization of the Pr3-generated products indicate that they contain at least one GXXPG sequence known to stimulate cellular effects after binding to the elastin receptor.     

Design of selective substrates of proteinase 3 using combinatorial chemistry methods

In this study, chemical synthesis of the selective chromogenic/fluorogenic substrates for proteinase 3 is described. The substrates’ sequence was obtained using combinatorial chemistry methods. Deconvolution of the tripeptide library against proteinase 3 with general formula ABZ-X₃-X₂-X₁-ANB-NH₂ yielded the active sequence. Selected peptide was further modified on its C terminus to investigate the impact of chromophore moiety modification on enzyme-substrate interaction. To determine specificity, activity of selected substrates was characterized against proteinase 3 and neutrophil elastase. Finally, the peptide ABZ-Tyr-Tyr-Abu-ANB-NH₂ displayed the highest value of specificity constant (k_cat/K_M = 189 × 10³ M⁻¹ s⁻¹) for proteinase 3. To the best of our knowledge, this is the first short peptide that undergoes selective proteolysis by proteinase 3 and displays no significant hydrolysis in the presence of human neutrophil elastase and cathepsin G.     

Differences in basal and ethanol-induced levels of opioid peptides in Wistar rats from five different suppliers

Mammalian cationic antimicrobial peptides have received increased attention over the last decade, due to their prokaryotic selectivity and decreased risk of microbial resistance. In addition, antimicrobial peptides display differential biological effects on mammalian immune cell function, such as migration, adhesion, and modulation of respiratory burst, which make them even more attractive as therapeutic agents. Synthetic combinatorial libraries provide a time-efficient and cost-effective source for these diverse molecules. The novel synthetic antimicrobial peptide, KSLW (KKVVFWVKFK-NH(2)), has been shown to display a broad spectrum of antimicrobial activity against Gram (+) and Gram (-) bacteria, fungi and viruses. In this study, we evaluated the alternative biological activity of the decapeptide on neutrophil migration and function. KSLW was demonstrated to be chemotactic for neutrophils in micromolar amounts, and neutrophil treatment with KSLW, after 1 min, resulted in significant increases in F-actin polymerization. KSLW was shown to inhibit oxygen radical production in PMA- and LPS-stimulated neutrophils. Future studies, to determine if KSLW regulates neutrophil phagocytosis, adhesion, and apoptosis, or examining the effect of KSLW on other mammalian cell types, such as cell populations of healing-impaired wounds, would provide significant insight for the potential therapeutic strategies offered by antimicrobial peptides.     

Limited Proteolysis of Angiogenin by Elastase Is Regulated by Plasminogen

Human neutrophil elastase cleaves angiogenin at the Ile-29/Met-30 peptide bond to produce two major disulfide-linked fragments with apparent molecular weights of 10,000 and 4000, respectively. Elastase-cleaved angiogenin has slightly increased ribonucleolytic activity, but has lost its ability to undergo nuclear translocation in endothelial cells, a process essential for angiogenic activity. Cleavage appears to alter the cell-binding properties of angiogenin, despite the fact that it occurs some distance from the putative receptor-binding site, since the elastase-cleaved protein fails to compete with its native counterpart for nuclear translocation in endothelial cells. Plasminogen specifically accelerates elastase proteolysis of angiogenin. It does not enhance elastase activity toward ribonuclease A or the synthetic peptide substrate MeOSuc-Ala-Ala-Pro-Val-pNA. Plasminogen-accelerated inactivation of angiogenin by elastase might be a significant event in the process of angiogenin-induced angiogenesis since (i) angiogenin and plasminogen circulate in plasma at high concentrations, (ii) angiogenin, especially when bound to actin, activates tissue plasminogen activator to generate plasmin from plasminogen, and (iii) elastase cleaves plasminogen to produce angiostatin, a potent inhibitor of angiogenesis and metastasis. Interrelationships among angiogenin, plasminogen, plasminogen activators, elastase, and angiostatin may provide a sensitive regulatory system to balance angiogenesis and antiangiogenesis.     

Alpha 1-proteinase inhibitor is a neutrophil chemoattractant after proteolytic inactivation by macrophage elastase

Mouse macrophage elastase, a metalloproteinase, catalytically inactivates human alpha 1-proteinase inhibitor (alpha 1-PI) by attacking a single peptide bond between Pro357 and Met358, resulting in Mr = 4,200 and 47,800 fragments. We show here that this proteolytically inactivated alpha 1-PI is a potent chemotactic factor for human neutrophils at a concentration of 1 nM. The chemotactic response is equivalent to that elicited by formyl-methionyl-leucyl-phenylalanine. Native alpha 1-PI does not stimulate chemotaxis. Purification of the two fragments of alpha 1-PI that result from proteolysis by macrophage elastase indicated that the Mr = 4,200 fragment is responsible for the chemotactic activity. However, the two proteolysis fragments do not dissociate from each other under physiologic conditions. Therefore, the ability of proteolytically inactivated alpha 1-PI to act as a mediator of inflammation is due to rearrangement of the alpha 1-PI molecule rather than to release of a cleavage fragment.     

Identification of a cross-linked peptide of a covalent complex between adrenodoxin reductase and adrenodoxin.

A cross-linked complex between bovine NADPH-adrenodoxin reductase (AR) and adrenodoxin (AD) was prepared with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and purified, as described previously [Hara, T. & Kimura, T. (1989) J. Biochem. 105, 594-600]. The covalent complex was S-pyridylethylated and digested with lysylendopeptidase, and the resulting peptides were separated by reversed-phase HPLC to identify the cross-linked peptide. Comparison of the HPLC chromatograms of the peptides showed that (i) two tandem peptides (K-4 and K-5) from AD and a peptide (K-1) from AR were missing in the chromatogram of the peptides of the covalent complex and (ii) a single new peak was observed in the chromatogram of the peptides from the covalent complex. Amino acid composition and sequence analyses showed that the newly observed peptide was a covalently cross-linked peptide formed between a peptide K-4-K-5 (Ile-25-Lys-98) derived from AD and a peptide K-1 (Ser-1-Lys-27) derived from AR, in which an amide bond had been formed between the epsilon-amino group of Lys-66 in AD and the gamma-carboxyl group of Glu-4 in AR. These results indicate that the binding site of AR with AD is localized in the amino-terminal part of AR and that of AD with AR is localized around Lys-66 of AD. The six clustered basic amino acid residues (His-24, Lys-27, His-28, His-29, Arg-31, and His-33) present in the amino-terminal portion of AR and the eight clustered acidic amino acid residues (Glu-65, Glu-68, Asp-72, Glu-73, Glu-74, Asp-76, Asp-79, and Asp-86) present in the middle part of AD may play an important role in the complex formation.     

Calcium dependent cysteine proteinase is a precursor of a chemotactic factor for neutrophils

A new chemotactic factor for neutrophils is generated from calcium dependent cysteine proteinase (calpain) I by autodigestion. An active peptide was isolated from the autodigest and its structure was determined to be an acetylated nonapeptide with the sequence: N-acetyl Ser-Glu-Glu-Ile-Ile-Thr-Pro-Val-Tyr. Compared with the entire sequence of human calpain I, the peptide was identical with the N-terminal amino acid sequence of the large subunit deduced from the cDNA sequence, except that the peptide was devoid of a methionine residue and acetylated at the N-terminus. The acetyl nonapeptide was synthesized and its chemotactic activity was reconfirmed. The biological significance and possible role of this calpain derived chemotactic factor in inflammation are discussed.     

The role of secretory leukocyte proteinase inhibitor and elafin (elastase-specific inhibitor/skin-derived antileukoprotease) as alarm antiproteinases in inflammatory lung disease

Secretory leukocyte proteinase inhibitor and elafin are two low-molecular-mass elastase inhibitors that are mainly synthesized locally at mucosal sites. It is thought that their physicochemical properties allow them to efficiently inhibit target enzymes, such as neutrophil elastase, released into the interstitium. Historically, in the lung, these inhibitors were first purified from secretions of patients with chronic obstructive pulmonary disease and cystic fibrosis. This suggested that they might be important in controlling excessive neutrophil elastase release in these pathologies. They are upregulated by 'alarm signals' such as bacterial lipopolysaccharides, and cytokines such as interleukin-1 and tumor necrosis factor and have been shown to be active against Gram-positive and Gram-negative bacteria, so that they have joined the growing list of antimicrobial 'defensin-like' peptides produced by the lung. Their site of synthesis and presumed functions make them very attractive candidates as potential therapeutic agents under conditions in which the excessive release of elastase by neutrophils might be detrimental. Because of its natural tropism for the lung, the use of adenovirus-mediated gene transfer is extremely promising in such applications.     

High fidelity processing and activation of the human α-defensin HNP1 precursor by neutrophil elastase and proteinase 3

The azurophilic granules of human neutrophils contain four α-defensins called human neutrophil peptides (HNPs 1-4). HNPs are tridisulfide-linked antimicrobial peptides involved in the intracellular killing of organisms phagocytosed by neutrophils. The peptides are produced as inactive precursors (proHNPs) which are processed to active microbicides by as yet unidentified convertases. ProHNP1 was expressed in E. coli and the affinity-purified propeptide isolated as two species, one containing mature HNP1 sequence with native disulfide linkages ("folded proHNP1") and the other containing non-native disulfide linked proHNP1 conformers (misfolded proHNP1). Native HNP1, liberated by CNBr treatment of folded proHNP1, was microbicidal against Staphylococcus aureus, but the peptide derived from misfolded proHNP1 was inactive. We hypothesized that neutrophil elastase (NE), proteinase 3 (PR3) or cathepsin G (CG), serine proteases that co-localize with HNPs in azurophil granules, are proHNP1 activating convertases. Folded proHNP1 was converted to mature HNP1 by both NE and PR3, but CG generated an HNP1 variant with an N-terminal dipeptide extension. NE and PR3 cleaved folded proHNP1 to produce a peptide indistinguishable from native HNP1 purified from neutrophils, and the microbicidal activities of in vitro derived and natural HNP1 peptides were equivalent. In contrast, misfolded proHNP1 conformers were degraded extensively under the same conditions. Thus, NE and PR3 possess proHNP1 convertase activity that requires the presence of the native HNP1 disulfide motif for high fidelity activation of the precursor in vitro.     

Synthetic peptides from the N-domains of CEACAMs activate neutrophils.

Four members of the carcinoembryonic antigen family, CEACAM1, CEACAM8, CEACAM6 and CEACAM3, recognized by CD66a, CD66b, CD66c and CD66d monoclonal antibodies (mAb), respectively, are expressed on human neutrophils. CD66a, CD66b, CD66c and CD66d mAb binding to neutrophils triggers an activation signal that regulates the adhesive activity of CD11/CD18, resulting in an increase in neutrophil adhesion to human umbilical vein endothelial cells. Molecular modeling of CEACAM1 using IgG and CD4 as models has been performed, and three peptides from the N-terminal domain were found to increase neutrophil adhesion to human umbilical vein endothelial cell monolayers. The peptides were 14 amino acids in length and were predicted to be present at loops and turns between beta-sheets. To better understand the amino acid sequences critical for this biological activity, in the present study we examined the other neutrophil CEACAMs and the highly homologous CEACAM, CEA. Molecular modeling of the N-terminal domains of human CEACAM8, -6, -3 and CEA was performed. Twenty peptides, each 14 amino acids in length, that were homologous to the previously reported peptides from the N-domains of CEACAM1, were synthesized and tested for their ability to alter neutrophil adhesion. Only one new peptide, from the N-domain of CEA, was found to increase neutrophil adhesion, and this peptide differed from the corresponding CEACAM1 peptide by only a single conservative amino acid substitution. Importantly, minor amino acid differences between active and inactive homologous peptides suggest regions of these peptides that are critical for biological activity. The data suggest that the regions SMPF of peptide CD66a-1, QLFG of peptide CD66a-2 and NRQIV of peptide CD66a-3 are critical for the activities of these peptides, and for the native CEACAMs.