Identification of proteases involved in the proteolysis of vascular endothelium cadherin during neutrophil transmigration

Transmigration of neutrophils across the endothelium occurs at the cell-cell junctions where the vascular endothelium cadherin (VE cadherin) is expressed. This adhesive receptor was previously demonstrated to be involved in the maintenance of endothelium integrity. We propose that neutrophil transmigration across the vascular endothelium goes in parallel with cleavage of VE cadherin by elastase and cathepsin G present on the surface of neutrophils. This hypothesis is supported by the following lines of evidence. 1) Proteolytic fragments of VE cadherin are released into the culture medium upon adhesion of neutrophils to endothelial cell monolayers; 2) conditioned culture medium, obtained after neutrophil adhesion to endothelial monolayers, cleaves the recombinantly expressed VE cadherin extracellular domain; 3) these cleavages are inhibited by inhibitors of elastase; 4) VE cadherin fragments produced by conditioned culture medium or by exogenously added elastase are identical as shown by N-terminal sequencing and mass spectrometry analysis; 5) both elastase- and cathepsin G-specific VE cadherin cleavage patterns are produced upon incubation with tumor necrosis factor alpha-stimulated and fixed neutrophils; 6) transendothelial permeability increases in vitro upon addition of either elastase or cathepsin G; and 7) neutrophil transmigration is reduced in vitro in the presence of elastase and cathepsin G inhibitors. Our results suggest that cleavage of VE cadherin by neutrophil surface-bound proteases induces formation of gaps through which neutrophils transmigrate.     

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.     

The cellular distribution of extracellular superoxide dismutase in macrophages is altered by cellular activation but unaffected by the naturally occurring R213G substitution

Extracellular superoxide dismutase (EC-SOD) is responsible for the dismutation of the superoxide radical produced in the extracellular space and known to be expressed by inflammatory cells, including macrophages and neutrophils. Here we show that EC-SOD is produced by resting macrophages and associated with the cell surface via the extracellular matrix (ECM)-binding region. Upon cellular activation induced by lipopolysaccharide, EC-SOD is relocated and detected both in the cell culture medium and in lipid raft structures. Although the secreted material presented a significantly reduced ligand-binding capacity, this could not be correlated to proteolytic removal of the ECM-binding region, because the integrity of the material recovered from the medium was comparable to that of the cell surface-associated protein. The naturally occurring R213G amino acid substitution located in the ECM-binding region of EC-SOD is known to affect the binding characteristics of the protein. However, the analysis of macrophages expressing R213G EC-SOD did not present evidence of an altered cellular distribution. Our results suggest that EC-SOD plays a dynamic role in the inflammatory response mounted by activated macrophages.     

The C-terminal proteolytic processing of extracellular superoxide dismutase is redox regulated

The antioxidant protein extracellular superoxide dismutase (EC-SOD) encompasses a C-terminal region that mediates interactions with a number of ligands in the extracellular matrix (ECM). This ECM-binding region can be removed by limited proteolysis before secretion, thus supporting the formation of EC-SOD tetramers with variable binding capacity. The ECM-binding region contains a cysteine residue (Cys219) that is known to be involved in an intersubunit disulfide bridge. We have determined the redox potential of this disulfide bridge and show that both EC-SOD dimers and EC-SOD monomers are present within the intracellular space. The proteolytic processing of the ECM-binding region in vitro was modulated by the redox status of Cys219, allowing cleavage under reducing conditions only. When wild-type EC-SOD or the monomeric variant Cys219Ser was expressed in mammalian cells proteolysis did not occur. However, when cells were exposed to oxidative stress conditions, proteolytic processing was observed for wild-type EC-SOD but not for the Cys219Ser variant. Although the cellular response to oxidative stress is complex, our data suggest that proteolytic removal of the ECM-binding region is regulated by the intracellular generation of an EC-SOD monomer and that Cys219 plays an important role as a redox switch allowing the cellular machinery to secrete cleaved EC-SOD.     

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.     

Cathelicidin family of antimicrobial peptides: proteolytic processing and protease resistance

Cathelicidins are a gene family of antimicrobial peptides produced as inactive precursors. Signal peptidase removes the N-terminal signal sequence, while peptidylglycine alpha-amidating monooxygenase often amidates and cleaves the C-terminal region. Removal of the cathelin domain liberates the active antimicrobial peptide. For mammalian sequences, this cleavage usually occurs through the action of elastase, but other tissue-specific processing enzymes may also operate. Once released, these bioactive peptides are susceptible to proteolytic degradation. We propose that some mature cathelicidins are naturally resistant to proteases due to their unusual primary structures. Among mammalian cathelicidins, proline-rich sequences should resist attack by serine proteases because proline prevents cleavage of the scissile bond. In hagfish cathelicidins, the unusual amino acid bromotryptophan may make the active peptides less susceptible to proteolysis for steric reasons. Such protease resistance could extend the pharmacokinetic lifetimes of cathelicidins in vivo, sustaining antimicrobial activity.     

The serpin MNEI inhibits elastase-like and chymotrypsin-like serine proteases through efficient reactions at two active sites.

MNEI (monocyte/neutrophil elastase inhibitor) is a 42 kDa serpin superfamily protein characterized initially as a fast-acting inhibitor of neutrophil elastase. Here we show that MNEI has a broader specificity, efficiently inhibiting proteases with elastase- and chymotrypsin-like specificities. Reaction of MNEI with neutrophil proteinase-3, an elastase-like protease, and porcine pancreatic elastase demonstrated rapid inhibition rate constants >10(7) M(-1) s(-1), similar to that observed for neutrophil elastase. Reactions of MNEI with chymotrypsin-like proteases were also rapid: cathepsin G from neutrophils (>10(6) M(-1) s(-1)), mast cell chymase (>10(5) M(-1) s(-1)), chymotrypsin (>10(6) M(-1) s(-1)), and prostate-specific antigen (PSA), which had the slowest rate constant at approximately 10(4) M(-1) s(-1). Inhibition of trypsin-like (plasmin, granzyme A, and thrombin) and caspase-like (granzyme B) serine proteases was not observed or highly inefficient (trypsin), nor was inhibition of proteases from the cysteine (caspase-1 and caspase-3) and metalloprotease (macrophage elastase, MMP-12) families. The stoichiometry of inhibition for all inhibitory reactions was near 1, and inhibitory complexes were resistant to dissociation by SDS, further indicating the specificity of MNEI for elastase- and chymotrypsin-like proteases. Determination of the reactive site of MNEI by N-terminal sequencing and mass analysis of reaction products identified two reactive sites, each with a different specificity. Cys(344), which corresponds to Met(358), the P(1) site of alpha1-antitrypsin, was the inhibitory site for elastase-like proteases and PSA, while the preceding residue, Phe(343), was the inhibitory site for chymotrypsin-like proteases. This study demonstrates that MNEI has two functional reactive sites corresponding to the predicted P(1) and P(2) positions of the reactive center loop. The data suggest that MNEI plays a regulatory role at extravascular sites to limit inflammatory damage due to proteases of cellular origin.     

Degradation of cartilage matrix proteoglycan by human neutrophils involves both elastase and cathepsin G

The granule proteases of human neutrophils are thought to be responsible for the connective tissue destruction associated with certain inflammatory diseases. Using a model system for the degradation of a macromolecular connective tissue substrate, purified neutrophil elastase and cathepsin G were both individually able to degrade cartilage matrix proteoglycan and this degradation was blocked by the appropriate specific inhibitors. Neutrophil granule lysate also produced cartilage matrix degradation but little inhibition of degradation occurred when either elastase or cathepsin G inhibitor was used alone. However, a combination of elastase and cathepsin G inhibitors each at 100 microM or each at 10 microM blocked cartilage matrix degradation by 89% +/- 1 and 65% +/- 9 (mean +/- SEM, n = 3), respectively. The magnitude of the cartilage degradation mediated by neutrophil lysate, and its sensitivity to specific inhibitors, was reproduced using purified elastase and cathepsin G at the concentrations at which they are present in neutrophil lysate. Human neutrophils stimulated with opsonized zymosan degraded cartilage matrix in a dose-dependent manner in the presence of serum antiproteases. Supernatants from stimulated neutrophils cultured in the presence of serum did not degrade cartilage matrix, indicating that neutrophil mediated degradation in the presence of serum was confined to the protected subjacent region between the inflammatory cell and the substratum. A combination of elastase and cathepsin G inhibitors each at 500 microM or each at 100 microM blocked subjacent cartilage matrix degradation by stimulated human neutrophils by 91% +/- 3 and 54% +/- 8 (mean +/- SEM, n = 5), respectively, whereas either the elastase or cathepsin G inhibitor alone was much less effective. These studies demonstrate that neutrophil-mediated cartilage matrix degradation is produced primarily by elastase and cathepsin G. Furthermore, these results support the hypothesis that inflammatory neutrophils form zones of close contact with substratum that exclude serum antiproteases and that this subjacent degradation of cartilage matrix by stimulated neutrophils can be blocked by a combination of synthetic elastase and cathepsin G inhibitors.     

Proteolytic regulation of the urokinase receptor/CD87 on monocytic cells by neutrophil elastase and cathepsin G

The urokinase receptor (CD87) participates to the pericellular proteolytic potential of migrating cells and to the recruitment of leukocytes during inflammation. It consists of three structurally homologous domains, with the C-terminal domain D3 attached to cell membranes through a GPI anchor. CD87 is susceptible to an endoproteolytic processing removing the N-terminal domain D1 and generating truncated D2D3 membrane species, thus modulating CD87-associated functions. Full-length or truncated CD87 can be also released from cells via juxtamembrane cleavage by phospholipases and/or by yet unidentified proteinases. Using a recombinant CD87 and the CD87-positive monocytic U937 cell line and isolated blood monocytes, we show by protein immunoblotting and flow immunocytometry that the human neutrophil serine-proteinases elastase and cathepsin G cleave CD87 within the D1-D2 linker sequence, while in addition cathepsin G is highly efficient in cleaving the C terminus of D3. The combination of cathepsin G and elastase provided by degranulated neutrophils results in enzymatic cooperation leading to the release from monocytic cells of a truncated D2D3 species resembling that previously described in pathological body fluids. Using mass spectrometry analysis, the proteolytic fragmentation of synthetic peptides mapping the D1-D2 linker and D3 C-terminal domains identifies potential cleavage sites for each enzyme and suggests the existence of a mechanism regulating the CD87(D1-D2)-associated chemotactic activity. Finally, isolated or combined elastase and cathepsin G drastically reduce the capacity of cells to bind urokinase. Secretable leukocyte serine-proteinases are thus endowed with high potential for the regulation of CD87 expression and function on inflammatory cells.     

Proteolytic cleavage by neutrophil elastase converts inactive storage proforms to antibacterial bactenecins.

Bac5 and Bac7, antibiotics of the bactenecin (proline/arginine-rich peptide) family, are stored as proforms in the large granules of bovine neutrophils [Zanetti, M., Litteri, L., Gennaro, R., Horstmann, H. and Romeo, D. (1990) J. Cell Biol. 111, 1363-1371]. These proforms have been purified to homogeneity from granule extracts by immunoaffinity and reverse-phase chromatography. While mature bactenecins efficiently kill Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium with minimal inhibitory concentrations of 6-12 micrograms/ml, proBac5 and proBac7 do not affect the growth of the same microorganisms, even at 500 micrograms/ml. Previous investigations have suggested that the conversion of probactenecins into mature antimicrobial peptides is catalyzed by a neutral serine protease stored in the azurophil granules. Purified proBac5 and proBac7 were thus treated with elastase, cathepsin G or proteinase 3, which constitute the pool of neutral serine proteases of the azurophils, and the reaction products were identified by Western blot analysis, mass spectrometry, and N-terminal sequence analysis. Of the three proteases, only elastase is able to catalyze the stepwise cleavage of probactenecins into the corresponding mature peptides, which have the same mass, N-terminal sequence and antibiotic activity of authentic Bac5 and Bac7. These results point to the importance of cooperation between azurophils and large granules in mounting a defense reaction.     

Processing of the human transferrin receptor at distinct positions within the stalk region by neutrophil elastase and cathepsin G

The ectodomain of the human transferrin receptor (TfR) is released as soluble TfR into the blood by cleavage within a stalk. The major cleavage site is located C-terminally of Arg-100; alternative cleavage sites are also present. Since the cleavage process is still unclear, we looked for proteases involved in TfR ectodomain release. In the supernatant of U937 histiocytic cells we detected alternatively cleaved TfR (at Glu-110). In membrane fractions of these cells we identified two distinct proteolytic activities responsible for TfR cleavage within the stalk at either Val-108 or Lys-95. Both activities could be inhibited by serine protease inhibitors, but not by inhibitors of any other class of proteases. Protein purification yielded a 28 kDa protein that generated the Val-108 terminus. The protease activity could be ascribed to neutrophil elastase according to the substrate specificity determined by amino acid substitution analysis of synthetic peptides, an inhibitor profile, the size of the protease and the use of specific antibodies. The results of analogous experiments suggest that the second activity is represented by another serine protease, cathepsin G. Thus, membrane-associated forms of neutrophil elastase and cathepsin G may be involved in alternative TfR shedding in U937 cells.     

Neutrophil elastase cleaves laminin-332 (laminin-5) generating peptides that are chemotactic for neutrophils

Proteolytic processing of laminin-332 by matrix metalloproteinase (MMP)-2 and MMP-14 has been shown to yield fragments that are promigratory for epithelial cells. During acute and chronic inflammation, proteases are elaborated by neutrophils and macrophages that can degrade basement membranes. We investigated the susceptibility of laminin-332 to degradation by the following neutrophil and macrophage proteases: neutrophil elastase (NE), cathepsin G, proteinase-3, and MMPs-2, -8, -9, and -12. Protease-specific differences were seen in the capacity to cleave the individual chains of laminin-332. NE and MMP-12 showed the greatest activity toward the gamma2 chain, generating a fragment similar in size to the gamma2x fragment generated by MMP-2. The digestion pattern of laminin-332 by degranulated neutrophils was nearly identical to that generated with NE alone. Digestion by supernatants of degranulated neutrophils was blocked by an inhibitor of NE, and NE-deficient neutrophils were essentially unable to digest laminin-332, suggesting that NE is the major neutrophil-derived protease that degrades laminin-332. In vivo, laminin gamma2 fragments were found in the bronchoalveolar lavage fluid of wild-type mice treated with lipopolysaccharide, whereas that obtained from NE-deficient mice showed a different cleavage pattern. In addition, NE cleaved a synthetic peptide derived from the region of human laminin gamma2 containing the MMP-2 cleavage site, suggesting that NE may generate laminin-332 fragments that are also promigratory. Both laminin-332 fragments generated by NE digestion and NE-digested laminin gamma2 peptide were found to be chemotactic for neutrophils. Collectively, these data suggest that degradation of laminin-332 by NE generates fragments with important biological activities.     

Evidence for a crucial role of neutrophil-derived serine proteases in the inactivation of interleukin-6 at sites of inflammation

The bioactivity of interleukin-6 (IL-6) was found to be dramatically reduced in fluids from sites of inflammation. Here, we provide evidence that the neutrophil-derived serine proteases elastase, proteinase 3 and cathepsin G are mainly involved in its degradation and subsequent inactivation. The initially hydrolyzed peptide bonds were detected to be Val(11)-Ala(12) and Leu(19)-Thr(20) (elastase), Phe(78)-Asn(79) (cathepsin G) and Ala(145)-Ser(146) (proteinase 3). The soluble IL-6 receptor elicits a protective effect against the IL-6 inactivation by cathepsin G only. The inactivation of IL-6 by neutrophil-derived serine proteases might act as a feedback mechanism terminating the IL-6-induced activation of neutrophils.     

Antiprotease targeting: altered specificity of alpha 1-antitrypsin by amino acid replacement at the reactive centre

Alpha-1 antitrypsin (alpha 1AT) is an efficient inhibitor of the human neutrophil proteases, elastase and cathepsin G. The reactive centre P1 residue (Met358) of alpha 1AT is important in defining the specificity of inhibition; furthermore, oxidation of this residue results in a loss of inhibitor activity. There is evidence that oxidative inactivation of alpha 1AT may be involved in the pathogenesis of pulmonary emphysema associated with cigarette smoking. We have studied the effect of a series of amino acid replacements at the active centre on the inhibition properties of alpha 1AT. The mutant proteins were produced in E. coli following in vitro mutagenesis of the alpha 1AT cDNA. Alpha-1-AT (Ile358), (Ala358) and (Val358) were efficient inhibitors of both neutrophil and pancreatic elastase, but not cathepsin G. Alpha-1-AT (Ala356, Val358) and alpha 1AT (Phe358) were specific for pancreatic elastase and cathepsin G respectively. Alpha-1-AT (Leu358) inhibited both neutrophil elastase and cathepsin G. These data show that, for effective inhibition, a potential cleavage site for the protease must be displayed at the alpha 1AT active centre. In each case, replacement of Met358 led to resistance to oxidative inactivation. Since alpha 1AT (Leu358) inhibits both neutrophil proteases and is resistant to oxidation, this variant may be of increased potential for the therapy of destructive lung disorders.     

Nucleotide-induced changes in the proteolytically sensitive regions of myosin subfragment 1

Limited proteolytic digestions of myosin subfragment 1 (S-1) with elastase, subtilisin, papain, and thermolysin yield fragments that correspond within 1-2K daltons to the 25K, 50K, and 20K fragments produced by trypsin. While papain and thermolysin cut preferentially at the 26K/70K junction, elastase and subtilisin cleave both the 26K/70K and the 75K/22K junctions in S-1. Using the above proteases as conformational probes, we have previously demonstrated that the binding of actin is sensed at both the 26K/50K and the 50K/22K junctions [Applegate, D., & Reisler, E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7109-7112]. We report here that the binding of nucleotides at the active site is also sensed at both junctions. Both 2 mM MgADP and 5 mM MgATP slow the rate of elastase and subtilisin cleavage of the 95K heavy chain. With elastase, the 3-fold decrease in the rate of cleavage induced by nucleotides is evidenced at both the 26K/50K and the 50K/22K junctions. The analysis of subtilisin digestions is complicated by Mg nucleotide induced cleavage at a new site to produce a 91K fragment. Using N-methyl-6-anilinonaphthalene-2-sulfonyl chloride (MnsCl) to fluorescently label the 26K peptide, we demonstrate that the additional cleavage site is approximately 4K daltons from the N-terminal portion of the 95K heavy chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cathepsin G-regulated Release of Formyl Peptide Receptor Agonists Modulate Neutrophil Effector Functions

Neutrophil serine proteases play an important role in inflammation by modulating neutrophil effector functions. We have previously shown that neutrophils deficient in the serine proteases cathepsin G and neutrophil elastase (CG/NE neutrophils) exhibit severe defects in chemokine CXCL2 release and reactive oxygen species (ROS) production when activated on immobilized immune complex. Exogenously added active CG rescues these defects, but the mechanism remains undefined. Using a protease-based proteomic approach, we found that, in vitro, the addition of exogenous CG to immune complex-stimulated CG/NE neutrophils led to a decrease in the level of cell-associated annexin A1 (AnxA1) and cathelin-related antimicrobial peptide (CRAMP), both known inflammatory mediators. We further confirmed that, in vivo, CG was required for the extracellular release of AnxA1 and CRAMP in a subcutaneous air pouch model. In vitro, CG efficiently cleaved AnxA1, releasing the active N-terminal peptide Ac2-26, and processed CRAMP in limited fashion. Ac2-26 and CRAMP peptides enhanced the release of CXCL2 by CG/NE neutrophils in a dose-dependent manner via formyl peptide receptor (FPR) stimulation. Blockade of FPRs by an antagonist, Boc2 (t-Boc-Phe-d-Leu-Phe-d-Leu-Phe), abrogates CXCL2 release, whereas addition of FPR agonists, fMLF and F2L, relieves Boc2 inhibition. Furthermore, the addition of active CG, but not inactive CG, also relieves Boc2 inhibition. These findings suggest that CG modulates neutrophil effector functions partly by controlling the release (and proteolysis) of FPR agonists. Unexpectedly, we found that mature CRAMP, but not Ac2-26, induced ROS production through an FPR-independent pathway.     

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.     

The intracellular proteolytic processing of extracellular superoxide dismutase (EC-SOD) is a two-step event

Extracellular superoxide dismutase (EC-SOD) is a tetramer composed of either intact (Trp(1)-Ala(222)) or proteolytically cleaved (Trp(1)-Glu(209)) subunits. The latter form is processed intracellularly before secretion and lacks the C-terminal extracellular matrix (ECM)-binding region ((210)RKKRRRESECKAA(222)-COOH). We have previously suggested that the C-terminal processing of EC-SOD is either a one-step mechanism accomplished by a single intracellular endoproteolytic event cleaving the Glu(209)-Arg(210) peptide bond or a two-step mechanism involving two proteinases (Enghild, J. J., Thogersen, I. B., Oury, T. D., Valnickova, Z., Hojrup, P., and Crapo, J. D. (1999) J. Biol. Chem. 274, 14818-14822). In the latter case, an initial endoproteinase cleavage occurs somewhere in the region between Glu(209) and Glu(216). A carboxypeptidase specific for basic amino acid residues subsequently trims the remaining basic amino acid residues to Glu(209). A naturally occurring mutation of EC-SOD substituting Arg(213) for Gly enabled us to test these hypotheses. The mutation does not prevent proteolysis of the ECM-binding region but prevents a carboxypeptidase B-like enzyme from trimming residues beyond Gly(213). The R213G mutation is located in the ECM-binding region, and individuals carrying this mutation have an increased concentration of EC-SOD in the circulatory system. In this study, we purified the R213G EC-SOD variant from heterozygous or homozygous individuals and determined the C-terminal residue of the processed subunit to be Gly(213). This finding supports the two-step processing mechanism and indicates that the R213G mutation does not disturb the initial endoproteinase cleavage event but perturbs the subsequent trimming of the C terminus.     

Extracellular superoxide dismutase exists as an octamer

Human extracellular superoxide dismutase (EC-SOD) is involved in the defence against oxidative stress induced by the superoxide radical. The protein is a homotetramer stabilised by hydrophobic interactions within the N-terminal region. During the purification of EC-SOD from human aorta, we noticed that material with high affinity for heparin-Sepharose formed not only a tetramer but also an octamer. Analysis of the thermodynamic stability of the octamer suggested that the C-terminal region is involved in formation of the quaternary structure. In addition, we show that the octamer is composed of both aEC-SOD and iEC-SOD folding variants. The presence of the EC-SOD octamer with high affinity may represent a way to influence the local concentration of EC-SOD to protect tissues specifically sensitive to oxidative damage.     

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.