Chemerin Activation by Serine Proteases of the Coagulation, Fibrinolytic, and Inflammatory Cascades
2005; Elsevier BV; Volume: 280; Issue: 41 Linguagem: Inglês
10.1074/jbc.m504868200
ISSN1083-351X
AutoresBrian A. Zabel, Samantha J. Allen, Paulina Kulig, Jessica A. Allen, Joanna Cichy, Tracy M. Handel, Eugene C. Butcher,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoProteases function at every level in host defense, from regulating vascular hemostasis and inflammation to mobilizing the “rapid responder” leukocytes of the immune system by regulating the activities of various chemoattractants. Recent studies implicate proteolysis in the activation of a ubiquitous plasma chemoattractant, chemerin, a ligand for the G-protein-coupled receptor CMKLR1 present on plasmacytoid dendritic cells and macrophages. To define the pathophysiologic triggers of chemerin activity, we evaluated the ability of serum- and inflammation-associated proteases to cleave chemerin and stimulate CMKLR1-mediated chemotaxis. We showed that serine proteases factor XIIa and plasmin of the coagulation and fibrinolytic cascades, elastase and cathepsin G released from activated neutrophil granules and mast cell tryptase are all potent activators of chemerin. Activation results from cleavage of the labile carboxyl terminus of the chemoattractant at any of several different sites. Activation of chemerin by the serine protease cascades that trigger rapid defenses in the body may direct CMKLR1-positive plasmacytoid dendritic cell and tissue macrophage recruitment to sterile sites of tissue damage, as well as trafficking to sites of infectious and allergic inflammation. Proteases function at every level in host defense, from regulating vascular hemostasis and inflammation to mobilizing the “rapid responder” leukocytes of the immune system by regulating the activities of various chemoattractants. Recent studies implicate proteolysis in the activation of a ubiquitous plasma chemoattractant, chemerin, a ligand for the G-protein-coupled receptor CMKLR1 present on plasmacytoid dendritic cells and macrophages. To define the pathophysiologic triggers of chemerin activity, we evaluated the ability of serum- and inflammation-associated proteases to cleave chemerin and stimulate CMKLR1-mediated chemotaxis. We showed that serine proteases factor XIIa and plasmin of the coagulation and fibrinolytic cascades, elastase and cathepsin G released from activated neutrophil granules and mast cell tryptase are all potent activators of chemerin. Activation results from cleavage of the labile carboxyl terminus of the chemoattractant at any of several different sites. Activation of chemerin by the serine protease cascades that trigger rapid defenses in the body may direct CMKLR1-positive plasmacytoid dendritic cell and tissue macrophage recruitment to sterile sites of tissue damage, as well as trafficking to sites of infectious and allergic inflammation. A network of serine proteases regulates the primary response to injury and infection in the host. Serine proteases of the coagulation and fibrinolytic cascades mediate the homeostatic response to blood vessel injury. Kallikrein and factor XIIa process kininogens to generate bradykinin, a potent vasodilator that triggers increased vascular permeability during inflammation. Serine proteases termed convertases release multiple pathogen-neutralizing components of activated complement. Serine protease cascades also regulate the recruitment of phagocytic and antigen-presenting cells to sites of inflammation and tissue damage. The complement cascade, for example, releases active components C5a and C3a, potent attractants for many leukocytes, including neutrophils and monocytes (1Fernandez H.N. Henson P.M. Otani A. Hugli T.E. J. Immunol. 1978; 120: 109-115PubMed Google Scholar, 2Falk W. Leonard E.J. Infect. Immun. 1980; 29: 953-959Crossref PubMed Google Scholar). Thus serine proteases are critical participants in rapid defense mechanisms in the body. We and others have recently identified chemerin as a potent chemoattractant for cells expressing the G-protein-linked receptor chemokine-like receptor 1 (CMKLR1), 5The abbreviations used are: CMKLR1, chemokine-like receptor 1; pDC, plasmacytoid dendritic cell; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; tPA, tissue-type plasminogen activator; uPA, urokinase-type plasminogen activator. 5The abbreviations used are: CMKLR1, chemokine-like receptor 1; pDC, plasmacytoid dendritic cell; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; tPA, tissue-type plasminogen activator; uPA, urokinase-type plasminogen activator. also known as ChemR23 or DEZ (3Wittamer V. Franssen J.D. Vulcano M. Mirjolet J.F. Le Poul E. Migeotte I. Brezillon S. Tyldesley R. Blanpain C. Detheux M. Mantovani A. Sozzani S. Vassart G. Parmentier M. Communi D. J. Exp. Med. 2003; 198: 977-985Crossref PubMed Scopus (677) Google Scholar, 4Meder W. Wendland M. Busmann A. Kutzleb C. Spodsberg N. John H. Richter R. Schleuder D. Meyer M. Forssmann W.G. FEBS Lett. 2003; 555: 495-499Crossref PubMed Scopus (154) Google Scholar, 5Zabel B.A. Silverio A.M. Butcher E.C. J. Immunol. 2005; 174: 244-251Crossref PubMed Scopus (207) Google Scholar). CMKLR1 is expressed in vitro by monocyte-derived macrophages and dendritic cells (3Wittamer V. Franssen J.D. Vulcano M. Mirjolet J.F. Le Poul E. Migeotte I. Brezillon S. Tyldesley R. Blanpain C. Detheux M. Mantovani A. Sozzani S. Vassart G. Parmentier M. Communi D. J. Exp. Med. 2003; 198: 977-985Crossref PubMed Scopus (677) Google Scholar, 5Zabel B.A. Silverio A.M. Butcher E.C. J. Immunol. 2005; 174: 244-251Crossref PubMed Scopus (207) Google Scholar, 6Samson M. Edinger A.L. Stordeur P. Rucker J. Verhasselt V. Sharron M. Govaerts C. Mollereau C. Vassart G. Doms R.W. Parmentier M. Eur. J. Immunol. 1998; 28: 1689-1700Crossref PubMed Scopus (194) Google Scholar) and in vivo by circulating plasmacytoid dendritic cells (pDCs) (5Zabel B.A. Silverio A.M. Butcher E.C. J. Immunol. 2005; 174: 244-251Crossref PubMed Scopus (207) Google Scholar) and tissue macrophages. 6B. A. Zabel, manuscript in preparation. 6B. A. Zabel, manuscript in preparation. pDCs are major producers of α-interferons and can differentiate into antigen-presenting cells capable of triggering T effector or suppressor responses (7Colonna M. Trinchieri G. Liu Y.J. Nat. Immunol. 2004; 5: 1219-1226Crossref PubMed Scopus (1329) Google Scholar). Tissue macrophages have a major role as phagocytes but, similar to pDCs, are also implicated in bridging innate and adaptive immune responses and in regulating immunity in sterile versus infectious tissue injury (8Stoy N. Pathobiology. 2001; 69: 179-211Crossref PubMed Scopus (30) Google Scholar). Importantly, chemerin is widely expressed and circulates in human plasma in an inactive state (5Zabel B.A. Silverio A.M. Butcher E.C. J. Immunol. 2005; 174: 244-251Crossref PubMed Scopus (207) Google Scholar). Active forms of chemerin have been isolated from human ascites fluid, hemofiltrate, and serum and are characterized by apparent proteolytic cleavage of carboxyl-terminal amino acids present in the relatively inactive full-length pro-chemoattractant (3Wittamer V. Franssen J.D. Vulcano M. Mirjolet J.F. Le Poul E. Migeotte I. Brezillon S. Tyldesley R. Blanpain C. Detheux M. Mantovani A. Sozzani S. Vassart G. Parmentier M. Communi D. J. Exp. Med. 2003; 198: 977-985Crossref PubMed Scopus (677) Google Scholar, 4Meder W. Wendland M. Busmann A. Kutzleb C. Spodsberg N. John H. Richter R. Schleuder D. Meyer M. Forssmann W.G. FEBS Lett. 2003; 555: 495-499Crossref PubMed Scopus (154) Google Scholar, 5Zabel B.A. Silverio A.M. Butcher E.C. J. Immunol. 2005; 174: 244-251Crossref PubMed Scopus (207) Google Scholar). Here we have identified serine proteases as potent triggers of chemerin activation. Serum enzymes can activate recombinant chemerin, and this activity was blocked by selective serine protease inhibition. The clotting-associated serine proteases, factors XIIa and VIIa, and the fibrinolysis-associated serine proteases plasmin and plasminogen activators can all activate chemerin. Inflammatory cell-associated serine proteases, including neutrophil granule elastase and mast cell tryptase, are activators as well. Moreover, we found that several different cleavage sites, present in endogenous serum chemerin or generated during processing with specific enzymes, are sufficient for activation of this potent leukocyte attractant. These findings add chemerin to the list of innate immune mediators whose activity is critically regulated by serine protease cascades and implicate chemerin as an important link between these mechanisms of blood and tissue hemostasis and the recruitment of specialized CMKLR1-expressing “rapid responder” macrophages and dendritic cells. In Vitro Transwell Chemotaxis—CMKLR1/L1.2 transfectants were generated as previously described (5Zabel B.A. Silverio A.M. Butcher E.C. J. Immunol. 2005; 174: 244-251Crossref PubMed Scopus (207) Google Scholar). 5-μm pore Transwell inserts (Costar) were used for the migration assays. 100 μl of cells were added to the top well, and test samples were added to the bottom well in a 600-μl volume. The chemotaxis medium consisted of RPMI 1640 plus 10% fetal calf serum plus additives. Migration was assayed for 2 h at 37 °C, the inserts were removed, and the cells that had migrated through the filter to the lower chamber were counted by flow cytometry. ∼2.5 × 105 cells/well were used as input, and the number of cells counted in 30 s was defined as the migration output. The results are reported as % input migration or % maximal migration. Chemerin Expression and Purification Using Baculovirus—Chemerin with a carboxyl-terminal His6 tag was cloned into pACGP67 (BD Biosciences) and transfected into Sf-9 cells. The expressed protein has the sequence NH2-ADPELTEA... LPRSPHHHHHH-COOH, where the underlined residues are non-native. After viral amplification, chemerin was expressed by adding high titer virus to shaker flasks containing Hi-5 insect cells in Ex-cell 420 media (JRH Biosciences). After incubation for 2–3 days at 27.5 °C, the supernatant was harvested by centrifugation, filtered to 0.22 μm, and concentrated at 4 °C using a tangential flow concentrator (Filtron) with a 3-kDa cutoff filter. After a >100-fold buffer exchange into 50 mm HEPES, 0.3 m NaCl, pH 8.0, chemerin was purified by running the solution over nickel-nitrilotriacetic acid, SP-Sepharose (Amersham Biosciences) and C-18 reverse phase high pressure liquid chromatography columns (Vydac). The protein was lyophilized and checked for purity using electrospray mass spectrometry. Chemerin Expression and Purification from Escherichia coli—Chemerin with a carboxyl-terminal His6 tag (having the sequence NH2-MELTEA...LPRSPHHHHHH-COOH, where the underlined residues are non-native) was expressed in TAP302 cells for 4 h at 37 °C. The cell pellets were spun down and detergent-solubilized by successive rounds of homogenization and spinning in the presence of 0.25% sodium deoxycholate. The insoluble inclusion body pellet was solubilized in a denaturing buffer (6 m guanidine HCl, 0.1 m sodium phosphate, 10 mm Tris, pH 8.0) and run over a nickel-nitrilotriacetic acid column using a decrease in pH to elute. The FoldIt screen (Hampton Research) was used to test suitable refolding conditions, and a modified version of buffer 11 was chosen as the best choice (50 mm HEPES, 0.3 m NaCl, 0.44 mm KCl, 2.2 mm MgCl2, 2.2 mm CaCl2, 550 mm l-arginine, 0.055% polyethylene glycol 8000, 1 mm reduced l-glutathione, 0.1 mm oxidized l-glutathione, pH 8.0). Chemerin was refolded by rapidly diluting into the refolding buffer at a final protein concentration of 0.1–0.2 mg/ml. After stirring for a few hours at 4 °C, the protein was rapidly diluted 20-fold further into column buffer (50 mm HEPES, 0.3 m NaCl, pH 8.0) and filtered. The protein was concentrated and purified using the tangential flow concentrator and chromatography, as described in the previous paragraph for baculovirus expression. Serum—The Institutional Review Board at Stanford University approved all human subject protocols, and informed consent was obtained for all donations. Serum was stripped of heparin-binding proteins (including chemerin) by collecting the “flow-through” after passage over a heparin-Sepharose column. An amount of E. coli-expressed chemerin showing <5% input migration was incubated with an equivalent volume of serum or plasma for 5 min at 37 °C and then tested in a chemotaxis assay with CMKLR1/L1.2 transfectants. Neutrophil-conditioned Media—Neutrophils were prepared from citrated peripheral blood obtained from healthy volunteers by density separation using Ficoll-Paque (Amersham Biosciences). The high density fraction containing neutrophils and erythrocytes was mixed (1:2 v/v) with 1% solution of polyvinyl alcohol in phosphate-buffered saline (Merck) and incubated for 20 min at room temperature. Neutrophils were collected from the upper phase and subjected to hypotonic lysis to remove contaminating red blood cells. Polymorphonuclear neutrophil (95% pure) were cultured for 20 h in serum-free RPMI 1640 medium. Conditioned media were collected, centrifuged, and normalized based on protein concentration, as determined by a BCA assay according to the manufacturer's specifications (Pierce). Conditioned media samples containing 14 μg of total protein were incubated for 5–10 min with recombinant chemerin. Serine Proteases and Inhibitors—Serum was preincubated with 2.5 × 10-5 m aprotinin or 4.6 × 10-3 m E-64 (Sigma) for 1 h before chemerin was added. Neutrophil-conditioned medium was preincubated with 4 × 10-3 m pefabloc SC plus (Roche Applied Science) or 1 × 10-5 m E-64 for 30 min before chemerin was added. Various concentrations of serine proteases were incubated with chemerin for 5–10 min at 37 °C and then tested in chemotaxis. In each case, digestion was arrested by placing the tubes on ice and immediately diluting the samples 1:50 into cold chemotaxis medium for assay. The active forms of the following clotting and complement enzymes were used at concentrations based on their respective physiologic blood zymogen levels, as listed here: 2.7 × 10-6 m thrombin (Sigma), 2.2 × 10-7 m factor Xa (Pierce), 1.0 × 10-8 m factor VIIa, 1.1 × 10-7 m factor IXa, 3.1 × 10-8 m factor XIa, 3.8 × 10-7 m factor XIIa, 4.7 × 10-7 m kallikrein (Enzyme Research Laboratories), 2.6 × 10-6 m plasmin (Sigma) (9Jandl J.H. Blood: Textbook of Hematology, 2nd Ed.. Little, Brown, and Company, Boston1996: 1213-1275Google Scholar), 6.0 × 10-8 m factor D, 3.8 × 10-7 m factor I, 5.7 × 10-7 m C1r, and 6.3 × 10-7 m C1s (Calbiochem) (10Sim R.B. Tsiftsoglou S.A. Biochem. Soc. Trans. 2004; 32: 21-27Crossref PubMed Scopus (152) Google Scholar). Factor B and C2 were used at concentrations based on their physiologic blood levels of 2.2 × 10-6 m and 2.9 × 10-7 m (Calbiochem), respectively (10Sim R.B. Tsiftsoglou S.A. Biochem. Soc. Trans. 2004; 32: 21-27Crossref PubMed Scopus (152) Google Scholar). Tissue-type (tPA, Calbiochem) and urokinase-type (uPA, American Diagnostica) plasminogen activators were used at 7.1 × 10-7 m and 1.5 × 10-7 m, respectively. Trypsin isolated from bovine pancreas (Calbiochem) was used at 2.1 × 10-7 m. Mast cell tryptase isolated from human lung (Calbiochem) was used at 5.2 × 10-11 to 5.2 × 10-9 m. Human neutrophil elastase and cathepsin G (Calbiochem) were used at 1 × 10-7 m, and α1-proteinase inhibitor and α1-antichymotrypsin were used at 1 × 10-6 m (BioCentrum). None of the enzymes or inhibitors displayed chemotactic activity when tested in Transwell chemotaxis using CMKLR1/L1.2 transfectants. Mass Spectrometry—MALDI-TOF and electrospray mass spectrometry were performed by the Stanford Protein and Nucleic Acid Biotechnology Facility, the Protein Chemistry Core Facility, University of Columbia, and the Berkeley Protein Core Facility. Tryptic mass values were used in a Mascot search (www.matrixscience.com) of public peptide databases. PeptideCutter was used to predict the mass values of various chemerin isoforms and to predict tryptic chemerin fragments (www.expasy.org). Serum Serine Proteases Activate Chemerin—Serum is significantly more potent than plasma in inducing the chemotaxis of CMKLR1-positive cells (5Zabel B.A. Silverio A.M. Butcher E.C. J. Immunol. 2005; 174: 244-251Crossref PubMed Scopus (207) Google Scholar), suggesting that clotting might activate proteases capable of cleaving plasma chemerin to an active form. To test this hypothesis, we stripped serum of endogenous heparin-binding proteins (including chemerin) by passing it over a heparin column. This treatment effectively removed the endogenous serum chemotactic activity (Fig. 1). However, incubation of the undiluted stripped serum with recombinant full-length pro-chemerin dramatically enhanced the chemotactic activity of the protein. The triggering event was rapid, as chemerin chemotactic activity peaked within 5 min at 37 °C (data not shown). Aprotinin, a general serine protease inhibitor (but not the cysteine protease inhibitor E-64), blocked chemerin activation by stripped serum, indicating that serum serine proteases are required for chemerin activation. Furthermore, the canonical serine protease trypsin also activated chemerin, consistent with a direct activity of serine proteases on the attractant (TABLE ONE).TABLE ONEIsolated serine proteases of the coagulation, fibrinolytic, and inflammatory cascades activate chemerin Recombinant full-length pro-chemerin was titered to yield low initial activity (<4.5 ± 0.4% CMKLR1 transfectant migration) and was incubated for 5–10 min at 37 °C with the indicated enzymes (basal migration was <1.7 ± 0.4). Plasmin was used at levels corresponding to physiologic blood plasminogen levels (2.6 × 10-6 m), as well as 10× higher. Enzyme concentrations corresponding to 10× the physiologic blood zymogen levels are displayed for factor VIIa, factor XIIa, and C1s. The concentrations used for uPA and tPA were those reported for mediating in vitro clot lysis (13Guimaraes A.H. Rijken D.C. Thromb. Haemostasis. 2004; 91: 473-479Crossref PubMed Google Scholar). For chemerin chemotactic activity, the mean of duplicate wells of representative experiments (of 2–4 performed with similar results) is shown with the range in each case. For percent of maximal migration, the intra-experimental chemotactic responses for chemerin alone were subtracted from each sample, and a ratio based on the best observed chemotactic response (10× plasmin) is displayed. Serine protease Concentration Chemerin chemotactic activity (input migration) Maximal migration m % % None <4.5 ± 0.4 0 Trypsin 2.1 × 10–7 33.6 ± 2.6 49.4 Plasmin 2.6 × 10–6 37.4 ± 1.6 55.5 Plasmin 2.6 × 10–5 65.3 ± 3.8 100 Factor VIIa 1.0 × 10–7 12.5 ± 0.4 15.8 Factor XIIa 3.8 × 10–6 44.1 ± 1.6 66.2 uPA 1.5 × 10–7 14.0 ± 0.4 15.2 tPA 7.1 × 10–7 14.7 ± 0.8 16.3 Elastase 1.0 × 10–7 15.5 ± 2.6 21.2 Cathepsin G 1.0 × 10–7 6.9 ± 0.5 7.4 Tryptase 5.2 × 10–9 40.9 ± 0.0 60.9 C1s 6.3 × 10–6 6.9 ± 0.6 6.7 Open table in a new tab Plasmin Is a Potent Activator of Chemerin—To identify the serum protease(s) involved, we incubated recombinant chemerin with factors VIIa, IXa, Xa, XIa, and XIIa, kallikrein, thrombin, or plasmin for 5 min at 37 °C and assayed for chemotactic activity. At physiologic blood plasminogen concentrations (and even concentrations 10× lower, not shown), plasmin, an abundant blood and tissue serine protease that cleaves fibrin and leads to clot lysis, was a potent chemerin-activator (TABLE ONE). At concentrations 10× higher than physiologic blood zymogen levels, plasmin in particular, but also coagulation cascade factors VIIa and XIIa were potent activators of chemerin (TABLE ONE). Although the effect was reduced when factors XIIa and VIIa were used at physiologic blood levels, they still generated significant activated chemerin, similar to the activating ability of serum (not shown). None of the other serine proteases of the coagulation cascade showed significant activity in the range tested (0.1–10× blood levels, data not shown). The serine proteases uPA and tPA cleave plasminogen to generate plasmin. Interestingly, these enzymes also activate chemerin (TABLE ONE). Although the enzyme concentrations required were higher (1000–10,000-fold) than their observed plasma zymogen levels (9Jandl J.H. Blood: Textbook of Hematology, 2nd Ed.. Little, Brown, and Company, Boston1996: 1213-1275Google Scholar), the uPA concentration was similar to the level required to cleave its primary physiologic target, plasminogen (11Vakili J. Standker L. Detheux M. Vassart G. Forssmann W.G. Parmentier M. J. Immunol. 2001; 167: 3406-3413Crossref PubMed Scopus (51) Google Scholar, 12Bangert K. Thorsen S. Thromb. Haemostasis. 2000; 84: 299-306Crossref PubMed Scopus (4) Google Scholar, 13Guimaraes A.H. Rijken D.C. Thromb. Haemostasis. 2004; 91: 473-479Crossref PubMed Google Scholar). Both plasminogen activators display increased abilities to activate plasminogen when in the bound state (14Ellis V. Dano K. J. Biol. Chem. 1993; 268: 4806-4813Abstract Full Text PDF PubMed Google Scholar, 15Hoylaerts M. Rijken D.C. Lijnen H.R. Collen D. J. Biol. Chem. 1982; 257: 2912-2919Abstract Full Text PDF PubMed Google Scholar), particularly tPA, which displays a kinetic acceleration of ∼50-fold in plasminogen activation in the presence of fibrin. Thus uPA and tPA may play a role in chemerin activation in vivo when concentrated on cell or matrix surfaces. To determine whether plasmin generates a discrete active cleavage product of chemerin, we digested recombinant chemerin with plasmin under controlled conditions and evaluated the products by polyacrylamide gel electrophoresis. Incubation with a minimal amount of plasmin (6.4 × 10-9 m) over 48 h at 37 °C generated a single primary proteolytic product, associated with a 20-fold increase in chemotactic potency compared with the starting material (data not shown). (Note that the recombinant chemerin contains a small amount of a spontaneously cleaved form as well; this form may be responsible for the low level of initial chemotactic activity prior to incubation with plasmin. Using a monoclonal antibody specific to the full-length form, Wittamer et al. (3Wittamer V. Franssen J.D. Vulcano M. Mirjolet J.F. Le Poul E. Migeotte I. Brezillon S. Tyldesley R. Blanpain C. Detheux M. Mantovani A. Sozzani S. Vassart G. Parmentier M. Communi D. J. Exp. Med. 2003; 198: 977-985Crossref PubMed Scopus (677) Google Scholar) showed that full-length chemerin, when purified from spontaneous cleavage products, had no detectable chemotactic activity.) Neutrophil Granule Proteases Cathepsin G and Elastase Activate Chemerin—Neutrophils are recruited early to sites of acute inflammation and, when activated, release an array of enzymes and factors that regulate the inflammatory process, including the recruitment of other leukocytes (16Tani K. Ogushi F. Shimizu T. Sone S. J. Med. Invest. 2001; 48: 133-141PubMed Google Scholar). To determine whether neutrophils release proteases that activate chemerin, we initially incubated recombinant full-length chemerin with neutrophil-conditioned media. The media displayed potent gelatinolytic activity, indicating the release of neutrophil granule proteinases (data not shown). Neutrophil-conditioned medium, itself, had no chemotactic activity for CMKLR1 transfectants, but it displayed significant chemerin-activating ability (Fig. 2). Pefabloc, a general serine protease inhibitor, significantly blocked chemerin activation (Fig. 2), indicating that serine proteases released upon neutrophil degranulation activate chemerin. The serine proteases elastase and cathepsin G are major components of primary (azurophil) granules of neutrophils. Both proteinases generated active chemerin (TABLE ONE). Inhibition of neutrophil elastase and cathepsin G by their selective inhibitors, α1-proteinase inhibitor and α1-antichymotrypsin, respectively, abrogated chemerin activation (data not shown). Direct enzymatic activity, therefore, is required for the protease-induced generation of the active chemoattractant. Serine Proteases of the Complement Cascade Are Weak Effectors—Serine proteases of the complement cascade trigger complement components that act as potent leukocyte attractants. Therefore, we evaluated the chemerin activation ability of factors B, D, and I, and C2, C1r, and C1s. At concentrations 10-fold higher than their blood zymogen levels (and within the range of the reported concentrations used for cleavage of their respective complement substrates, i.e. factor I cleavage of C3b (17Ekdahl K.N. Nilsson U.R. Nilsson B. J. Immunol. 1990; 144: 4269-4274PubMed Google Scholar), C1s cleavage of C2 (18Nagasawa S. Stroud R.M. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 2998-3001Crossref PubMed Scopus (78) Google Scholar), factor D cleavage of factor B (19Taylor F.R. Bixler S.A. Budman J.I. Wen D. Karpusas M. Ryan S.T. Jaworski G.J. Safari-Fard A. Pollard S. Whitty A. Biochemistry. 1999; 38: 2849-2859Crossref PubMed Scopus (21) Google Scholar), and C1r cleavage of the zymogen form of C1s (20Kardos J. Gal P. Szilagyi L. Thielens N.M. Szilagyi K. Lorincz Z. Kulcsar P. Graf L. Arlaud G.J. Zavodszky P. J. Immunol. 2001; 167: 5202-5208Crossref PubMed Scopus (36) Google Scholar)), only C1s had a weak but detectable effect on chemerin potency (TABLE ONE). Mast Cell Tryptase Is a Potent Chemerin Activator—Mast cell granules contain heparin, histamine, and numerous proteinases, the most abundant being the serine protease tryptase. Mast cell tryptase also circulates in the body, and although serum tryptase levels are quite low in healthy individuals (<1 × 10-10 m), they can be 10–100-fold higher in patients undergoing anaphylactic reactions (21Dybendal T. Guttormsen A.B. Elsayed S. Askeland B. Harboe T. Florvaag E. Acta Anaesthesiol. Scand. 2003; 47: 1211-1218Crossref PubMed Scopus (66) Google Scholar). Although low concentrations of tryptase (5.2 × 10-11 m) did not activate chemerin, higher concentrations (5.2 × 10-9 m) served as a potent activator (TABLE ONE). Identifying the Carboxyl-terminal Processing Site for Endogenous Serum Chemerin—Wittamer et al. (3Wittamer V. Franssen J.D. Vulcano M. Mirjolet J.F. Le Poul E. Migeotte I. Brezillon S. Tyldesley R. Blanpain C. Detheux M. Mantovani A. Sozzani S. Vassart G. Parmentier M. Communi D. J. Exp. Med. 2003; 198: 977-985Crossref PubMed Scopus (677) Google Scholar, 22Wittamer V. Gregoire F. Robberecht P. Vassart G. Communi D. Parmentier M. J. Biol. Chem. 2004; 279: 9956-9962Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) demonstrated that proteolytic cleavage in the carboxyl terminus of chemerin results in its activation. To identify the site of cleavage of serum chemerin, we isolated the active peptide from serum as described previously (5Zabel B.A. Silverio A.M. Butcher E.C. J. Immunol. 2005; 174: 244-251Crossref PubMed Scopus (207) Google Scholar) and carried out mass spectrometric (MALDI-TOF) analysis of tryptic peptides. Peptides identified covered 55% of the protein sequence. In addition to peptides with canonical tryptic cleavage sites, one peptide displayed a mass value of 1669.7 Da, corresponding to a non-tryptic peptide comprising amino acids 141–155 from the carboxyl terminus of chemerin (Fig. 3, A and B). This peptide defines the carboxyl-terminal processing site of serum chemerin NH2...FA ↓ FSKALPRS... COOH (Fig. 3, B and C). In corroboration, MALDI-TOF mass spectrometry of a chemerin-enriched fraction from human serum displayed a peak with a mass value of 15648.1 Da, within 5.2 Da of the predicted value of the truncated form above (15642.9 Da) (data not shown). This cleavage site is distinct from those defined previously by Meder et al. (4Meder W. Wendland M. Busmann A. Kutzleb C. Spodsberg N. John H. Richter R. Schleuder D. Meyer M. Forssmann W.G. FEBS Lett. 2003; 555: 495-499Crossref PubMed Scopus (154) Google Scholar) from hemofiltrate and Wittamer et al. (3Wittamer V. Franssen J.D. Vulcano M. Mirjolet J.F. Le Poul E. Migeotte I. Brezillon S. Tyldesley R. Blanpain C. Detheux M. Mantovani A. Sozzani S. Vassart G. Parmentier M. Communi D. J. Exp. Med. 2003; 198: 977-985Crossref PubMed Scopus (677) Google Scholar) from ascites (Fig. 3C) but has been recently reported by Busmann et al. (23Busmann A. Walden M. Wendland M. Kutzleb C. Forssmann W.G. John H. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2004; 811: 217-223Crossref PubMed Scopus (6) Google Scholar) as a processed form present in Chinese hamster ovary cell supernatant. Distinct Carboxyl-terminal Chemerin Cleavage Sites for Plasmin, Elastase, and Tryptase—To define the sites of chemerin cleavage by specific activating enzymes, we carried out controlled digestion of the recombinant full-length molecule to generate the active form(s) and used electrospray and MALDI-TOF mass spectrometry to define the cleavage products. This approach identified a single dominant plasmin carboxyl-terminal peptide cleavage site as NH2... FAFSK ↓ ALPRS-COOH. This is a canonical serine protease (trypsin) cleavage site, as predicted by PeptideCutter software (www.expasy.org). Several carboxyl-terminal cleavage sites were observed following controlled activation with neutrophil elastase, consistent with its less discriminating activity and its preference for cleaving bonds adjacent to small, often hydrophobic residues. One of these is the same as that of the endogenously active chemerin in serum, NH2... FA ↓ FSKALPRS... COOH. As summarized in Fig. 3C, mast cell tryptase also generated several distinct carboxyl-terminal cleavage products, including the serum form. The ubiquitous distribution of pro-chemerin is reminiscent of the prevalent expression of tissue factor in non-vascular sites. Tissue factor is the most potent activator of the clotting cascade and provides a “hemostatic envelope” (24Drake T.A. Morrissey J.H. Edgington T.S. Am. J. Pathol. 1989; 134: 1087-1097PubMed Google Scholar), allowing for the rapid detection and correction of breaches in vascular integrity. Similarly, chemerin may se
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