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S-Nitrosylated human α1-protease inhibitor
Biochimica et Biophysica Acta 1477 (2000) 90^97 www.elsevier.com/locate/bba
Review
S-Nitrosylated human K1-protease inhibitor Yoichi Miyamoto, Takaaki Akaike, Hiroshi Maeda * Department of Microbiology, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan Received 1 November 1999; accepted 1 December 1999
Abstract K1 -Protease inhibitor (K1 PI) is an acute phase plasma protein, and possesses a single cysteine residue at position 232. A single cysteinyl sulfhydryl of human K1 PI is found to be readily S-nitrosylated by nitric oxide (NO) in vitro without affecting the inhibitory capacity against bovine trypsin or elastase, a major target protease of K1 PI in vivo. S-Nitroso-K1 PI (S-NOK1 PI) was also formed by the reaction of K1 PI with NO produced excessively by a murine macrophage cell line (RAW264 cells) upon infection with Salmonella typhimurium and in an ex vivo perfusion system of the liver obtained from lipopolysaccharide-treated rats. S-NO-K1 PI (1039 ^1036 M) induces a dose-dependent relaxation of the ring preparation of rabbit aorta. Also, S-NO-K1 PI but not K1 PI shows a potent inhibitory effect on platelet aggregation. Unprecedented observation is that S-NO-K1 PI showed a potent bacteriostatic effect against a wide range of bacteria at the concentration of 1^10 WM, which was 10^1000-fold stronger than that of NO and other S-nitrosylated compounds including S-nitrosylated albumin and S-nitrosylated glutathione. These results suggest that S-NO-K1 PI is produced as an NO sink under inflammatory conditions, where production of both K1 PI and NO is highly up-regulated, and it may function as a soluble factor which consists of an innate defense system through not only the protease inhibitory activity but also its antibacterial activity and facilitating the peripheral blood flow. Therefore, S-nitrosylation of K1 PI occurring under physiological conditions in vivo should diversify the biological functions contributing to cytoprotective effects of K1 PI. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: K1 -Protease inhibitor; S-Nitrosylation; Antibacterial activity; EDRF; Nitric oxide trapping
1. Introduction K1 -Protease inhibitor (K1 PI), also referred to as K1 antitrypsin, is the most abundant serine protease inhibitor found in human blood circulation [1]. Its plasma level ranges from 30 to 60 WM (or 1.5^3.0 mg/ml blood) in the physiological state, which increases in the acute phase of a number of disorders including in£ammation and bacterial infection [2].
* Corresponding author. Fax: +81 (96) 3628362; E-mail: [email protected]
De¢ciency of K1 PI is reportedly associated with pulmonary emphysema, liver cirrhosis, glomerulo-nephritis, rheumatoid arthritis, and so on [1]. One of the primary roles of K1 PI is regarded as regulation of neutrophil elastase which degrades matrix tissues, leading to tissue injury in in£ammation and infections [1]. K1 PI with a molecular weight of 53 000 is a single polypeptide consisting of 394 amino acids containing three carbohydrate chains [3]. The molecular mechanism of inactivation of serine proteases by K1 PI is well studied and it is now known that one molecule of K1 PI binds to one molecule of protease forming a
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complex, following cleavage of the peptide bond between methionine-358 and serine-359 in the active center of this inhibitor [4]. Oxidation of methionine-358 results in the fall of inhibitory activity against proteases [5]. K1 PI has no disul¢de bridge but a single cysteine residue localized at position 232. The biochemical role of this sulfhydryl group remained obscure, except that some studies suggested that mixed disul¢de formation with other sulfhydryls such as glutathione (GSH) modulates the mode of its binding to human neutrophil elastase [6]. In recent years, accumulating evidence shows diverse biological functions of nitric oxide (NO) and related nitrogen intermediates. Among a series of NO metabolites with di¡erent redox states, nitrosonium cation (NO ) preferentially exists as S-nitrosothiols [7], i.e., adducts of NO and sulfhydryl (thiolate anion) such as cysteine, GSH and proteinous sulfhydryls. S-Nitrosothiols are considered to modulate various extracellular and intracellular signal transductions in neuronal and cardiovascular systems, and cell apoptosis. In this context, we developed recently a speci¢c and sensitive method to quantify S-nitrosothiols using high performance liquid chromatography (HPLC) coupled with a chromogenic £ow reactor system [8], which enables us to investigate the formation and degradation of S-nitrosothiols much more precisely and conveniently than ever. In this article, we describe the S-nitrosylation of K1 PI and novel biological functions of S-nitrosylated K1 PI (S-NO-K1 PI). 2. Nitrosylation of thiol compounds It is now conceivable that nitrosylation of thiols is involved in modulation of various biological events, such as functional regulation of receptors [9], ion channels [10,11], synaptic vesicle fusion [12], intracellular [13] and extracellular [14^17] signal transduction, and transcription factors [18^20]. S-Nitrosylation of some of the enzymes having thiols as their active centers is also regarded as an important way of regulation of their activities by interacting with NO [21^24]. Once S-nitrosylation occurs in biological systems, it is believed that NO in the nitroso compound is readily transferred to the other sulf-
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hydryl moiety of various thiol-containing molecules, e.g., proteins and GSH. Accordingly, it seems likely that S-nitrosylation is one of the important posttranscriptional modi¢cations of the proteins and peptides, which may be involved in NO-dependent signal transductions. Although the mechanism of biologically relevant nitrosothiol formation had been unknown, our recent study using an HPLC-based S-nitroso analyzer indicates that S-nitrosylation can be e¡ectively catalyzed by the copper ion of ceruloplasmin, a major multicopper-containing plasma protein, under physiological conditions [25]. Here, we employed our HPLC-£ow reactor analysis to examine S-nitrosylation of K1 PI. Our nitrosothiol detection system is an HPLC analysis coupled with £ow reactors for Hg2 -induced nitrosothiol decomposition followed by a colorimetric assay of NO3 2 using Griess reagent, which forms a diazo-dye having a strong absorbance at 540 nm at acidic pH: the extinction coe¤cient at 540 nm is 53 000 M31 cm31 [26]. Speci¢cally, we apply a gel ¢ltration HPLC technique (column size, 8U300 mm, Diol120; YMC, Kyoto, Japan) to separate various nitrosothiols from NO3 2 , and in the subsequently connected £ow reactor each RS-NO eluted was decomposed by Hg2 to form NO3 2 which was then reacted with Griess reagent in the second £ow reactor for the reaction forming azo-dye to be detected at 540 nm. The system we developed is schematically illustrated in Fig. 1. Using this HPLC-£ow reactor system, we found that K1 PI is e¤ciently S-nitrosylated in vitro. The S-nitrosylation reaction of K1 PI was found to be 10 times or more e¤cient than those of albumin or GSH, which indicates the possibility that K1 PI is one of the major targets of the nitrosonium cation in vivo and may became a reservoir of NO as well. To con¢rm the biological relevance of the nitrosylation of K1 PI, S-nitrosylation of plasma by an NO releasing reagent propylamine NONOate (p-NONOate, CH3 N[N(O)NO]3 (CH2 )3 NH 2 CH3 ) was examined. Two moles of NO are spontaneously generated by one molecule of p-NONOate at neutral pH. Fresh human plasma obtained from healthy volunteers was treated with 25 WM p-NONOate for 30 min at 37³C. S-Nitroso compounds formed in the reaction mixture were analyzed with the HPLC-£ow reactor system to identify RS-NO fraction in the plasma as described
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Fig. 1. Schematic illustration of the HPLC-£ow reactor system for the measurement of various nitrosothiols. S-Nitroso compounds including nitroso proteins eluted from the HPLC column were decomposed with Hg2 and detected as NO3 2 after reaction with Griess reagent. A deproteinization column was used in the system particularly for the S-NO-protein determination before access to the detector [8].
above. After 30 min incubation, 3.25 þ 0.10 WM of proteins were found to be S-nitrosylated. Furthermore, to test which protein in the human plasma is e¡ectively S-nitrosylated, NO-treated plasma was incubated with anti-human K1 PI polyclonal goat antibodies (IgG fractions) or non-immunized goat IgG for 1 h at 37³C and the resultant immune complexes of K1 PI were removed by centrifugation. After immunoprecipitation with 7.5 and 30 mg/ml of antihuman K1 PI polyclonal antibody, the amount of S-nitrosylated proteins was decreased to 83.0 and 80.7% of control plasma treated with non-immunized IgG, respectively, indicating at least 19% of S-nitrosylated plasma proteins (0.62 WM) was assumed to be S-NO-K1 PI. Because the concentration of K1 PI in human plasma is 30^60 WM (1.5^3.0 mg/ml), more than 1^2% of K1 PI was thought to be S-nitrosylated by 50 WM NO in the reaction system used in this experiment.
of 1U106 cells/well in antibiotic-free medium supplemented with 10% fetal bovine serum. The medium was removed and the cells were incubated for 1 h at 37³C in a CO2 incubator with 0.2 ml of the fresh medium containing Salmonella typhimurium LT2 (1U106 CFU/0.2 ml). The bacteria remaining outside the cells were removed by washing twice with KrebsRinger phosphate bu¡er (KRP; 15.6 mM sodium phosphate bu¡er, pH 7.4, including 120 mM NaCl, 4.8 mM KCl, 0.54 mM CaCl2 , 1.2 mM MgSO4 , 11 mM glucose), and killed by the treatment with 50 Wg/ml gentamicin in the medium for 1 h at 37³C. After further incubation for various periods at 37³C in the medium containing 15 Wg/ml gentamicin, the medium was replaced with KRP containing 1 mM L-arginine in the presence or absence of K1 PI, or bovine serum albumin (BSA), followed by incubation for 45 min at 37³C for nitrosothiol formation. The supernatant was applied to the HPLC-£ow re-
3. S-Nitrosylation of K1 PI in biological systems It is well known that the production of NO increases to a great extent through induction of inducible NO synthase (iNOS) in the in£ammatory condition or during microbial infections. Induction of iNOS is stimulated generally by lipopolysaccharide (LPS) and proin£ammatory cytokines such as interferon-Q (IFN-Q). A mouse macrophage cell line RAW264 cell is known to express iNOS in response to stimulation by IFN-Q and LPS [8]. RAW264 cells were cultured overnight in 24-well plates at a density
Fig. 2. S-Nitrosylation of K1 PI by S. typhimurium-infected RAW264 cells. RAW264 cells infected with S. typhimurium LT2 were incubated with or without 100 WM K1 PI or BSA. (A) Time course of NO production by cells either stimulated with IFN-Q and LPS (dotted line) or infected with S. typhimurium (squares). (B) S-NO-K1 PI (shaded columns) and S-NO-BSA (open columns) formed in the supernatant quanti¢ed by the HPLC-£ow reactor system. Data are mean values of three different experiments.
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4. Serpin activity of S-NO-K K1 PI
Fig. 3. Nitrosylation of K1 PI in an ex vivo organ perfusion system of rat livers treated with LPS (E. coli). E. coli LPS was injected i.p. (8.4 mg/kg) to male Wistar rats (200^220 g) 14 h prior to obtain the liver. The liver was perfused with 30 ml Krebs-Henseleit bicarbonate bu¡er (pH 7.4) containing 1 mM L-arginine and 50 WM K1 PI for 1 h at 37³C, and the perfusate was applied to the HPLC-£ow reactor system for nitrosothiol measurement.
actor system to quantify the S-nitroso proteins produced in the cell culture (Fig. 2). The broken line indicates the time course of NO production quanti¢ed by measuring nitrite anion after addition of IFN-Q and LPS, which reached a maximum level at 12 h after addition of these stimulants. This cell itself did not produce a signi¢cant amount of NO. On the other hand, NO production was induced in the cells infected by S. typhimurium without any other stimulation as shown by the squares. Then, K1 PI was added at 100 WM which is a physiologically conceivable concentration of the protein to the cell culture infected by Salmonella, and nitrosylated products in the supernatant were quanti¢ed. One hundred to 200 nM of S-NO-K1 PI was detected at 12 and 18 h after infection, which was almost 10-fold higher than that of nitrosoalbumin. Nitrosylation of K1 PI was also detected in an ex vivo organ perfusion system of the liver excised from rats treated with LPS from Escherichia coli. E. coli LPS was injected i.p. (8.4 mg/kg) to the rats (male Wistar rats weighing 200^220 g) 14 h prior to obtain the liver. The liver was perfused with 30 ml Krebs-Henseleit bicarbonate bu¡er (pH 7.4) containing 1 mM L-arginine and 50 WM K1 PI at 37³C. After 1 h perfusion, S-NO-K1 PI production was clearly observed by the HPLC-£ow reactor analysis (Fig. 3).
We examined the e¡ect of nitrosation on the inhibitory activity of K1 PI against porcine pancreatic trypsin. The inhibitory e¡ects of K1 PI and S-NOK1 PI on porcine pancreatic trypsin were assayed using its substrate, benzoyl-DL-arginine p-nitroanilide hydrochloride, essentially according to the methods described elsewhere [27,28]. Although it is reported that chemical modi¢cation of the thiol group in K1 PI a¡ects the inhibitory action for human neutrophil elastase, S-nitrosylation did not in£uence the inhibitory activity of K1 PI for trypsin. 5. Novel functions of K1 PI generated after S-nitrosylation 5.1. Vasorelaxation One of the well-known biological functions of NO and nitrosothiols is relaxation of blood vessels. Therefore, we examined the vasorelaxing activity of S-NO-K1 PI using ring preparations of rabbit aorta. The rings were mounted in an organ bath and precontracted with 0.1 WM phenylephrine, after which S-NO-K1 PI-induced relaxation was measured. S-NOK1 PI at nM to WM concentrations exhibited relaxation of the precontracted aorta ring in a concentration-dependent manner (Fig. 4). On the contrary, native K1 PI without S-nitrosylation did not have any vasorelaxing activity at all. It is reported that
Fig. 4. Vasorelaxing activity of S-NO-K1 PI and K1 PI in the ring preparations of rabbit aorta. The rings were mounted in organ baths and precontracted with 0.1 WM phenylephrine, after which relaxation induced by the graded concentrations of S-NO-K1 PI and K1 PI was monitored. Data are means þ S.E. (n = 4).
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S-nitrosothiols mediate vasorelaxation without liberation of NO [29]. Transnitrosylation from S-NOK1 PI to vascular smooth muscle cells might occur either via a pure chemical reaction or an enzymatic reaction as reported recently [30], which needs to be elucidated more critically. 5.2. Antibacterial e¡ect NO has been implicated as a host-derived antimicrobial molecule [31]. NO per se, however, has minimal antimicrobial e¡ects as we reported previously [32]. Alternatively, it is now accepted that its reactive metabolites, such as peroxynitrite and NO2 (or NO2 Cl) are responsible for NO-dependent antimicrobial as well as cytotoxic activities [32^35]. On the other hand, there are several pieces of earlier works on the antibacterial activities of S-nitrosocysteine and other S-nitrosylated compounds with low molecular weights [36,37]. S-Nitrosylated albumin [38] and homocysteine-conjugated albumin [39], and S-nitrosoglutathione (GS-NO) [40,41] were also reported recently to show antimicrobial activities. It is well documented that the production of K1 PI and NO derived from iNOS are up-regulated by the bacterial infection [42]. Intriguingly, not only hepatocytes but also monocytes and macrophages, that are the main sources of iNOS, are responsible for increased production of K1 PI in the state of in£ammation [43]. Therefore, it is highly likely that S-NOK1 PI is produced at the site of bacterial infection. When we examined the antibacterial activities of S-NO-K1 PI and other NO-related compounds, we found a marked growth suppressing activity of S-NO-K1 PI against a number of Gram-positive and Gram-negative bacteria in comparison with other nitrosothiols and NO (Fig. 5). Speci¢cally, S-NO-K1 PI at low WM range inhibited the growth of various bacteria, while an mM level of GS-NO is needed to exhibit the same extent of antibacterial activity (Fig. 5). The mode of antibacterial action of S-NO-K1 PI seems to be bacteriostatic rather than bactericidal, which is a clear contrast to that of peroxynitrite, a strong bactericidal metabolite of NO [33]. Our preliminary experiments indicated an e¤cient transnitrosylation from S-NO-K1 PI to the bacterial cells, which was much faster than that from S-NO-albumin or GS-NO (not shown). The amount of S-NO-
Fig. 5. Antibacterial activity of S-NO-K1 PI and GS-NO. The bacteria indicated were suspended in KRP at the density of 1U106 CFU/ml and treated with various concentrations of S-NO-K1 PI or GS-NO for 1 h at 37³C. The bacterial suspension (20 Wl) was then added to brain-heart infusion broth (180 Wl) and additionally cultured for 6 h at 37³C to observe the bacterial growth by measuring the turbidity (absorbance at 610 nm). The 50% growth inhibitory concentration is shown for each bacterium examined.
K1 PI produced and/or its rate of production at the site of infection (not in the systemic circulation) seems to be an important point to be clari¢ed in future studies. 5.3. Other possible biological activities Similar to other nitrosothiols such as GS-NO [44], S-NO-K1 PI has a potent inhibitory e¡ect on platelet aggregation. It is also reported that nitrosothiols inhibit neutrophil adhesion to the endothelial surface and attenuate its accumulation in various organs with in£ammatory and ischemia-reperfusion injuries [45]. Thus, S-NO-K1 PI may exert tissue protecting activity from neutrophil-mediated tissue injury through inhibition of both neutrophil accumulation and its elastase. It seems therefore interesting to assess the e¡ects of S-NO-K1 PI on the neutrophilmediated tissue injury induced by infection and ischemia. Now we are examining such protective effects of S-NO-K1 PI on the ischemia-reperfusion injury in the liver, which will be reported elsewhere before long. Apoptosis is one of the most fundamental cellular events that is critically involved in various physiological and pathological phenomena. Accumulating evidence indicates that apoptosis takes place in a variety of infectious diseases including viral and bacterial infections and that it plays an important role in their
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pathogenesis [46,47]. Caspases, particularly caspase3, are regarded as key denominators in executing apoptosis [48,49]. Since caspases belong to cysteine proteases, the active center cysteinyl sulfhydryl residue might be susceptible to chemical modi¢cations by NO or its reactive metabolites. In fact, it is currently reported that NO suppresses apoptosis through inhibition of caspase-3 by S-nitrosylation of its active center [50^52]. As we described earlier in this paper, S-NO-K1 PI is a prominent nitrosonium (NO ) carrier (donor) possibly formed in the in£ammatory and infectious foci. Thus, an antiapoptotic e¡ect of S-NO-K1 PI is a critical issue of great interest which remains to be clari¢ed.
[3]
[4]
[5]
[6]
[7] [8]
6. Conclusion In this article, we describe that K1 PI is e¤ciently S-nitrosylated in vitro and also in biological systems. Besides retaining the inhibitory activity for serine proteases, the resultant S-NO-K1 PI showed new diverse biological functions, i.e., vasorelaxation and bacteriostatic activity. As a result of increased production of both NO and K1 PI in the in£ammatory condition or at a site of infection, S-NO-K1 PI is likely to be formed and it exhibits protective e¡ects on the hosts via its bacteriostatic activity and facilitating peripheral blood £ow.
[9]
[10]
[11]
[12]
[13]
Acknowledgements We thank Ms. Rie Yoshimoto for preparing the manuscript. This work was supported by a grant-inaid for Scienti¢c Research from the Ministry of Education, Science, Sports and Culture of Japan, and grants from the Ministry of Health and Welfare of Japan.
[14]
[15]
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[33] L. Zhu, C. Gunn, J.S. Beckman, Bactericidal activity of peroxynitrite, Arch. Biochem. Biophys. 298 (1992) 452^457. [34] T. Akaike, M. Suga, H. Maeda, Free radicals in viral pathogenesis: molecular mechanisms involving superoxide and NO, Proc. Soc. Exp. Biol. Med. 217 (1998) 64^73. [35] A. van der Vliet, J.P. Eiserich, M.K. Shigenaga, C.E. Cross, Reactive nitrogen species and tyrosine nitration in the respiratory tract, Am. J. Respir. Crit. Care Med. 160 (1999) 1^9. [36] K. Incze, J. Farkas, V. Miha¨lyi, E. Zuka¨l, Antibacterial effect of cysteine-nitrosothiol and possible precursors thereof, Appl. Microbiol. 27 (1974) 202^205. [37] S.L. Morris, J.N. Hansen, Inhibition of Bacillus cereus spore outgrowth by covalent modi¢cation of a sulfhydryl group by nitrosothiol and iodoacetate, J. Bacteriol. 148 (1981) 465^ 471. [38] S. Mnaimneh, M. Ge¡ard, B. Veyret, P. Vincendeau, Albumin nitrosylated by activated macrophages possesses antiparasitic e¡ects neutralized by anti-NO-acetylated-cysteine antibodies, J. Immunol. 158 (1997) 308^314. [39] J.F. Ewing, D.V. Young, D.R. Janero, D.S. Garvey, T.A. Grinnell, Nitrosylated bovine serum albumin derivatives as pharmacologically active nitric oxide congeners, J. Pharmacol. Exp. Ther. 283 (1997) 947^954. [40] M.A. De Groote, D. Granger, Y. Xu, G. Campbell, R. Prince, F.C. Fang, Genetic and redox determinants of nitric oxide cytotoxicity in a Salmonella typhimurium model, Proc. Natl. Acad. Sci. USA 92 (1995) 6399^6403. [41] M.A. De Groote, T. Testerman, Y. Xu, G. Stau¡er, F.C. Fang, Homocysteine antagonism of nitric oxide-related cytostasis in Salmonella typhimurium, Science 272 (1996) 414^ 417. [42] K. Umezawa, T. Akaike, S. Fujii, M. Suga, K. Setoguchi, A. Ozawa, H. Maeda, Induction of nitric oxide synthesis and xanthine oxidase and their roles in the antimicrobial mechanism against Salmonella typhimurium infection in mice, Infect. Immun. 65 (1997) 2932^2940. [43] D.H. Perlmutter, L.T. May, P.B. Sehgal, Interferon L 2/interleukin 6 modulates synthesis of K 1-antitrypsin in human mononuclear phagocytes and in human hepatoma cells, J. Clin. Invest. 84 (1989) 138^144. [44] M.W. Radomski, D.D. Ress, A. Dutra, S. Moncada, S-Nitroso-glutathione inhibits platelet activation in vitro and in vivo, Br. J. Pharmacol. 107 (1992) 745^749. [45] P. Kubes, M. Suzuki, D.N. Granger, Nitric oxide: an endogenous modulator of leukocyte adhesion, Proc. Natl. Acad. Sci. USA 88 (1991) 4651^4655. [46] A. Zychlinsky, P. Sansonetti, Perspectives series: host/pathogen interactions. Apoptosis in bacterial pathogenesis, J. Clin. Invest. 100 (1997) 493^495. [47] H. Everett, G. McFadden, Apoptosis: an innate immune response to virus infection, Trends Microbiol. 7 (1999) 160^165. [48] M. Enari, R.V. Talanian, W.W. Wong, S. Nagata, Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis, Nature 380 (1996) 723^726.
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A.M. Zeiher, A. Mulsch, S. Dimmeler, Nitric oxide inhibits caspase-3 by S-nitrosation in vivo, J. Biol. Chem. 274 (1999) 6823^6826. [52] J.B. Mannick, A. Hausladen, L. Liu, D.T. Hess, M. Zeng, Q.X. Miao, L.S. Kane, A.J. Gow, J.S. Stamler, Fas-induced caspase denitrosylation, Science 284 (1999) 651^654.
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Review
S-Nitrosylated human K1-protease inhibitor Yoichi Miyamoto, Takaaki Akaike, Hiroshi Maeda * Department of Microbiology, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan Received 1 November 1999; accepted 1 December 1999
Abstract K1 -Protease inhibitor (K1 PI) is an acute phase plasma protein, and possesses a single cysteine residue at position 232. A single cysteinyl sulfhydryl of human K1 PI is found to be readily S-nitrosylated by nitric oxide (NO) in vitro without affecting the inhibitory capacity against bovine trypsin or elastase, a major target protease of K1 PI in vivo. S-Nitroso-K1 PI (S-NOK1 PI) was also formed by the reaction of K1 PI with NO produced excessively by a murine macrophage cell line (RAW264 cells) upon infection with Salmonella typhimurium and in an ex vivo perfusion system of the liver obtained from lipopolysaccharide-treated rats. S-NO-K1 PI (1039 ^1036 M) induces a dose-dependent relaxation of the ring preparation of rabbit aorta. Also, S-NO-K1 PI but not K1 PI shows a potent inhibitory effect on platelet aggregation. Unprecedented observation is that S-NO-K1 PI showed a potent bacteriostatic effect against a wide range of bacteria at the concentration of 1^10 WM, which was 10^1000-fold stronger than that of NO and other S-nitrosylated compounds including S-nitrosylated albumin and S-nitrosylated glutathione. These results suggest that S-NO-K1 PI is produced as an NO sink under inflammatory conditions, where production of both K1 PI and NO is highly up-regulated, and it may function as a soluble factor which consists of an innate defense system through not only the protease inhibitory activity but also its antibacterial activity and facilitating the peripheral blood flow. Therefore, S-nitrosylation of K1 PI occurring under physiological conditions in vivo should diversify the biological functions contributing to cytoprotective effects of K1 PI. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: K1 -Protease inhibitor; S-Nitrosylation; Antibacterial activity; EDRF; Nitric oxide trapping
1. Introduction K1 -Protease inhibitor (K1 PI), also referred to as K1 antitrypsin, is the most abundant serine protease inhibitor found in human blood circulation [1]. Its plasma level ranges from 30 to 60 WM (or 1.5^3.0 mg/ml blood) in the physiological state, which increases in the acute phase of a number of disorders including in£ammation and bacterial infection [2].
* Corresponding author. Fax: +81 (96) 3628362; E-mail: [email protected]
De¢ciency of K1 PI is reportedly associated with pulmonary emphysema, liver cirrhosis, glomerulo-nephritis, rheumatoid arthritis, and so on [1]. One of the primary roles of K1 PI is regarded as regulation of neutrophil elastase which degrades matrix tissues, leading to tissue injury in in£ammation and infections [1]. K1 PI with a molecular weight of 53 000 is a single polypeptide consisting of 394 amino acids containing three carbohydrate chains [3]. The molecular mechanism of inactivation of serine proteases by K1 PI is well studied and it is now known that one molecule of K1 PI binds to one molecule of protease forming a
0167-4838 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 9 ) 0 0 2 6 4 - 2
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complex, following cleavage of the peptide bond between methionine-358 and serine-359 in the active center of this inhibitor [4]. Oxidation of methionine-358 results in the fall of inhibitory activity against proteases [5]. K1 PI has no disul¢de bridge but a single cysteine residue localized at position 232. The biochemical role of this sulfhydryl group remained obscure, except that some studies suggested that mixed disul¢de formation with other sulfhydryls such as glutathione (GSH) modulates the mode of its binding to human neutrophil elastase [6]. In recent years, accumulating evidence shows diverse biological functions of nitric oxide (NO) and related nitrogen intermediates. Among a series of NO metabolites with di¡erent redox states, nitrosonium cation (NO ) preferentially exists as S-nitrosothiols [7], i.e., adducts of NO and sulfhydryl (thiolate anion) such as cysteine, GSH and proteinous sulfhydryls. S-Nitrosothiols are considered to modulate various extracellular and intracellular signal transductions in neuronal and cardiovascular systems, and cell apoptosis. In this context, we developed recently a speci¢c and sensitive method to quantify S-nitrosothiols using high performance liquid chromatography (HPLC) coupled with a chromogenic £ow reactor system [8], which enables us to investigate the formation and degradation of S-nitrosothiols much more precisely and conveniently than ever. In this article, we describe the S-nitrosylation of K1 PI and novel biological functions of S-nitrosylated K1 PI (S-NO-K1 PI). 2. Nitrosylation of thiol compounds It is now conceivable that nitrosylation of thiols is involved in modulation of various biological events, such as functional regulation of receptors [9], ion channels [10,11], synaptic vesicle fusion [12], intracellular [13] and extracellular [14^17] signal transduction, and transcription factors [18^20]. S-Nitrosylation of some of the enzymes having thiols as their active centers is also regarded as an important way of regulation of their activities by interacting with NO [21^24]. Once S-nitrosylation occurs in biological systems, it is believed that NO in the nitroso compound is readily transferred to the other sulf-
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hydryl moiety of various thiol-containing molecules, e.g., proteins and GSH. Accordingly, it seems likely that S-nitrosylation is one of the important posttranscriptional modi¢cations of the proteins and peptides, which may be involved in NO-dependent signal transductions. Although the mechanism of biologically relevant nitrosothiol formation had been unknown, our recent study using an HPLC-based S-nitroso analyzer indicates that S-nitrosylation can be e¡ectively catalyzed by the copper ion of ceruloplasmin, a major multicopper-containing plasma protein, under physiological conditions [25]. Here, we employed our HPLC-£ow reactor analysis to examine S-nitrosylation of K1 PI. Our nitrosothiol detection system is an HPLC analysis coupled with £ow reactors for Hg2 -induced nitrosothiol decomposition followed by a colorimetric assay of NO3 2 using Griess reagent, which forms a diazo-dye having a strong absorbance at 540 nm at acidic pH: the extinction coe¤cient at 540 nm is 53 000 M31 cm31 [26]. Speci¢cally, we apply a gel ¢ltration HPLC technique (column size, 8U300 mm, Diol120; YMC, Kyoto, Japan) to separate various nitrosothiols from NO3 2 , and in the subsequently connected £ow reactor each RS-NO eluted was decomposed by Hg2 to form NO3 2 which was then reacted with Griess reagent in the second £ow reactor for the reaction forming azo-dye to be detected at 540 nm. The system we developed is schematically illustrated in Fig. 1. Using this HPLC-£ow reactor system, we found that K1 PI is e¤ciently S-nitrosylated in vitro. The S-nitrosylation reaction of K1 PI was found to be 10 times or more e¤cient than those of albumin or GSH, which indicates the possibility that K1 PI is one of the major targets of the nitrosonium cation in vivo and may became a reservoir of NO as well. To con¢rm the biological relevance of the nitrosylation of K1 PI, S-nitrosylation of plasma by an NO releasing reagent propylamine NONOate (p-NONOate, CH3 N[N(O)NO]3 (CH2 )3 NH 2 CH3 ) was examined. Two moles of NO are spontaneously generated by one molecule of p-NONOate at neutral pH. Fresh human plasma obtained from healthy volunteers was treated with 25 WM p-NONOate for 30 min at 37³C. S-Nitroso compounds formed in the reaction mixture were analyzed with the HPLC-£ow reactor system to identify RS-NO fraction in the plasma as described
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Fig. 1. Schematic illustration of the HPLC-£ow reactor system for the measurement of various nitrosothiols. S-Nitroso compounds including nitroso proteins eluted from the HPLC column were decomposed with Hg2 and detected as NO3 2 after reaction with Griess reagent. A deproteinization column was used in the system particularly for the S-NO-protein determination before access to the detector [8].
above. After 30 min incubation, 3.25 þ 0.10 WM of proteins were found to be S-nitrosylated. Furthermore, to test which protein in the human plasma is e¡ectively S-nitrosylated, NO-treated plasma was incubated with anti-human K1 PI polyclonal goat antibodies (IgG fractions) or non-immunized goat IgG for 1 h at 37³C and the resultant immune complexes of K1 PI were removed by centrifugation. After immunoprecipitation with 7.5 and 30 mg/ml of antihuman K1 PI polyclonal antibody, the amount of S-nitrosylated proteins was decreased to 83.0 and 80.7% of control plasma treated with non-immunized IgG, respectively, indicating at least 19% of S-nitrosylated plasma proteins (0.62 WM) was assumed to be S-NO-K1 PI. Because the concentration of K1 PI in human plasma is 30^60 WM (1.5^3.0 mg/ml), more than 1^2% of K1 PI was thought to be S-nitrosylated by 50 WM NO in the reaction system used in this experiment.
of 1U106 cells/well in antibiotic-free medium supplemented with 10% fetal bovine serum. The medium was removed and the cells were incubated for 1 h at 37³C in a CO2 incubator with 0.2 ml of the fresh medium containing Salmonella typhimurium LT2 (1U106 CFU/0.2 ml). The bacteria remaining outside the cells were removed by washing twice with KrebsRinger phosphate bu¡er (KRP; 15.6 mM sodium phosphate bu¡er, pH 7.4, including 120 mM NaCl, 4.8 mM KCl, 0.54 mM CaCl2 , 1.2 mM MgSO4 , 11 mM glucose), and killed by the treatment with 50 Wg/ml gentamicin in the medium for 1 h at 37³C. After further incubation for various periods at 37³C in the medium containing 15 Wg/ml gentamicin, the medium was replaced with KRP containing 1 mM L-arginine in the presence or absence of K1 PI, or bovine serum albumin (BSA), followed by incubation for 45 min at 37³C for nitrosothiol formation. The supernatant was applied to the HPLC-£ow re-
3. S-Nitrosylation of K1 PI in biological systems It is well known that the production of NO increases to a great extent through induction of inducible NO synthase (iNOS) in the in£ammatory condition or during microbial infections. Induction of iNOS is stimulated generally by lipopolysaccharide (LPS) and proin£ammatory cytokines such as interferon-Q (IFN-Q). A mouse macrophage cell line RAW264 cell is known to express iNOS in response to stimulation by IFN-Q and LPS [8]. RAW264 cells were cultured overnight in 24-well plates at a density
Fig. 2. S-Nitrosylation of K1 PI by S. typhimurium-infected RAW264 cells. RAW264 cells infected with S. typhimurium LT2 were incubated with or without 100 WM K1 PI or BSA. (A) Time course of NO production by cells either stimulated with IFN-Q and LPS (dotted line) or infected with S. typhimurium (squares). (B) S-NO-K1 PI (shaded columns) and S-NO-BSA (open columns) formed in the supernatant quanti¢ed by the HPLC-£ow reactor system. Data are mean values of three different experiments.
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4. Serpin activity of S-NO-K K1 PI
Fig. 3. Nitrosylation of K1 PI in an ex vivo organ perfusion system of rat livers treated with LPS (E. coli). E. coli LPS was injected i.p. (8.4 mg/kg) to male Wistar rats (200^220 g) 14 h prior to obtain the liver. The liver was perfused with 30 ml Krebs-Henseleit bicarbonate bu¡er (pH 7.4) containing 1 mM L-arginine and 50 WM K1 PI for 1 h at 37³C, and the perfusate was applied to the HPLC-£ow reactor system for nitrosothiol measurement.
actor system to quantify the S-nitroso proteins produced in the cell culture (Fig. 2). The broken line indicates the time course of NO production quanti¢ed by measuring nitrite anion after addition of IFN-Q and LPS, which reached a maximum level at 12 h after addition of these stimulants. This cell itself did not produce a signi¢cant amount of NO. On the other hand, NO production was induced in the cells infected by S. typhimurium without any other stimulation as shown by the squares. Then, K1 PI was added at 100 WM which is a physiologically conceivable concentration of the protein to the cell culture infected by Salmonella, and nitrosylated products in the supernatant were quanti¢ed. One hundred to 200 nM of S-NO-K1 PI was detected at 12 and 18 h after infection, which was almost 10-fold higher than that of nitrosoalbumin. Nitrosylation of K1 PI was also detected in an ex vivo organ perfusion system of the liver excised from rats treated with LPS from Escherichia coli. E. coli LPS was injected i.p. (8.4 mg/kg) to the rats (male Wistar rats weighing 200^220 g) 14 h prior to obtain the liver. The liver was perfused with 30 ml Krebs-Henseleit bicarbonate bu¡er (pH 7.4) containing 1 mM L-arginine and 50 WM K1 PI at 37³C. After 1 h perfusion, S-NO-K1 PI production was clearly observed by the HPLC-£ow reactor analysis (Fig. 3).
We examined the e¡ect of nitrosation on the inhibitory activity of K1 PI against porcine pancreatic trypsin. The inhibitory e¡ects of K1 PI and S-NOK1 PI on porcine pancreatic trypsin were assayed using its substrate, benzoyl-DL-arginine p-nitroanilide hydrochloride, essentially according to the methods described elsewhere [27,28]. Although it is reported that chemical modi¢cation of the thiol group in K1 PI a¡ects the inhibitory action for human neutrophil elastase, S-nitrosylation did not in£uence the inhibitory activity of K1 PI for trypsin. 5. Novel functions of K1 PI generated after S-nitrosylation 5.1. Vasorelaxation One of the well-known biological functions of NO and nitrosothiols is relaxation of blood vessels. Therefore, we examined the vasorelaxing activity of S-NO-K1 PI using ring preparations of rabbit aorta. The rings were mounted in an organ bath and precontracted with 0.1 WM phenylephrine, after which S-NO-K1 PI-induced relaxation was measured. S-NOK1 PI at nM to WM concentrations exhibited relaxation of the precontracted aorta ring in a concentration-dependent manner (Fig. 4). On the contrary, native K1 PI without S-nitrosylation did not have any vasorelaxing activity at all. It is reported that
Fig. 4. Vasorelaxing activity of S-NO-K1 PI and K1 PI in the ring preparations of rabbit aorta. The rings were mounted in organ baths and precontracted with 0.1 WM phenylephrine, after which relaxation induced by the graded concentrations of S-NO-K1 PI and K1 PI was monitored. Data are means þ S.E. (n = 4).
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S-nitrosothiols mediate vasorelaxation without liberation of NO [29]. Transnitrosylation from S-NOK1 PI to vascular smooth muscle cells might occur either via a pure chemical reaction or an enzymatic reaction as reported recently [30], which needs to be elucidated more critically. 5.2. Antibacterial e¡ect NO has been implicated as a host-derived antimicrobial molecule [31]. NO per se, however, has minimal antimicrobial e¡ects as we reported previously [32]. Alternatively, it is now accepted that its reactive metabolites, such as peroxynitrite and NO2 (or NO2 Cl) are responsible for NO-dependent antimicrobial as well as cytotoxic activities [32^35]. On the other hand, there are several pieces of earlier works on the antibacterial activities of S-nitrosocysteine and other S-nitrosylated compounds with low molecular weights [36,37]. S-Nitrosylated albumin [38] and homocysteine-conjugated albumin [39], and S-nitrosoglutathione (GS-NO) [40,41] were also reported recently to show antimicrobial activities. It is well documented that the production of K1 PI and NO derived from iNOS are up-regulated by the bacterial infection [42]. Intriguingly, not only hepatocytes but also monocytes and macrophages, that are the main sources of iNOS, are responsible for increased production of K1 PI in the state of in£ammation [43]. Therefore, it is highly likely that S-NOK1 PI is produced at the site of bacterial infection. When we examined the antibacterial activities of S-NO-K1 PI and other NO-related compounds, we found a marked growth suppressing activity of S-NO-K1 PI against a number of Gram-positive and Gram-negative bacteria in comparison with other nitrosothiols and NO (Fig. 5). Speci¢cally, S-NO-K1 PI at low WM range inhibited the growth of various bacteria, while an mM level of GS-NO is needed to exhibit the same extent of antibacterial activity (Fig. 5). The mode of antibacterial action of S-NO-K1 PI seems to be bacteriostatic rather than bactericidal, which is a clear contrast to that of peroxynitrite, a strong bactericidal metabolite of NO [33]. Our preliminary experiments indicated an e¤cient transnitrosylation from S-NO-K1 PI to the bacterial cells, which was much faster than that from S-NO-albumin or GS-NO (not shown). The amount of S-NO-
Fig. 5. Antibacterial activity of S-NO-K1 PI and GS-NO. The bacteria indicated were suspended in KRP at the density of 1U106 CFU/ml and treated with various concentrations of S-NO-K1 PI or GS-NO for 1 h at 37³C. The bacterial suspension (20 Wl) was then added to brain-heart infusion broth (180 Wl) and additionally cultured for 6 h at 37³C to observe the bacterial growth by measuring the turbidity (absorbance at 610 nm). The 50% growth inhibitory concentration is shown for each bacterium examined.
K1 PI produced and/or its rate of production at the site of infection (not in the systemic circulation) seems to be an important point to be clari¢ed in future studies. 5.3. Other possible biological activities Similar to other nitrosothiols such as GS-NO [44], S-NO-K1 PI has a potent inhibitory e¡ect on platelet aggregation. It is also reported that nitrosothiols inhibit neutrophil adhesion to the endothelial surface and attenuate its accumulation in various organs with in£ammatory and ischemia-reperfusion injuries [45]. Thus, S-NO-K1 PI may exert tissue protecting activity from neutrophil-mediated tissue injury through inhibition of both neutrophil accumulation and its elastase. It seems therefore interesting to assess the e¡ects of S-NO-K1 PI on the neutrophilmediated tissue injury induced by infection and ischemia. Now we are examining such protective effects of S-NO-K1 PI on the ischemia-reperfusion injury in the liver, which will be reported elsewhere before long. Apoptosis is one of the most fundamental cellular events that is critically involved in various physiological and pathological phenomena. Accumulating evidence indicates that apoptosis takes place in a variety of infectious diseases including viral and bacterial infections and that it plays an important role in their
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pathogenesis [46,47]. Caspases, particularly caspase3, are regarded as key denominators in executing apoptosis [48,49]. Since caspases belong to cysteine proteases, the active center cysteinyl sulfhydryl residue might be susceptible to chemical modi¢cations by NO or its reactive metabolites. In fact, it is currently reported that NO suppresses apoptosis through inhibition of caspase-3 by S-nitrosylation of its active center [50^52]. As we described earlier in this paper, S-NO-K1 PI is a prominent nitrosonium (NO ) carrier (donor) possibly formed in the in£ammatory and infectious foci. Thus, an antiapoptotic e¡ect of S-NO-K1 PI is a critical issue of great interest which remains to be clari¢ed.
[3]
[4]
[5]
[6]
[7] [8]
6. Conclusion In this article, we describe that K1 PI is e¤ciently S-nitrosylated in vitro and also in biological systems. Besides retaining the inhibitory activity for serine proteases, the resultant S-NO-K1 PI showed new diverse biological functions, i.e., vasorelaxation and bacteriostatic activity. As a result of increased production of both NO and K1 PI in the in£ammatory condition or at a site of infection, S-NO-K1 PI is likely to be formed and it exhibits protective e¡ects on the hosts via its bacteriostatic activity and facilitating peripheral blood £ow.
[9]
[10]
[11]
[12]
[13]
Acknowledgements We thank Ms. Rie Yoshimoto for preparing the manuscript. This work was supported by a grant-inaid for Scienti¢c Research from the Ministry of Education, Science, Sports and Culture of Japan, and grants from the Ministry of Health and Welfare of Japan.
[14]
[15]
[16]
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