of February 19, 2018.This information is current as Experimental Sepsis

Association with Nitric Oxide Synthase-3 in Implications following Its PhysicalNitration and Pathophysiological Site-Specific Carboxypeptidase B1 Tyrosine

Ronald P. MasonLeesa J. Deterding, Kenneth B. Tomer, Maria Kadiiska andSuchandra Bhattacharjee, Krisztian Stadler, Jean Corbett, Saurabh Chatterjee, Olivier Lardinois, Marcelo G. Bonini,

http://www.jimmunol.org/content/183/6/4055doi: 10.4049/jimmunol.0900593August 2009;

2009; 183:4055-4066; Prepublished online 28J Immunol 

Referenceshttp://www.jimmunol.org/content/183/6/4055.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2009 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Site-Specific Carboxypeptidase B1 Tyrosine Nitration andPathophysiological Implications following Its PhysicalAssociation with Nitric Oxide Synthase-3 in ExperimentalSepsis1

Saurabh Chatterjee,2* Olivier Lardinois,* Marcelo G. Bonini,* Suchandra Bhattacharjee,*Krisztian Stadler,* Jean Corbett,* Leesa J. Deterding,† Kenneth B. Tomer,† Maria Kadiiska,*and Ronald P. Mason*

LPS-induced sepsis results in oxidative modification and inactivation of carboxypeptidase B1 (CPB1). In this study, immunopre-cipitated CPB1 was probed for tyrosine nitration using monoclonal nitrotyrosine-specific Abs in a murine model of LPS-inducedsepsis. Tyrosine nitration of CPB1 was significantly reduced in the presence of NO synthase (NOS) inhibitors and the xanthineoxidase (XO) inhibitor allopurinol and in NOS-3 knockout (KO) mice. CPB1 tyrosine nitration and loss of activity by the concertedaction of NOS-3 and XO were also confirmed in vitro using both the NO donor 3-morpholinosydnonimine and peroxynitrite.Liquid chromatography/tandem mass spectrometry data indicated five sites of tyrosine nitration in vitro including Tyr248, thetyrosine at the catalytic site. The site- and protein-specific nitration of CPB1 and the possible high nitration yield to inactivate itwere elucidated by confocal microscopy. The studies indicated that CPB1 colocalized with NOS-3 in the cytosol of sinus-lining cellsin the red pulp of the spleen. Further analysis of CPB1-immunoprecipitated samples indicated immunoreactivity to a monoclonalNOS-3 Ab, suggesting protein complex formation with CPB1. XO and NOS inhibitors and NOS-3 KO mice injected with LPS haddecreased levels of C5a in spleens of septic mice, indicating peroxynitrite as a possible cause for CPB1 functional alteration. Thus,CPB1 colocalization, coupling, and proximity to NOS-3 in the sinus-lining cells of spleen red pulp could explain the site-specifictyrosine nitration and inactivation of CPB1. These results open up new avenues for the investigation of several enzymes involvedin inflammation and their site-specific oxidative modifications by protein-protein interactions as well as their role in sepsis. TheJournal of Immunology, 2009, 183: 4055–4066.

T he possible formation of peroxynitrite and the resultantposttranslational nitration of protein tyrosine residues areassociated with the pathogenesis of a series of diseases,

including acute and chronic inflammatory processes, sepsis, isch-emia-reperfusion, and neurodegenerative diseases (1, 2). Nitrationof tyrosine residues by radical mechanisms is always tyrosyl- andNO- or nitrogen dioxide-dependent (3). Despite the number ofdiseases and pathological conditions in which tyrosine nitrationhas been observed, our knowledge of specific enzyme targets islimited, especially where a distinctive association is found betweentyrosine nitration and functional alterations.

Tyrosine nitration does not depend on the abundance or numberof tyrosine residues present on a particular protein (2). The com-monalities among various nitrated proteins include glutamate oraspartate residues in the vicinity of Tyr residues and/or the pres-

ence of turn-inducing amino acids such as proline or glycine (4–6). Tyrosine nitration yields in proteins, organs, and disease con-ditions have typically been low; the poor yield has raised questionsabout nitration as a posttranslational modification in the molecularbasis of disease (2).

Carboxypeptidase B (CPB)3 in tissue, designated CPB1, wasinitially described as a pancreatic metallocarboxypeptidase and isa marker for acute pancreatitis. This stable protease has high ho-mology with plasma CPB and has substrates in common with it.This was assessed in recent studies where supplementation of thematrix with additional thrombin-activatable fibrinolysis inhibitor(TAFI) or CPB produced a reduction in capillary tube formation(7). Plasma CPB, CPU (carboxypeptidase U), or active TAFI (des-ignated TAFIa) has a half-life of 8 min and plays a role in inflam-mation (8–10). Earlier work from this laboratory has identifiedCPB1 in the septic spleen and found it to form a radical in thepresence of xanthine oxidase (XO) and NO synthase (NOS)-3.This study further investigates the nature of the radical and itsposttranslational modification.

In this work, we address the site-specific nature of protein ty-rosyl radical formation and nitration and the higher nitration yield

*Free Radical Metabolism Group, Laboratory of Pharmacology and †Laboratory ofStructural Biology, National Institute of Environmental Health Sciences, NationalInstitutes of Health, Research Triangle Park, NC 27709

Received for publication February 23, 2009. Accepted for publication July 20, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work has been supported by the Intramural Research Program of the NationalInstitutes of Health and by National Institute of Environmental Health Sciences GrantZ01 ES050139-13.2 Address correspondence and reprint requests to Dr. Saurabh Chatterjee, Free Rad-ical Metabolism Group, Laboratory of Pharmacology, National Institute of Environ-mental Health Sciences, National Institutes of Health, 111 T. W. Alexander Drive,Research Triangle Park, NC 27709. E-mail address: chatterjees2@niehs.nih.gov

3 Abbreviations used in this paper: CPB, carboxypeptidase B; DMPO, 5-dimethyl-1-pyrroline N-oxide; FeTPPS, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) chloride; KO, knockout; L-NIO, N5-(1-imino-3-butenyl)-L-ornithine; MGTA, DL-2-mercaptomethyl-3-guanidinoethylthiopropionic acid; NOS, NO synthase;SIN-1, 3-morpholinosydnonimine; TAFI, thrombin-activatable fibrinolysis inhib-itor; TRIM, 1-(2-trifluoromethylphenyl)imidazole; 1400W, N-3-(aminomethyl)-benzylacetamide � 2HCl; XO, xanthine oxidase.

The Journal of Immunology

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generated in CPB1, a zinc-containing tissue metalloprotein, fol-lowing LPS-induced systemic inflammation. Our previous workhas shown that LPS-induced systemic inflammation leads to theformation of CPB1 radicals, which are mediated by XO and en-dothelial NOS (NOS-3) with a concomitant loss of enzyme activity(11). Immuno-spin trapping of the CPB1 radical was a significantstep in the demonstration of the involvement of NOS-3- and XO-derived oxidizing species in vivo. However, molecular and post-translational footprints of reactive oxygen species- and reactivenitrogen species-based oxidative stress needed to be identified. Weused relatively specific NOS inhibitors to identify the relativecontributions of different NOS isoforms and peroxynitrite decom-position catalysts to identify the role of peroxynitrite in CPB1-tyrosine nitration. The CPB1 inhibitor DL-2-mercaptomethyl-3-guanidinoethylthiopropionic acid (MGTA) was used to study theinvolvement of the catalytic process in the nitration of CPB1.

Furthermore, to understand the molecular basis of CPB1, wehave examined the role of 3-morpholinosydnonimine (SIN-1) andperoxynitrite in tyrosine oxidation and nitration in vitro. Impor-tantly, it has been shown that carboxypeptidase M cleaves peptidesat crucial arginine residues and contributes to the arginine pool(12). We hypothesized that CPB1 sensitivity to nitration was dueto its proximity to NOS-3 activated during inflammatory stress,providing it with a crucial substrate in inflammatory conditions.This hypothesis has now been supported by the colocalization ofNOSs, XO, and CPB1 in the spleen. We also studied the enzymeinactivation of CPB1 and its correlation with nitrotyrosine forma-tion and identified the sites of the tyrosine residues nitrated. Wereport the coupling of NOS-3 with CPB1, the formation of NO viaNOS-3, and the role of XO in producing O2

., whose concertedaction with NOS-3-derived NO leads to tyrosine nitration ofCPB1. The above-mentioned events might result in higher nitra-tion yields sufficient to inactivate CPB1 in sepsis.

Materials and MethodsMaterials

LPS (Escherichia coli strain 55:B5), porcine CPB, SIN-1 hydrochloride,and allopurinol were obtained from Sigma-Aldrich. The spin trap 5,5-di-methyl-1-pyrroline N-oxide (DMPO) was obtained from Alexis Biochemi-cals. Trypsin (from bovine pancreas, a modified sequencing grade) andchymotrypsin (from bovine pancreas, a modified sequencing grade) wereobtained from Roche Molecular Biochemicals. All other chemicals were ofanalytical grade and were purchased from Sigma-Aldrich or Roche Mo-lecular Biochemicals. All aqueous solutions were prepared using waterpassed through a Picopure2 UV Plus system (Hydro Services and Supplies)equipped with a 0.2-�m pore size filter. Absorption spectra were recordedon a Cary 100 UV-visible spectrometer (Varian). HPLC was conducted onan Agilent ChemStation 1100 series liquid chromatography system (Agi-lent Technologies) equipped with a control module, binary pump, manualinjector, and diode array UV-visible detector. HPLC fractions were col-lected using a fraction collector, Bio-Rad model 2110.

Mice

Adult male, pathogen-free, 8- to 10-wk-old C57BL6/J mice (The JacksonLaboratory) weighing 23–27 g on arrival were used in these experiments.The animals were housed for 1 wk, one to a cage, before any experimentaluse. Experiments using mice that contained the disrupted NOS-2 (NOS-2�/�; B6.129P2-Nos2tm1Lau/J), gp91phox (gp91phox�/�; B6.129S6-Cybbtm1Din) and NOS-3 (NOS-3�/�; B6.129P2-Nos3tm1Unc/J backcrossedonto the C57BL6/J for 12 generations) genes were treated identically. Thecontrol animals for knockout (KO) experiments were age-matched mice ofC57BL6/J origin in the case of NADPH oxidase and NOS-3 andB6129PF2/J origin in the case of NOS-2 that had normal NOS-2, NADPHoxidase, and NOS-3 activity. Mice had ad libitum access to food and waterand were housed in a temperature-controlled room at 23–24°C with a 12-hlight/dark schedule. All animals were treated in strict accordance withguidance on the humane care and use of laboratory animals from the Na-tional Institutes of Health, and the experiments were approved by the in-

stitutional review board of the National Institute of Environmental HealthSciences.

LPS-induced systemic inflammation model

Systemic inflammation was induced in mice following LPS administrationas described previously (11, 13). Briefly, mice received a bolus infusion ofLPS (6 and 12 mg/kg) at 0 h. A sham group was also included in whichnormal mice received saline in place of LPS. LPS was dissolved in pyro-gen-free saline and administered through the i.p. route. At 6 h, mice fromthe sham group and the LPS groups were sacrificed. The spleens werecollected and snap-frozen in liquid nitrogen. Tissues were homogenized inphosphate buffer containing 100 �M diethylenetriaminepentaacetic acidand centrifuged at 3000 rpm at 4°C for 20 min. The samples were storedat �80°C until further use.

Administration of allopurinol, NOS inhibitors, CPB inhibitor,and the peroxynitrite scavenger FeTPPS

Allopurinol, a specific inhibitor of XO, the nonselective NOS-3 inhibitorsN5-(1-imino-3-butenyl)-L-ornithine (L-NIO) and vinyl-L-NIO (CaymanChemical), the putatively selective inhibitor of neuronal NOS-1, 1-(2-tri-fluoromethylphenyl)imidazole (TRIM; Calbiochem), and the NOS-2 inhib-itor N-3-(aminomethyl)benzylacetamide � 2HCl (1400W; Sigma-Aldrich)were administered in a single bolus dose of 20 mg/kg through the i.p. route30 min before LPS treatment. In different experiments, the peroxynitritedecomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyri-nato iron (III) chloride (FeTPPS; Calbiochem) and the CPB1 inhibitorMGTA (Sigma-Aldrich), an inhibitor of CPB, were administered at bolusdoses of 30 and 100 mg/kg and 20 mg/kg, respectively, through the i.p.route 15 min before LPS administration.

Immunoprecipitation and immunoblotting

CPB1 was immunoprecipitated with polyclonal anti-CPB1 Ab (R&D Sys-tems) using the Seize X mammalian immunoprecipitation kit (Pierce Bio-medical) with some modifications. Solubilized spleen cell homogenatesadjusted to a protein concentration of 150 �g per sample were preclearedby adding 200 �l of protein A/G-agarose followed by incubation for 1 h atroom temperature. The homogenate was then incubated overnight with 30�l of polyclonal anti-mouse CPB1 Ab (0.1 �g/�l), and the Ag-antibodymixture was further incubated with the protein A/G-agarose slurry over-night. Immune complexes were eluted with elution buffer according to themanufacturer’s instructions. Anti-CPB1 immunoprecipitates were sub-jected to SDS/PAGE on 4–12% Bis-Tris gels (Invitrogen) and electroblot-ted onto nitrocellulose membranes. Abs for the corresponding Westernblots used in these experiments were mouse monoclonal anti-nitrotyrosine(1/1000 dilution; Abcam). In some experiments, lysates were subjected toimmunoblotting without immunoprecipitation. The Abs used in these ex-periments were anti-mouse polyclonal CPB1 (1/1000 dilution; R&D Sys-tems), mouse monoclonal anti-NOS-3 (1/1000; Cell Signaling), and puri-fied rat anti-mouse C5a (1/2000, BD PharMingen). The immunocomplexedmembranes were probed (1 h at room temperature) with either goat anti-rabbit (1/5000; Upstate Biotechnologies), donkey anti-goat (1/3000; R&DSystems), or goat anti-mouse (1/5000; Pierce) HRP-conjugated secondaryAbs. Immunoreactive proteins were detected using ECL (Immobilon West-ern chemiluminiscence HRP substrate; Millipore). The images were sub-jected to densitometry analysis using LabImage 2006 Professional one-dimensional gel analysis software from Kaplean Bio-Imaging Solutions.

Carboxypeptidase activity assay

Carboxypeptidase activity of immunoprecipitates was measured using theActichrome TAFI activity kit (American Diagnostica) according to theprocedure described by the manufacturer with some modifications. Briefly,the reaction was initiated by adding 50 �l of the TAFI substrate to 25 �lof anti-CPB1 immunoprecipitates. After incubation for 30 min at 37°C, thereaction was stopped by the addition of sulfuric acid and the releasedchromogenic product was measured at 490 nm. The A490 of blanks, whichconsisted of complete mixtures incubated for 0 min, was subtracted fromeach value. CPB concentration in immunoprecipitates was estimated byELISA using anti-CPB1 Ab (1/1000; R&D Systems). A calibration curvewas constructed with known concentrations of recombinant mouse CPB1(R&D Systems).

The activity of purified porcine CPB was estimated by monitoring theformation of hippuric acid liberated from hippuryl-L-arginine (14, 15). For-mation of hippuric acid was monitored at 254 nm under the following assayconditions: 1 mM hippuryl-L-arginine, 4 nM CPB, 100 mM NaCl, and 25mM Tris-HCl (pH 7.65) at 25°C. The concentration of purified porcineCPB solutions was determined at 278 nm (� � 7.35 � 104 M�1cm�1) (14).

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Confocal microscopy

For confocal studies, C57BL/B6 wild-type mice were injected with LPS at12 mg/kg and sacrificed 6 h after LPS administration. Spleens were col-lected and washed in PBS and the tissues were fixed in 10% neutral buff-ered formalin. After fixation, spleens were removed and placed in 30%sucrose for 24 h. Tissues were then sliced on a microtome into 20-�msections, placed in PBS, and then permeabilized with 0.1% Surfact-AmpsX-100 detergent solution (Pierce Biomedical) for 1 h. After blocking with0.1% BSA in PBS, NOS-3 was stained using monoclonal anti-NOS-3 Ab(BD Transduction Laboratories) as the primary Ab and Alexa Fluor 488anti-mouse conjugated with fluorescein (Molecular Probes-Invitrogen) asthe secondary Ab. CPB1 localization was probed by incubating the sameslides with anti-CPB1 polyclonal Ab and Alexa Fluor 594 anti-goat sec-ondary Ab. Secondary controls were used to determine background fluo-rescence by applying the secondary Ab only (data not shown). Slices weremounted on microscope cover glasses (22 � 22 mm; 1-mm thickness)(Erie Scientific) and sections were analyzed under a confocal laser micro-scope (Zeiss).

C5a chemotaxis assay

Cell migration assay was conducted using the CytoSelect 96-well cellmigration assay kit (Cell Biolabs) following the manufacturer’s proto-col. HL-60 cells were stimulated for 72 h with 1000 U/ml IFN-� to haveC5a receptor expression and were used for the assay. Mouse rC5a(R&D Chemicals) was used as a positive control and incubated in thefeeder layer. A group where no rC5a was added served as the negativecontrol.

Experiments with pancreatic CPB, mouse rCPB1 peroxynitrite,and SIN-1 in vitro

To illustrate the possible posttranslational modification of CPB in vitro,porcine CPB was incubated with different concentrations of SIN-1 andperoxynitrite in the presence or absence of 25 mM sodium bicarbonate.Peroxynitrite was prepared in the laboratory according to the method de-scribed by Bonini et al., (16, 17). CPB (100 �M) was dissolved in phos-phate buffer (pH 7.4) and incubated with 300, 1,250, or 2,500 �M per-oxynitrite or peroxynitrite donor SIN-1. In experiments with mouse rCPB,1 �M mouse rCPB1 was dissolved in phosphate buffer (pH 7.4) and in-

cubated with 3, 12.5, or 25 �M peroxynitrite. Samples consisting of onlyenzyme, only peroxynitrite, or only SIN-1 formed the negative controls.The reaction mixture was incubated at 37°C for 1 h. The resultant reactionmixture was then separated by SDS-PAGE and tested for nitrotyrosineimmunoreactivity by Western analysis.

Sample preparation and proteolytic digestion

Typically, incubations contained 100 �M CPB in 50 mM potassium phos-phate buffer (pH 7.4) and were conducted at 37°C in the presence or ab-sence of 25 mM sodium bicarbonate, as indicated in the figure legends. Thereactions were initiated by the addition of different concentrations of per-oxynitrite (3–25 equivalents) and allowed to proceed for 30 min, afterwhich the solutions were passed over a Sephadex G-25 gel filtration col-umn (GE Healthcare Bio-Sciences) previously equilibrated and eluted with100 mM Tris-HCl (pH 8.5). The samples were denatured with 6 M gua-nidine hydrochloride in 100 mM Tris-HCl (pH 8.5) at 60°C for 30 min,reduced with 5 mM DTT at 37°C for 30 min, and alkylated with 25 mMiodoacetamide at 37°C for 30 min. The resulting solutions were thenloaded onto a previously equilibrated PD-10 gel filtration column andeluted with 100 mM Tris-HCl (pH 8.5). After passage through the PD-10column, carboxyamidated samples were digested using a 20:1 substrate-to-protease ratio for 16 h at 37°C (trypsin) or for 16 h at 25°C (chymo-trypsin). Reactions were stopped by injecting the final mixture directly ontoa reverse-phase HPLC column.

Reverse-phase HPLC analysis of proteolytic hydrolysates

Digested samples (200 �l) were injected directly onto a Vydac 218TP54,4.6 mm � 250 mm, C18 reverse-phase HPLC column. Peptides wereeluted at a flow rate of 0.8 ml/min with a linear gradient from 100% solventA (0.1% trifluoroacetic acid in water) to 50% solvent B (0.085% trifluoro-acetic acid in acetonitrile) over 80 min. A rapid gradient to 90% solvent Band then back to 100% solvent A was used to regenerate the column. Theeluent was monitored at 214, 280, and 365 nm with an Agilent HP1100diode array detector. HPLC fractions were collected with a fraction col-lector, and the fractions containing nitrated peptides (as judged by theappearance of chromatographic peaks at 365 nm) were pooled, lyophilized,and stored at �70°C for subsequent analysis.

FIGURE 1. Immunoblotting ofimmunoprecipitated CPB1 fromspleen tissue homogenates with anti-nitrotyrosine Ab. A, Spleen tissueproteins were isolated from controland LPS-treated mice and then immu-noprecipitated with anti-CPB1 Aband immunoblotted with an anti-nitrotyrosine Ab. A 47-kDa band cor-responding to CPB1 is shown by ablack arrow. Equal loading was con-firmed using a direct ELISA of CPB1present in the immunoprecipitate. TheCPB1 band density was comparedwith nitrotyrosine band density fornormalization. B, Densitometric anal-ysis of normalized band densitiesagainst anti-CPB1 immunoblots. �,p � 0.05 when compared with sham.Similar results were obtained in threeindependent experiments. C, In a con-trol experiment the specificity ofCPB1 tyrosine nitration was con-firmed by preabsorbing the anti-CPB1 antiserum with recombinantmouse CPB1 (i, lane 2; ii, lane 4),BSA (i, lane 1) and normal rabbit se-rum (ii, lane 3).

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FIGURE 2. CPB1 tyrosine nitration is a result of peroxynitrite formation by dual action of NOS-3 and XO. A, Spleen proteins from sham-, LPS- andLPS plus FeTPPS (peroxynitrite decomposition catalyst)-treated mice were immunoprecipitated with anti-CPB1 Ab, separated by SDS-PAGE, and elec-troblotted on a nitrocellulose membrane. Western blot analysis was performed using an Ab specific to 3-nitrotyrosine (inset). The bar chart shows the bandintensity of the immunoreactive proteins. B, Spleen proteins from sham-, LPS-, LPS plus L-NIO-, and LPS plus vinyl-L-NIO-treated mice (relatively specificNOS-3 inhibitors) and LPS plus allopurinol-treated mice (XO-specific inhibitor) were immunoprecipitated with anti-CPB1 Ab, separated by SDS-PAGE,and electroblotted on a nitrocellulose membrane. Western blot analysis was performed using an Ab specific to 3-nitrotyrosine. C, Graphical representationof the band intensities of immunoreactive proteins. Equal loading was confirmed using a direct ELISA of CPB1 present in the immunoprecipitate. The CPB1band density was compared with nitrotyrosine band density for normalization. �, p � 0.05 when compared with sham; #, p � 0.05 when compared withLPS (12 mg/kg)-treated mice. D, Spleen proteins from sham, LPS-, LPS plus L-NIO-, LPS plus 1400W (NOS-2 inhibitor)-, and LPS plus TRIM (NOS-1inhibitor)-treated mice were immunoprecipitated with anti-CPB1 Ab, separated by SDS-PAGE, and electroblotted on a nitrocellulose membrane. Westernblot analysis was performed using an Ab specific to 3-nitrotyrosine. E, Graphical representation of the band intensities of immunoreactive proteins is alsoprovided. Equal loading was confirmed using a direct ELISA of CPB1 present in the immunoprecipitate. The CPB1 band density was compared withnitrotyrosine band density for normalization. �, p � 0.05 when compared with sham; #, p � 0.05 when compared with LPS (12 mg/kg)-treated mice. F,Spleen proteins from sham- and LPS-treated wild-type (WT) and NOS-3 KO LPS mice were immunoprecipitated with anti-CPB1 Ab, separated bySDS-PAGE, and electroblotted on a nitrocellulose membrane. Western blot analysis was performed using an Ab specific to 3-nitrotyrosine (3-NT). G,Graphical representation of the band intensities of immunoreactive proteins. Equal loading was confirmed using a direct ELISA of CPB1 present in the

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Electrospray mass spectrometry

A Micromass quadrupole/time-of-flight micro hybrid tandem mass spec-trometer (Waters Micromass) was used for the acquisition of the electro-spray ionization mass spectra and tandem mass spectra. All experimentswere performed in the positive ion mode. Lyophilized HPLC fractions ofnitrated peptides were resuspended in a minimal volume (�50 �l) of 50:50water:acetonitrile containing 0.1% formic acid and infused into the massspectrometer at �300 nl/min using a pressure injection vessel (18). Theneedle voltage was �3,200 volts and the collision energy was 10 eV for themass spectrometry analyses. Collision-induced dissociation experimentsused argon with collision energy between 30 and 50 electron volts. Dataanalysis was accomplished with a MassLynx data system and MaxEntdeconvolution software supplied by the manufacturer.

Statistical analyses

All in vivo experiments were repeated three times with three mice pergroup (n � 3). All in vitro experiments were repeated three times and thestatistical analysis was conducted using the Microcal Origin software pack-age. Quantitative data from Western blotting as depicted from the relativeintensity of the bands were analyzed by performing a one-tailed Student’st test. Values of p � 0.05 were considered statistically significant.

ResultsLPS-induced septic shock produces CPB1 tyrosine nitrationin vivo

CPB1 forms a DMPO-trappable protein radical in response to LPStreatment as detected by immuno-spin trapping (11). To detectwhether the CPB1 radical correlated with the nitration of tyrosineresidues, which is dependent on tyrosyl radical formation (19),spleen homogenates from sham and LPS (6 and 12 mg/kg) treat-ments were probed for 3-nitrotyrosine immunoreactivity (data notshown). The data shows anti-nitrotyrosine Ab immunoblotting ofspleen tissue homogenates from control and LPS-treated mice. Anumber of proteins showed detectable immunoreactivity towardthe Ab at 6 h, but not in 3 or 24 h, in both the control and theLPS-treated mice. The most prominently nitrated protein had amolecular mass of �47 kDa. MALDI-TOF analysis of this 47-kDaband performed in an earlier study (11) resulted in the detection of�10 proteins, one of which was CPB1.

To determine whether the CPB1 protein was nitrated, mousespleen tissue lysates were immunoprecipitated with the anti-CPB1Ab. The immunoprecipitates were then immunoblotted with theanti-nitrotyrosine Ab. Western blot analysis of the immunoprecipi-tates (Fig. 1A) indicated the presence of only one nitrated proteinband of �47 kDa. The results also indicated that CPB1 nitrationwas significantly higher in the LPS-treated group than in the sham-treated group at 6 h (Fig. 1B). This result suggests that the 47-kDanitrated protein detected in the splenocyte lysate is indeed CPB1.As an additional control of specificity for the nitration of CPB1,anti-CPB1 antiserum was preabsorbed with mouse rCPB1. Thepreabsorbed Ab was used for immunoprecipitation of CPB1. Anti-CPB1 Ab was also preabsorbed with BSA and normal rabbit serumfor negative controls. Results indicated that immunoprecipitationof CPB1 using a CPB1 Ab preabsorbed with mouse rCPB1 hadlittle or no reactivity to anti-nitrotyrosine Ab (Fig. 1C, lanes 2 and4), whereas immunoprecipitation of CPB1 using an Ab preab-sorbed with either BSA or normal rat serum showed distinct bandson the Western blot (Fig. 1C, lanes 1 and 3).

Tyrosine nitration of CPB1 is apparently due to formation ofperoxynitrite and is mediated by XO and NOS-3

It is known that protein tyrosine nitration can be mediated by per-oxynitrite (1). Superoxide and NO in a diffusion-controlled reac-tion yield peroxynitrite that, in biological systems, promptly reactswith carbon dioxide, leading to the formation of carbonate (CO3

.)and nitrogen dioxide (NO2

.) radicals (16, 20, 21, 22). Althoughthere are no direct methods for detecting peroxynitrite in vivo,there are reports of certain classes of iron porphyrins that act asperoxynitrite decomposition catalysts (23).

To determine whether peroxynitrite plays a role in LPS-inducedtyrosine nitration of CPB1, we treated mice with FeTPPS, a por-phyrin peroxynitrite scavenger, before LPS treatment. Proteinsfrom spleen homogenates were immunoprecipitated with an anti-CPB1 Ab and analyzed by Western blotting using an anti-nitrotyrosine Ab. As depicted in Fig. 2A, administration of FeTPPSsignificantly inhibited the nitration of the protein, supporting theinvolvement of peroxynitrite.

Peroxynitrite is a powerful oxidant that is formed by the diffu-sion-limited reaction between nitrogen monoxide (NO�) and su-peroxide (O2

.). To identify the possible O2. and NO� generators in

LPS-treated mice, we used allopurinol, a specific XO inhibitor,1400 W, a specific inhibitor for NOS-2, TRIM, a putatively spe-cific inhibitor for NOS-1, and vinyl-L-NIO and L-NIO, two non-selective inhibitors of all NOS isoforms. As seen in Fig. 2B, ad-ministration of the XO inhibitor allopurinol and two nonselectiveinhibitors of NOS, the isoforms L-NIO and vinyl-L-NIO, signifi-cantly inhibited the nitration of CPB1 (Fig. 2, B and C), whereasthe administration of 1400W, a specific inhibitor for NOS-2, andTRIM, a relatively specific inhibitor for NOS-1, did not inhibitnitration (Fig. 2, D and E). Because the inhibitors showing markedisoform selectivity toward NOS-1 and NOS-2 did not inhibit thenitration of CPB1, whereas, in strong contrast, the nonselectiveinhibitors were highly effective, it is thus reasonable to suspect thatNOS-3 plays a central role in the nitration process.

In the septic spleen, NOS-2 has been shown to be a major sourceof NO, whereas at early sepsis (4–6 h after LPS administration)NOS-3 and XO were major sources of NO� and O2

., respectively(11, 24). The respective roles of the three enzymes in the CPB1nitration process were delineated using gene KO mice. Westernblot analysis of anti-CPB1 immunoprecipitates indicated that thenitration of tyrosine residues was not blocked in either NOS-2 KOmice (Fig. 2, F and G, lane 4 from the left) or NADPH oxidase KOmice (data not shown). In contrast, no staining was observed foreither sham or LPS-treated NOS-3 KO mice (Fig. 2, F and G, lanes2 and 3 from the left). These data indicate that NOS-3 is the pri-mary NOS isoform responsible for CPB1 nitration in vivo.

To investigate the involvement of CPB1 in the catalysis of itsself-nitration in vivo, we used MGTA, an inhibitor of CPB1, whichbinds to the catalytic site of the enzyme. Tyrosine nitration ofimmunoprecipitated CPB1 was completely blocked by the admin-istration of this inhibitor in LPS-treated mice as shown in Fig. 2,H and I.

immunoprecipitate. The CPB1 band density was compared with nitrotyrosine band density for normalization. �, p � 0.05 when compared with sham ofNOS-3 KO; #, p � 0.05 when compared with sham-treated wild-type (WT) mice. H, Spleen proteins from sham-, LPS-, and LPS plus MGTA (CPB inhibitorthat binds to the catalytic site)-treated mice were immunoprecipitated with an anti-CPB1 Ab, separated by SDS-PAGE, and electroblotted on a nitrocellulosemembrane. Western blot analysis was performed using an Ab specific to 3-nitrotyrosine (3-NT). I, Graphical representation of the band intensities ofimmunoreactive proteins. Equal loading was confirmed using a direct ELISA of CPB1 present in the immunoprecipitate. The CPB1 band density wascompared with nitrotyrosine band density for normalization. �, p � 0.05 when compared with sham; #, p � 0.05 when compared with LPS (12 mg/kg)-treated mice.

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FIGURE 3. Peroxynitrite-mediated tyrosine nitration inactivates CPB1 in the septic spleen and in vitro. A, CPB1 activity in spleen tissue homog-enates from LPS, LPS plus allopurinol, LPS plus L-NIO, 1400W, LPS plus vinyl-L-NIO, and NOS-3 KO mice was measured using an ActichromeTAFI activity kit with slight modifications, and activity indices were calculated using sham-treated values (activity index � treated activity/shamactivity). B, Tyrosine nitration mediated by peroxynitrite as a function of loss of activity of CPB. Porcine CPB (100 �M) was incubated with differentconcentrations of peroxynitrite. Samples were separated by SDS-PAGE, and the proteins were electroblotted on a nitrocellulose membrane. Theblotted proteins were probed for 3-nitrotyrosine immunoreactivity using a 3-nitrotyrosine Ab (Anti 3-NT). C, Graphical representation of the bandintensities of immunoreactive proteins. Equal loading was confirmed using Coomassie blue-stained bands on the SDS-polyacrylamide gel. Further-more, the CPB1 band density of the Western blot with anti-CPB Ab was compared with the nitrotyrosine band density for normalization. �, p �0.05 when compared with the CPB only group; $, p � 0.05 when compared with the CPB plus ONOO� (0.6 mM) group; #, p � 0.001 when comparedwith the CPB plus ONOO� (1.25 mM) group. D, Percentage activity of CPB as a function of 0, 3, 6, 12.5, and 25 molar equivalents (eqs) ofperoxynitrite (Per). E, Mouse recombinant CPB1 (1 �M) was incubated with different concentrations of peroxynitrite. Samples were separated bySDS-PAGE and the proteins were electroblotted on a nitrocellulose membrane. The blotted proteins were probed for 3-nitrotyrosine immunoreac-tivity using a 3-nitrotyrosine Ab (Anti 3-NT). F, Graphical representation of the band intensities of immunoreactive proteins. Equal loading wasconfirmed using Ponceau-stained bands on the nitrocellulose membrane. Furthermore, the CPB1 band density of the Western blot with the anti-CPB1Ab was compared with the nitrotyrosine band density for normalization. �, p � 0.05 when compared with the recombinant mouse CPB1 only group;#, p � 0.001 when compared with the CPB plus ONOO� (25 �M) group.

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LPS-induced peroxynitrite formation and nitration lead toinactivation of CPB1 in vivo and in vitro

To study the biological consequences of CPB1 tyrosine nitration,we measured the enzyme activity of CPB1. The activity index ofimmunoprecipitated CPB1 in LPS-treated mice and LPS plus1400W-treated mice was significantly lower than that in the sham-treated mice ( p � 0.05) (Fig. 3A). Administration of allopurinol,L-NIO, or vinyl-L-NIO to LPS-treated mice significantly increasedactivity compared with the LPS-treated group alone ( p � 0.05).LPS-treated mice lacking the NOS-3 gene also had activity com-parable to that of the corresponding sham-treated NOS-3 KO mice(Fig. 3A). The results obtained in vivo were further confirmed withparallel measurements of peroxynitrite-mediated nitration of CPBin vitro and loss of activity. When porcine CPB and/or mouserCPB1 was treated with 3, 6, 12.5, or 25 molar equivalents ofperoxynitrite, there was a concentration-dependent increase in ty-rosine nitration (Fig. 3, B, C, E, and F). The increased tyrosinenitration was associated with parallel decreases in the activity ofCPB, showing �50% loss of activity with 12.5 molar equivalentsof peroxynitrite (Fig. 3D).

LPS-induced tyrosine nitration of CPB1 and inactivation arelinked to colocalization and protein complex formation withNOS-3

The loss of CPB activity in the presence of authentic peroxynitritein vitro (50% activity loss with 12.5 equivalents or 1.25 mM per-oxynitrite) raises the question of the possible existence of suchconcentrations of peroxynitrite in inflammatory physiology. Typ-ically, protein tyrosine nitration is a relatively widespread in vivomodification, with its overall yield (expressed as moles of 3-nitro-tyrosine/mole of tyrosine) being typically low. The low yield posesserious doubts about whether these modifications are relevant tothe molecular basis of disease in pathophysiological states (2). Toestablish the connection between the inactivation of CPB1 in vivoand the concentration of peroxynitrite required to inactivate CPBin vitro, we sought to determine the localization of CPB1 in thespleen. We propose that a close proximity to the availability of NOand O2

. sources can create sufficiently high nitration yields in asite-specific manner so as to inactivate the enzyme. It has beenshown that NO production in rat lung microvascular endothelialcells is stimulated more efficiently by arginine released from car-boxypeptidase substrates than by free arginine after LPS stimula-tion, thus indicating a mechanism by which the arginine supply forNO production in inflammatory conditions may be maintained(12).

These observations prompted us to investigate whether subcel-lular colocalization of two enzymes in the splenic vascular bedresulted in a steady flow of NO and the formation of higher con-centrations of peroxynitrite. This could lead to high nitration yieldsthat are not obtained even in inflammatory microenvironmentswhere the enzymes are widely separated. Confocal microscopyresults indicated that levels of both CPB1 and NOS-3 were high inthe septic spleen and that CPB1 colocalized with NOS-3 in thesinus-lining cells of the red pulp. NOS-3 was mostly localized inthe membrane in sham-treated spleens (Fig. 4AI), and LPS treat-ment resulted in the translocation of NOS-3 to the cytosol andcolocalization with CPB1 (Fig. 4AII) (25). Cells lining the venoussinusoids also showed significant colocalization of CPB1 andNOS-3 (Fig. 4AIII). We did not observe the same colocalizationpattern in the white pulp of the spleen (data not shown).

To further address whether the colocalization resulted from thebinding of CPB1 to NOS-3 following its activation and transloca-tion into the cytosol in LPS- treated mice, immunoprecipitates with

CPB1 were probed with monoclonal anti-NOS-3 Ab. In both thesham-treated and LPS-treated groups there was an immunoreactiveband at 140 kDa corresponding to NOS-3, whereas in the NOS-3KO group no immunoreactivity was observed (Fig. 4B). The in-tensity of the bands was analyzed by band analysis software, andthe band intensity from the LPS-treated group was significantlyhigher than that from the sham-treated group (data not shown).These data strongly suggest that CPB1 existed in a form boundwith NOS-3 in the sham- and LPS-treated spleens, but not inNOS-3 KO mice (Fig. 4B).

CPB1 oxidative inactivation leads to accumulation of C5a in theLPS-treated spleen

It has recently been shown that thrombin-activatable CPB is cat-alytically more efficient than plasma carboxypeptidase N (CPN),the major plasma anaphylatoxin inhibitor, in inhibiting bradykinin,activated complements C3a and C5a, and thrombin-cleaved os-teopontin in vitro (26). CPB inactivates these active inflammatorymediators by specific cleavage of the carboxyl-terminal arginines(10). Similarly, pro-CPB-deficient mice displayed enhanced pul-monary inflammation with C5a-induced alveolitis (10). Based onthe above evidence, we chose to probe the functional consequencesof LPS treatment in the spleen. When we separated spleen tissue

FIGURE 4. NOS-3 colocalization and protein complex formation withCPB1 contribute to its tyrosine nitration. A, Blocks I, II, and III representconfocal imaging of spleen sections from sham- and LPS-treated mice. Thesections are focused on the splenic red pulp from the regions surroundingthe venous sinusoids. Block III focuses on specific sinus lining cells of thered pulp. Each block represents staining in red (CPB1), green (NOS-3), andyellow (colocalized regions). B, Spleen proteins from NOS-3 KO(LPS�NOS-3 k/o; negative control)-, sham-, and wild type (LPS�WT)-treated mice were immunoprecipitated with anti-CPB1 Ab, separated bySDS-PAGE, and electroblotted on a nitrocellulose membrane. Western blotanalysis was performed using a mAb specific to mouse NOS-3.

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FIGURE 5. CPB1 tyrosine nitration results in C5a accumulation in the mouse spleen following LPS administration. A, Spleen proteins from sham-, LPS-,LPS plus NOS-2 inhibitor 1400W-, LPS plus L-NIO-, LPS plus vinyl-L-NIO (relatively specific NOS-3 inhibitors)-, and LPS plus allopurinol (XOinhibitor)-treated mice were separated by SDS-PAGE and electroblotted on a nitrocellulose membrane. Western blot analysis was performed using an Abspecific to C5a. B, Bar graph represents the normalized relative band intensities of the Western blot analysis of the proteins immunoreactive to C5a Ab.C, Spleen proteins from sham (wild type), LPS (wild type), sham (NOS-3 KO) and LPS (NOS-3 KO) were separated by SDS-PAGE and electroblottedon a nitrocellulose membrane. Western blot analysis was performed using an Ab specific to C5a. D, Bar graph represents the normalized relative bandintensities of the Western blot analysis of the proteins immunoreactive to C5a Ab.�, p � 0.05 when compared with the sham-treated wild-type (WT) mice;#, p � 0.05 when compared with the sham-treated KO mice. E, Chemotaxis of HL-60 cells in response to rC5a. Also, rC5a was incubated with both mouserCPB1 and nitro-CPB1 to assess the effect of nitration of CPB1 on C5a-induced chemotaxis. �, p � 0.05 when compared with rC5a alone; #, p � 0.05when compared with rC5a plus mouse rCPB1. F, Chemotaxis of HL-60 cells in response to immunoprecipitated C5a from LPS- (wild type)-, XO inhibitor-,NOS-3 inhibitor-, NOS-2 inhibitor-, and LPS-treated KO mice spleen tissue homogenates was measured in terms of relative fluorescent intensity by aCytoSelect 96-well cell migration assay following the manufacturer’s protocol (Cell Biolabs). �, p � 0.05 when compared with sham-treated mice alone;#, p � 0.05 when compared with LPS-treated wild-type (WT) mice.

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homogenate proteins by SDS-PAGE and immunoblotted themagainst the C5a Ab, we found that LPS-treated and LPS plus1400W-treated mouse spleen tissue homogenates showed signifi-cant levels of C5a compared with no detectable levels in sham-treated spleens (Fig. 5, A and B). Accumulation of C5a under theseconditions would lead to severe inflammatory reactions as thoseseen in sepsis and septic shock.

The XO inhibitor allopurinol and the NOS-3 inhibitors L-NIOand vinyl-L-NIO, which significantly decreased tyrosine nitrationof CPB1 in spleens of LPS-treated mice (Fig. 2B), also decreasedthe levels of C5a in the LPS-treated spleen (Fig. 5, A and B).Furthermore, levels of C5a were significantly increased in NOS-3KO mice compared with the corresponding KO sham treatment,but were significantly lower than those in LPS-treated wild-typemice (Fig. 5, C and D).

Because polyclonal anti-serum cannot distinguish between C5a(74 aa) and C5a des Arg (73 aa), neutrophil chemotaxis assay wasperformed to evaluate concentrations of C5a relative to its lessactive (5%) des Arg form (27, 28). In the first set of experiments,HL-60 cell chemotactic response to purified C5a was examined.Results indicated that at 2 � 10�9 M, mouse rC5a could inducechemotaxis of HL-60 cells, whereas preincubation of C5a withmouse rCPB1 reduced chemotactic response by up to 40% (Fig.5E). Little chemotactic activity was detectable in the absence ofC5a. Furthermore, when nitro-mouse rCPB1 was incubated withmouse rC5a, chemotaxis was comparable to the control/unmodi-fied C5a. In a second set of experiments, the chemotactic responseof HL-60 cells to immunoprecipitated C5a from spleen tissue ho-mogenates was evaluated. These experiments were performed us-ing equal amounts of immunoprecipitated C5a as assessed byELISA. The results indicated that the chemotactic response wassignificantly higher for samples from LPS- and LPS plus 1400W-treated mice than that observed for sham, allopurinol, L-NIO, Vi-nyl-L-NIO, and LPS KO mouse samples (Fig. 5F).

In vitro characterization of peroxynitrite-induced chemicalmodifications of CPB1

To study the molecular basis of tyrosine oxidation and nitration ofCPB by peroxynitrite in vitro, porcine CPB (100 �M) was incu-bated with peroxynitrite (300 �M) in the presence or absence of 25mM sodium bicarbonate. Carbonate radicals (CO3

.) can be formedbiologically by the decomposition of the peroxynitrite-carbon di-oxide adduct (ONOOCO2

�) or by enzymatic activities, i.e., per-oxidase activity of copper-zinc-dependent superoxide dismutase(CuZnSOD) and XO turnover in the presence of bicarbonate (29,30, 31). Peroxynitrite-dependent tyrosine nitration is likely to oc-cur through the initial reaction of peroxynitrite with carbon dioxideor metal centers, leading to secondary nitrating species (19, 32).We found that a reaction mixture containing sodium bicarbonate asa bicarbonate source showed several immunoreactive bands whencompared with samples incubated with only peroxynitrite. CPB(32 and 35 kDa) and mouse CPB1 (43 and 48 kDa) migrate asdoublets on an SDS-polyacrylamide gel. Apart from the monomer(35 kDa) and a slightly less intense band at 30–32 kDa, anotherdistinct band of �70 kDa was immunoreactive to an Ab specific to3-nitrotyrosine (Fig. 6).

Following this observation, we used SDS-PAGE to analyze areaction mixture containing both peroxynitrite and SIN-1 as a po-tential mimetic of O2

./NO generation. The analysis revealed theformation of dimers, characterized by the 70-kDa Coomassie blue-stained band, compared with none in a sample containing onlyCPB (Fig. 7A). The dimers were inhibited in a dose-dependentmanner when incubated with 10 and 100 mM DMPO (Fig. 7A). Ithas been reported that the formation of dimers is the result of

dityrosine crosslinks that form by the reaction of two tyrosyl rad-icals. Dityrosine crosslinks are considered to be stable markers forthe oxidative modification of proteins (33). To identify the natureof the dimer, we measured the fluorescence of the intact proteincontaining the reaction mixture at 410 nm. The spectra showed apattern that is distinct for dityrosine formation in samples incu-bated with SIN-1, but was absent in samples containing CPB alone(Fig. 7B). When the reaction mixture was digested with trypsin andrun through a reverse-phase HPLC column, a spectrum indicativeof dityrosine crosslink formation was found in one of the peptides(data not shown).

Nitration of the catalytic site tyrosines (Tyr248 and Tyr198) as anindex of tyrosine oxidation and inactivation of CPB

To examine the possible sites of tyrosine nitration of CPB, theenzyme was incubated with 12.5 molar equivalents of peroxyni-trite, digested with either chymotrypsin or trypsin, and subjected toreverse-phase HPLC. The fractions corresponding to the absor-bance at 365 nm were collected and subjected to tandem massspectrometry analysis. At least 11 peptides with five sites of ni-tration on tyrosine residues were identified. These include Tyr92,Tyr210, Tyr277, Tyr248, and Tyr198. Of these, Tyr248 and Tyr198 arelocated in the catalytic site of CPB (Table I). The observed nitra-tion of Tyr248 was consistent with previous findings showing thatTyr248 is more reactive and undergoes nitration with an 8-foldmolar excess of tetranitromethane (34).

DiscussionWe have previously shown that CPB1 produces a DMPO-trap-pable protein radical in vivo in LPS-induced systemic inflamma-tion and that this radical production is mediated by the dual role ofNOS-3 and XO (11). In the present work, we report the site-spe-cific nitration of splenic CPB1, possibly mediated by peroxynitrite,in vivo and in vitro in LPS-induced acute inflammation in mice, amodel that resembles systemic inflammation response syndromeor SIRS.

We chose to use FeTPPS, a relatively specific peroxynitrite de-composition catalyst in vivo (35), to investigate the involvement of

FIGURE 6. Role of carbonate radical formation in CPB tyrosine nitra-tion. CPB (100 �M) was incubated with 300 �M peroxynitrite in the ab-sence or presence of 25 mM sodium bicarbonate. The protein was thenseparated by SDS-PAGE and electroblotted on a nitrocellulose membrane.Western blot analysis was performed using an Ab specific to 3-nitroty-rosine (Anti 3-NT). The Coomassie blue-stained protein on the SDS-poly-acrylamide gel is included as a loading control. The Western blot analysisis a representative of three independent experiments.

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peroxynitrite in CPB1 nitration. We limited our studies with FeT-PPS in vivo to relating the tyrosine nitration process of CPB1 toperoxynitrite formation in LPS-induced systemic inflammation.

In peroxynitrite formation in sepsis, NO from NOS is known toplay a very important role (36). In this study, the involvement ofNOS-3 as a principal source of peroxynitrite at 6 h was confirmed

by using both NOS-3 and NOS-2 KO mice. NOS-2 KO miceshowed no significant difference in immunoreactivity to 3-nitroty-rosine Ab, suggesting the important role played by NOS-3 in per-oxynitrite formation and nitration of CPB1.

Tyrosine nitration at catalytic sites has been shown to result inloss of activity in many enzyme targets (2). To determine whether

FIGURE 7. Formation of dity-rosine dimer as a footprint of tyrosineradical formation on CPB by per-oxynitrite. A, CPB (0.1 mM) was in-cubated with different concentrationsof SIN-1 and peroxynitrite in thepresence or absence of DMPO. Thereaction mixture was then separatedby SDS-PAGE and stained with Coo-massie Blue. B, CPB (0.1 mM) wasincubated with SIN-1 (0.3 mM) for1 h. The reaction mixture was thenchecked for an increase in fluores-cence at 410 nm, characteristic of di-tyrosine dimer formation. The Coo-massie blue-stained gel photographand the dityrosine dimer detection byfluorescence are representative of atotal of three experiments.

Table I. Summary of identified peptides originating from proteolytic digestion of CPB and nitrated tyrosine residuesa

Protease Elution Time Position of YNO2 Sequence

Trypsin 45.6 210 (Y)SYDYKLPENNAELNNLA(K)Trypsin 48.3 248 (K)YTYGPGATTIYPAAGGSDDWAYDQGIK(Y)Trypsin 52.6 92 (R)EAVLTYGYESHMTEFLN(K)Trypsin 58.79 277 (R)YGFILPESQIQATCEETMLAIK(Y)Trypsin 61.4 277 (R)YGFILPESQIQATCEETMLAI(K)Chymotrypsin 30.9 277 (F)ELRDKGRY(G)Chymotrypsin 40.84 198 (Y)LTIHSY(S)Chymotrypsin 41.43 210 (Y)SYDYKLPENNAELNNL(A)Chymotrypsin 41.77 92 (Y)GYESHMTEF(L)Chymotrypsin 41.77 248 (T)IYPAAGGSDDW(A)Chymotrypsin 41.83 92 (Y)GYESHMTEF(L)Chymotrypsin 42.06 248 (T)IYPAAGGSDDW(A)

a Summary of identified peptides originating from proteolytic digestion of porcine CPB. Tryptic and chymotryptic digestions of untreated CPB andCPB reacted with peroxynitrite were subjected to reverse-phase HPLC. Absorbance at 365 nm was used to monitor the elution of the nitrated peptidefragment. The fractions showing intense signals at 365 nm were collected and further characterized by tandem mass spectrometry. The nitrated tyrosineresidues (YNO2) are underlined in the “Sequence” column.

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the catalytic site tyrosines of CPB1 were targets of nitration invivo, we blocked the catalytic site by administering MGTA, aspecific inhibitor of CPB-like metalloproteases that binds effi-ciently to the catalytic site (12, 37). Tyrosine nitration of CPB1was completely blocked by MGTA (Fig. 2D). CPB-like enzymesare known to contribute to the arginine pool by providing the sub-strate to NOS (12). By binding to the catalytic site, MGTA de-creased the production of arginine from peptide substrates and theproduction of NO. This effect might result in decreased peroxyni-trite formation and complete inhibition of tyrosine nitration ofCPB1. Also, blocking of the tyrosine nitration of CPB1 after ad-ministration of MGTA would suggest that most of the target ni-tration sites may be located in the catalytic subunit of the enzyme,because MGTA binding would have masked the catalytic site ty-rosines in CPB1. This evidence clearly pointed to the tyrosinenitration of CPB1 in vivo, but it did not define the specificity of thenitration process in CPB1, a zinc-containing metalloprotein. Anal-ysis of the results from experiments using porcine CPB tissue (Ta-ble I) clearly showed the existence of at least five sites of nitrationon tyrosine residues.

There has been considerable speculation recently that proteintyrosine nitration is only a biomarker rather than an event thatalters protein function. Our in vitro data with CPB and peroxyni-trite suggests that nitration yields sufficient to cause �50% loss ofactivity require a 12.5 molar excess or 1.25 mM peroxynitrite (Fig.3B), whereas CPB1 loses �50% of its activity in pathophysiolog-ical conditions that mimic sepsis (Fig. 3). Porcine CPB was foundto be nitrated at Tyr248 with an 8-fold molar excess of tetrani-tromethane, with enzymatic activity toward both basic and nonba-sic substrates falling to �30% of the control (34). The relativeyield of protein 3-nitrotyrosine formation, even for proteins con-sidered to be preferential targets for nitration, is, in general, low.As a result, the biological relevance of protein tyrosine nitrationhas been questioned, mainly in the context of the loss of proteinfunction (38). We attribute the loss of activity of CPB1 in thespleen to its proximal association and binding to NOS-3 in theearly septic spleen. To define the specific nature of CPB1 nitrationand inactivation in vivo, we chose to examine the proximal asso-ciation and protein-protein coupling in a cellular compartment as apossible way to nitrate specific tyrosines with a high nitrationyield. The results, which demonstrated the colocalization, cotrans-location, and coupling of CPB1 with NOS-3 (Fig. 4), supportedour assertion that CPB1 nitration is protein- and site-specific. Also,our data strongly suggest a novel argument that tyrosine nitration

of CPB1 affects its physiological functions owing to its proximalassociation with NOS-3 in the sinus lining cells of the spleen.

The 3-nitrotyrosine moiety is often localized to specific tissueregions and cell types in different inflammatory disease processes,indicating a close association between sites of production of ni-trating species and susceptible target proteins (39). Specific CPB1tyrosine nitration and loss of activity in septic mice is presumablydue to the higher nitration yield attained in the local microenvi-ronment (Fig. 8). This possibility is supported by the fact thatnitration was significantly decreased in the presence of allopurinol,a specific inhibitor of XO. Proximal association of CPB1 withNOS-3 and sufficient superoxide generation formed via XO couldgenerate peroxynitrite, resulting in nitration of one or more ty-rosine residues that are important for catalysis of CPB1 (Fig. 8).

Liquid chromatography/tandem mass spectrometry analysis ofnitro-CPB showed five potential sites of nitration of tyrosine res-idues, including Tyr248 and Tyr198, which are located in proximityto the zinc atom in the catalytic site. A loss of function of CPB asobserved with 12.5 molar equivalents or 1.25 mM peroxynitrite(Fig. 3B) could be attributed to the nitration of Tyr248 and Tyr198

(Table I).The significant inactivation of CPB1 in vivo was probed further

for any functional alteration of the enzyme. The hydrolysis of pep-tide bonds at the C terminus of peptides and proteins conducted bycarboxypeptidases may be a step in the degradation of some sub-strate molecules or result in the maturation of others. The physi-ological effect of these enzymes, as for every type of protease, isthus varied and site- and organism-dependent (40). The presenceof significantly increased levels of CPB1 has been identified in thespleens of septic mice (11). Because enzymes of the carboxypep-tidase family, especially CPB2 or TAFI, have been known tocleave basic arginine and lysine residues from peptides like C5aand bradykinin and regulate inflammation (10), the functional sig-nificance of enzyme inactivation was probed in septic mice. Theresults of increased accumulation of C5a in LPS-administeredmice (Fig. 5) and their regulation by NOS-3 and XO inhibitorsmay indicate a broader role for tyrosine nitration of CPB1.

In conclusion, we report the posttranslational tyrosine nitrationof CPB1 with significant loss of its activity and concomitant ac-cumulation of C5a in the spleen. The pathological consequencesobserved might be a result of high nitration rates due to the prox-imal association of CPB1 and NOS-3 in the sinus lining cells of thered pulp.

FIGURE 8. Tyrosine nitration of CPB1 in the sinus lining cells of the spleen. Proximal association and binding of CPB1 and NOS-3 possibly resultedin higher nitration yield in the local milieu, leading to loss of CPB1 activity. This would possibly lead to the accumulation of inflammatory mediators likeC5a in the spleen, thus amplifying the systemic inflammatory response and altering immune pathology.

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AcknowledgmentsWe acknowledge Tiwanda Marsh, Jeoffrey Hulbert, and Jeff Tucker forexcellent technical assistance. We also thank Dr. Ann Motten, Dr. MarilynEhrenshaft, and Mary Mason for helping with the editing of thismanuscript.

DisclosuresThe authors have no financial conflict of interest.

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