HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: jimmunol.0900593v1 183/6/4055    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Google Scholar Articles by Chatterjee, S. Articles by Mason, R. P. PubMed PubMed Citation Articles by Chatterjee, S. Articles by Mason, R. P.

Site-Specific Carboxypeptidase B1 Tyrosine Nitration and Pathophysiological Implications following Its Physical Association with Nitric Oxide Synthase-3 in Experimental Sepsis¹

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^*

^* Free Radical Metabolism Group, Laboratory of Pharmacology and Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709

LPS-induced sepsis results in oxidative modification and inactivation of carboxypeptidase B1 (CPB1). In this study, immunoprecipitated CPB1 was probed for tyrosine nitration using monoclonal nitrotyrosine-specific Abs in a murine model of LPS-induced sepsis. Tyrosine nitration of CPB1 was significantly reduced in the presence of NO synthase (NOS) inhibitors and the xanthine oxidase (XO) inhibitor allopurinol and in NOS-3 knockout (KO) mice. CPB1 tyrosine nitration and loss of activity by the concerted action 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, the tyrosine at the catalytic site. The site- and protein-specific nitration of CPB1 and the possible high nitration yield to inactivate it were elucidated by confocal microscopy. The studies indicated that CPB1 colocalized with NOS-3 in the cytosol of sinus-lining cells in the red pulp of the spleen. Further analysis of CPB1-immunoprecipitated samples indicated immunoreactivity to a monoclonal NOS-3 Ab, suggesting protein complex formation with CPB1. XO and NOS inhibitors and NOS-3 KO mice injected with LPS had decreased 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-specific tyrosine nitration and inactivation of CPB1. These results open up new avenues for the investigation of several enzymes involved in inflammation and their site-specific oxidative modifications by protein-protein interactions as well as their role in sepsis.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work has been supported by the Intramural Research Program of the National Institutes of Health and by National Institute of Environmental Health Sciences Grant Z01 ES050139-13.

² Address correspondence and reprint requests to Dr. Saurabh Chatterjee, Free Radical Metabolism Group, Laboratory of Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T. W. Alexander Drive, Research Triangle Park, NC 27709. E-mail address: chatterjees2{at}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 inhibitor; TRIM, 1-(2-trifluoromethylphenyl)imidazole; 1400W, N-3-(aminomethyl)benzylacetamide · 2HCl; XO, xanthine oxidase.