Site-specific carboxypeptidase B1 tyrosine nitration and pathophysiological implications following its physical association with nitric oxide synthase-3 in experimental sepsis. J Immunol 2009 Sep 15;183(6):4055-66
Date
09/01/2009Pubmed ID
19717511Pubmed Central ID
PMC2804978DOI
10.4049/jimmunol.0900593Scopus ID
2-s2.0-70349334296 (requires institutional sign-in at Scopus site) 28 CitationsAbstract
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 Tyr(248), 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.
Author List
Chatterjee S, Lardinois O, Bonini MG, Bhattacharjee S, Stadler K, Corbett J, Deterding LJ, Tomer KB, Kadiiska M, Mason RPMESH terms used to index this publication - Major topics in bold
AnimalsBinding Sites
Carboxypeptidase B
Lipopolysaccharides
Mice
Mice, Knockout
Nitric Oxide Synthase Type III
Nitrosation
Protein Binding
Sepsis
Spleen
Tandem Mass Spectrometry
Tyrosine