Site-specific carboxypeptidase B1 tyrosine nitration and pathophysiological implications following its physical association with nitric oxide synthase-3 in experimental sepsis.
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.
Chatterjee
Saurabh
S
Free Radical Metabolism Group, Laboratory of Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Lardinois
Olivier
O
Bonini
Marcelo G
MG
Bhattacharjee
Suchandra
S
Stadler
Krisztian
K
Corbett
Jean
J
Deterding
Leesa J
LJ
Tomer
Kenneth B
KB
Kadiiska
Maria
M
Mason
Ronald P
RP
D000818:Animals; D001665:Binding Sites; D043424:Carboxypeptidase B / Q000032:analysis / Q000378:metabolism*; D008070:Lipopolysaccharides; D051379:Mice; D018345:Mice, Knockout; D052250:Nitric Oxide Synthase Type III / Q000032:analysis / Q000172:deficiency / Q000378:metabolism*; D015538:Nitrosation; D011485:Protein Binding; D018805:Sepsis / Q000139:chemically induced / Q000201:enzymology / Q000378:metabolism*; D013154:Spleen / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D014443:Tyrosine / Q000378:metabolism
D008070:Lipopolysaccharides; D014443:Tyrosine; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D043424:Carboxypeptidase B