We have previously shown that CPB1 produces a DMPO-trappable protein radical in vivo
in LPS-induced systemic inflammation and that this radical production is mediated by the dual role of NOS-3 and xanthine oxidase (11
). In the present work, we report the site-specific nitration of splenic CPB1, possibly mediated by peroxynitrite, in vivo
and in vitro
in LPS-induced acute inflammation in mice, a model that resembles systemic inflammation response syndrome, or SIRS.
We chose to use FeTPPS, a relatively specific peroxynitrite decomposition catalyst in vivo
), to investigate the involvement of peroxynitrite in CPB1 nitration. We limited our studies with FeTPPS in vivo
to relating the tyrosine nitration process of CPB1 to peroxynitrite formation in LPS-induced systemic inflammation.
In peroxynitrite formation in sepsis, nitric oxide from NOS is known to play a very important role (36
). Here, the involvement of NOS-3 as a principal source of peroxynitrite at 6 h was confirmed using both NOS-3 and NOS-2 knockout mice. NOS-2 knockout mice showed no significant difference in immunoreactivity to 3-nitrotyrosine antibody, suggesting the important role played by NOS-3 in peroxynitrite formation and nitration of CPB1.
Tyrosine nitration at catalytic sites has been shown to result in loss of activity in many enzyme targets (2
). To determine whether the catalytic site tyrosines of CPB1 were targets of nitration in vivo
, we blocked the catalytic site by administering MGTA, a specific inhibitor of carboxypeptidase B-like metalloproteases that binds efficiently to the catalytic site (12
). Tyrosine nitration of CPB1 was completely blocked by MGTA (). Carboxypeptidase B-like enzymes are known to contribute to the arginine pool by providing the substrate to NOSs (12
). By binding to the catalytic site, MGTA decreased the production of arginine from peptide substrates and the production of NO. This effect might result in decreased peroxynitrite formation and complete inhibition of tyrosine nitration of CPB1. Also, blocking of tyrosine nitration of CPB1 after administration of MGTA would suggest that most of the target nitration sites may be located in the catalytic subunit of the enzyme, since MGTA binding would have masked the catalytic site tyrosines in CPB1. This evidence clearly pointed to the tyrosine nitration of CPB1 in vivo
, but did not define the specificity of the nitration process in CPB1, a zinc-containing metalloprotein. Analysis of the results from experiments using porcine CPB tissue () clearly showed the existence of at least five sites of nitration on tyrosine residues.
There has been considerable speculation recently that protein tyrosine nitration is only a biomarker rather than an event that alters protein function. Our in vitro
data with CPB and peroxynitrite suggests that nitration yields sufficient to cause <50% loss of activity require a 12.5 molar excess or 1.25 mM peroxynitrite (, right panel), while CPB1 loses >50% of its activity in pathophysiological conditions that mimic sepsis (). Porcine CPB was found to be nitrated at Tyr-248 with an 8-fold molar excess of tetranitromethane, with enzymatic activity towards both basic and non-basic substrates falling to less than 30% of the control (34
). The relative yield of protein 3-NT formation, even for proteins considered to be preferential targets for nitration, is, in general, low. As a result, the biological relevance of protein tyrosine nitration has been questioned, mainly in the context of the loss of protein function (38
). We attribute the loss of activity of CPB1 in the spleen to its proximal association and binding to NOS-3 in the early septic spleen. In order to define the specific nature of CPB1 nitration and inactivation in vivo
, we chose to examine the proximal association and protein-protein coupling in a cellular compartment as a possible way to nitrate specific tyrosines with a high nitration yield. The results, which demonstrated the co-localization, co-translocation and coupling of CPB1 with NOS-3 (), supported our 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 proximal association with NOS-3 in the sinus lining cells of the spleen.
The 3-nitrotyrosine moiety is often localized to specific tissue regions and cell types in different inflammatory disease processes, indicating a close association between sites of production of nitrating species and susceptible target proteins (39
). Specific CPB1 tyrosine nitration and loss of activity in septic mice is presumably due to the higher nitration yield attained in the local microenvironment (). This possibility is supported by the fact that nitration was significantly decreased in the presence of allopurinol, a specific inhibitor of xanthine oxidase. Proximal association of CPB1 with NOS-3 and sufficient superoxide generation formed via xanthine oxidase could generate peroxynitrite, resulting in nitration of one or more tyrosine residues that are important for catalysis of CPB1 ().
Fig. 8 Tyrosine nitration of CPB1 in the sinus lining cells of the spleen. Proximal association and binding of CPB1 and NOS-3 possibly resulted in higher nitration yield in the local milieu, leading to loss of CPB1 activity. This would possibly lead to accumulation (more ...)
LC/MS/MS analysis of nitro CPB showed five potential sites of nitration of tyrosine residues, including Tyr-248 and Tyr-198, which are located in proximity to the Zn atom in the catalytic site. A loss of function of CPB as observed with 12.5 molar equivalents or 1.25 mM peroxynitrite () could be attributed to the nitration of Tyr 248 and Tyr 198 ().
The significant inactivation of CPB1 in vivo
was probed further for any functional alteration of the enzyme. The hydrolysis of peptide bonds at the C-terminus of peptides and proteins carried out by carboxypeptidases may be a step in the degradation of some substrate molecules or result in the maturation of others. The physiological effect of these enzymes, as for every type of protease, is thus varied and site- and organism-dependent (40
). The presence of significantly increased levels of CPB1 has been identified in the spleens of septic mice (11
). Since enzymes of the carboxypeptidase family, especially CPB2 or TAFI, have been known to cleave basic arginine and lysine residues from peptides like C5a and bradykinin and regulate inflammation (10
), the functional significance of enzyme inactivation was probed in septic mice. The results of increased accumulation of C5a in LPS-administered mice () and their regulation by NOS-3 and XO inhibitors may indicate a broader role for tyrosine nitration of CPB1.
In conclusion, we report the post-translational tyrosine nitration of CPB1 with significant loss of its activity and concomitant accumulation of C5a in the spleen. The pathological consequences observed might be a result of high nitration rates due to the proximal association of CPB1 and NOS-3 in the sinus lining cells of the red pulp.