Nitration of annexin II tetramer.
Annexin II tetramer (AII(t)) is a member of the Ca(2+)- and phospholipid-binding protein family and is implicated in membrane fusion during surfactant secretion. It had previously been shown that high concentrations of nitric oxide (NO) inhibit surfactant secretion from lung type II cells. NO reacts with superoxide (O(2)(-)) to form peroxynitrite (ONOO(-)), a tyrosine nitrating agent, which is found in lungs under certain pathological conditions. It is therefore hypothesized that nitration of AII(t) by ONOO(-) may be a mechanism for the NO inhibition of regulated exocytosis. We therefore performed in vitro studies to test effects of ONOO(-) on AII(t). Western blot analysis using anti-nitrotyrosine antibodies showed a dose-dependent nitration of tyrosine residues in AII(t) treated with ONOO(-). Nitration occurred on the core domain of the p36 subunit, as well as on the p11 subunit. ONOO(-) also caused the formation of dimers between p36 and p11 subunits which were stable in the presence of heating, SDS, and beta-mercaptoethanol. AII(t)-mediated liposome aggregation was inhibited by ONOO(-) with an IC(50) of approximately 30 microM. The inhibition was abolished by urate (a scavenger of ONOO(-) and *OH), but not by mannitol (a scavenger of *OH) or superoxide dismutase (a scavenger of O(2)(-)) and appeared to be specific to AII(t), since ONOO(-) only slightly influenced annexin I-mediated liposome aggregation. The conformational change of AII(t) induced by Ca(2+) had no effect on the inhibition. Furthermore, ONOO(-) only partially inhibited the binding of AII(t) to membranes. Nitration of AII(t) also occurred in intact A549 cells, a lung epithelial cell line, treated with ONOO(-). The results of this study suggest that AII(t)-mediated liposome aggregation was inhibited by nitration of the protein.
Rowan
William H
WH
Department of Physiology, East Carolina University, Greenville, North Carolina 27858, USA.
Sun
Peng
P
Liu
Lin
L
D000818:Animals; D017306:Annexin A2 / Q000378:metabolism*; D001704:Biopolymers / Q000378:metabolism*; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008081:Liposomes; D008168:Lung / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism*; D011233:Precipitin Tests
D017306:Annexin A2; D001704:Biopolymers; D008081:Liposomes; D009566:Nitrates