Nitration of Annexin II Tetramer

Annexin II tetramer (AIIt) is a member of the Ca2+- 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 (O2 -) to form peroxynitrite (ONOO-), a tyrosine nitrating agent, which is found in lungs under certain pathological conditions. It is therefore hypothesized that nitration of AIIt 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 AIIt. Western blot analysis using anti-nitrotyrosine antibodies showed a dose-dependent nitration of tyrosine residues in AIIt 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 β-mercaptoethanol. AIIt-mediated liposome aggregation was inhibited by ONOO- with an IC50 of ∼30 μM. 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 O2 -) and appeared to be specific to AIIt, since ONOO- only slightly influenced annexin I-mediated liposome aggregation. The conformational change of AIIt induced by Ca2+ had no effect on the inhibition. Furthermore, ONOO- only partially inhibited the binding of AIIt to membranes. Nitration of AIIt also occurred in intact A549 cells, a lung epithelial cell line, treated with ONOO-. The results of this study suggest that AIIt-mediated liposome aggregation was inhibited by nitration of the protein.