Interaction of annexins with membranes: the N-terminus as a governing parameter as revealed with a chimeric annexin

The modulating effect of the variable N-terminus of annexins on the properties of these Ca2+-binding proteins was investigated. To this end, the interaction of annexin V and a mutant annexin, I(N)V(C), consisting of the N-terminus of annexin I (amino acids 1-45) and the core of annexin V (19-320), with large unilamellar phosphatidylserine (PS) vesicles was examined. In contrast to annexin V, the mutant annexin mediated Ca2+-dependent aggregation of the lipid vesicles at neutral pH. However, annexin V induces Ca2+-dependent aggregation at mild acidic pH. Moreover, both proteins can engage in hydrophobic interactions with PS vesicles, which results in release of the vesicle contents. These membrane-perturbing properties are expressed by both annexins in the absence of Ca2+ and occur at neutral and mild acidic pH. Interestingly, addition of Ca2+ inhibits annexin V-induced release, but sustains the release induced by the mutant annexin I(N)V(C). The Ca2+-dependent effects on the release of vesicle contents are reversed upon EDTA addition. Conformational changes revealed by binding of the hydrophobic probe, 4,4'-bis(1-anilino-8-naphthalensulfonate), underly the observed Ca2+-modulated effects on leakage. However, low-pH-mediated aggregation by annexin V does not seem to be related to macroscopic conformational changes. Annexin I(N)V(C) also affects Ca2+-induced fusion of PS vesicles, displaying synergistic properties in conjunction with Ca2+ at neutral pH. By contrast, annexin V does not display similar properties at mild acidic pH, in spite of its ability to aggregate vesicles under such conditions. Since the cores of annexins V and I(N)V(C) are identical, the present results emphasize the role of the N-terminus in governing annexin-membrane interaction properties. It is furthermore of interest that, in addition, the properties of annexins might be regulated by pH, which would extend their physiological range of operation.