Polymorphonuclear leukocytes (PMN) are essential for the maintenance of normal host defenses against infection. Central to this function is the phenomenon of degranulation, whereby PMN cytoplasmic granules fuse with, and discharge their contents into, phagocytic vacuoles. The objective of the proposed research is to determine whether the phenomenon of degranulation in human PMN is mediated by a calcium-binding, cytosol protein that is similar to, or identical with, synexin. The hypothesis will be tested that PMN synexin, in the presence of calcium and arachidonic acid, promotes fusion of specific and azurophil granules membranes with those portions of the PMN plasma membrane that surround ingested particles. In preliminary studies, we documented that bovine liver synexin, in the presence of calcium, causes isolated human PMN cytoplasmic granules to aggregate. In addition, we isolated and partially purified a protein from human PMN cytosol that also promotes calcium-dependent granule aggregation, and that resembles bovine liver synexin antigenically, physicochemically, and with respect to its other biologic activities. The PMN cytosol protein (PMN synexin) not only promotes calcium-dependent fusion of phospholipid vesicles (liposomes), but also promotes calcium- and arachidonic acid-dependent fusion of PMN specific granules with liposomes. We wish to extend these studies and to characterize further the physiocochemical properties and biologic activities of PMN synexin. We will determine whether PMN synexin binds in a calcium-dependent fashion to granule membranes. We also examine whether PMN synexin is phosphorylated following exposure of intact cells to stimuli that provoke degranulation. Finally, in experiments using phagocytic vacuoles isolated from PMN cytoplasts, we will determine whether PMN synexin promotes calcium- and arachidonic acid-dependent fusion of isolated granules with the cytoplasmic surface of the PMN plasma membrane. The studies outlined in this proposal should yield significant new information concerning the roles played by calcium and arachidonic acid, as well as the role played by synexin, in promoting fusion of PMN cytoplasmic granule membranes with those portions of the PMN plasma membrane that surround ingested particles. In addition, information derived from these studies should enhance our understanding of the phenomenon of stimulus-secretion coupling in PMN and in other cell types.
| Ernst, J D; Hoye, E; Blackwood, R A (1989) Use of a novel strategy for the preparation and characterization of an antipeptide antibody capable of recognizing members of the annexin family. Biochem Biophys Res Commun 161:959-64 |
