Human neutrophil annexin I promotes granule aggregation and modulates Ca(2+)-dependent membrane fusion.
The mechanism and cofactor requirements of exocytotic membrane fusion in neutrophils are unknown. Cytosolic proteins have been implicated in membrane fusion events. We assessed neutrophil cytosol for the presence of fusogenic proteins using a liposome fusion assay (lipid mixing). A fusogenic 36-kD protein containing amino acid sequence homology with human annexin I was purified from the cytosol of human neutrophils. This protein also shared functional characteristics with annexin I: it associated with and promoted lipid mixing of liposomes in a Ca(2+)-dependent manner at micromolar Ca2+ concentrations. The 36-kD protein required diacylglycerol to promote true fusion (contents mixing) at the same Ca2+ concentrations used for lipid mixing. The 36-kD protein exhibited a biphasic dose-response curve, by both promoting and inhibiting Ca(2+)-dependent lipid-mixing between liposomes and a plasma membrane fraction. The 36-kD protein also promoted Ca(2+)-dependent increases in aggregation of a specific granule fraction, as measured by a turbidity increase. Antiannexin I antibodies depleted the 36-kD protein from the cytosol by greater than 70% and diminished its ability to promote lipid mixing. Antiannexin I antibodies also decreased by greater than 75% the ability of neutrophil cytosol to promote Ca(2+)-dependent aggregation of the specific granules. These data suggest that annexin I may be involved in aggregation and fusion events in neutrophils.
Francis
J W
JW
Department of Pediatrics, University of Michigan School of Medicine, Ann Arbor 48109.
Balazovich
K J
KJ
Smolen
J E
JE
Margolis
D I
DI
Boxer
L A
LA
D017302:Annexins; D015153:Blotting, Western; D002118:Calcium / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D003594:Cytoplasmic Granules / Q000502:physiology* / Q000648:ultrastructure; D003600:Cytosol / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D008081:Liposomes; D008561:Membrane Fusion; D008970:Molecular Weight; D009504:Neutrophils / Q000502:physiology* / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry; D010743:Phospholipids / Q000378:metabolism
D017302:Annexins; D002135:Calcium-Binding Proteins; D008081:Liposomes; D010446:Peptide Fragments; D010743:Phospholipids; D002118:Calcium