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A truncated form of annexin I, formed during Ca2+-induced translocation to neutrophil specific granules and secretory vesicles/plasma membranes, is generated through the action of an endogenous membrane protease. The cleavage of annexin I is inhibited by the metalloprotease inhibitor 1,10-phenanthroline as well as by Triton X-100 and dithiothreitol, classifying the protease as a membrane-bound, thiol-dependent metalloprotease. The cleavage site is located close to the N-terminal of annexin I, leaving a truncated form of the molecule, des1-8 annexin I, that contains the Ca2+-binding sites, as well as a number of phosphorylation sites of importance for the function of the protein. When assessing binding capacity to different neutrophil organelles, full-length annexin I bound to azurophil granules, specific granules, and secretory vesicles/plasma membranes, while des1-8 annexin I only bound to specific granules and secretory vesicles/plasma membranes, but not to azurophil granules (C. Sjölin, C. Dahlgren, Biochim. Biophys. Acta 1281 (1996) 227-234). This implies that there are different mechanisms of binding to neutrophil organelles of full-length annexin I and the truncated form, and that cleavage of annexin I might be of regulatory importance for the degranulation process.

In an earlier study, annexin XI was found to be present in the cytosol of neutrophil granulocytes (Blood (1996) 87, 4817). The protein was isolated by calcium-dependent translocation to specific granules and was found to be a 42-kDa truncated form of annexin XI. Using human autoantibodies directed against annexin XI we have now reinvestigated the ability of full size annexin XI to translocate to different neutrophil organelles isolated by subcellular fractionation. The autoantisera used recognised a protein of 55-kDa in neutrophil cytosol and comparison with a whole cell lysate indicated that the larger portion of the cellular content of this protein is localised to the cytosol. Azurophil granules, specific granules and secretory vesicles/plasma membrane were isolated by subcellular fractionation on Percoll gradients, mixed respectively with neutrophil cytosol and the calcium concentration was raised. Immunoblotting showed that annexin XI translocated to specific granules and secretory vesicles/plasma membrane at 100 micromol/l calcium. When raising the concentration of calcium to 1 mmol/l, annexin XI translocated to the azurophil granules as well. Periphagosomal translocation of annexin XI occurred during phagocytosis of yeast particles, implying that this protein plays a role in the events associated with the phagocytic process.

A neutrophil annexin I-related protein, detected after translocation of cytosolic proteins to specific granules and secretory vesicles/plasma membrane (Sjölin et al. (1994) Biochem. J. 300, 325-330), has been characterized with respect to origin and organelle-binding properties. The annexin I-related protein is formed as a result of annexin I cleavage, and this occurs during translocation of annexin I to the specific granules and secretory vesicles/plasma membrane, but not when annexin I is translocated to azurophil granules. The cleavage required calcium and it was facilitated in the presence of specific granules or secretory vesicles/plasma membrane, but not in the presence of azurophil granules. We conclude that the membranes of specific granules and secretory vesicles/plasma membrane contain a protease which is able to cleave annexin I into a truncated 38 kDa fragment, which retains the ability to bind to these organelles. The azurophil granules lack the capacity to cleave annexin I as well as the ability to bind the 38 kDa fragment. These findings may implicate a role for annexin I in the divergent regulation of exocytosis of the different neutrophil granules.