The function of annexin I in human neutrophils. Studies of membrane binding

Abstract

The neutrophil granulocyte plays an important role in host defense against infections induced by invading microorganisms. The defense mechanism is dependent on the migration of neutrophils from the blood stream to the infected area, where the intruder is phagocytosed and killed. Neutrophil activation is associated with a mobilization of intracellular granules and vesicles. These organelles contain both matrix proteins that upon activation are secreted to the extracellular milieu or into the phagosome, and membrane localized receptors that become exposed on the cell surface. Neutrophil secretion is highly regulated and depends on intracellular Ca2+-levels, suggesting that Ca2+-regulated proteins may be part of the regulatory network.The aim of this study was to investigate the role of Ca2+-regulated proteins in the secretion of neutrophil organelles. The study was focused on the role of the Ca2+-dependent and membrane-binding proteins annexin I and grancalcin, found in the cytosol of resting neutrophils.To study the Ca2+-regulated binding of annexin I to different neutrophil membrane compartments (i.e. azurophil granules, specific granules, gelatinase granules and secretory vesicles/plasma membrane) in vitro, these organelles were isolated by subcellular fractionation on different types of Percoll gradients. The binding properties of annexin I and grancalcin to specialized detergent insoluble membrane domains, or lipid rafts, were studied by isolating these domains from the plasma membrane/secretory vesicles by cold Triton X-100 extraction and Percoll gradient centrifugation.Annexin I has beside its cytosolic localization also been found extracellularly, secreted via an unknown pathway. By using the subcellular fractionation technique, annexin I was shown to be exclusively localized to the cytosol of human neutrophils and absent from the granules. This implies that the secretion of annexin I to the extracellular milieu is independent of degranulation.Annexin I has earlier been shown to bind to all subcellular organelles isolated from neutrophils in the presence of Ca2+. The present study shows that the binding is accompanied by the cleavage of the annexin I molecule in the N-terminal domain, generating a truncated annexin I molecule, annexin Ides1-8. The cleavage is mediated by a membrane-localized metalloprotease present in some subcellular organelles, but excluded from others. The cleavage of annexin I affects the binding of the protein to membranes by decreasing the Ca2+ requirement. This suggests that the membrane-binding properties of annexin I is regulated by the N-terminal cleavage of the protein by effecting the Ca2+ affinity.Annexin I and grancalcin both bind to detergent insoluble membrane domains within the plasma membrane in the presence of Ca2+, suggesting that lipid rafts are of regulatory importance in neutrophil secretion.In conclusion, the Ca2+-dependent binding of annexin I and grancalcin to detergent insoluble membrane domains and the functional modification achieved through endogenous cleavage of annexin I suggest an involvement of these processes during regulation of neutrophil secretion.