Background
Annexin A7 is a Ca2+- and phospholipid-binding protein, which was isolated as the agent that mediated aggregation of chromaffin granules and fusion of phospholipid membranes in the presence of Ca2+. This activity led to the proposal of annexin A7's involvement in the exocytotic secretion of catecholamines [1]. The protein belongs to a family of evolutionarily conserved proteins of a bipartite structure with a variable N-terminal and a conserved C-terminal domain. The C-terminal domain is responsible for the Ca2+- and phospholipid-binding, the N-terminal domain appears to confer functional diversity [2-4]. The binding to negatively charged phospholipids is thought to be mediated by Ca2+ ions.
Annexin A7 is unique in that it carries an extraordinarily long and hydrophobic amino terminus with more than 100 amino acids. Alternative splicing gives rise to two isoforms of 47 and 51 kDa. Both isoforms differ by an additional cassette exon located in the first third of the N-terminal domain. Most tissues harbor only the 47 kDa isoform, both forms are found in brain and heart, while the large isoform is exclusively expressed in mature skeletal muscle.
At the cellular level annexin A7 can be detected in the cytosol, at the plasma membrane, around the nucleus, at vesicular structures including adrenal chromaffin granules, and at the t-tubule system [5,6]. Annexin A7 translocates to membranes in a Ca2+-dependent fashion and, when intracellular Ca2+ levels rise, sequentially redistributes to the plasma and the nuclear membrane as well as to intracellular vesicles. Furthermore, annexin A7 associates with lipid rafts [7]. Lipid rafts play a key role in membrane budding and in vesiculation processes such as endo- and exocytosis [8-10].
Two binding partners of annexin A7 have been identified, sorcin and galectin-3 [11-13]. Sorcin is a Ca2+-binding protein and belongs to the penta EF-hand protein family [14]. Like annexin A7 it binds to membranes in a Ca2+-dependent manner. Sorcin also has been described as interaction partner of the ryanodine receptor, and appears to modulate its function [15]. The influence of the sorcin/annexin A7 interaction on the ryanodine receptor is unknown. Sorcin and annexin A7 are coexpressed in all tissues examined so far [11]. The binding of annexin A7 and sorcin is Ca2+-dependent and occurs at micromolar Ca2+ concentrations. The binding sites have been localised to the amino terminal GGYY and GYGG motifs in sorcin and to the GYPP motif in the amino terminus of annexin A7. The proteins bind to each other with a stoichiometry of two sorcin molecules per annexin A7 molecule [12].
Galectin-3 is a multifunctional oncogenic protein with an anti-apoptotic activity found in the extracellular space, in the nucleus and cytoplasm and in mitochondria. Cytoplasmic galectin-3 correlates with tumor progression and protects mitochondrial integrity. Down regulation of annexin A7 prevents galectin-3 translocation to the perinuclear membrane and increases galectin-3 secretion. For annexin A7 a role for galectin-3 trafficking, apoptosis regulation, and mitochondrial integrity was proposed [13].
The cellular role of annexin A7 is not well understood. It is thought to regulate and stabilize membrane domains and to have a role in Ca2+ homeostasis and Ca2+-dependent signaling pathways. These proposals are supported by data obtained from analysis of annexin A7 deficient mouse mutants. Two annexin A7 129Sv null mice strains were generated independently using a different strategy. The one of Srivastava et al. [16] is lethal. Heterozygous mice exhibit an insulin secretion defect and tumor phenotypes. The null strain reported by Herr et al. [17] is viable, healthy, and shows no insulin secretion defect or other obvious defects. However, in isolated cardiomyocytes the frequency-induced shortening is disturbed. Here we have focused on the analysis of red blood cells and platelets of the anxA7-/- mutant. Generally, red blood cells and platelets were thought not to contain annexin A7 and only recently the 47 kDa isoform has been reported as component of red blood cells [7].
Red blood cells undergo various biochemical or morphological changes that appear to be Ca2+-dependent processes [18,19]. One of them is the release of hemoglobin-containing exovesicles occurring in vivo as well as in vitro [7,20]. This process is thought to represent a protective method of the red blood cell against an attack by for example complement components. Red blood cells which lack the ability to vesiculate cause a disease with red blood cell destruction and haemoglobinuria [21]. There exist two types of vesicles, micro- and nanovesicles with a size of 180 nm and 60 nm, respectively. They are enriched in cholesterol and sphingolipid rich lipid raft domains that are associated with proteins like acetylcholinesterase, cell surface proteins including a complement receptor, and the lipid raft proteins stomatin and flotillin, but they lack any cytoskeletal protein [7]. Furthermore, they contain sorcin and annexin A7 attached to the lipid rafts. Both proteins are more abundant in nanovesicles. The vesicle formation goes along with several other changes in the red blood cell like cytoskeletal rearrangements and changes in the phospholipid orientation in the cellular membrane.