Annexin A7 expression in red blood cells, red blood cell derived exovesicles and in platelets
Salzer et al. recently reported the presence of the 47 kDa isoform of annexin A7 and its partner sorcin in micro- and nanovesicles derived from red blood cells where they are located in the lumen and are also enriched in membrane rafts [7]. We have now extended these findings and show here that the 51 kDa isoform is present as well. In western blots of human red blood cells the 47 and 51 kDa isoforms were detected using mAb 203–217 (Fig. 1). The 47 kDa isoform was detected in silver stained gels and its identity with annexin A7 confirmed by peptide mass fingerprinting (data not shown). The 51 kDa isoform was only detected in western blots.
Figure 1  Expression of annexin A7 in human red blood cells (1) and human platelets (2). Protein homogenates were separated by SDS-PAGE (12 % acrylamide). The resulting western blot was probed with mAb 203–217, visualization was by a secondary peroxidase coupled antibody followed by ECL. Both isoforms are detected in red blood cells, in platelets only the small isoform is present. Its presence in vesicles led to the suggestion, that, along with raft domains, annexin A7 plays a role in membrane organization and the vesiculation process. To examine this, we analysed the ability of red blood cells derived from the annexin A7 knock out mice (anxA7-/-) to form exovesicles. Because of better accessibility and larger available amounts that led to clear results, we included our data obtained with human blood. Independent of the presence of annexin A7 both types of exovesicles, micro- and nanovesicles, were released after Ca2+/ionophore treatment as determined by acetylcholine esterase activity and microscopic examination. Annexin A7 is present in both vesicle types where it is more enriched in nanovesicles (Fig. 2). The 51 kDa isoform is only detectable in nanovesicles. The quantity of both vesicle types did not differ between wild type and knock out red blood cell vesicles as determined by the AChE-values (A405 nanovesicles: ~0.18; A405 microvesicles: ~0.9; both, for wt and ko). It appears that annexin A7, although it has been described to fuse membranes, is not a key component in the process of the formation of red blood cell exovesicles. When we probed for the presence of sorcin in wild type and mutant vesicles we found that the sorcin levels were reduced in the mutant nanovesicles (Fig. 2).
Figure 2  Enrichment of annexin A7 and sorcin in exovesicles derived from red blood cells. Vesicles from wild type (wt) and anxA7-/- mutant (ko) were generated with Ca2+/ionophore treatment, isolated by differential centrifugation and analysed by immunoblotting with mAb 203–217 and a sorcin polyclonal antibody. In general, annexin A7 and sorcin are more abundant in nanovesicles. The 51 kDa isoform of annexin A7 (arrows) is only observed in nanovesicles. Samples of both vesicles types were normalized according to their acetylcholine esterase activity. Human red blood cells (co) were used for control and normalized independently. To study the distribution of annexin A7 in red blood cells we used self forming iodixanol density gradients. Both isoforms were present in the soluble fraction as well as in the gradient fractions where they are assumed to be associated with membranes. These membranes are exclusively plasma membranes as red blood cells are free of any organelles. The distribution was however not homogeneous throughout the gradient. This could reflect the binding of annexin A7 to membrane subdomains that have different lipid or lipid/protein composition as discussed by Salzer et al. [7]. Likewise, sorcin does not exhibit a homogeneous distribution in the gradient. Moreover, it segregates into vesicles which are not associated with annexin A7 (Fig. 3). We also tested platelets for annexin A7 expression and detected the 47 kDa isoform (Fig. 1).
Figure 3  Association of annexin A7 and sorcin with distinct red blood cell plasma membrane fractions. Lysed human red blood cells were added to a self forming iodixanol density gradient. The density of the gradient increases from left (1.06 g/ml) to right (1.20 g/ml). Fractions were analysed by immunoblotting using mAb 203–217 and the sorcin polyclonal antibody.