Abstract
The storage of erythrocyte concentrates (ECs) induces lesions that notably affect metabolism, protein activity, deformability of red blood cells (RBCs), as well as the release of oxygen. Band 3 is one of the proteins affected during the ex vivo aging of RBCs. This membrane protein is an anion transporter, an anchor site for the cytoskeleton and other membrane proteins as well as a binding site for glycolytic enzymes and bears blood group antigens. In the present study, band 3 complexes were isolated from RBCs stored for 7 and 42 days in average (n = 3), as well as from microvesicles (n = 3). After extraction of membrane proteins with a deoxycholate containing buffer, band 3 complexes were co-immunoprecipitated on magnetic beads coated with two anti-band 3 antibodies. Both total membrane protein extracts and eluates (containing band 3 complexes) were separated on SDS-PAGE and analyzed by bottom-up proteomics. It revealed that three proteins were present or absent in band 3 complexes stemming from long-stored or short-stored ECs, respectively, whereas the membrane protein contents remained equivalent. These potential markers for storage-induced RBC aging are adenylosuccinate lyase (ADSL), α-adducin and flotillin-2, and were further analyzed using western blots. ADSL abundance tended to increase during storage in both total membrane protein and band 3 complexes, whereas α-adducin mainly tended to stay onto the membrane extract. Interestingly, flotillin-2 was equivalently present in total membrane proteins whereas it clearly co-immunoprecipitated with band 3 complexes during storage (1.6-fold-change, p = 0.0024). Moreover, flotillin-2 was enriched (almost threefold) in RBCs compared to microvesicles (MVs) (p < 0.001) and the amount found in MVs was associated to band 3 complexes. Different types of band 3 complexes are known to exist in RBCs and further studies will be required to better understand involvement of this protein in microvesiculation during the storage of RBCs.
Highlights
The organization of the red blood cell (RBC) membrane is tightly associated to band 3 macrocomplexes and cytoskeleton
The knowledge on the composition and organization of the multiprotein complexes in RBC membrane has evolved during the last decades, from the membrane skeleton (Byers and Branton, 1985) to the binding of Rh proteins (Bruce et al, 2003), the role of protein 4.1 (Salomao et al, 2008), protein 4.2 (Satchwell et al, 2009), adducins (Anong et al, 2009), dematin involved in actin binding (Khan et al, 2008) and in junctional complex integrity (Lu et al, 2016), and ankyrin in band 3 tetramer stability (Satchwell et al, 2016)
Six erythrocyte concentrate (EC) were followed, total membrane proteins were extracted using deoxycholic acid (DC)-based buffer and band 3 complexes were isolated from RBCs at day 6 and day 42, and from MVs using Co-IP
Summary
The organization of the RBC membrane is tightly associated to band 3 macrocomplexes and cytoskeleton. The multiple role of these complexes encompasses the cell deformability required to fulfill the RBC function of O2 delivery to tissue and organs (Mohandas and Gallagher, 2008; Kodippili et al, 2009; Burton and Bruce, 2011), in gas exchange process (Bruce et al, 2003) and in metabolism regulation (Chu et al, 2008) These functions are altered during the storage of RBCs under blood banking conditions (i.e., stored at 4◦C up to 42 or 56 days depending on the various additive solutions used). These storage lesions induce the aggregation and degradation of band 3, leading to the formation of neoantigens recognized by the macrophages for the cell clearance (Bosman et al, 2008), accumulation of hemoglobin, antioxidant and metabolic enzymes at the membrane such as peroxiredoxin-2 (Antonelou et al, 2010; Rinalducci et al, 2011), degradation of proteins and decrease in spectrins and ankyrin contents (D’Amici et al, 2007; Bosman et al, 2008), accumulation of oxidized proteins (in particular at the cytoskeleton) (Antonelou et al, 2010; Delobel et al, 2012; Delobel et al, 2016)
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