Abstract
Red blood cells (RBCs) begin their circulatory life as reticulocytes (Retics) after their egress from the bone marrow where, as R1 Retics, they undergo significant rearrangements in their membrane and intracellular components, via autophagic, proteolytic, and vesicle-based mechanisms. Circulating, R2 Retics must complete this maturational process, which involves additional loss of significant amounts of membrane and selected membrane proteins. Little is known about the mechanism(s) at the basis of this terminal differentiation in the circulation, which culminates with the production of a stable biconcave discocyte. The membrane of R1 Retics undergoes a selective remodeling through the release of exosomes that are enriched in transferrin receptor and membrane raft proteins and lipids, but are devoid of Band 3, glycophorin A, and membrane skeletal proteins. We wondered whether a similar selective remodeling occurred also in the maturation of R2 Retics. Peripheral blood R2 Retics, isolated by an immunomagnetic method, were compared with mature circulating RBCs from the same donor and their membrane protein and lipid content was analyzed. Results show that both Band 3 and spectrin decrease from R2 Retics to RBCs on a “per cell” basis. Looking at membrane proteins that are considered as markers of membrane rafts, flotillin-2 appears to decrease in a disproportionate manner with respect to Band 3. Stomatin also decreases but in a more proportionate manner with respect to Band 3, hinting at a heterogeneous nature of membrane rafts. High resolution lipidomics analysis, on the contrary, revealed that those lipids that are typically representative of the membrane raft phase, sphingomyelin and cholesterol, are enriched in mature RBCs with respct to Retics, relative to total cell lipids, strongly arguing in favor of the selective retention of at least certain subclasses of membrane rafts in RBCs as they mature from Retics. Our hypothesis that rafts serve as additional anchoring sites for the lipid bilayer to the underlying membrane-skeleton is corroborated by the present results. It is becoming ever more clear that a proper lipid composition of the reticulocyte is necessary for the production of a normal mature RBC.
Highlights
Reticulocytes (Retics) are the result of the enucleation in the bone marrow of their immediate nucleated precursor, the orthochromatic erythroblast, and still retain several components in their membrane and cytoplasm with an excess of approximately 20% of plasma membrane that must be removed
We have recently shown that this is not the case, as the membrane skeleton is apparently lost in parallel with the lipid bilayer during red blood cell (RBC) aging (Ciana et al, 2017a,b), suggesting that the membrane is lost by/removed from aging RBCs in a form that is different from the well-known and characterized spectrin-free vesicles obtained in vitro
When looking at the membrane of young and old circulating RBCs, we have found that flotillin-2, a membrane raft component, is lost disproportionately with respect to the loss of membrane that takes place during RBC aging
Summary
Reticulocytes (Retics) are the result of the enucleation in the bone marrow of their immediate nucleated precursor, the orthochromatic erythroblast, and still retain several components in their membrane and cytoplasm with an excess of approximately 20% of plasma membrane that must be removed. We have recently shown that this is not the case, as the membrane skeleton is apparently lost in parallel with the lipid bilayer during RBC aging (Ciana et al, 2017a,b), suggesting that the membrane is lost by/removed from aging RBCs in a form that is different from the well-known and characterized spectrin-free vesicles obtained in vitro. A recent model proposes that, in the peculiar environment of the oscillatory splenic flow, conditions may arise whereby RBCs can spontaneously release vesicles through a novel deformation mode, called “infolding” (Asaro et al, 2018). Support to this theoretical model from experimental evidence obtained in vivo is still lacking. Unknown is the mechanism by which the membrane and the membrane skeleton are removed, but probably require the active intervention of other tissues/organs (spleen, liver, endothelium)
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