Reticulocyte and red blood cell deformation triggers specific phosphorylation events

Abstract

The capacity to undergo substantial deformation is a defining characteristic of the red blood cell (RBC), facilitating transit through the splenic interendothelial slits and microvasculature. Establishment of this remarkable property occurs during a process of reticulocyte maturation that begins with egress through micron-wide pores in the bone marrow and is completed within the circulation. The requirement to undertake repeated cycles of deformation necessitates that both reticulocytes and erythrocytes regulate membrane-cytoskeletal protein interactions in order to maintain cellular stability. In the absence of transcriptional activity, modulation of these interactions in RBCs is likely to be achieved primarily through specific protein posttranslational modifications, which at present remain undefined. In this study, we use high-throughput methods to define the processes that underlie the response to deformation and shear stress in both reticulocytes and erythrocytes. Through combination of a bead-based microsphiltration assay with phosphoproteomics we describe posttranslational modification of RBC proteins associated with deformation. Using microsphiltration and microfluidic biochip-based assays, we explore the effect of inhibiting kinases identified using this dataset. We demonstrate roles for GSK3 and Lyn in capillary transit and maintenance of membrane stability following deformation and show that combined inhibition of these kinases significantly decreases reticulocyte capacity to undergo repeated deformation. Finally, we derive a comprehensive and integrative phosphoproteomic dataset that provides a valuable resource for further mechanistic dissection of the molecular pathways that underlie the RBC’s response to mechanical stimuli and for the study of reticulocyte maturation.

Post-translational modification as a response to cellular stress induced by hemoglobin oxidation in sickle cell disease

“…Upregulation of band-3 and interacting partners in SCD RBC (compared to controls) was also confirmed in earlier studies linking phosphorylation of these proteins with oxidative stress in humans 31,33 . Because we see a higher relative abundance of both PTMs in SCD patients, our proteomic data and orthogonal studies suggest that both PTMs of this band-3 interaction network are associated with MP formation.…”

Reticulocyte Maturation

“…In addition, reticulocytes have a highly active protein kinase [ 114 ] and more phosphorylated peptides are derived from reticulocytes when passed through a micro-filtration system compared to RBCs [ 115 ]. Phosphorylation regulates cytoskeletal and membrane interactions which contribute to membrane stability.…”

Reticulocyte Maturation and Variant Red Blood Cells

“…Indeed, we have also shown that native reticulocytes, when incubated in either plasma or media at 37°C for ten days, reduce their levels of RNA and TfR and develop an increased ratio of R2:R1 reticulocytes (data not shown). Cultured reticulocytes can be induced to begin the maturation process when put under shear stress ( Moura et al, 2019 ) and appear to mature fully in the circulation of a mouse model ( Kupzig et al, 2017 ) but completing the maturation process in vitro has yet to be achieved. Proteomic analysis comparing native reticulocytes to cultured reticulocytes shows their proteomic profile to be very similar ( Moura et al, 2018 ) but there must be an essential factor missing from the cRBCs (perhaps a plasma protein or the lack of macrophages) that restricts their development in vitro .…”