Proteome of Stored RBC Membrane and Vesicles from Heterozygous Beta Thalassemia Donors

Abstract

Genetic characteristics of blood donors may impact the storability of blood products. Despite higher basal stress, red blood cells (RBCs) from eligible donors that are heterozygous for beta-thalassemia traits (βThal+) possess a differential nitrogen-related metabolism, and cope better with storage stress compared to the control. Nevertheless, not much is known about how storage impacts the proteome of membrane and extracellular vesicles (EVs) in βThal+. For this purpose, RBC units from twelve βThal+ donors were studied through proteomics, immunoblotting, electron microscopy, and functional ELISA assays, versus units from sex- and aged-matched controls. βThal+ RBCs exhibited less irreversible shape modifications. Their membrane proteome was characterized by different levels of structural, lipid raft, transport, chaperoning, redox, and enzyme components. The most prominent findings include the upregulation of myosin proteoforms, arginase-1, heat shock proteins, and protein kinases, but the downregulation of nitrogen-related transporters. The unique membrane proteome was also mirrored, in part, to that of βThal+ EVs. Network analysis revealed interesting connections of membrane vesiculation with storage and stress hemolysis, along with proteome control modulators of the RBC membrane. Our findings, which are in line with the mild but consistent oxidative stress these cells experience in vivo, provide insight into the physiology and aging of stored βThal+ RBCs.

Red Blood Cell Proteasome in Beta-Thalassemia Trait: Topology of Activity and Networking in Blood Bank Conditions

“…Network analysis was performed for the topological visualization of the statistically significant correlations between a great variety (>300 nodes) of omics (RBC membrane proteome) and physiological (e.g., cellular fragility, redox equilibrium) parameters of the βThal + and control RBC units [ 22 , 23 ], including the proteasome (components and activities). This approach helps to elucidate the possible associations between parameters of stored RBCs that may fuel future more targeted research concerning the regulation of proteasome activity, interactions, recruitment to the membrane, and release during storage.…”

“…Apart from the correlations with intracellular ROS thoroughly analyzed in this study ( Figure 2 ), the levels of proteasome activities in the βThal + cytosol were positively connected to additional redox-related variables (e.g., glutathione S-transferase, protein carbonylation; see Figure 6 and Figure 7 ). Of note, some cellular and membrane protein features of βThal + stored RBCs [ 22 , 23 ] were also present in their activity interactomes in contrast to controls: band 3, phospholipid scramblase-1, osmotic fragility ( Figure 6 ), calpain, arginase-1, RHAG, ATPase Na + /K + ( Figure 7 ). On the other hand, the control networks of cytosolic activities were dominated by calcium-related (cytosolic calcium concentration, calreticulin) and internal or membrane-associated components (e.g., day 42: positive with stomatin and myosin; negative with glycophorin A, CD44), the cytosolic concentration of Hb, and several enzymes.…”

“…The hematological (MCV, MCH etc. ), physiological (e.g., hemolysis, osmotic fragility, antioxidant capacity, reactive oxygen species (ROS)), and protein (e.g., carbonylation) parameters used for the correlation analyses (scatterplots and biological networks) originate from specific experiments that were performed in the same cohorts of donors as thoroughly analyzed and reported in recent publications related to this project [ 22 , 23 ]. Intracellular levels of reactive oxygen species (ROS) were assayed with the help of the redox-sensitive and membrane-permeable probe 5-(and-6)-chloromethyl-2′,7′-dichloro-dihydro-fluorescein diacetate, acetyl ester (CM-H 2 DCFDA, Invitrogen, Molecular Probes, Eugene, OR, USA), as extensively described before [ 26 ].…”

“…ROS accumulation was also determined following stimulation of the cells with the oxidants tert-butyl-hydroperoxide (tBHP), diamide, and phenylhydrazine. Proteomic analysis was performed in 12 membrane samples of each group in early (day 7) and late (day 42) storage, as well as in 5 EV samples of each group, through a FASP digestion prior to analysis via nano-UHPLC-MS/MS (Evosep One system coupled to timsTOF Pro mass spectrometer-Bruker Daltonics, Bremen, Germany), as extensively described before [ 23 ].…”

“…In our previous works, we observed lower reactive oxygen species (ROS) levels in late storage and a shift of the metabolism toward the Pentose Phosphate Pathway (PPP), suggesting a higher antioxidant potential compared to controls [ 22 ]. Furthermore, a recent comparison of the proteome of βThal + and control membranes revealed enrichment of the former in a number of “repair or destroy” molecules during storage, such as chaperone HSP70 and proteasomal subunits, among others [ 23 ]. The effectiveness of these compiled protective pathways is also supported by reports of lower levels of protein carbonylation, lipid peroxidation, and other oxidative stress indices, such as the metabolite allantoin during storage in this group [ 22 ].…”