Reticulocyte Maturation and Variant Red Blood Cells.

The bone marrow produces billions of reticulocytes daily. These reticulocytes mature into red blood cells by reducing their plasma membrane by 20% and ejecting or degrading residual internal organelles, membranes and proteins not required by the mature cell. This process occurs by autophagy, protein degradation and vesiculation but is not well understood. We previously reported that Southeast Asian Ovalocytic RBCs demonstrate incomplete reticulocyte maturation and we have now extended this study to a number of other variant RBCs. By comparing the profile of a pure reticulocyte preparation of cultured red cells with these variant cells, we show that the largest of these cells, the overhydrated hereditary stomatocytosis cells, are the least mature, they barely reduced their plasma membrane and contain large amounts of proteins that should have been reduced or removed. Intermediate sized variant RBCs appear to be more mature but retain some endoplasmic reticulum and residual membrane proteins. We propose that the size and composition of these variant cell types correlate with the different stages of reticulocyte maturation and provide insight into the reticulocyte maturation process.

Blood group type A secretors are associated with a higher risk of COVID-19 cardiovascular disease complications.

The SARS-CoV-2 virus causes COVID-19, an infection capable of causing severe disease and death but which can also be asymptomatic or oligosymptomatic. We investigated whether ABO blood group or secretor status was associated with COVID-19 severity. We investigated secretor status because expression of ABO glycans on secreted proteins and non-erythroid cells are controlled by a fucosyltransferase (FUT2), and inactivating FUT2 mutations result in a non-secretor phenotype which protects against some viral infections. Data combined from healthcare records and our own laboratory tests (n = 275) of hospitalized SARS-CoV-2 polymerase chain reaction positive patients confirmed higher than expected numbers of blood group A individuals compared to O (RR = 1.24, CI 95% [1.05, 1.47], p = 0.0111). There was also a significant association between group A and COVID-19-related cardiovascular complications (RR = 2.56, CI 95% [1.43, 4.55], p = 0.0011) which is independent of gender. Molecular analysis revealed that group A non-secretors are significantly less likely to be hospitalized than secretors. Testing of convalescent plasma donors, among whom the majority displayed COVID-19 symptoms and only a small minority required hospitalization, group A non-secretors were slightly over-represented. Our findings showed that group A non-secretors are not resistant to infection by SARS-CoV-2, but are more likely to experience a less severe form of associated disease.

An immortalized adult human erythroid line facilitates sustainable and scalable generation of functional red cells.

With increasing worldwide demand for safe blood, there is much interest in generating red blood cells in vitro as an alternative clinical product. However, available methods for in vitro generation of red cells from adult and cord blood progenitors do not yet provide a sustainable supply, and current systems using pluripotent stem cells as progenitors do not generate viable red cells. We have taken an alternative approach, immortalizing early adult erythroblasts generating a stable line, which provides a continuous supply of red cells. The immortalized cells differentiate efficiently into mature, functional reticulocytes that can be isolated by filtration. Extensive characterization has not revealed any differences between these reticulocytes and in vitro-cultured adult reticulocytes functionally or at the molecular level, and importantly no aberrant protein expression. We demonstrate a feasible approach to the manufacture of red cells for clinical use from in vitro culture.

Superior survival of ex vivo cultured human reticulocytes following transfusion into mice.

The generation of cultured red blood cells from stem cell sources may fill an unmet clinical need for transfusion-dependent patients, particularly in countries that lack a sufficient and safe blood supply. Cultured red blood cells were generated from human CD34+ cells from adult peripheral blood or cord blood by ex vivo expansion, and a comprehensive in vivo survival comparison with standard red cell concentrates was undertaken. Significant amplification (>105-fold) was achieved using CD34+ cells from both cord blood and peripheral blood, generating high yields of enucleated cultured red blood cells. Following transfusion, higher levels of cultured red cells could be detected in the murine circulation compared to standard adult red cells. The proportions of cultured blood cells from cord or peripheral blood sources remained high 24 hours post-transfusion (82±5% and 78±9%, respectively), while standard adult blood cells declined rapidly to only 49±9% by this time. In addition, the survival time of cultured blood cells in mice was longer than that of standard adult red cells. A paired comparison of cultured blood cells and standard adult red blood cells from the same donor confirmed the enhanced in vivo survival capacity of the cultured cells. The study herein represents the first demonstration that ex vivo generated cultured red blood cells survive longer than donor red cells using an in vivo model that more closely mimics clinical transfusion. Cultured red blood cells may offer advantages for transfusion-dependent patients by reducing the number of transfusions required.

Protein distribution during human erythroblast enucleation in vitro.

Enucleation is the step in erythroid terminal differentiation when the nucleus is expelled from developing erythroblasts creating reticulocytes and free nuclei surrounded by plasma membrane. We have studied protein sorting during human erythroblast enucleation using fluorescence activated cell sorting (FACS) to obtain pure populations of reticulocytes and nuclei produced by in vitro culture. Nano LC mass spectrometry was first used to determine the protein distribution profile obtained from the purified reticulocyte and extruded nuclei populations. In general cytoskeletal proteins and erythroid membrane proteins were preferentially restricted to the reticulocyte alongside key endocytic machinery and cytosolic proteins. The bulk of nuclear and ER proteins were lost with the nucleus. In contrast to the localization reported in mice, several key erythroid membrane proteins were detected in the membrane surrounding extruded nuclei, including band 3 and GPC. This distribution of key erythroid membrane and cytoskeletal proteins was confirmed using western blotting. Protein partitioning during enucleation was investigated by confocal microscopy with partitioning of cytoskeletal and membrane proteins to the reticulocyte observed to occur at a late stage of this process when the nucleus is under greatest constriction and almost completely extruded. Importantly, band 3 and CD44 were shown not to restrict specifically to the reticulocyte plasma membrane. This highlights enucleation as a stage at which excess erythroid membrane proteins are discarded in human erythroblast differentiation. Given the striking restriction of cytoskeleton proteins and the fact that membrane proteins located in macromolecular membrane complexes (e.g. GPA, Rh and RhAG) are segregated to the reticulocyte, we propose that the membrane proteins lost with the nucleus represent an excess mobile population of either individual proteins or protein complexes.

The ins and outs of human reticulocyte maturation: autophagy and the endosome/exosome pathway.

The maturation of reticulocytes into functional erythrocytes is a complex process requiring extensive cytoplasmic and plasma membrane remodeling, cytoskeletal rearrangements and changes to cellular architecture. Autophagy is implicated in the sequential removal of erythroid organelles during erythropoiesis, although how this is regulated during late stages of erythroid differentiation, and the potential contribution of autophagy during reticulocyte maturation, remain unclear. Using an optimized ex vivo differentiation system for human erythropoiesis, we have observed that maturing reticulocytes are characterized by the presence of one or few large vacuolar compartments. These label strongly for glycophorin A (GYPA/GPA) which is internalized from the plasma membrane; however, they also contain organellar remnants (ER, Golgi, mitochondria) and stain strongly for LC3, suggesting that they are endocytic/autophagic hybrid structures. Interestingly, we observed the release of these vacuoles by exocytosis in maturing reticulocytes, and speculate that autophagy is needed to concentrate the final remnants of the reticulocyte endomembrane system in autophagosome/endosome hybrid compartments that are primed to undergo exocytosis.

Maturing reticulocytes internalize plasma membrane in glycophorin A-containing vesicles that fuse with autophagosomes before exocytosis.

The erythrocyte is one of the best characterized human cells. However, studies of the process whereby human reticulocytes mature to erythrocytes have been hampered by the difficulty of obtaining sufficient numbers of cells for analysis. In the present study, we describe an in vitro culture system producing milliliter quantities of functional mature human adult reticulocytes from peripheral blood CD34(+) cells. We show that the final stage of reticulocyte maturation occurs by a previously undescribed mechanism in which large glycophorin A-containing vesicles forming at the cytosolic face of the plasma membrane are internalized and fuse with autophagosomes before expulsion of the autophagosomal contents by exocytosis. Early reticulocyte maturation is characterized by the selective elimination of unwanted plasma membrane proteins (CD71, CD98, and ß1 integrin) through the endosome-exosome pathway. In contrast, late maturation is characterized by the generation of large glycophorin A-decorated vesicles of autophagic origin.

Critical band 3 multiprotein complex interactions establish early during human erythropoiesis.

Band 3, the major anion transport protein of human erythrocytes, forms the core of a multiprotein complex in the erythrocyte membrane. Here we studied the spatiotemporal mechanisms of band 3 multiprotein complex assembly during erythropoiesis. Significant pools of intracellular band 3 and Rh-associated glycoprotein (RhAG) were found in the basophilic erythroblast. These intracellular pools decreased in the polychromatic erythroblast, whereas surface expression increased and were lowest in the orthochromatic erythroblast and reticulocytes. Protease treatment of intact cells to remove extracellular epitopes recognized by antibodies to band 3 and RhAG was used to study surface delivery kinetics and intracellular complex composition from the proerythroblast stage to the enucleated reticulocyte. Newly synthesized band 3 and protein 4.2 interact initially in the early stages of the secretory pathway and are found associated at the plasma membrane from the basophilic stage of erythropoiesis. Although we could successfully coimmunoprecipitate Rh with RhAG from plasma membrane pools at a similar stage, no intracellular interaction between these proteins was detectable. Knockdown of RhAG during early erythropoiesis was accompanied by a concomitant drop in membrane expression of Rh polypeptides. These data are consistent with assembly of major components of the band 3 macrocomplex at an early stage during erythropoiesis.

The ability of GAP1IP4BP to function as a Rap1 GTPase-activating protein (GAP) requires its Ras GAP-related domain and an arginine finger rather than an asparagine thumb.

GAP1(IP4BP) is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs) that includes GAP1(m), CAPRI, and RASAL. Composed of a central Ras GAP-related domain (RasGRD), surrounded by amino-terminal C2 domains and a carboxy-terminal PH/Btk domain, these proteins, with the notable exception of GAP1(m), possess an unexpected arginine finger-dependent GAP activity on the Ras-related protein Rap1 (S. Kupzig, D. Deaconescu, D. Bouyoucef, S. A. Walker, Q. Liu, C. L. Polte, O. Daumke, T. Ishizaki, P. J. Lockyer, A. Wittinghofer, and P. J. Cullen, J. Biol. Chem. 281:9891-9900, 2006). Here, we have examined the mechanism through which GAP1(IP4BP) can function as a Rap1 GAP. We show that deletion of domains on either side of the RasGRD, while not affecting Ras GAP activity, do dramatically perturb Rap1 GAP activity. By utilizing GAP1(IP4BP)/GAP1(m) chimeras, we establish that although the C2 and PH/Btk domains are required to stabilize the RasGRD, it is this domain which contains the catalytic machinery required for Rap1 GAP activity. Finally, a key residue in Rap1-specific GAPs is a catalytic asparagine, the so-called asparagine thumb. By generating a molecular model describing the predicted Rap1-binding site in the RasGRD of GAP1(IP4BP), we show that mutagenesis of individual asparagine or glutamine residues that lie in close proximity to the predicted binding site has no detectable effect on the in vivo Rap1 GAP activity of GAP1(IP4BP). In contrast, we present evidence consistent with a model in which the RasGRD of GAP1(IP4BP) functions to stabilize the switch II region of Rap1, allowing stabilization of the transition state during GTP hydrolysis initiated by the arginine finger.

Epigenetic silencing of a Ca(2+)-regulated Ras GTPase-activating protein RASAL defines a new mechanism of Ras activation in human cancers.

Ras has achieved notoriety as an oncogene aberrantly activated in multiple human tumors. Approximately 30% of all human tumors express an oncogenic form of this GTPase that is locked in an active conformation as a result of being insensitive to Ras GTPase-activating proteins (GAPs), proteins that normally regulate the inactivation of Ras by enhancing its intrinsic GTPase activity. Besides oncogenic mutations in Ras, signaling by wild-type Ras is also frequently deregulated in tumors through aberrant coupling to activated cell surface receptors. This indicates that alternative mechanisms of aberrant wild-type Ras activation may be involved in tumorigenesis. Here, we describe another mechanism through which aberrant Ras activation is achieved in human cancers. We have established that Ras GTPase-activating-like protein (RASAL), a Ca(2+)-regulated Ras GAP that decodes the frequency of Ca(2+) oscillations, is silenced through CpG methylation in multiple tumors. With the finding that ectopic expression of catalytically active RASAL leads to growth inhibition of these tumor cells by Ras inactivation, we have provided evidence that epigenetically silencing of this Ras GAP represents a mechanism of aberrant Ras activation in certain cancers. Our demonstration that RASAL constitutes a tumor suppressor gene has therefore further emphasized the importance of Ca(2+) in the regulation of Ras signaling and has established that deregulation of this pathway is an important step in Ras-mediated tumorigenesis.

Studying the spatial and temporal regulation of Ras GTPase-activating proteins.

Two classes of proteins govern Ras activation. Guanine-nucleotide exchange factors (Ras GEFs) catalyze the activation of Ras by inducing the dissociation of GDP to allow association of the more abundant GTP, whereas GTPase-activating proteins (Ras GAPs), bind to the GTP-bound form and, by enhancing the intrinsic GTPase activity, catalyze Ras inactivation. A wide range of Ras GEFs and Ras GAPs have been identified from the various genome projects, and in a few instances, the mechanisms by which signals originating from activated receptors converge on specific GEFs and GAPs have been mapped. However, for most Ras GEFs and GAPs we have a poor understanding of their regulation. Here we focus on describing methods used to study the regulation of the GAP1 family of Ras GAPs. In particular, we emphasize how by combining biochemical, molecular, and imaging techniques, one can determine some of the complex array of mechanisms that have evolved to modulate the spatial and temporal dynamics of Ras regulation through these various Ras GAPs. By combining biochemical, molecular, and imaging techniques, we describe the visualization of the diverse and dynamic mechanisms through which stimulation of cell surface receptors leads to the regulation of these proteins. Thus, although each member of the GAP1 family performs the same basic biological function, that is, they function as Ras GAPs, each is designed to respond and decode signals from distinct second messenger pathways.

GAP1 family members constitute bifunctional Ras and Rap GTPase-activating proteins.

GAP1(IP4BP) is a member of the GAP1 family of Ras GTPase-activating proteins (Ras GAPs) that includes GAP1(m), CAPRI, and RASAL. Composed of a central Ras GAP domain, surrounded by amino-terminal C(2) domains and a carboxyl-terminal pleckstrin homology/Bruton's tyrosine kinase domain, GAP1(IP4BP) has previously been shown to possess an unexpected GAP activity on the Ras-related protein Rap, besides the predicted Ras GAP activity (Cullen, P. J., Hsuan, J. J., Truong, O., Letcher, A. J., Jackson, T. R., Dawson, A. P., and Irvine, R. F. (1995) Nature 376, 527-530). Here we have shown that GAP1(IP4BP) is indeed an efficient Ras/Rap GAP, having K(m)s of 213 and 42 microm and estimated k(cat)s of 48 and 16 s(-1) for Ras and Rap, respectively. For this dual activity, regions outside the Ras GAP domain are required, as the isolated domain (residues 291-569) retains a pronounced Ras GAP activity yet has very low activity toward Rap. Interestingly, mutagenesis of the Ras GAP arginine finger, and surrounding residues important in Ras binding, inhibit both Ras and Rap GAP activity of GAP1(IP4BP). Although the precise details by which GAP1(IP4BP) can function as a Rap GAP remain to be determined, these data are consistent with Rap associating with GAP1(IP4BP) through the Ras-binding site within the Ras GAP domain. Finally, we have established that such dual Ras/Rap GAP activity is not restricted to GAP1(IP4BP). Although GAP1(m) appears to constitute a specific Ras GAP, CAPRI and RASAL display dual activity. For CAPRI, its Rap GAP activity is modulated upon its Ca(2+)-induced association with the plasma membrane.

The frequencies of calcium oscillations are optimized for efficient calcium-mediated activation of Ras and the ERK/MAPK cascade.

Ras proteins are binary switches that, by cycling through inactive GDP- and active GTP-bound conformations, regulate multiple cellular signaling pathways, including those that control growth and differentiation. For some time, it has been known that receptor-mediated increases in the concentration of intracellular free calcium ([Ca(2+)](i)) can modulate Ras activation. Increases in [Ca(2+)](i) often occur as repetitive Ca(2+) spikes or oscillations. Induced by electrical or receptor stimuli, these repetitive Ca(2+) oscillations increase in frequency with the amplitude of receptor stimuli, a phenomenon critical for the induction of selective cellular functions. Here, we show that Ca(2+) oscillations are optimized for Ca(2+)-mediated activation of Ras and signaling through the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. We present additional evidence that Ca(2+) oscillations reduce the effective Ca(2+) threshold for the activation of Ras and that the oscillatory frequency is optimized for activation of Ras and the ERK/MAPK pathway. Our results describe a hitherto unrecognized link between complex Ca(2+) signals and the modulation of the Ras/ERK/MAPK signaling cascade.

Identification of a Ras GTPase-activating protein regulated by receptor-mediated Ca2+ oscillations.

Receptor-mediated increases in the concentration of intracellular free calcium ([Ca2+]i) are responsible for controlling a plethora of physiological processes including gene expression, secretion, contraction, proliferation, neural signalling, and learning. Increases in [Ca2+]i often occur as repetitive Ca2+ spikes or oscillations. Induced by electrical or receptor stimuli, these repetitive Ca2+ spikes increase their frequency with the amplitude of the receptor stimuli, a phenomenon that appears critical for the induction of selective cellular functions. Here we report the characterisation of RASAL, a Ras GTPase-activating protein that senses the frequency of repetitive Ca2+ spikes by undergoing synchronous oscillatory associations with the plasma membrane. Importantly, we show that only during periods of plasma membrane association does RASAL inactivate Ras signalling. Thus, RASAL senses the frequency of complex Ca2+ signals, decoding them through a regulation of the activation state of Ras. Our data provide a hitherto unrecognised link between complex Ca2+ signals and the regulation of Ras.

Bst-2/HM1.24 is a raft-associated apical membrane protein with an unusual topology.

An expression screen of a rat cDNA library for sequences encoding Golgi-localized integral membrane proteins identified a protein with an apparent novel topology, i.e. with both an N-terminal transmembrane domain and a C-terminal glycosyl-phosphatidylinositol (GPI) anchor. Our data are consistent with this. Thus, the protein would have a topology that, in mammalian cells, is shared only by a minor, but pathologically important, topological isoform of the prion protein (PrP). The human orthologue of this protein has been described previously (BST-2 or HM1.24 antigen) as a cell surface molecule that appears to be involved in early pre-B-cell development and which is present at elevated levels at the surface of myeloma cells. We show that rat BST-2/HM1.24 has both a cell surface and an intracellular (juxtanuclear) location and is efficiently internalized from the cell surface. We also show that the cell surface pool of BST-2/HM1.24 is predominantly present in the apical plasma membrane of polarized cells. The fact that rat BST-2/HM1.24 apparently possesses a GPI anchor led us to speculate that it might exist in cholesterol-rich lipid microdomains (lipid rafts) at the plasma membrane. Data from several experiments are consistent with this localization. We present a model in which BST-2/HM1.24 serves to link adjacent lipid rafts within the plasma membrane.

Analyzing the role of the putative inositol 1,3,4,5-tetrakisphosphate receptor GAP1IP4BP in intracellular Ca2+ homeostasis.

Inositol 1,3,4,5-tetrakisphosphate (IP(4)) has been linked to a potential role in the regulation of intracellular free Ca(2+) concentration ([Ca(2+)](i)) following cellular stimulation with agonists that activate phosphoinositide-specific phospholipase C. However, despite many studies, the function of IP(4) remains unclear and indeed there is still some debate over whether it has a function at all. Here we have used various molecular approaches to address whether manipulation of the potential IP(4) receptor, GAP1(IP4BP), affects [Ca(2+)](i) following cellular stimulation. Using single cell imaging, we show that the overexpression of a constitutively active and a potential dominant negative form of GAP1(IP4BP) appear to have no effect on Ca(2+) mobilization or Ca(2+) entry following stimulation of HeLa cells with histamine. In addition, through the use of small interfering RNA duplexes, we have examined the effect of suppressing endogenous GAP1(IP4BP) production on [Ca(2+)](i). In HeLa cells in which the endogenous level of GAP1(IP4BP) has been suppressed by approximately 95%, we failed to observe any effect on Ca(2+) mobilization or Ca(2+) entry following histamine stimulation. Thus, using various approaches to manipulate the function of endogenous GAP1(IP4BP) in intact HeLa cells, we have been unable to observe any detectable effect of GAP1(IP4BP) on [Ca(2+)](i).