ALBERT

Description: Invasion of the red blood cell (RBC) by the Plasmodium parasite defines the start of malaria disease pathogenesis. To date, experimental investigations into invasion have focused predominantly on the role of parasite adhesins or signaling pathways and the identity of binding receptors on the red cell surface. A potential role for signaling pathways within the erythrocyte, which might alter red cell biophysical properties to facilitate invasion, has largely been ignored. The parasite erythrocyte-binding antigen 175 (EBA175), a protein required for entry in most parasite strains, plays a key role by binding to glycophorin A (GPA) on the red cell surface, although the function of this binding interaction is unknown. Here, using real-time deformability cytometry and flicker spectroscopy to define biophysical properties of the erythrocyte, we show that EBA175 binding to GPA leads to an increase in the cytoskeletal tension of the red cell and a reduction in the bending modulus of the cell’s membrane. We isolate the changes in the cytoskeleton and membrane and show that reduction in the bending modulus is directly correlated with parasite invasion efficiency. These data strongly imply that the malaria parasite primes the erythrocyte surface through its binding antigens, altering the biophysical nature of the target cell and thus reducing a critical energy barrier to invasion. This finding would constitute a major change in our concept of malaria parasite invasion, suggesting it is, in fact, a balance between parasite and host cell physical forces working together to facilitate entry.

Description: Culture systems for human in vitro erythropoiesis are now well established. Using our 3-stage feeder-free erythroid culture system we can efficiently differentiate erythroid cells from adult and cord blood (CB) CD34+ cells with 〉105 fold expansion, enucleation rates of up to 95% and producing packed reticulocyte yields of 〉12ml post leukofiltration1. The final preparations for a first in man clinical trial of adult cultured reticulocytes produced under good manufacturing practice (RESTORE) are underway. Although we have shown that it is possible to modify the CD34+ derived cells using lentivirus for reengineering or additions to the medium, the finite proliferative capacity of CD34+ cells in culture currently limits yield. We therefore took the alternative approach of immortalising early erythroid cells differentiated from adult bone marrow (BM) CD34+ cells, creating the BEL-A (Bristol Erythroid Line Adult) line2, a sustainable erythroid cell source that recapitulates normal adult erythropoiesis, terminally differentiating to generate enucleated reticulocytes that express normal levels of adult globin. We have created a further 13 lines from BM, adult peripheral blood, CB and iPSC CD34+ cells, demonstrating reproducibility of the approach and its application to create lines from more accessible stem cell sources. Analysis of surface marker and globin profiles demonstrate the lines follow a similar differentiation profile to their respective primary cell source, with comparative proteomics confirming cell source representation of the lines. Lines always established at the pro-erythroblast/early basophilic stage, even when later stage erythroid cells were present in populations. As well as proof of principal as an alternative transfusion product and improved tools for studying erythropoiesis, such lines have far reaching additional applications, a number of which we are now exploring: Diagnostic and 'Universal' transfusion products: Presently serological testing by blood group reference laboratories relies on donated blood, which represent a finite resource and for some blood group phenotypes can be difficult to source. We used CRISPR-Cas9 gene editing to remove blood group antigens in order to generate a sustainable bank of cell lines with useful blood group phenotypes for diagnostic purposes3. Building on this, with the aim of developing a more compatible "universal" transfusion product to meet the needs of chronically transfused patients and those with rare blood group phenotypes, we used combinatorial gene targeting to create sublines deficient in multiple antigens responsible for the most common transfusion incompatibilities (ABO [Bombay], Rh [Rhnull], Kell [K0], Duffy [Fynull], GPB [S-s-U-]). Individual and multiple blood group knockout lines retained the ability to undergo terminal differentiation and enucleation, also illustrating the capacity for coexistence of multiple rare blood group phenotypes within viable reticulocytes3. Cytokine independent lines Cytokines represent a substantial cost contribution to erythroid culture systems. We therefore exploited activating mutations found in patient c-Kit and EPOR that cause hypersensitivity to ligand, to create cytokine independent lines thus increasing economic viability of cultured red cells. Bi-allelic EPOR or c-kit edits were introduced into BEL-A with confirmation and exploration of mechanism on differentiation in the absence, or with substantially reduced levels of cytokines. Model disease systems In addition to potential therapeutic applications we are also creating lines as model cellular disease systems for studying molecular mechanisms and as drug screening platforms, via CRISPR-Cas9 gene editing of BEL-A and by directly immortalising patient stem cells. To date we have made b-thalassemia major, HbE thalassemia and lines with KLF1 mutations. Furthermore, we have shown BEL-A reticulocytes support invasion and growth of Plasmodium falciparum and are utilising the line to study mechanisms of malaria parasite invasion4. Kupzig S, Parsons SF, Curnow E, Anstee DJ, Blair A. Superior survival of ex vivo cultured human reticulocytes following transfusion into mice. 2016;102:476-483Trakarnsanga K, Griffiths RE, Wilson MC, et al. An immortalized adult human erythroid line facilitates sustainable and scalable generation of functional red cells. Nat. Commun. 2017;8:14750Hawksworth J, Satchwell TJ, Meinders M, et al. Enhancement of red blood cell transfusion compatibility using CRISPR-mediated erythroblast gene editing. EMBO Mol. Med. 2018; 10:e8454Satchwell TJ, Wright K, Haydn-Smith K, et al. Genetic manipulation of cell line derived reticulocytes enables dissection of host malaria invasion requirements. Nat. Comm. 2019;10:3806 Disclosures No relevant conflicts of interest to declare.

Description: Congenital dyserythropoietic anemia (CDA) type II is the most frequent type of congenital dyserythropoietic anemia; it is transmitted in an autosomal recessive fashion and is characterized by ineffective erythropoiesis, peripheral hemolysis, bi-multinuclearity in the erythroblasts, and hypoglycosylation of red blood cell (RBC) membrane proteins such as band 3. The disease is generally caused by biallelic mutations in the SEC23B gene. However, there are a small portion of patients with clinical and hematologic features of CDA II that are negative for mutations in SEC23B, suggesting that alternative etiologies for such disturbed erythropoiesis exist. We identified two siblings of Italian origin who had dyserythropoiesis with a chronic macrocytic anemia. Their parents were healthy with normal hematologic parameters. No history of consanguinity for at least three generations was noted. The affected siblings had anisopoikylocytosis on peripheral blood smear with stomatocytes (8-9%), spherocytes (4-5%), rare ovalocytes, and dacryocytes. RBCs osmotic fragility was increased but the red cells had normal eosin-5-maleimide (EMA)-binding. Serum ferritin and transferrin saturation were increased in only one sibling. Bone marrow morphology revealed erythroid hyperplasia (myeloid: erythroid ratio = 0.6) with binuclearity and megaloblastic changes, as well as occasional cytoplasmic bridging between cells at different stage of maturation; electron microscopy of bone marrow erythroblasts showed multiple membranes that ran parallel to the plasma membrane or that were grouped in stacked segments, possibly attributable to residual endoplasmic reticulum (ER) cisternae. SDS-PAGE analysis of RBC ghosts from both siblings demonstrated hypoglycosylation of band 3 and GLUT1, as well as residual residual Protein Disulphide Isomerase (PDI) positive ER remnants, as observed in classical CDA II cases. However, in contrast to CDAII, the Ham's test performed with 15 normal serum samples was negative, and no mutations were detected in the SEC23B gene. To uncover the underlying etiologies, whole-exome sequencing was conducted on all available family members. After filtering for common variants, only a single gene had biallelic mutations in the affected siblings, which were transmitted from the unaffected heterozygous parents. The identified mutations resided in the PARP4 gene, which encodes a poly-ADP ribose polymerase enzyme, and were predicted to be deleterious. We demonstrate that knockdown of PARP4 using shRNA in primary human erythroid progenitors results in impaired erythroid differentiation and increased apoptosis. In addition, morpholino-mediated knockdown of the PARP4 orthologue in the zebrafish resulted in dyserythropoiesis and anemia in developing embryos. Sequencing of PARP4 in additional rare cases of CDA II without an identified molecular basis will help to uncover the frequency and spectrum of PARP4 mutations leading to dyserythropoiesis. The finding of a new gene implicated in a similar type of CDA with features such as redundant ER membranes offers the potential for more mechanistic dissection of the role of both SEC23B and PARP4 in erythroid development and suggests that new insight can be gained into the underlying pathophysiology of both normal and disordered erythropoiesis through the study of such rare cases. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 812 Band 3 forms the core of a large multiprotein complex in the erythrocyte membrane, the Band 3 macrocomplex, which also includes proteins of the Rhesus complex (Rh and RhAG). Mutations in genes encoding proteins within this complex can result in hereditary spherocytosis with varying severity. The effect of distinct mutations and deficiencies in proteins of the Band 3 macrocomplex has been studied in detail in mature erythrocytes. This revealed important functional and structural properties of individual proteins and their relationships with other proteins within the Band 3 macrocomplex. Nevertheless, considerably less is know about the spatio-temporal mechanisms that direct the formation of the Band 3 macrocomplex, and that may explain the aberrations in the complex observed in spherocytosis. Therefore, we studied expression and mutual interactions of proteins of the band3 macrocomplex during development of proerythroblasts to reticulocytes. Using confocal microscopy and western blotting, significant pools of intracellular Band 3 and RhAG were found in the basophilic normoblast. These intracellular pools gradually decreased in the polychromatic normoblast and were absent or low in the orthochromatic normoblast and reticulocytes, while surface expression increased. We used pronase treatment of intact cells to remove extracellular epitopes of BRIC 6 (Band 3 antibody) and LA1818 (RhAG antibody) to study the mechanism by which the intracellular pool of Band 3 and RhAG contributes to formation of the Band 3 complex on the cell surface. Pronase treatment of cells incubated with cycloheximide to block protein synthesis resulted in a reduced but still significant reappearance of BRIC6 (Band 3) and LA1818 (RhAG) epitopes on the plasma membrane confirming the presence of intracellular Band 3 and RhAG pools. It also showed that the bulk of Band 3 and RhAG is synthesized and trafficked to the membrane between the early basophilic and polychromatic stage. Immuneprecipitation of Band 3 from cell lysates of pronase treated cells pre-treated with brefeldin A to collapse the Golgi showed no increase in co-immuneprecipitated protein 4.2 albeit an increase in intracellular Band 3 expression. This suggests that protein 4.2 and Band 3 interact in the first Golgi compartment or late ER. In addition, pre-treatment of cells with cycloheximide prior to pronase treatment resulted in depletion of the intracellular Band 3 and co-immuneprecipitated protein 4.2 pool indicating that Band 3 and protein 4.2 traffic as a complex to the plasma-membrane. We were unable to co-immuneprecipitate Rh or Band 3 with intracellular pools of RhAG, whereas Rh was co-immuneprecipitated with RhAG from the plasma-membrane and from total cell lysates. Knockdown of RhAG in differentiating erythroblasts revealed a concomitant drop in membrane expression of Rh, leaving Band 3 unaffected, indicating that plasma-membrane expression of Rh but not Band 3 is dependent on RhAG. In conclusion, despite the described association between the RhAG complex and the Band 3 complex in erythrocytes, the data suggest that the Band 3-protein 4.2 complex traffics and assembles independently from Rh and RhAG during erythroid differentiation. The experiments suggest that Rh and RhAG do not traffic as a complex to the plasma-membrane but probably assemble in the plasma-membrane. The RhAG knockdown experiments suggest that the dependency of Rh on RhAG as observed in Rhnull syndrome erythrocytes (“Rh regulator type”) originates early during erythropoiesis. Band3 surface expression was not affected upon RhAG knock down, which re-produced the unperturbed Band 3 levels seen in these patients. Disclosures: No relevant conflicts of interest to declare.

Description: CD47 is a ubiquitously expressed ‘Marker of Self’ that protects cells from phagocytosis, through recognition by SIRPα on macrophages (Oldenborg et al Science 2000). CD47 was originally isolated on ovarian tumour cells (Poels et al J Natl Cancer Inst 1986) and has subsequently been detected on leukemic stem cells, where increased CD47 levels ensure immune evasion (Jaiswal et al Cell 2009). CD47 is also a ‘Marker of Self’ on red cells, but is reduced at the cell surface in certain patients with Hereditary Spherocytosis. In red cells, ~60% of CD47 is connected to the cytoskeleton (Dahl et al Blood 2004). Cytoskeletal connectivity of CD47 in the red cell membrane is dependent on the band 3 complex associated protein 4.2, demonstrated by an ~80% reduction in CD47 levels in protein 4.2 null red cells (Mouro-Chanteloup et al Blood 2003). Previous work (van den Akker et al Haematologica 2009) established that CD47 becomes dependent on protein 4.2 at the basophilic erythroblast stage (48 hours post-differentiation), but it is unknown what interactions support CD47 membrane stability prior to protein 4.2 expression during expansion and early erythroid differentiation. CD47 mRNA is alternatively spliced giving rise to four potential isoforms. The most abundant isoforms are form 2, expressed in all bone-marrow derived cells, and form 4 (and form 3), found predominantly in neural tissues (Reinhold et al J Cell Sci 1995). CD47 isoform 2 is the only form expressed on mature red cells, but we hypothesized that expression of other CD47 isoforms with different trafficking or binding characteristics could explain the independence of CD47 prior to band 3 complex assembly. Using specific polyclonal antibodies to multiple CD47 isoforms, we demonstrate that isoform 2 is expressed prior to and throughout in vitroerythroid differentiation. CD47 isoforms 3 and 4 were detected by western blotting until the late polychromatic erythroblast stage (96 hours post-differentiation), but only CD47 isoform 2 was detected at the cell surface. Therefore, we next hypothesised that CD47 must interact with another protein or exhibit different trafficking characteristics to maintain its membrane stability early during terminal differentiation. To identify a candidate protein or associated protein complex, CD47 was immunoprecipitated from expanding erythroblasts (Exp), proerythroblasts (T0), and basophilic erythroblasts (T48), and analysed via Nano-LC mass spectroscopy. In Exp and T0 erythroblasts, CD47 pulled down actin and multiple actin-associated proteins. These interactions were not observed in T48 erythroblasts, corresponding to the time during terminal differentiation when CD47 is dependent on protein 4.2. To confirm a dependence on actin for CD47 membrane stability, well-characterised drugs that disrupt actin dynamics were employed. CD47 expression at the cell membrane, as judged by flow cytometry, was markedly reduced within 30 minutes using actin stabilising drugs (Cytochalasin D (5µM): Exp 13.7±5.4% versus T48 0.5±5.7%; Latrunculin A (1µM): Exp 18.9±3.5% versus T48 9.9±5.9%, of the DMSO control), and destabilising drug (Jasplakinolide (1µM): Exp 24.2±1.9% versus T48 -6±1.8%, of the DMSO control), until the basophilic erythroblast stage. In K562 cells, which predominantly express CD47 isoforms 3 and 4, a larger actin dependency is observed (37±14.9% reduction in CD47 with Cytochalasin D versus a DMSO control) suggesting that dependence on actin by CD47 is not isoform specific. In summary, we propose a role for actin in the maintenance of CD47 at the cell surface before and during early erythroid differentiation. We have shown that CD47 isoform 2 is the major isoform present at the cell surface and that this version is initially dependent on the actin cytoskeleton for its membrane stability by an as yet undetermined mechanism. Once band 3 complex assembly initiates at the surface of the basophilic erythroblast (48 hours post-differentiation), CD47 is selectively incorporated via an interaction with protein 4.2, and is preferentially retained whilst the actin cytoskeleton remodels. In addition to explaining how CD47 expression is maintained during the formation of the red cell membrane, this work raises the possibility that the dependence on actin by CD47 for its membrane stability in hematopoietic stem cells may be exploited for the development of therapeutics that render the leukemic cells susceptible to phagocytosis. Disclosures No relevant conflicts of interest to declare.

Description: The bicarbonate/chloride exchanger protein band 3 is the most abundant protein in the erythrocyte membrane and forms the core of a major multiprotein complex required for vertical association between the plasma membrane and the underlying spectrin cytoskeleton. A wealth of knowledge, derived from a host of varied studies including in vitro binding assays, work on mature erythrocytes and in other cellular systems have identified a number of binding partners including ankyrin, adducin and protein 4.2 amongst others. However, studies of the role that band 3 and the establishment of its connectivity with the cytoskeleton play both in assembly of multiprotein complexes during erythropoiesis and in particular in protein retention during enucleation have been understandably limited by the technical challenges associated with study of this protein within its unique native cellular context. The complete absence of band 3 in human erythrocytes has only been reported once, in a Portuguese patient with severe hereditary spherocytosis and distal renal tubular acidosis resulting from homozygosity for a V488M band 3 mutation (band 3 Coimbra). In this study, we used in vitro culture of erythroblasts derived from this patient as well as shRNA mediated depletion of band 3 to investigate the development of a band 3 deficient erythrocyte membrane and to specifically assess the formation, stability and retention of band 3 dependent protein complexes in the absence of this core protein during erythropoiesis and erythroblast enucleation. We demonstrate that the mutant band 3 Coimbra protein is expressed at very low but detectable levels during erythropoiesis but does not reach the cell surface and is not rescued by interaction with wild type protein. Failure to traffic to the plasma membrane and rapid degradation during erythropoiesis accounts for the absence of band 3 in Coimbra erythrocytes. The absence of plasma membrane expression of band 3 results in secondary deficiencies of a host of band 3 associated membrane proteins that we quantitatively show result predominantly from reduced plasma membrane expression during erythropoiesis compounded by impaired retention in the nascent reticulocyte membrane during erythroblast enucleation. In order to explore the importance of the capacity of band 3 to associate with the cytoskeleton for surface expression of this protein and its associated multiprotein complex binding proteins, immature band 3 Coimbra patient erythroblasts were lentivirally transduced with N terminally GFP-tagged wild type band 3 or band 3 mutants with absent or impaired ability to associate with the cytoskeleton. We demonstrate for the first time the ability to restore expression of band 3 to normal levels in this uniquely compromised patient and to rescue key secondary protein deficiencies arising from the absence of band 3 in reticulocytes. Exogenous expression levels of band 3, monitored by GFP intensity, correlate directly with degree of rescue of a variety of band 3 associated proteins. When expressed in band 3 deficient Coimbra erythroblasts, the band 3 membrane domain, which is unable to associate with the cytoskeleton, exhibits an increased partitioning to the plasma membrane surrounding the extruded nuclei compared to wild type band 3 and fails to rescue reticulocyte membrane retention of band 3 associated proteins. Expression of the kidney isoform of band 3, which is unable to bind ankyrin but retains the binding site for the cytoskeletal accessory protein, protein 4.2 results in partial rescue of the protein 4.2 dependent CD47 only. This demonstrates the importance of band 3 association with the cytoskeleton for efficient retention of band 3 associated proteins during erythroblast enucleation. Interestingly, whilst both exhibit reduced reticulocyte membrane retention relative to wild type, a significant proportion of both band 3 membrane domain and kidney band 3 is retained in the reticulocyte membrane following erythroblast enucleation indicating that cytoskeletal attachment of band 3 is not the sole determinant of partitioning during this complex process. This study advances our understanding of the mechanisms by which the properties of band 3 influence the sculpting and composition of the erythrocyte membrane and highlights the role of this protein as a core for assembly and stabilisation of key membrane proteins in both the early and late stages of terminal erythroid differentiation. Disclosures No relevant conflicts of interest to declare.

Description: We report on a 40 year old patient with mild hereditary spherocytosis (RBC: 4.43×1012/dL; Reticulocyte count: 253×109/dL; Hb: 14g/dL), whose red blood cells completely lack protein 4.2. Genetic analysis showed that the patient was a double heterozygote for EPB42 deletions; one allele lacked exon 9 but the sequence remained in frame (protein 4.2 Chartres I) and the other allele contained a di-nucleotide deletion resulting in a premature stop signal (protein 4.2 Chartres II). Homology modelling showed that the hairpin region that forms the proposed band 3 binding site is still present in both mutants. However, the deletion of exon 9 removes a large portion of Domain 2 (core domain) of protein 4.2, potentially removing a band 3 binding groove, and the truncation mutant lacks a portion of the core domain and the whole domains 3 and 4. Therefore, these mutations are likely to destabilize protein 4.2 either directly, or indirectly by disturbing the interaction of protein 4.2 with band 3. Flow cytometry, SDS-PAGE and Western blotting of erythrocyte membranes showed a significant reduction of 70–80 % in CD47 levels, altered Rh associated glycoprotein (RhAG) mobility, reduced GPA/GPB heterodimers, and a 3 fold increase in CD44 levels as reported previously for protein 4.2 null red cells. We stored mature red cells at 4 degrees Celsius over 35 days and found that CD47 continues to be lost in microvesicles as the red cell ages, consistent with a weaker link of CD47 with the cytoskeleton. We investigated band 3 complex stability by performing co-immunoprecipitations and found that lower amounts of band 3 were co-immunoprecipitated using an anti-ankyrin antibody in Chartres red cells compared to wild type, suggesting that the association of band 3 with the cytoskeleton is severely affected. Furthermore, less band 3 was co-immunoprecipitated with an anti-RhAG antibody, consistent with a disturbance of the association of the Rh complex with band 3. We next investigated the stage during erythropoiesis at which the observed changes in band 3 macrocomplex proteins occur. To this end we expanded and differentiated erythroid progenitors from peripheral blood of wild type and the Chartres patient using a three culture system modified from Leberbauer et al. (2005). Synchronous differentiation of a pure erythroid progenitor pool (60% enucleation) demonstrated that protein 4.2 co-immunoprecipitated with band 3 early on in erythroid progenitor differentiation. However, in protein 4.2 Chartres progenitors the mutant forms of protein 4.2 were not expressed at any stage during erythropoiesis, demonstrating that both protein 4.2 mutants are unstable and rapidly degraded. Surprisingly, flow cytometry and western blot analysis revealed that CD47, RhAG, band 3, CD44, and GPA/GPB levels are all similar compared to wild type during erythroid differentiation. Thus, despite the absence of protein 4.2 throughout erythropoiesis, the final changes in the Rh/band3 complex observed in patient’s erythrocytes are not observed. Overall our results suggest that protein 4.2 Chartres is unstable probably due to specific 4.2 mutations that either cause disruption of the band 3 binding sites or an intrinsic instability of these individual mutant proteins. The association of band 3 and ankyrin also appears to be altered in protein 4.2 Chartres suggestive of a weakening of the band 3 cytoskeleton linkage, which could also contribute to the HS phenotype. Importantly, the absence of protein 4.2 not only disturbs ankyrin recruitment to band 3 but also affects association of band 3 with RhAG and disturbs GPA/GPB complexes, which demonstrates the importance of protein 4.2 in the process of band 3 complex formation. Most strikingly, our work demonstrates that the loss of CD47 and the other alterations observed in the band 3/Rh complex in protein 4.2 Chartres must occur late during red blood cell progenitor maturation, presumably after enucleation.

Description: 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.