Complete absence of GLUT1 does not impair human terminal erythroid differentiation.

The Glucose transporter 1 (GLUT1) is one of the most abundant proteins within the erythrocyte membrane and is required for glucose and dehydroascorbic acid (Vitamin C precursor) transport. It is widely recognized as a key protein for red cell structure, function, and metabolism. Previous reports highlighted the importance of GLUT1 activity within these uniquely glycolysis-dependent cells, in particular for increasing antioxidant capacity needed to avoid irreversible damage from oxidative stress in humans. However, studies of glucose transporter roles in erythroid cells are complicated by species-specific differences between humans and mice. Here, using CRISPR-mediated gene editing of immortalized erythroblasts and adult CD34+ hematopoietic progenitor cells, we generate committed human erythroid cells completely deficient in expression of GLUT1. We show that absence of GLUT1 does not impede human erythroblast proliferation, differentiation, or enucleation. This work demonstrates for the first-time generation of enucleated human reticulocytes lacking GLUT1. The GLUT1-deficient reticulocytes possess no tangible alterations to membrane composition or deformability in reticulocytes. Metabolomic analyses of GLUT1-deficient reticulocytes reveal hallmarks of reduced glucose import, downregulated metabolic processes and upregulated AMPK-signalling, alongside alterations in antioxidant metabolism, resulting in increased osmotic fragility and metabolic shifts indicative of higher oxidant stress. Despite detectable metabolic changes in GLUT1 deficient reticulocytes, the absence of developmental phenotype, detectable proteomic compensation or impaired deformability comprehensively alters our understanding of the role of GLUT1 in red blood cell structure, function and metabolism. It also provides cell biological evidence supporting clinical consensus that reduced GLUT1 expression does not cause anaemia in GLUT1 deficiency syndrome.

Ex vivo culture of adult CD34+ stem cells using functional highly porous polymer scaffolds to establish biomimicry of the bone marrow niche.

Haematopoiesis, the process of blood production, occurs from a tiny contingent of haematopoietic stem cells (HSC) in highly specialised three-dimensional niches located within the bone marrow. When haematopoiesis is replicated using in vitro two-dimensional culture, HSCs rapidly differentiate, limiting self-renewal. Emulsion-templated highly porous polyHIPE foam scaffolds were chosen to mimic the honeycomb architecture of human bone. The unmodified polyHIPE material supports haematopoietic stem and progenitor cell (HSPC) culture, with successful culture of erythroid progenitors and neutrophils within the scaffolds. Using erythroid culture methodology, the CD34+ population was maintained for 28 days with continual release of erythroid progenitors. These cells are shown to spontaneously repopulate the scaffolds, and the accumulated egress can be expanded and grown at large scale to reticulocytes. We next show that the polyHIPE scaffolds can be successfully functionalised using activated BM(PEG)2 (1,8-bismaleimido-diethyleneglycol) and then a Jagged-1 peptide attached in an attempt to facilitate notch signalling. Although Jagged-1 peptide had no detectable effect, the BM(PEG)2 alone significantly increased cell egress when compared to controls, without depleting the scaffold population. This work highlights polyHIPE as a novel functionalisable material for mimicking the bone marrow, and also that PEG can influence HSPC behaviour within scaffolds.

Band 3 mutations, renal tubular acidosis and South-East Asian ovalocytosis in Malaysia and Papua New Guinea: loss of up to 95% band 3 transport in red cells.

We describe three mutations of the red-cell anion exchangerband 3 (AE1, SLC4A1) gene associated with distalrenal tubular acidosis (dRTA) in families from Malaysia and Papua NewGuinea: Gly(701)-->Asp (G701D), Ala(858)-->Asp(A858D) and deletion of Val(850) (DeltaV850). The mutationsA858D and DeltaV850 are novel; all three mutations seem to berestricted to South-East Asian populations. South-East Asianovalocytosis (SAO), resulting from the band 3 deletion of residues400-408, occurred in many of the families but did not itselfresult in dRTA. Compound heterozygotes of each of the dRTA mutationswith SAO all had dRTA, evidence of haemolytic anaemia and abnormal red-cell properties. The A858D mutation showed dominant inheritance and therecessive DeltaV850 and G701D mutations showed a pseudo-dominantphenotype when the transport-inactive SAO allele was also present. Red-cell and Xenopus oocyte expression studies showed that theDeltaV850 and A858D mutant proteins have greatly decreased aniontransport when present as compound heterozygotes (DeltaV850/A858D,DeltaV850/SAO or A858D/SAO). Red cells with A858D/SAO had only 3% ofthe SO(4)(2-) efflux of normal cells, thelowest anion transport activity so far reported for human red cells. The results suggest dRTA might arise by a different mechanism for eachmutation. We confirm that the G701D mutant protein has an absoluterequirement for glycophorin A for movement to the cell surface. Wesuggest that the dominant A858D mutant protein is possibly mis-targetedto an inappropriate plasma membrane domain in the renal tubular cell,and that the recessive DeltaV850 mutation might give dRTA because ofits decreased anion transport activity.

Red-cell glycophorin A-band 3 interactions associated with the movement of band 3 to the cell surface.

We have examined the mechanism by which glycophorin A (GPA) facilitates the movement of the human red-cell anion exchanger (band 3, AE1) to the cell surface. GPA itself forms stable dimers in membranes and detergent solution. Four mutants of human GPA with impaired dimerization were prepared (L75I, I76A, G79L and G83L). All four GPA mutants enhanced band 3 translocation to the Xenopus oocyte plasma membrane in the same way as wild-type GPA, showing that the GPA monomer is sufficient to mediate this process. Cell-surface expression of the natural band 3 mutant G701D has an absolute requirement for GPA. GPA monomers also rescued the cell-surface expression of this mutant band 3. Taking into account other evidence, we infer that the site of GPA interaction with band 3 is located outside the GPA dimerization interface but within the GPA transmembrane span. The results of examination of the cell-surface expression of GPA and band 3 in different K562 erythroleukaemia cell clones stably transfected with band 3 are consistent with the movement of GPA and band 3 to the cell surface together. We discuss the pathways by which band 3 moves to the cell surface in the presence and the absence of GPA, concluding that GPA has a role in enhancing the folding and maturation of band 3. We propose that GPA functions in erythroid cells to assist with the incorporation of large amounts of properly folded band 3 into the membrane within a limited time span during erythroid maturation.

Keratinocyte superoxide generation.

We have demonstrated using the reduction of cytochrome c, that the keratinocyte cell line H357 generates superoxide at significant rates (8.36 nmol/h/10[6] cells). The rate of superoxide release decreased as the cells reached confluence. Superoxide production was increased more than twofold following preincubation with IL-1beta, or by the addition of the Ca2+ ionophore, Ionomycin. Other stimuli known to activate the NADPH oxidase of phagocytes were ineffective, but the regulatory cytokine IFNgamma lowered the rate of release. Inhibitors of lipoxygenase function decreased the rate of superoxide production, whereas inhibitors of cyclo-oxygenase, xanthine oxidase, or NADPH oxidase failed to inhibit. The addition of NADH or NADPH to whole cells increased the rate threefold.