Lethal alpha-thalassaemia created by gene targeting in mice and its genetic rescue.

Mutations at the alpha-globin locus are the most common class of mutations in humans, with deletion of all four adult alpha-globin genes resulting in the perinatal lethal condition haemoglobin Barts hydrops fetalis. Using gene targeting in mice, we have deleted a 16 kilobase region encompassing both adult alpha-globin genes. Animals homozygous for this deletion become hydropic and die late in gestation mimicking humans with hydrops fetalis. Introduction of a human alpha-globin transgene rescued these animals from perinatal death thus demonstrating the utility of this murine model in the development of cellular and gene based approaches for treating this human genetic disease.

The gene encoding ribosomal protein S19 is mutated in Diamond-Blackfan anaemia.

Diamond-Blackfan anaemia (DBA) is a constitutional erythroblastopenia characterized by absent or decreased erythroid precursors. The disease, previously mapped to human chromosome 19q13, is frequently associated with a variety of malformations. To identify the gene involved in DBA, we cloned the chromosome 19q13 breakpoint in a patient with a reciprocal X;19 chromosome translocation. The breakpoint occurred in the gene encoding ribosomal protein S19. Furthermore, we identified mutations in RPS19 in 10 of 40 unrelated DBA patients, including nonsense, frameshift, splice site and missense mutations, as well as two intragenic deletions. These mutations are associated with clinical features that suggest a function for RPS19 in erythropoiesis and embryogenesis.

Erythrocyte membrane deformability and stability: two distinct membrane properties that are independently regulated by skeletal protein associations.

Skeletal proteins play an important role in determining erythrocyte membrane biophysical properties. To study whether membrane deformability and stability are regulated by the same or different skeletal protein interactions, we measured these two properties, by means of ektacytometry, in biochemically perturbed normal membranes and in membranes from individuals with known erythrocyte abnormalities. Treatment with 2,3-diphosphoglycerate resulted in membranes with decreased deformability and decreased stability, whereas treatment with diamide produced decreased deformability but increased stability. N-ethylmaleimide induced time-dependent changes in membrane stability. Over the first minute, the stability increased; but with continued incubation, the membranes became less stable than control. Meanwhile, the deformability of these membranes decreased with no time dependence. Biophysical measurements were also carried out on pathologic erythrocytes. Membranes from an individual with hereditary spherocytosis and a defined abnormality in spectrin-protein 4.1 association showed decreased stability but normal deformability. In a family with hereditary elliptocytosis and an abnormality in spectrin self-association, the membranes had decreased deformability and stability. Finally, membranes from several individuals with Malaysian ovalocytosis had decreased deformability but increased stability. Our data from both pathologic membranes and biochemically perturbed membranes show that deformability and stability change with no fixed relationship to one another. These findings imply that different skeletal protein interactions regulate membrane deformability and stability. In light of these data, we propose a model of the role of skeletal protein interactions in deformability and stability.

Signal transduction by glycophorin A: role of extracellular and cytoplasmic domains in a modulatable process.

Binding of ligands to the extracellular region of the erythrocyte transmembrane protein glycophorin A induces a decrease in membrane deformability. Since the property of membrane deformability is regulated by the skeletal proteins on the cytoplasmic side of the membrane, this suggests that ligand binding may initiate a transmembrane signal. To further study this process, we examined which domains of the extracellular region of glycophorin are involved in signal transduction and whether the cytoplasmic domain of the molecule is necessary for transmitting the signal. Using the ektacytometer, we compared the effect on deformability of four monoclonal antibodies that detect different epitopes on glycophorin A. We found that 9A3 (which recognized the amino terminus of glycophorin) caused a 5.8-fold increase in rigidity, R-10 and 10F7 (which recognized epitopes in the mid-region of the extracellular domain) caused a 10.8-fold increase in rigidity and B14 (which binds to glycophorin close to the membrane) caused a 18-fold increase in rigidity. Further, a direct relationship was observed between the degree of antibody-induced rigidity and the amount of glycophorin A that became associated with the skeletal proteins in a Triton shell assay. In Miltenberger V erythrocytes, which contain a hybrid sialoglycoprotein with no cytoplasmic domain, antibody binding did not induce an increase in rigidity. These results imply that glycophorin A is capable of a modulatable form of transmembrane signaling that is determined by the extracellular domain to which the ligand binds, and the cytoplasmic domain of glycophorin A is crucial for this process.

Mechanochemistry of protein 4.1's spectrin-actin-binding domain: ternary complex interactions, membrane binding, network integration, structural strengthening.

Mechanical strength of the red cell membrane is dependent on ternary interactions among the skeletal proteins, spectrin, actin, and protein 4.1. Protein 4.1's spectrin-actin-binding (SAB) domain is specified by an alternatively spliced exon encoding 21 amino acid (aa) and a constitutive exon encoding 59 aa. A series of truncated SAB peptides were engineered to define the sequences involved in spectrin-actin interactions, and also membrane strength. Analysis of in vitro supramolecular assemblies showed that gelation activity of SAB peptides correlates with their ability to recruit a critical amount of spectrin into the complex to cross-link actin filaments. Also, several SAB peptides appeared to exhibit a weak, cooperative actin-binding activity which mapped to the first 26 residues of the constitutive 59 aa. Fluorescence-imaged microdeformation was used to show SAB peptide integration into the elastic skeletal network of spectrin, actin, and protein 4.1. In situ membrane-binding and membrane-strengthening abilities of the SAB peptides correlated with their in vitro gelation activity. The findings imply that sites for strong spectrin binding include both the alternative 21-aa cassette and a conserved region near the middle of the 59 aa. However, it is shown that only weak SAB affinity is necessary for physiologically relevant action. Alternatively spliced exons can thus translate into strong modulation of specific protein interactions, economizing protein function in the cell without, in and of themselves, imparting unique function.

Structural protein 4.1 in the nucleus of human cells: dynamic rearrangements during cell division.

Structural protein 4.1, first identified as a crucial 80-kD protein in the mature red cell membrane skeleton, is now known to be a diverse family of protein isoforms generated by complex alternative mRNA splicing, variable usage of translation initiation sites, and posttranslational modification. Protein 4.1 epitopes are detected at multiple intracellular sites in nucleated mammalian cells. We report here investigations of protein 4.1 in the nucleus. Reconstructions of optical sections of human diploid fibroblast nuclei using antibodies specific for 80-kD red cell 4.1 and for 4.1 peptides showed 4.1 immunofluorescent signals were intranuclear and distributed throughout the volume of the nucleus. After sequential extractions of cells in situ, 4.1 epitopes were detected in nuclear matrix both by immunofluorescence light microscopy and resinless section immunoelectron microscopy. Western blot analysis of fibroblast nuclear matrix protein fractions, isolated under identical extraction conditions as those for microscopy, revealed several polypeptide bands reactive to multiple 4.1 antibodies against different domains. Epitope-tagged protein 4.1 was detected in fibroblast nuclei after transient transfections using a construct encoding red cell 80-kD 4.1 fused to an epitope tag. Endogenous protein 4.1 epitopes were detected throughout the cell cycle but underwent dynamic spatial rearrangements during cell division. Protein 4.1 was observed in nucleoplasm and centrosomes at interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, as well as in the midbody during cytokinesis. These results suggest that multiple protein 4.1 isoforms may contribute significantly to nuclear architecture and ultimately to nuclear function.

The 13-kD FK506 binding protein, FKBP13, interacts with a novel homologue of the erythrocyte membrane cytoskeletal protein 4.1.

We have identified a novel generally expressed homologue of the erythrocyte membrane cytoskeletal protein 4.1, named 4.1G, based on the interaction of its COOH-terminal domain (CTD) with the immunophilin FKBP13. The 129-amino acid peptide, designated 4.1G-CTD, is the first known physiologic binding target of FKBP13. FKBP13 is a 13-kD protein originally identified by its high affinity binding to the immunosuppressant drugs FK506 and rapamycin (Jin, Y., M.W. Albers, W.S. Lane, B.E. Bierer, and S.J. Burakoff. 1991. Proc. Natl. Acad. Sci. USA. 88:6677- 6681); it is a membrane-associated protein thought to function as an ER chaperone (Bush, K.T., B.A. Henrickson, and S.K. Nigam. 1994. Biochem. J. [Tokyo]. 303:705-708). We report the specific association of FKBP13 with 4.1G-CTD based on yeast two-hybrid, in vitro binding and coimmunoprecipitation experiments. The histidyl-proline moiety of 4.1G-CTD is required for FKBP13 binding, as indicated by yeast experiments with truncated and mutated 4.1G-CTD constructs. In situ hybridization studies reveal cellular colocalizations for FKBP13 and 4.1G-CTD throughout the body during development, supporting a physiologic role for the interaction. Interestingly, FKBP13 cofractionates with the red blood cell homologue of 4.1 (4.1R) in ghosts, inside-out vesicles, and Triton shell preparations. The identification of FKBP13 in erythrocytes, which lack ER, suggests that FKBP13 may additionally function as a component of membrane cytoskeletal scaffolds.

Molecular maps of red cell deformation: hidden elasticity and in situ connectivity.

Fluorescence-imaged micropipette aspiration was used to map redistribution of the proteins and lipids in highly extended human red blood cell membranes. Whereas the fluid bilayer distributed uniformly (+/- 10 percent), the underlying, solidlike cytoskeleton of spectrin, actin, and protein 4.1 exhibited a steep gradient in density along the aspirated projection, which was reversible on release from deformation. Quantitation of the cytoskeletal protein density gradients showed that skeletal elasticity is well represented by a grafted polymer network with a ratio of surface dilation modulus to shear modulus of approximately 2:1. Fractionally mobile integral proteins, such as band 3, and highly mobile receptors, such as CD59 as well as glycophorin C in protein 4.1-deficient cells, appeared to be squeezed out of areas dense in the underlying network and enriched in areas of network dilation. This complementary segregation demonstrates patterning of cell surface components by cytoskeletal dilation.

Transgenic knockout mice with exclusively human sickle hemoglobin and sickle cell disease.

To create mice expressing exclusively human sickle hemoglobin (HbS), transgenic mice expressing human alpha-, gamma-, and betaS-globin were generated and bred with knockout mice that had deletions of the murine alpha- and beta-globin genes. These sickle cell mice have the major features (irreversibly sickled red cells, anemia, multiorgan pathology) found in humans with sickle cell disease and, as such, represent a useful in vivo system to accelerate the development of improved therapies for this common genetic disease.

Neurobehavioral deficits in mice lacking the erythrocyte membrane cytoskeletal protein 4.1.

The erythrocyte membrane cytoskeletal protein 4.1 (4.1R) is a structural protein that confers stability and flexibility to erythrocytes via interactions with the cytoskeletal proteins spectrin and F-actin and with the band 3 and glycophorin C membrane proteins. Mutations in 4.1R can cause hereditary elliptocytosis, a disease characterized by a loss of the normal discoid morphology of erythrocytes, resulting in hemolytic anemia [1]. Different isoforms of the 4.1 protein have been identified in a wide variety of nonerythroid tissues by immunological methods [2-5]. The variation in molecular weight of these different 4.1 isoforms, which range from 30 to 210 kDa [6], has been attributed to complex alternative splicing of the 4.1R gene [7]. We recently identified two new 4.1 genes: one is generally expressed throughout the body (4. 1G) [8] and the other is expressed in central and peripheral neurons (4.1N) [9]. Here, we examined 4.1R expression by in situ hybridization analysis and found that 4.1R was selectively expressed in hematopoietic tissues and in specific neuronal populations. In the brain, high levels of 4.1R were discretely localized to granule cells in the cerebellum and dentate gyrus. We generated mice that lacked 4.1R expression; these mice had deficits in movement, coordination, balance and learning, in addition to the predicted hematological abnormalities. The neurobehavioral findings are consistent with the distribution of 4.1R in the brain, suggesting that 4.1R performs specific functions in the central nervous system.

Sickle erythrocyte adherence to vascular endothelium. Morphologic correlates and the requirement for divalent cations and collagen-binding plasma proteins.

Increased adherence of sickle erythrocytes to vascular endothelium has been suggested by Hebbel and his colleagues to play a role in vasocclusive events of sickle cell disease. To define the role of cell membrane changes and plasma factors in cell adherence, a micropipette technique previously developed by us to obtain a direct, quantitative measure of cell adherence was used to evaluate the adhesivity of different morphologic classes of sickle cells to endothelial cells in various suspending media. Irregularly shaped, deformable sickle cells were four- to fivefold more adherent than discoid sickle cells, whereas rigid irreversibly sickled cells were least adherent. Sickle erythrocytes adhered to endothelial cells when suspended in autologous citrated or heparinized plasma but were totally nonadherent when suspended in autologous EDTA plasma. Removal of the divalent cation chelator and addition of calcium to EDTA plasma restored its ability to promote adhesion, implying an absolute requirement for divalent cations in sickle cell adherence. Sickle cells also did not adhere to endothelial cells in protein-free media containing divalent cations, suggesting an additional requirement for plasma proteins. Removal of collagen-binding proteins from citrated sickle plasma resulted in a three- to fivefold reduction in its ability to promote cell adhesion, suggesting an important role for these plasma proteins in sickle cell adherence. The results of this study imply that sickle cell adherence to vascular endothelium is a complex process in which temporal changes in the numbers of cells identified to be most adhesive and the plasma concentration of protein(s) involved in the adhesive process determine the extent of in vivo sickle cell adherence.

Modulation of erythrocyte membrane material properties by Ca2+ and calmodulin. Implications for their role in regulation of skeletal protein interactions.

Skeletal proteins of the red blood cell apparently play an important role in regulating membrane material properties of deformability and stability. However, the role of various intracellular constituents in regulating membrane properties has not been clearly defined. To determine whether Ca2+ and calmodulin might play a role in this regulation, we measured the membrane stability and deformability of resealed ghosts prepared in the presence of varying concentrations of Ca2+ and calmodulin (CaM). For membranes resealed in the presence of Ca2+ and physiologic concentrations of CaM (2-8 microM), membrane stability decreased with increasing Ca2+ concentrations (greater than 1.0 microM). Moreover, Ca2+ and CaM-induced alterations in membrane stability were completely reversible. In the absence of CaM, an equivalent decrease in membrane stability was seen only when Ca2+ concentration was two orders of magnitude higher (greater than 100 microM). Calmodulin did not alter membrane stability in the absence of Ca2+. Compared with these changes in membrane stability, membrane deformability decreased only at Ca2+ concentrations greater than 100 microM, and calmodulin had no effect on Ca2+-induced decrease in membrane deformability. Examination of the effects of Ca2+ and CaM on various membrane interactions have enabled us to suggest that spectrin-protein 4.1-actin interaction may be one of the targets for the effect of Ca2+ and CaM. These results imply that Ca2+ and calmodulin can regulate membrane stability through modulation of skeletal protein interactions, and that these protein interactions are of a dynamic nature on intact membranes.

Static and dynamic rigidities of normal and sickle erythrocytes. Major influence of cell hemoglobin concentration.

Static and dynamic deformabilities of erythrocytes are important determinants of microcirculatory blood flow. To determine the influence of increased cellular hemoglobin concentration on these properties, we quantitated static and dynamic deformabilities of isolated subpopulations of oxygenated normal and sickle erythrocytes with defined cell densities using micromechanical manipulations of individual cells. The rheological properties measured to characterize static deformability were membrane extensional rigidity and bending rigidity. To characterize dynamic deformability of the cells, we measured the time constants for rapid elastic recovery from extensional and bending deformations. The extensional rigidity of sickle cells increased with increasing cell hemoglobin concentration while that of normal cells was independent of the state of cell hydration. Moreover, sickle cells were found to exhibit inelastic behavior at much lower cell hemoglobin concentrations than normal cells. In contrast, the dynamic rigidity of both normal and sickle cells was increased to the same extent at elevated hemoglobin concentrations. Moreover, this increase in dynamic rigidity with increasing cellular dehydration was much more pronounced than that seen for static rigidity. Both the increased static and dynamic rigidities of the dehydrated sickle cells could be greatly improved by hydrating the cells. This suggests that increased bulk hemoglobin concentration, which is perhaps inordinately increased adjacent to the membrane, plays a major role in regulating the rigidity of sickle cells. In addition, irreversible membrane changes also appear to accompany cell dehydration in vivo, resulting in increased membrane shear rigidity and plastic flow. We expect that the marked increases in rigidity of dehydrated sickle cells observed here may have a major influence on the dynamics of their circulation in the microvasculature.

Deficiency of skeletal membrane protein band 4.1 in homozygous hereditary elliptocytosis. Implications for erythrocyte membrane stability.

Erythrocytes from three patients with severe hemolytic anemia, marked erythrocyte fragmentation, and elliptocytic poikilocytosis, were studied in terms of both their membrane protein composition and their mechanical characteristics. Erythrocytes from the patients' parents and one minimally affected and one normal sibling were also studied. Morphologic observations implied that the severely affected patients suffered from homozygous hereditary elliptocytosis because erythrocytes of both parents and the one minimally affected sibling showed moderate elliptocytosis on smear, whereas those of an unaffected sibling had normal morphology. The parallel findings of markedly reduced levels of band 4.1 in the erythrocyte membrane proteins of the patients and an intermediate reduction in the cells of the parents and the putative heterozygous sibling, suggest that the elliptocytic shape of the cells was related to the reduced levels of band 4.1. Additional studies showed marked abnormalities in cellular deformability and membrane fragility in the erythrocytes from the homozygous patients. Importantly, these changes were also closely proportional to the reduced levels of band 4.1, suggesting a central role for this protein in the maintenance of normal membrane stability and normal cell shape. It seems likely that this role for band 4.1 is intimately related to its known biochemical connection to the "membrane skeleton" through its linkage with spectrin and actin.

Decreased membrane mechanical stability and in vivo loss of surface area reflect spectrin deficiencies in hereditary spherocytosis.

Whereas marked variations in the clinical manifestations of hereditary spherocytosis have long been recognized, we have only recently begun to define the molecular basis for this heterogeneity. An important unanswered question is whether decreased spectrin results in reduced membrane mechanical stability, and if this reduction in membrane mechanical stability can be related to in vivo surface area loss. Using the ektacytometer, we quantitated membrane surface area and stability in erythrocytes from 18 individuals with hereditary spherocytosis and deficiencies of spectrin (30-80% of normal spectrin level). Membrane mechanical stability was reduced and the magnitude of the reductions correlated with the spectrin content. Moreover, the reductions in mechanical stability correlated with in vivo loss of membrane surface area. These data indicate that decreased spectrin content results in reduced membrane mechanical stability and surface area loss in vivo. We conclude that partial deficiencies of spectrin, reductions in membrane mechanical stability, and loss of membrane surface area are directly related and are major features determining the heterogeneous clinical manifestations of hereditary spherocytosis.

Hydration of sickle cells using the sodium ionophore Monensin. A model for therapy.

Mean cell hemoglobin concentration (MCHC) is thought to have an important influence in sickle cell disease, both through the strong dependence of sickling rates on hemoglobin S concentration, and through the profoundly limiting effect of high MCHC on the rheologic competence of oxygenated, irreversibly sickled cells (ISC). Recent studies have tested the ability of antidiuretic hormone to reduce sickle cell MCHC by reducing plasma sodium (Na) and osmolality. An alternative means of reducing MCHC is to elevate intracellular cation content, rather than to depress extracellular cation concentration. In an effort to do this, we have treated sickle cells with Monensin, an antibiotic that selectively enhances membrane Na permeability. At submicromolar concentrations, Monensin substantially reduced the MCHC of whole sickle blood and isolated ISC, causing an improvement in cell deformability. Monensin's effectiveness in producing a controlled increase in erythrocyte water content suggests that agents that selectively increase membrane Na permeability could be therapeutically useful.

Erythrocyte membrane rigidity induced by glycophorin A-ligand interaction. Evidence for a ligand-induced association between glycophorin A and skeletal proteins.

Erythrocyte skeletal proteins are known to play an important role in determining membrane deformability. In order to see whether transmembrane proteins also influence deformability and, if so, whether this influence is mediated by an interaction with the membrane skeleton, we examined the effect on deformability of ligands specific for transmembrane proteins. We found membrane deformability markedly reduced in erythrocytes that were pretreated with glycophorin A-specific ligands. In contrast, ligands specific for band 3 and A and B blood group antigens had no effect. The increase in membrane rigidity appeared to depend upon a transmembrane event and not upon a rigidity-inducing lattice on the outside surface of the cell in that a monovalent Fab of antiglycophorin IgG caused decreased deformability. We therefore looked for a ligand-induced association of glycophorin and the skeletal proteins and found, in Triton X-100-insoluble residues, a partitioning of glycophorin with the skeletal proteins only after preincubation with a ligand specific for glycophorin. We then studied cells and resealed membranes with skeletal protein abnormalities. In spectrin-deficient and protein 4.1-deficient erythrocytes and in 2,3-diphosphoglycerate-treated resealed membranes, the antiglycophorin IgG was only one-third as effective in decreasing deformability as it was in normal cells. Thus, normal skeletal proteins appear to be essential for liganded glycophorin to affect membrane deformability maximally. Taken together, these observations indicate that there is a ligand-induced interaction between glycophorin A and skeletal proteins and that this interaction can directly influence membrane deformability.

Effect of human beta (s)-globin chains on cellular properties of red cells from beta-thalassemic mice.

The transgenic mouse system provides an in vivo setting in which to examine the effects on mouse red cells of hemoglobin genes that have been genetically introduced into the animals' genome. In this report, we have analyzed the cellular properties of red cells from homozygous beta-thalassemic mice (Hbbth-1/Hbbth-1), homozygous beta-thalassemic transgenic mice containing a human beta-sickle (beta(s)) gene (Hbb(th-1)/Hbb(th-1) + beta(s)), and normal animals. The presence of human beta(s)-globin chains in red cells from the Hbbth-1/Hbb(th-1) + beta(s) transgenic animals was noted to have a significant effect on cellular deformability and density distribution, as well as on the degree of anemia in these animals. We conclude from these studies that red cell deformability and density distribution is a sensitive means for assessing at the cellular level the effects of globin genes genetically introduced into whole organisms. In addition, these studies suggest that small decreases in the amount of excess alpha-globin chains can significantly ameliorate the severity of anemia in the beta-thalassemic mouse.

Deformability of oxygenated irreversibly sickled cells.

The deformability characteristics of isolated subpopulations of irreversibly sickled cells (ISC) have been studied in an ektacytometer. Analysis of laser diffraction patterns of well-oxygenated cells subjected to shear stress in solutions of varying osmolality has demonstrated a profound influence of mean corpuscular hemoglobin concentration and intracellular viscosity on the deformability of ISC. Virtually undeformable at 290 mosM, ISC became almost totally deformable at 130 mosM. In addition, when ISC membranes were loaded with normal hemoglobin at low concentration, they deformed easily in isotonic medium, as did resealed normal cell membranes. The restoration of deformability of ISC upon reduction of their hemoglobin concentration and internal viscosity to normal levels suggests that altered membrane properties are not the primary determinant of decreased deformability in these cells. Rather, cellular dehydration induced by previous sickling would appear to contribute in a major way to their abnormal rheological behavior.

Concurrent sickle cell anemia and alpha-thalassemia. Effect on pathological properties of sickle erythrocytes.

The concurrence of sickle cell anemia and alpha-thalassemia results in less severe hemolytic anemia apparently as a result of reduced intraerythrocytic concentration of hemoglobin S and its retarded polymerization. We have evaluated the effect of alpha-globin gene number on several interrelated properties of sickle erythrocytes (RBC) that are expected to correlate with the hemolytic and rheologic consequences of sickle cell disease. The irreversibly sickled cell number, proportion of very dense sickle RBC, and diminished deformability of sickle RBC each varied directly with alpha-globin gene number. Sickle RBC density was a direct function of the mean corpuscular hemoglobin concentration (MCHC). Even in nonsickle RBC, alpha-globin gene number varied directly with RBC density. Despite differences in alpha-globin gene number, sickle RBC of the same density had the same degree of deformability and dehydration. These data indicate that the fundamental effect of alpha-thalassemia is to inhibit the generation of sickle RBC having high density and MCHC, and that the other beneficial effects of sickle RBC are secondary to this process. The less consistent effect on overall clinical severity reported for subjects with this concurrence may reflect an undefined detrimental effect of alpha-thalassemia, possibly on the whole blood viscosity or on sickle RBC membrane-mediated adherence phenomena.