A Stem Cell Strategy Identifies Glycophorin C as a Major Erythrocyte Receptor for the Rodent Malaria Parasite Plasmodium berghei.

The clinical complications of malaria are caused by the parasite expansion in the blood. Invasion of erythrocytes is a complex process that depends on multiple receptor-ligand interactions. Identification of host receptors is paramount for fighting the disease as it could reveal new intervention targets, but the enucleated nature of erythrocytes makes genetic approaches impossible and many receptors remain unknown. Host-parasite interactions evolve rapidly and are therefore likely to be species-specific. As a results, understanding of invasion receptors outside the major human pathogen Plasmodium falciparum is very limited. Here we use mouse embryonic stem cells (mESCs) that can be genetically engineered and differentiated into erythrocytes to identify receptors for the rodent malaria parasite Plasmodium berghei. Two proteins previously implicated in human malaria infection: glycophorin C (GYPC) and Band-3 (Slc4a1) were deleted in mESCs to generate stable cell lines, which were differentiated towards erythropoiesis. In vitro infection assays revealed that while deletion of Band-3 has no effect, absence of GYPC results in a dramatic decrease in invasion, demonstrating the crucial role of this protein for P. berghei infection. This stem cell approach offers the possibility of targeting genes that may be essential and therefore difficult to disrupt in whole organisms and has the potential to be applied to a variety of parasites in diverse host cell types.

Duffy antigen is important for the lethal effect of the lethal strain of Plasmodium yoelii 17XL.

We studied the potential role of the Duffy antigen and glycophorin A as receptors for rodent malaria parasite invasion of erythrocytes. Parasitemia increased exponentially after infection with Plasmodium berghei NK65, P. chabaudi, and P. vinckei in Duffy antigen knockout, glycophorin A knockout, and wild-type mice, indicating that the Duffy antigen and glycophorin A are not essential for these malaria parasites. However, parasitemia of the Duffy antigen knockout mice infected with P. yoelii 17XL remained constant from day 5 to 14 after infection, and then decreased, resulting in autotherapy. The treatment of P. yoelii 17XL-infected Duffy antigen knockout mice with anti-CD4 antibody increased the parasitemia 15 days after infection and the mice eventually died, indicating that CD-4-positive cells play an important role in the clearance of P. yoelii 17XL at the late stage of the infection.

Altered structure and anion transport properties of band 3 (AE1, SLC4A1) in human red cells lacking glycophorin A.

We have studied the properties of band 3 in different glycophorin A (GPA)-deficient red cells. These red cells lack either both GPA and glycophorin B (GPB) (M(k)M(k) cells) or GPA (En(a-) cells) or contain a hybrid of GPA and GPB (MiV cells). Sulfate transport was reduced in all three red cell types to approximately 60% of that in normal control red cells as a result of an increased apparent K(m) for sulfate. Transport of the monovalent anions iodide and chloride was also reduced. The reduced iodide transport resulted from a reduction in the V(max) for iodide transport. The anion transport site was investigated by measuring iodide fluorescence quenching of eosin-5-maleimide (EMA)-labeled band 3. The GPA-deficient cells had a normal K(d) for iodide binding, in agreement with the unchanged K(m) found in transport studies. However, the apparent diffusion quenching constant (K(q)) was increased, and the fluorescence polarization of band 3-bound EMA decreased in the variant cells, suggesting increased flexibility of the protein in the region of the EMA-binding site. This increased flexibility is probably associated with the decrease in V(max) observed for iodide transport. Our results suggest that band 3 in the red cell can take up two different structures: one with high anion transport activity when GPA is present and one with lower anion transport activity when GPA is absent.

Complete deficiency of glycophorin A in red blood cells from mice with targeted inactivation of the band 3 (AE1) gene.

Glycophorin A is the major transmembrane sialoglycoprotein of red blood cells. It has been shown to contribute to the expression of the MN and Wright blood group antigens, to act as a receptor for the malaria parasite Plasmodium falciparum and Sendai virus, and along with the anion transporter, band 3, may contribute to the mechanical properties of the red blood cell membrane. Several lines of evidence suggest a close interaction between glycophorin A and band 3 during their biosynthesis. Recently, we have generated mice where the band 3 expression was completely eliminated by selective inactivation of the AE1 anion exchanger gene, thus allowing us to study the effect of band 3 on the expression of red blood cell membrane proteins. In this report, we show that the band 3 -/- red blood cells contain protein 4.1, adducin, dematin, p55, and glycophorin C. In contrast, the band 3 -/- red blood cells are completely devoid of glycophorin A (GPA), as assessed by Western blot and immunocytochemistry techniques, whereas the polymerase chain reaction (PCR) confirmed the presence of GPA mRNA. Pulse-label and pulse-chase experiments show that GPA is not incorporated in the membrane and is rapidly degraded in the cytoplasm. Based on these findings and other published evidence, we propose that band 3 plays a chaperone-like role, which is necessary for the recruitment of GPA to the red blood cell plasma membrane.

Reduced invasion and growth of Plasmodium falciparum into elliptocytic red blood cells with a combined deficiency of protein 4.1, glycophorin C, and p55.

In this investigation, we have measured the invasion and growth of the malaria parasite Plasmodium falciparum into elliptocytic red blood cells (RBCs) obtained from subjects with homozygous hereditary elliptocytosis. These elliptocytic RBCs have been previously characterized to possess molecular defects in protein 4.1 and glycophorin C. Our results show that the invasion of Plasmodium falciparum into these protein 4.1 (-) RBCs is significantly reduced. Glycophorin C (-) Leach RBCs were similarly resistant to parasite invasion in vitro. The intracellular development of parasites that invaded protein 4.1 (-) RBCs was also dramatically reduced. In contrast, no such reduction of intracellular parasite growth was observed in the glycophorin C (-) Leach RBCs. In conjunction with our recent finding that a third protein termed p55 is also deficient in protein 4.1 (-) and glycophorin C (-) RBCs, the present data underscore the importance of the membrane-associated ternary complex between protein 4.1, glycophorin C, and p55 during the invasion and growth of malaria parasites into human RBCs.

Perturbation of red blood cell membrane rigidity by extracellular ligands.

It is known that binding of extracellular antibodies against the major sialoglycoprotein, glycophorin A, reduced the deformability of the red blood cell membrane. This has been taken to result from new or altered interactions between the glycophorin A and the membrane skeleton. We have shown by means of the micropipette aspiration technique that antibodies against the preponderant transmembrane protein, band 3, induce similar effects. A definite but much smaller reduction in elasticity of the membrane is engendered by univalent Fab fragments of the anti-band 3 antibodies. By examining cells genetically devoid of glycophorin A or containing a variant of this constituent, truncated at the inner membrane surface, we have shown that the anti-band 3 antibodies do not act through the band 3-associated glycophorin A. We examined the effect of anti-glycophorin A antibodies on homozygous Wr(a+b-) cells, in which an amino acid replacement in band 3 annihilates the Wright b (Wrb) epitope (comprising sequence elements of glycophorin A and band 3) and thus, by implication disrupts or perturbs the band 3-glycophorin A interaction; these cells show a much smaller response to an anti-glycophorin A antibody than do normal controls. We infer that in this case anti-glycophorin A antibodies exert their rigidifying effect through the associated band 3. Another anti-glycophorin A antibody, directed against an epitope remote from the membrane surface, however, increases the rigidity of both Wr(a+b-) and normal cells. This implies that not all antibodies act in the same manner in modifying the membrane mechanical properties. The effect exerted by anti-band 3 antibodies appears not to be transmitted through the band 3-ankyrin-spectrin pathway because the rigidifying effect of the intact antibody persists at alkaline pH, at which there is evidence that the ankyrin-band 3 link is largely dissociated. The large difference between the effects of saturating concentrations of the divalent and univalent anti-band 3 antibodies implies the existence of an overriding effect on rigidity, resulting from the bifunctionality of the intact antigen. Freeze-fracture electron microscopy shows that the anti-band 3 promotes the formation of small clusters of intra-membrane proteins. Extracellular ligands may in general act by promoting strong or transient interactions between integral membrane proteins, thereby impeding local distortion of the membrane skeletal network in response to shear.

Altered band 3 structure and function in glycophorin A- and B-deficient (MkMk) red blood cells.

The anion transport activity of the human erythrocyte anion transporter (band 3; AE1) has been examined in both normal and glycophorin A (GPA)-deficient (MkMk) human red blood cells (RBCs). The sulfate transport activity of MkMk cells (from two ethnically diverse sources) was approximately 60% that of normal erythrocytes under the transport assay conditions used. However, MkMk and normal RBCs contained similar amounts of band 3. The reduction in sulfate transport activity was shown to be caused by an increase in the apparent Km for sulfate in MkMk RBCs, suggesting the band 3 in the MkMk RBCs has a lowered binding affinity for sulfate anions. The size of the N-glycan chain on band 3 of the MkMk cells was larger than that on band 3 from normal RBCs. In contrast, the size of the N-glycan chain on the glucose transporter (GLUT1) from MkMk cells was smaller than that on GLUT1 from normal cells. The possible role of GPA in the biosynthesis and anion transport activity of band 3 in normal RBCs is discussed.

Studies on the glycoprotein associated with Rh (rhesus) blood group antigen expression in the human red blood cell membrane.

The blood group Rh antigens are associated with non-glycosylated 30-kDa erythrocyte membrane proteins (the Rh30 polypeptides) and the Rh glycoprotein. We used antipeptide antibodies to study the Rh glycoprotein in human erythrocyte membranes. The Rh glycoprotein was present in Rhnull U+ve cells. However, the N-glycan chain of the Rh glycoprotein in Rhnull U+ve cells was smaller than in normal cells. In contrast, the N-glycan chain of the Rh glycoprotein was larger than normal in glycophorin B-deficient red cells. We suggest that this observation reflects a lower rate of movement of newly synthesized Rh glycoprotein through intracellular membranes to the cell surface in the absence of glycophorin B, and that in normal red cells glycophorin B facilitates the movement of the Rh protein complex to the cell surface. Our results provide evidence for the intracellular interaction of at least three proteins, the Rh glycoprotein, Rh30 polypeptides, and glycophorin B during the biosynthesis and cell surface expression of the Rh complex. These observations are likely to be important for the successful design of expression systems for the blood group Rh antigens.

Absence of high-affinity band 4.1 binding sites from membranes of glycophorin C- and D-deficient (Leach phenotype) erythrocytes.

We investigated the role of glycophorins C and D in the association of band 4.1 with the erythrocyte membrane by measuring the binding of band 4.1 to erythrocyte inside-out vesicles stripped of endogenous band 4.1. Vesicles were prepared from either normal erythrocytes or erythrocytes completely lacking glycophorins C and D (Leach phenotype). Band 4.1 binding to vesicles from normal erythrocytes gave rise to a nonlinear Scatchard plot, indicative of two classes of binding sites: a low-capacity, high-affinity class of sites (about 10% of the total) and a high-capacity, low-affinity class of sites. Vesicles prepared from Leach erythrocytes had a binding capacity for band 4.1 that was, on average, 32% lower than that of vesicles from normal erythrocytes. This difference was caused by the complete absence of the high-affinity binding sites as well as by a decrease in the number of low-affinity binding sites. Reduction of membrane phosphatidylinositol 4,5-biphosphate (PIP2) content by adenosine triphosphate depletion or activation of phosphoinositidase C resulted in a decrease in band 4.1 binding capacity to a similar extent in both control and Leach vesicles. The principal effect of PIP2 depletion was a reduction in the number of low-affinity band 4.1 binding sites in control and Leach vesicles. The fact that PIP2 depletion induced a decrease in band 4.1 binding to Leach vesicles shows that glycophorin C or D is not required for the formation of PIP2-sensitive band 4.1 binding sites, and may not be involved in PIP2-sensitive band 4.1 binding sites even when they are present. Our studies give new insights into the involvement of glycophorins and of PIP2 in modulating cytoskeletal-membrane interactions.

Weak N activity of En(a-) human erythrocyte membranes.

The propositus's erythrocytes with phenotype En(a-), which was found for the first time in a Japanese family, reacted more weakly with anti-N serum than the ordinary phenotype N erythrocytes. The En(a-) erythrocytes lack the major membrane sialoglycoprotein (glycophorin A) as demonstrated by Bio-Gel 1.5m gel filtration from active sialoglycoproteins, which were isolated from En(a-) erythrocyte membranes by the method of lithium diiodosalicylate (LIS)-phenol extraction. It is suggested from observation via enzyme-linked immunosorbent assay (ELISA) that N activity is derived from the glycophorin B molecule on En(a-) erythrocyte membranes.

Molecular analysis of glycophorin C deficiency in human erythrocytes.

Human erythrocyte glycophorin C plays a functionally important role in maintaining erythrocyte shape and regulating membrane mechanical stability. We report here the characterization of the glycophorins C and D deficiency in erythrocytes of the Leach phenotype. Glycophorin C gene is encoded by 4 exons. Amplification of reticulocyte cDNA from Leach phenotype and normal individuals generated a 140-bp fragment when using primers spanning exons 1 and 2. However, no polymerase chain reaction (PCR) products were detected in the Leach phenotype using primers flanking either exons 1 and 3 or exons 1 and 4, suggesting that the 3' end of the mRNA was missing or altered. Exon 4 also appeared to be missing from Leach genomic DNA, based on both Southern hybridization and PCR. These results indicate that an absence of glycophorin C and glycophorin D in erythrocytes from these Leach phenotype individuals is a consequence of a deletion or marked alteration of exon 3 and exon 4 of their glycophorin C gene. Surprisingly, the mutant gene encodes an mRNA stable enough to be detected in circulating reticulocytes. Although this mRNA could encode an N-terminal fragment of glycophorin C, these protein isoform(s) would not be expressed in the membrane because they lack the transmembrane and cytoplasmic domains.

Serological and biochemical studies on En(a-) human erythrocytes in a Japanese family.

The propositus erythrocytes with phenotype En(a-), which was found in the first example of a Japanese family, reacted with anti-N serum weaker than the ordinary phenotype N erythrocytes. When the erythrocyte membranes of the propositus were subjected to SDS-PAGE, no glycophorin A was observed on the gel by PAS staining, whereas glycophorin B band was observed. The S and the s antigens of the propositus erythrocytes were appeared to be normal. These results suggested that N activity of the propositus erythrocytes may be derived from glycophorin B components on the erythrocyte membranes. The amounts of bound sialic acid of the erythrocyte membranes were significantly lower in the En(a-) erythrocytes than the ordinary OMN erythrocytes. Neither the OMN nor the En(a-) erythrocytes showed the agglutinability to Arachis hypogaea lectin. The number of lectin receptor sites on the En(a-) erythrocyte membranes was significantly lower than on the OMN erythrocyte membranes for Limulus polyphemus, Triticum vurgaris and Bauhinia purpurea lectins. These results provide further support for the contention that En(a-) cells lack the glycophorin A as major erythrocytes sialoglycoprotein on the membranes.

Molecular basis for elliptocytosis associated with glycophorin C and D deficiency in the Leach phenotype.

Glycophorin C (GPC) and glycophorin D (GPD) are highly glycosylated integral membrane proteins of human erythrocytes encoded by the same gene and associated with expression of Gerbich blood group system antigens. GPC/D deficiency (the Leach phenotype) is a rare condition usually found after identification of Gerbich blood group system antibodies in persons undergoing prenatal or pretransfusion evaluation. In all cases, the Leach phenotype has been associated with elliptocytosis. Characterization of the molecular basis of this phenotype in three previously uninvestigated families has shown that the most common genetic basis of GPC/D deficiency is deletion of exons 3 and 4 of the GPC gene. However, in one family, the Leach phenotype appeared due to a deletion of one nucleotide in exon 3, causing a frameshift mutation in the messenger RNA and premature generation of a stop codon. The GPC and GPD protein sequences are therefore interrupted in the extracellular domain and probably intracellularly degraded.

Erythrocyte glycophorin B deficiency may occur by two distinct gene alterations.

The genomic DNA from rare persons whose erythrocytes are deficient in glycophorin B (GPB) (S-s-U- phenotype), was examined by Southern hybridizations using glycophorin B probes and was subdivided into two main categories. In the type I variant (Fav., M.H., S.K.), we found that the S-s-U- condition is generated by a large gene deletion extending from exons B2 to B4 of glycophorin B gene. Conversely, in the type II variant (Del.), the entire gene is present, and its promoter is almost similar to common Glycophorin A (GPA) and GPB as well as to type I promoters, except for four-point mutations, which do not occur in potential cis-acting elements. We concluded that the same phenotypic glycophorin B deficiency may occur by different gene alterations, including either a gene deletion or a mutation that might alter transcription or translation of the gene.

Effects of deficiencies of glycophorins C and D on the physical properties of the red cell.

Red cells of the rare Leach phenotype lack the membrane glycophorins C and D, and a proportion of the red cells are elliptocytes. Judging from tests on suspensions of red cell ghosts sheared rotationally in an ektacytometer, it has previously been suggested that these membranes are relatively fragile and poorly deformable. We have carried out analyses of individual red cells to investigate possible factors which underlie the physical changes in these glycophorin-deficient cells. Micropipette analysis of the red cell membrane showed that the rigidity and viscosity were normal, both for elliptocytes and discocytes, for three donors deficient in glycophorins C and D. Red cell transit times through 5 microns pores, measured electronically for 2000 individual cells, showed no differences from controls. It was confirmed that the index of deformation obtained using an ektacytometer was reduced, but our results suggest that this arises from shape rather than membrane changes. The elliptocytes were found to have a lower volume and surface area than discocytes from the same donor (measured by micropipette aspiration of single red cells), and were rarely found in less dense red cell fractions. No reticulocytes were found to be elliptical. These data suggest that the elliptocytes are older red cells, and are formed from red cells which are initially released into the circulation with normal shape. Their elongated shape might arise from permanent distortion of the unstable membrane by shear forces in the circulation.

A novel gene member of the human glycophorin A and B gene family. Molecular cloning and expression.

A new gene closely related to the glycophorin A (GPA) and glycophorin B (GPB) genes has been identified in the normal human genome as well as in that of persons with known alterations of GPA and/or GPB expression. This gene, called glycophorin E (GPE), is transcribed into a 0.6-kb message which encodes a 78-amino-acid protein with a putative leader peptide of 19 residues. The first 26 amino acids of the mature protein are identical to those of M-type glycophorin A (GPA), but the C-terminal domain (residues 27-59) differs significantly from those of glycophorins A and B (GPA and GPB). The GPE gene consists of four exons distributed over 30 kb of DNA, and its nucleotide sequence is homologous to those of the GPA and GPB genes in the 5' region, up to exon 3. Because of branch and splice site mutations, the GPE gene contains a large intron sequence partially used as exons in GPA and GPB genes. Compared to its counterpart in the GPB gene, exon 3 of the GPE gene contains several point mutations, an insertion of 24 bp, and a stop codon which shortens the reading frame. Downstream from exon 3, the GPE and the GPB sequences are virtually identical and include the same Alu repeats. Thus, it is likely that the GPE and GPB genes have evolved by a similar mechanism. From the analysis of the GPA, GPB and GPE genes in glycophorin variants [En(a-), S-s-U- and Mk], it is proposed that the three genes are organized in tandem on chromosome 4. Deletion events within this region may remove one or two structural gene(s) and may generate new hybrid structures in which the promoter region of one gene is positioned upstream from the body of another gene of the same family. This model of gene organization provides a basis with which to explain the diversity of the glycophorin gene family.

Studies on human red-cell membrane glycophorin A and glycophorin B genes in glycophorin-deficient individuals.

1. Genomic DNA derived from individuals who lack glycophorin A (GPA), glycophorin B (GPB) or both of these proteins was subjected to Southern-blot analysis using GPA and GPB cDNA probes. 2. Bands on the Southern blots were assigned to the GPA gene, GPB gene or to a putative pseudogene. 3. Genomic DNA derived from an individual of the Mk phenotype was shown to have deletions in the GPA and GPB genes. The simplest model for the results obtained is that a single deletion spans the GPA and GPB genes in the individual studied.

Molecular analysis of glycophorin A and B gene structure and expression in homozygous Miltenberger class V (Mi. V) human erythrocytes.

In the Miltenberger class V (Mi. V) condition, red cells lack glycophorin A (GPA) and glycophorin B (GPB) but carry instead an unusual glycoprotein thought to be a hybrid molecule produced by the unequal crossing-over between the closely linked genes encoding for GPA and GPB. By Western blot analysis with rabbit anti-GPA antibodies specific for discrete domains of GPA, it was found that the Mi. V glycoprotein (donor F. M.) contains approximately 60 amino acid residues of GPA at its N-terminus. As a preliminary approach to the molecular analysis of this variant the restriction maps of the GPA and GPB genes were established by Southern blot analysis of genomic DNA and from genomic clones isolated from a human leukocyte library constructed in lambda EMBL4. The GPA and GPB genes cover about 30 kb of DNA and are organized into seven exons (A-1-A-7) and five exons (B-1-B-5), respectively. In addition to the normal genes, a third gene (named inv), closely resembling the GPA and GPB genes, was also identified. In the homozygous Mi. V individual the normal GPA and GPB genes were absent, but an unusual form of gene structure was detected by Southern blot analysis. The Mi. V glycoprotein gene was composed of exon B-1 of the GPB gene followed by exons A-2 and A-3 of the GPA gene and the exons B-3, B-4 and B-5 of the GPB gene. Exon B-1 can be distinguished from exon A-1 of GPA since it is located within a different restriction fragment, but both encode the same amino acid sequence (N-terminal region of the signal peptides). Using the polymerase chain reaction, the junction between exon A-3 and exon B-3 was confirmed by amplification of the DNA region where the putative crossing-over has occurred and it was deduced that the Mi. V glycoprotein is a hybrid molecule composed of amino acid residues 1-58 from GPA fused to amino acid residues 27-72 of GPB. In addition, the finding that part of the signal peptide and the 5'-untranslated region are derived from GPB suggests that the genetic background of the Mi. V variant is rather complex and may involve a cascade of recombination or gene conversion events.

Alteration of the genes for glycophorin A and B in glycophorin-A-deficient individuals.

Glycophorins A and B are homologous glycoproteins of the red cell membrane which carry the blood-group MN and Ss antigens, respectively, and are encoded by two distinct genes closely linked on chromosome 4, which are probably derived from each other by duplication during evolution. The lack of glycophorin A is associated with the rare phenotype En(a-), indicating individuals who are defective for MN antigens, as well as for the Ena antigens, also located on this glycoprotein. The En(a-) condition is heterogenous and includes two categories of variants exemplified by the Finnish and the English types referred to as En(Fin) and En(UK), respectively. By Southern blot and preliminary genomic clone analyzes we have compared the status of the genes for glycophorins A and B, as well as that of the gene encoding glycophorin C, another unrelated red cell membrane glycoprotein, in the En(a-) variants and in the En(a+) control donors. Our data indicate that the En(Fin) variant is homozygous for a complete deletion of the glycophorin A gene without any detectable abnormality of the genes encoding glycophorins B or C. In the genome of the En(UK) variant, with the presumed genotype Mk/En(UK), and where the Mk condition abolishes the expression of MN and Ss antigens, we have identified several abnormalities of the glycophorin A and B genes, but the glycophorin C gene was unaffected. Our results strongly support the view that in Mk chromosome the glycophorin A and B genes are largely deleted, whereas the En(UK) chromosome probably contains a gene fusion product encoding a hybrid glycoprotein AM-B, composed of the N-terminal portion of a blood group M-type glycophorin A and of the C-terminal portion of glycophorin B. The determination of the 5' and 3' limits of the hybrid gene and elucidation of the mechanism involved will require sequencing of the rearranged DNA of the variant and a full knowledge of the organization of the glycophorin A and B genes.