Expression of the Rh/RhAG complex is reduced in Mi.III erythrocytes

Background and Objectives Miltenberger blood group antigen subtype III (Mi.III) is characterized by expression of a glycophorin B‐A‐B hybrid (Gp.Mur) on the erythrocyte surface. The two alleles of glycophorin B are substituted with the B–A–B hybrid alleles in homozygous Mi.III (Mi.III^+/+), and thus, Mi.III^+/+ erythrocytes lack glycophorin B (GPB) and express Gp.Mur only. Because GPB is a major component of the Rh complex on RBCs, in this study, we explored how the absence of GPB might affect Rh expression in Mi.III RBCs. Materials and Methods (1) Mi.III+ RBCs were serologically identified and further differentiated their homozygosity or heterozygosity by immunoblot or direct sequencing. (2) RhD and RhCcEe mRNA was cloned, and their sequences analysed. (3) The expression levels of Rh antigen, Rh‐associated glycoprotein (RhAG) and the U antigen in MI.III vs. non‐Mi.III RBCs were assessed by flow cytometry and Western blot. Results Compared with the non‐Mi.III samples, the surface expression of the Rh antigen was reduced to 76·4% in Mi.III^+/+ RBCs and 93·6% in Mi.III^+/?. RhAG expression was also significantly reduced in Mi.III^+/+, but not in Mi.III^+/?. The U antigen expression in Mi.III^+/? was only 14·9% relative to the control RBCs, while GPB was half the level of the controls. The mRNA sequences of Rh polypeptides from Mi.III+ samples were identical to the NCBI reference sequences. Conclusion Substitution of GPB with Gp.Mur significantly reduced the expression of Rh antigen and RhAG on the Mi.III^+/+ erythrocyte membrane. The Mi.III phenotype is predicted to induce considerable structural variations within the band 3/Rh‐associated macrocomplexes.     

Severe Hemolytic Disease of the Newborn due to Anti-Vw and Detection of Glycophorin A Antigens on the Miltenberger I Sialoglycoprotein by Western Blotting

Anti-Vw detecting an antigen on Miltenberger I (Mi I) variant glycophorin A (GPA) has rarely been reported as a cause of hemolytic disease of the newborn (HDN). We report an infant with severe HDN due to anti-Vw. Examination of the Vw+ erythrocytes of the father and paternal grandmother by sodium dodecylsulphate polyacrylamide gel electrophoresis showed an extra trypsin-sensitive, periodic-acid-Schiff staining band, consistent with Mi I variant GPA. Staining of Western blots by monoclonal antibodies showed that normal paternal GPA expressed blood group M, while Mi I variant GPA expressed blood group N. Mi I variant GPA expressed the trypsin-sensitive antigenic determinant detected by MoAb 10F7, indicating that the alterations known to occur in the trypsin-sensitive fragment of Mi I variant GPA do not affect expression of the antigen detected by 10F7.     

Miltenberger Class IX of the MNS Blood Group System

Mi.IX is a new phenotype in the Miltenberger series of the MNS blood group system with a frequency of 0.43% in Denmark. Mi.IX red cells are Mur+ but do not express any of the other established Miltenberger determinants. They react with a new antibody, anti-DANE, which defines a determinant present on Mi.IX cells but not on cells of other Miltenberger phenotypes. Four Mi.IX propositi have been found. Their families show that MiIX is inherited with a MS complex (lod score 3.69 at theta = 0.00) which produces a trypsin-resistant M antigen. DANE has been allotted the ISBT number 002032 (MNS32). Serological and immunochemical studies with human and monoclonal antibodies to various determinants on glycophorin A (GPA) suggest that Mi.IX is associated with an aberrant GPA molecule that lacks the trypsin cleavage site at amino-acid residue 39, retains the chymotrypsin cleavage site at residue 34 and has an apparent Mr of about 1,000 less than normal GPA. It is proposed that this Mi.IX molecule has an amino acid and possibly also a glycosylation change in the region of amino-acid residues 35-39.     

Direct evidence for the existence of Miltenbergera antigen

The Miltenberger (Mi) subsystem, which originally consisted of four phenotypes, now has 11 phenotypes. The antigens of this subsystem belong to the MNS blood group system. The Mia antigen has been reported to be present on red blood cells with several Miltenberger phenotypes, namely: Mi.I, Mi.II, Mi.III, Mi.IV, Mi.VI and Mi.X. However, the existence of the Mia antigen as a separate entity has been in question and difficult to prove with polyclonal reagents. We report the first monoclonal anti-Mia (GAMA210), whose epitope is TNDKHKRD or QTNDMHKR, and thereby confirm the existence of the Mia antigen.     

Changes in the blood group Wright antigens are associated with a mutation at amino acid 658 in human erythrocyte band 3: a site of interaction between band 3 and glycophorin A under certain conditions [see comments]

The Wright (Wr) blood group antigens, Wra and Wrb, have been suggested to be determined by alleles of the same gene. The Wrb antigen appears to involve both red blood cell (RBC) band 3 and glycophorin A (GPA). We have examined the cDNA sequences of the band 3 and GPA of one of the two known Wr(a+b-) individuals. We show that this individual is homozygous for the mutation Glu658-->Lys in band 3, but has normal GPA. Putative heterozygotes with Wr(a+b+) RBCs have both Glu and Lys at residue 658 of band 3, whereas the common Wr(a-b+) RBC phenotype only have band 3 with Glu658. The Wra and Wrb antigens are determined by the amino acid at residue 658 of band 3 and are antithetical. Examination of the amino acid sequence and Wrb antigen expression of GPA-related hybrid glycophorins suggests that Arg61 of GPA interacts with Glu658 of band 3 to form the Wrb antigen. We suggest that the interaction is stabilized by the presence of anti-Wrb antibodies and that this site of association between GPA and band 3 may be responsible for the previously reported ability of anti-GPA antibodies to decrease the deformability of RBCs.     

A British family possessing two variants of the MNSs blood group system, Mv and a new class within the Miltenberger complex

Abstract. A naturally-occurring anti-M^v was identified. The family of the M^v propositus was studied and shown to contain another variant of the MNSs blood group system. A weak N antigen was associated with a reaction with anti-Hil, part of the Miltenberger complex. A new class (V) was revealed which is Vw(-) Mi(a-) Mur(-) Hil(+).     

Red cell antigens on band 3 and glycophorin A

Band 3 and glycophorin A (GPA) are the two most abundant integral proteins of the red cell membrane, being present in approximately 10(6) copies per cell. The main functions of band 3 are membrane anion transport and maintenance of red cell membrane stability through interaction with the cytoskeleton. GPA plays an important role in prevention of red cell aggregation in the circulation and contribution to the glycocalyx. The extracellular domains of both proteins are highly polymorphic. Band 3 carries the antigens (currently 19) of the Diego blood group system and GPA and glycophorin B the antigens (currently 43) of the MNS system. There is substantial evidence that band 3 and GPA associate in the red cell membrane and the Wr(b) antigen, although a product of the band 3 gene, is known to require a complex of GPA and band 3 for normal expression. The discovery of a novel GPA mutation (Ala65-->Pro) giving rise to aberrant Wr(b) expression has been informative with regard to the site of interaction of the two proteins. The extensive array of GPA-related antigens is largely due to genetic events between two closely linked genes and different genetic mechanisms can give rise to the same antigen. This is in contrast to the antigens on band 3 which are exclusively due to single nucleotide mutations in the band 3 gene.     

Functional Cell Surface Expression of Band 3,the Human Red Blood Cell Anion Exchange Protein (AE1), in K562 Erythroleukemia Cells: Band 3Enhances the Cell Surface Reactivity of Rh Antigens

Human K562 erythroleukemia cells were transfected with human band 3 (anion exchanger 1 [AE1]) cDNA, using the pBaberetroviral vector. Stable K562 clones expressing band 3 were isolatedby flow cytometry, and surface expression was quantified byimmunoblotting. The function of band 3 expressed at the cell surfacewas demonstrated in chloride transport assays. K562 cells expressingband 3 also displayed high levels of the Wr^b blood groupantigen, confirming the role of band 3 in Wr^b expression,and an increase in the low levels of endogenous Rh antigen activity. Wealso performed coexpression experiments with K562 clones that hadpreviously been transduced with cDNAs encoding RhD or RhcEpolypeptides. The transfection and expression of band 3 in these clonessubstantially increased the levels of RhD and cE antigen activityexpressed on the cells and also increased the reactivity of the cellswith antibody to the endogenous Rh glycoprotein (RhGP, Rh50). Theincreased reactivity of Rh antigens may result from cell surface orintracellular interactions of band 3 with the protein complex whichcontains the Rh polypeptides and RhGP, or from indirect effects of band3 on the membrane environment. This work establishes a system for cellsurface expression of band 3 in a mammalian cell line, which willenable further studies of the protein and its interactions with othermembrane components.     

A novel variety of the Dantu gene complex (Dantu MD) detected in a Caucasian

Summary The first Caucasian (MD) shown to exhibit the low-frequency MNSs system antigen, Dantu was detected due to an increased tendency of erythrocytes to be aggregated by substances that promote red cell agglutination. The donor was found to exhibit a novel variety of the Dantu gene complex (Dantu ^MD), as judged from biochemical, immunochemical, and serological studies. The glycophorin (GP) A level of MD's erythrocyte membranes were slightly decreased (about 17%) but GP B was not significantly different from normal. GP A and GP B of MD's cells were shown to carry M and N or S and s antigens, respectively, indicating that MD exhibits two genes encoding GP A and two genes encoding GP B. MD's cells contain a Dantu-, N- and s-specific GP B-GP A hybrid GP (molar ratio to GP A approx. 0.6 : 1.0). Partial amino-acid sequence analysis indicates that the structure of this molecule is rather similar to, or completely identical with, that of the hybrid GP in Dantu^NE erythrocytes. The residues 1–39 or 40–99 of the latter molecule correspond to the residues 1–39 of s-specific GP B and the residues 72–131 of GP A, respectively. Statistical evidence suggests that MD exhibits a single gene encoding the hybrid GP. Thus, MD appears to be heterozygous for a typical anti-Lepore type gene complex that seems to comprise genes for GP A, GP B, and the GP B-GP A hybrid. The diminished GP A level and a decreased galactose-oxidase labelling of the major membrane protein (anion channel protein, band 3) in MD's cells is in accordance with previous data suggesting that band 3 might form a complex with GP A and the Dantu-specific hybrid GP. This complex formation may be necessary for optimum incorporation of the latter molecules into the membrane.This work was supported by grants from the Deutsche Forschungsgemeinschaft, the Fonds der chemischen Industrie, the Ministerium für Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the Institut National de la Sante et de la Recherche Medicale and through a Heisenberg-research fellowship by the Deutsche Forschungsgemeinschaft to W. Dahr Preliminary results of this work were presented at the 38th Annual Meeting of the American Association of Blood Banks [26]     

Erythroid band 3 variants and disease

This review describes some of the naturally occurring band 3 (AEI) variants and their association with disease. Southeast Asian Ovalocytic (SAO) band 3, an inactive and misfolded protein, is probably only maintained in certain populations because it provides protection against the cerebral form of malaria. Many mutations that cause instability of band 3, either at the mRNA or protein level, result in hereditary spherocytosis (HS). Some polymorphisms alter amino acid residues in the extracellular loops of band 3 and are associated with blood group antigens. A truncated form of AEI is expressed in kidney cells and certain AEI mutations are associated with distal renal tubular acidosis (dRTA). The molecular basis of these variants and their effect on the structure and function of band 3 are discussed. The association between band 3 and glycophorin A (GPA) and the structure/function changes of band 3 in the absence of GPA are also described.     

Wright (a+b−) human erythrocytes and plasmodium falciparum malaria

Summary We find Wr(a+b-) erythrocytes of donor M. Fr., which appear to carry a rare glycophorin A variant, to be fully susceptible to invasion by nine isolates of Plasmodium falciparum. Thus we fail to confirm the previous publication on the refractoriness of these erythrocytes. In addition the serum of donor M. Fr., which is known to contain anti-Wr^b directed against an epitope located on glycophorin A in close proximity to the erythrocyte membrane, was not found to inhibit P. falciparum invasion of blood group 0 Rh^– red blood cells.Despite this, different lines of evidence still indicate that glycophorin A is one of the receptors for erythrocyte invasion by P. falciparum. The Wr^b epitope, however, does not appear to represent a distinct receptor site, which is in contrast to previous suggestions.A preliminary communication of this work has been published recently [9]     

Human red blood cell Wright antigens: a genetic and evolutionary perspective on glycophorin A-band 3 interaction

The Wright (Wra/Wrb) blood group polymorphism is defined by an allelic change (Lys658Glu) in the band 3 protein; nevertheless, the Wrb antigen apparently requires glycophorin A (GPA) for surface presentation. To gain insight into the structural basis for this protein-protein interaction and delineate its relationship with Wrb antigen expression, we investigated GPA and band 3 sequence polymorphisms occurring in rare humans and nonhuman primates. The lack of GPA or amino acid residues 59 through 71 of GPA results in the absence of Wrb from human red blood cells (RBCs) exhibiting the MkMk, En(a-), or MiV phenotype. However, the SAT homozygous cells carried a Glu658 form of band 3 and a hybrid glycophorin with the entire GPA extramembrane domain from residues 1 through 71, yet expressed no Wrb antigen. This finding suggests that formation of the Wrb antigenic structure is dependent on protein folding and that the transmembrane junction of GPA is important in maintaining the required conformation. Comparative analyses of GPA and band 3 homologues led to the identification in the interacting regions of conserved and dispensable amino acid residues that correlated with the Wrb positive or negative status on nonhuman primates. In particular, the chimpanzee RBCs cells expressed Wrb and the Glu658 form of band 3, which is identical to humans, but their GPA contained the Gly rather than Arg residue at position 61. Taken together, the results suggest that (1) Arg61 of GPA and the proposed Arg61-Glu658 charge pair are not crucial for Wrb antigen exhibition and (2) the role of GPA for interaction with band 3, including Glu658, probably involves a number of amino acid residues located in the alpha-helical region and transmembrane junction.     

Degradation of glycophorin A of human erythrocytes in patients with myelo- or lymphoproliferative disorders: possible role of neutrophil proteases

We have previously reported that glycophorin A (GPA) of human erythrocytes (carrying blood group M and N determinants) was totally digested by incubation of erythrocytes with human neutrophil elastase (HNE) and cathepsin G (CathG). The membrane-bound GPA fragments fractionated by SDS-PAGE gave characteristic patterns of bands detected by immunoblotting with the monoclonal antibody PEP80. Erythrocytes were incubated with HNE and CathG at low enzyme concentrations, similar to those found in vivo . Characteristic electrophoretic patterns of bands derived from a partial GPA digestion were observed and these patterns were different for both enzymes and different from those obtained after total GPA digestion. GPA was also partially digested by incubation of erythrocytes with granulocytes in the presence of Ca²⁺ and calcium ionophore and electrophoretic pattern of digestion products was similar to that obtained with low doses of HNE. No GPA digestion products were detected after treatment of erythrocytes with plasmin and kallikrein. Untreated erythrocytes of 21 patients with various myelo- or lymphoproliferative disorders were tested by SDS-PAGE of RBC membranes and immunoblotting with the anti-GPA PEP80 antibody. GPA degradation products, resembling those formed by a mild CathG treatment of control RBC, were detected in nine patients. GPA fragmentation was in some cases accompanied by a reduced expression of blood group MN determinants. No distinct relation was observed between the occurrence of GPA degradation in erythrocytes and increases in plasma concentrations of HNE-α₁-proteinase inhibitor (α₁-PI) complex considered to be an indication of a release of neutrophil proteinases in vivo . However, the results suggested that a partial GPA degradation in haematological proliferative disorders may occur due to limited proteolysis by neutrophil proteinases, most likely by CathG.     

Glycophorin A dimerization and band 3 interaction during erythroid membrane biogenesis: in vivo studies in human glycophorin A transgenic mice

Band 3 and glycophorin A (GPA) are the 2 most abundant integral proteins in the human erythrocyte membrane. Earlier studies suggested that the 2 proteins may associate not only in the mature erythrocyte membrane, but also during their posttranslational processing and intracellular trafficking. The purpose of this study was to directly examine the GPA-band 3 interaction in vivo and determine the nature of this association during erythroid membrane biogenesis. Transgenic mice were generated expressing the human glycophorin A gene and were used to examine how the induction of human GPA expression affected the levels of murine GPA and band 3 expression in the red cell membrane. Murine GPA expression was reduced in erythrocytes expressing human GPA, whereas the level of band 3 expression remained constant, implying a tight coupling of band 3 and GPA expression in the membrane of mature red cells. In vivo GPA dimerization was not modulated solely by the GPA transmembrane motif, but the distance between this motif and the basic residues on the cytoplasmic side of the transmembrane domain may also be important. In addition, GPA monomers with varying degrees of glycosylation dimerized, providing clear evidence that carbohydrate structures on the extracellular domain do not affect dimerization. The association between the multiple transmembrane-spanning protein, band 3, and the single transmembrane-spanning sialoglycoprotein, GPA, may serve as a model for interactions of other multi-pass and single-pass polypeptides during membrane biogenesis.     

Synthetic peptides homologous to human glycophorins of the Miltenberger complex of variants of MNSs blood group system specify the epitopes for Hil, SJL, Hop, and Mur antisera.

The antigenic epitopes of the MNSs blood groups are localized on alpha and delta glycophorins (glycophorins A and B) of the erythrocyte surface. Hil, SJL, Mur, and Hop antisera define the Miltenberger (Mi) complex of MiV, MiJ.L., MiIII, and MiVI variant serologic phenotypes of this blood group system. We report here the location of the epitopes for antibodies in these antisera. The antigens of these Mi classes are variant glycophorins that are hybrids of alpha and delta glycophorins in alpha-delta and delta-alpha-delta arrangements. The hybrid junctions give rise to novel polypeptide sequences not present in the parent glycophorins; in MiIII and MiVI this also includes an expressed sequence of the delta pseudoexon. These sequences are identical in the above Mi-glycophorins occurring in erythrocytes that share a common Mi determinant. Four peptides of 10 to 14 amino acids each were constructed to be homologous to the identical sequences; they were designated, "Hil", "SJL", "Mur", and "Hop" to reflect the common determinant. The peptides were tested for inhibition of reaction of appropriate cells with the relevant antisera. The Hil peptide, outlining the alpha-delta s junction region in MiIII, MiV, and MiVI glycophorins, inhibited the reaction of respective erythrocytes (red blood cells [RBCs]) with anti-Hil. The SJL peptide, which differs from the Hil peptide by a single Thr----Met substitution, was specific for inhibition of the reaction of MiJ.L. RBCs with anti-SJL (an example of anti-S specific for such RBCs). The Hop peptide, which corresponds to the delta-alpha junction in MiVI glycophorin, inhibited the hemagglutination of MiVIII RBCs by anti-Hop. MiVI and MiVIII glycophorins share an identical sequence at that site. The Mur peptide, corresponding to a portion of the expressed pseudoexon sequence in MiIII and MiVI glycophorins, was specific for inhibition of the reaction of MiIII and MiVI RBCs with anti-Mur. The peptides had no effect on the hemagglutination of control MNSs RBCs by their respective antisera nor of unrelated Mi classes RBCs by antisera that distinguish these classes. We conclude that the alpha-delta junction in MiIII, MiV, and MiVI glycophorins outlines the epitopes for anti-Hil, the alpha-delta junction in MiJ.L. outlines the epitope for anti-SJL, the delta-alpha junction in MiVI constitutes the epitope for anti-Hop, and the expressed delta pseudoexon sequence in MiIII and MiVI constitutes the epitope for anti-Mur.     

Recombinant Miltenberger I and II human blood group antigens: the role of glycosylation in cell surface expression and antigenicity of glycophorin A

Glycophorin A is a heavily glycosylated glycoprotein (1 N-linked and 15 O-linked oligosaccharides) and is highly expressed on the surface of human red blood cells. It is important in transfusion medicine because it carries several clinically relevant human blood group antigens. To study further the role of glycosylation in surface expression of this protein, four mutations were separately introduced into glycophorin A cDNA by site-directed mutagenesis. Each of these mutations blocks N- linked glycosylation at Asn26 of this glycoprotein by affecting the Asn- X-Ser/Thr acceptor sequence. Two of these mutations are identical to the amino acid polymorphisms found at position 28 in the Mi.I and Mi.II Miltenberger blood group antigens. The mutated recombinant glycoproteins were expressed in transfected wild-type and glycosylation- deficient Chinese hamster ovary (CHO) cells. When expressed in wild- type CHO cells and analyzed on Western blots, each of the four mutants had a faster electrophoretic mobility than wild-type glycophorin A, corresponding to a difference of approximately 4 Kd. This change is consistent with the absence of the N-linked oligosaccharide at Asn26. Each of the four mutants was highly expressed on the surface of CHO cells, confirming that, in the presence of normal O-linked glycosylation, the N-linked oligosaccharide is not necessary for cell surface expression of this glycoprotein. To examine the role of O- linked glycosylation in this process, the Mi.I mutant cDNA was transfected into the IdlD glycosylation-deficient CHO cell line. When the transfected IdlD cells were cultured in the presence of N- acetylgalactosamine alone, only intermediate levels of cell surface expression were seen for Mi.I mutant glycophorin A containing truncated O-linked oligosaccharides. In contrast, when cultured in the presence of galactose alone, or in the absence of both galactose and N- acetylgalactosamine, Mi.I mutant glycophorin A lacking both N-linked and O-linked oligosaccharides was not expressed at the cell surface. This extends previous results (Remaley et al, J Biol Chem 266:24176, 1991) showing that, in the absence of O-linked glycosylation, some types of N-linked glycosylation can support cell surface expression of glycophorin A. The glycophorin A mutants were also used for serologic testing with defined human antisera. These studies showed that the recombinant Mi.I and Mi.II glycoproteins appropriately bound anti-Vw and anti-Hut, respectively. They also demonstrated that these antibodies recognized the amino acid polymorphisms encoded by Mi.I and Mi.II rather than cryptic peptide antigens uncovered by the lack of N- linked glycosylation.     

Molecular basis for the human erythrocyte glycophorin specifying the Miltenberger class I (MiI) phenotype.

Human glycophorin Mil (HGpMil) is a structural variant of the MNSs blood group system that specifies the Miltenberger class I phenotype. We report here the molecular basis of the HGpMil gene identified in a white family in which the first homozygote was encountered. Immunoblotting analysis showed the expression of HGpMil and HGpB but the absence of HGpA on the homozygous Mil erythrocytes. Southern blot analysis detected no gross alterations in gene structure or band intensity. Genomic sequences encompassing exons II and III of the HGpMil gene were amplified by single-copy polymerase chain reaction. Restriction digestion and direct DNA sequence analysis showed that HGpMil gene is derived from an alpha N allele of HGpA and differs from the latter in the third exon by a single nucleotide change. In HGpMil, the presence of a deoxythymidine at the second position of codon 28 (ATG) not only resulted in a methionine substitution but also altered the consensus sequence for N-glycosylation from Asn-Asp-Thr to Asn-Asp-Met. These data are consistent with the occurrence of Mil on the red blood cell membrane as a variant deficient in the asparagine-linked carbohydrate unit. Significantly, this particular point mutation lies in between the two half-sites of a direct repeat that has been implicated to facilitate the recombination events leading to several other glycophorin genes of the Miltenberger series. Based on this relatedness, we propose an untemplated nucleotide replacement resulting from a gene conversion event as the molecular basis for the origin of HGpMil gene.     

Glycophorin A-mediated haemolysis of normal human erythrocytes: evidence for antigen aggregation in the pathogenesis of immune haemolysis

The inexplicable severity of anti-Pr autoimmune haemolytic anaemia led us to test the hypothesis that the haemolysis was primarily due to a change in the function of glycophorin A, on which the Pr antigen is located. The lectins Maclura pomifera and wheat germ agglutinin that bind to glycophorin A induced the haemolysis of normal erythrocytes in vitro. Lectin binding led to an increase in erythrocyte membrane permeability to sodium and potassium, the former resulting in an influx of water and subsequent haemolysis. The response was glycophorin A specific as Concanavalin A, which binds to band 3, did not cause haemolysis and peanut agglutinin only did so after removal of erythrocyte sialic acid. The lectin-induced cation leak was not mediated by activation of cation channels as the inhibitors, tetrodotoxin, amiloride and 4,4' disothiocyanate stilbene 2,2'disulphonate, had no effect, suggesting that the haemolysis was due to exacerbation of the inherent cation permeability of the erythrocyte membrane. A human IgAK anti-Pr autoantibody and a mouse anti-human glycophorin A antibody increased erythrocyte permeability to sodium. The role of glycophorin A in stabilizing and, upon aggregation, destabilizing the phospholipid bilayer is discussed. Our findings may help explain the severity of anti-Pr autoimmune haemolytic anaemia and other pathophysiological changes in human erythrocytes.     

Relationship of the human erythrocyte Wrb antigen to an interaction between glycophorin A and band 3

The Wrb antigen is a high-frequency human erythrocyte antigen invariably absent from En (a-) erythrocytes, which lack glycophorin A. However, glycophorin A from En (a+) Wr (a+b-) red cells has an amino acid sequence identical to that of glycophorin A from Wr (b+) erythrocytes. Evidence has suggested that the Wrb antigen may require the interaction of glycophorin A with either a lipid moiety or with another erythrocyte-integral membrane protein, band 3. We have investigated the role of band 3 in Wrb expression using murine monoclonal antibodies (MoAbs) with Wrb specificity. These antibodies reacted by radioimmunoassay (RIA) only with cells expressing both glycophorin A and band 3. In immunoprecipitation studies, Wrb antibodies immunoprecipitated both band 3 and glycophorin A, while antibodies specific for band 3 or glycophorin precipitated only the protein with which they were reactive. These data strongly suggest that band 3 is the other membrane component necessary for expression of Wrb and that band 3 and glycophorin A are closely associated in the erythrocyte membrane.