Partial Purification of ABO Blood Group Antigens: Sodium Deoxycholate Fractionation of Human Erythrocyte Membranes

Abstract. A method has been developed for the fractionation of human erythrocyte membranes, using sodium deoxycholate, EDTA, differential ultracentrifugation, and DEAE-cellulose column chromatography. Serologic studies demonstrate the presence of ABO blood group antigens in a 50,000 g supernatant. This fraction is further purified on DEAE-cellulose, and in type AB ghost preparations results in the separation of A and B antigens. Phytohemagglutinin binding activity is also in the same supernatant and after DEAE chromatography is located in the fraction that exhibited A activity. Rh and MN activity is associated with a larger membrane fragment which is pelleted at 30,000 g . The S, s, and I antigens are present in a soluble form, and remain in the supernatant following centrifugation at 100,000 g .     

Molecular biology of the Rh antigens

The RBC Rh antigens are of large clinical importance, but until recently have been poorly understood at a molecular level. The Rh polypeptides are a family of nonglycosylated Mr 30- to 32-Kd transmembrane proteins that are core structural components of the Rh antigens and have been purified and partially characterized biochemically. Rh polypeptides are present in RBCs from normal humans and other mammalian species and are probably required for normal membrane integrity, because they appear to be missing from the RBCs of the rare Rhnull individuals that express several membrane defects. The Rh polypeptides contain an exofacial free sulfhydryl that is important for Rh antigenic reactivity and several intracellular sulfhydryls that appear to be palmitylated, but most of the molecule appears to reside between the leaflets of the phospholipid bilayer. The cDNA coding for a 416-amino acid Rh polypeptide was recently isolated but was not found to share sequence homology with any known protein, and Northern analysis indicated that Rh is erythroid specific. The Rh antigens within the native membranes are thought to exist as a complex of Rh polypeptides and multiple other membrane components, including certain Rh-related glycoproteins. While it is thought that this assembly may be important for the Rh antigenic reactivity, the structural basis of this remains to be established. While the physiologic role of Rh is yet to be defined, several clues indicate that it may play a role in the organization of membrane phospholipids or synthesis or membrane expression of various glycoproteins. While our knowledge of Rh is still very incomplete, recent research has significantly advanced the molecular understanding of these important blood group antigens.     

Immunochemical characterization of rhesus proteins with antibodies raised against synthetic peptides

Rabbit polyclonal antibodies were raised against synthetic peptides corresponding to hydrophilic regions of the human Rhesus (Rh) IX cDNA- encoded polypeptide predicted to be extracellularly or intracellularly exposed in the topologic model of the Rh blood group protein. Four antibodies encompassing residues 33-45 (MPC1), 224-233 (MPC4), 390-404 (MPC6), and 408-416 (MPC8) were characterized and compared with a polyclonal anti-Rh protein obtained by immunization with purified Rh proteins. All antibodies had specificity for authentic Rh polypeptides and reacted on Western blot with Rh proteins immunoprecipitated with human monoclonal anti-RhD, -c, and -E. MPC1, but not the other antibodies, agglutinated all human erythrocytes except Rhnull and Rhmod cells, which either lack totally or are severely deficient in Rh proteins, respectively. Immunoblotting analysis with membrane proteins from common and rare variants showed that MPC1 and MPC8 reacted in Western blot with 32-Kd Rh polypeptides from all common red blood cells except those from Rhnull and Rhmod, indicating that peptide regions 33- 45 and 408-416 may be common to several if not all Rh proteins, whatever the Rh blood group specificity. MPC4 reacted only with membrane preparations from cells carrying the E antigen, whereas MPC6 recognized preferentially the Rh proteins from E and Ee preparations, suggesting that the protein encoded by the RhIXb cDNA carries the E and/or e antigen(s). Immunoadsorption experiments using inside-out or right-side-out sealed vesicules from DccEE red blood cells as competing antigen showed that the MPC6 and MPC8 antibodies bound only to the cytoplasmic side of the erythrocyte membrane, thus providing evidence for the intracellular orientation of the C-terminal 27 residues of the Rh polypeptides. Attempts to transiently or stably express the Rh polypeptides. Attempts to transiently or stably express the Rh cDNA in eukaryotic cells were largely unsuccessful, suggesting that Rh antigen expression at the cell surface requires correct transport and/or folding of the Rh proteins, possibly as a complex with one-membrane proteins of the Rh cluster that are lacking in Rhnull cells.     

Molecular biology and genetics of the Rh blood group system

The Rh (Rhesus) blood group system is the most complex of the known human blood group polymorphisms. The expression of its antigens is controlled by a two-component genetic system consisting of RH and RHAG loci, which encode Rh30 polypeptides and Rh50 glycoprotein, respectively. Over the past decade, there has been a rapid advance in knowledge of the biochemistry, molecular biology, and genetics of the Rh genes and proteins. The primary structures of D and CcEe antigens have become well understood and the molecular genetic basis of a vast array of phenotype polymorphisms has been delineated. The identification of various molecular defects associated with Rh deficiency syndrome clarifies the nature of the amorph, suppressor, and modifier genes. The observed mutation spectrum defines a basic set of components essential for Rh complex assembly in the erythrocyte membrane. The resulting molecular information, combined with new experimental tools, is helping to dissect the fine structure of Rh antigens in terms of epitope mapping. The discovery of novel Rh homologs in primitive organisms and in nonerythroid tissues opens new avenues of research beyond the scope of erythrocytes and Rh antigens. This review provides an update on the Rh family in antigen expression, phenotype diversity, and disease association.     

Erythrocyte Rh antigens increase with red cell age

Certain blood group antigens may play a role in the removal of senescent red cells from the circulation. In order to elucidate this hypothesis, we have investigated the effect of red cell aging on Rh blood group antigens. Red cells from volunteer donors were separated into subpopulations of uniformly defined densities on discontinuous Stractan density gradients. The number of the five major Rh antigen sites of each fraction was determined both by enzyme-linked immunosorbent assay (ELISA) and by hemagglutination-scoring technique. All Rh antigens tested increased with cell age (p less than 0.01). The data indicate that there is a positive correlation between red cell age and the Rh blood group antigen site number.     

Membrane orientation of Rh(D) polypeptide and partial localization of its epitope-containing domain.

We have previously shown that the effects of various enzyme treatments on Rh antigen-containing polypeptides in situ could be monitored by an antibody preparation which recognizes only these polypeptides following Western blotting. We now have prepared antibodies that specifically react with either the N- or C-terminal ends of Rh-related proteins. Using all three, we have established that the C-terminus of Rh(D) polypeptide is at the cell surface, whereas its N-terminal domain is situated at the cytoplasmic side of the red blood cell membrane. Chymotrypsin digestion of ghosts derived from (-D-/-D-) cells that are devoid of Rh (C/c) and (E/e) antigens produces three major Rh(D)-related fragments: the 20-Kd fragment contains the molecule's C-terminal domain, the 17-Kd fragment its N-terminus, and the 13-Kd fragment neither. However, only the 17-Kd fragment forms an immune-complex with human polyclonal anti-D, indicating that it contains the Rh(D) antigenic domain. Other findings presented here provide further evidence for a unique folding of Rh(D) polypeptide within the cell membrane and suggest that Rh(C/c) and (E/e) polypeptides, when present, may form complexes with it.     

Identification of autoantigens in autoimmune haemolytic anaemia by a non-radioisotope immunoprecipitation method

In human autoimmune anaemia (AIHA), warm autoantibodies frequently appear to have serological specificity for the Rh complex, but to date, immunochemical techniques have failed to demonstrate that the antibodies react with Rh-associated polypeptides. We describe the immunoprecipitation of red blood cell (RBC) autoantigens, using biotin labelling and a luminescent detection method. In three cases of warm AIHA, a band of 32 kD and a diffuse zone of 38-51 kD or 40-51 kD were specifically precipitated by eluted RBC autoantibody. This pattern corresponds closely with that precipitated by two Rh-specific monoclonal antibodies, BRIC 207 and AB5. Antibody from the three eluates also showed serological specificity for the Rh complex in a haemagglutination assay against a panel of RBC with a range of Rh phenotypes, including rare -D-/-D- and Rh null cells. Eluted autoantibody from another warm AIHA patient immunoprecipitated a peptide of 67 kD that did not correspond in apparent molecular mass either with Rh-associated bands, or with major RBC membrane proteins or sialoglycoproteins (SGP). The haemagglutination assay showed that this eluate contained both Rh-specific and Rh-unrelated antibody. Warm autoantibody eluted from the RBC of a clinically normal, but direct antiglobulin test positive, blood donor was serologically unreactive with the Rh complex, and immunoprecipitated unknown peptides of 26, 29, 35, 48 and 51 kD, together with a band of 90 kD that comigrated with SGP alpha 2 (glycophorin A). In six further warm AIHA cases, no antigens were precipitated by autoantibody-containing RBC eluates. Overall, the results demonstrate that autoantibodies bind to Rh polypeptides in some, but not all, patients with warm AIHA, suggesting that the aetiology of the disease may vary.     

Genetic basis of the RhD-positive and RhD-negative blood group polymorphism as determined by Southern analysis.

Several lines of evidence have previously indicated that the RhD, c, and E blood group antigens are most likely carried by three distinct but homologous red blood cell membrane proteins. To determine whether these polypeptides are encoded by one or several related genes, we have performed Southern blot analysis of genomic DNA prepared from donors of different Rh phenotypes. Using an entire Rh cDNA probe and several exon-specific probes covering the cloned gene from its 5' to 3' ends, we have shown that the Rh locus carried by the genome of RhD-positive individuals is composed of two different but strongly related genes of identical general organization whether they expressed the C or c and E or e antigens, and, surprisingly, even when they do not express these epitopes, as in the D-- phenotype. The only antigenic variation found to be associated with a consistent genomic polymorphism corresponded to the RhD-positive/RhD-negative phenotypes. Indeed, one of the two Rh genes was completely lacking when the genomes of several unrelated RhD-negative donors were analyzed. From the present study we conclude that one of the two genes of the Rh locus encodes the RhC/c and RhE/e polypeptides while the other encodes the RhD protein. The absence of any D gene and of its postulated allelic form d in the RhD-negative genome explains finally why no Rhd antigen has ever been shown.     

Evidence That the Human Blood Group Antigens Gya and Hy Are Carried on a Novel Glycosylphosphatidylinositol-Linked Erythrocyte Membrane Glycoprotein

Immunoblotting under non-reducing conditions with purified human anti-Gya and anti-Hy locates both antigens to an erythrocyte membrane glycoprotein of apparent Mr 46,750-57,500. The antigens are destroyed on intact red cells by the enzymes pronase, trypsin and chymotrypsin, and by treatment with reducing agents. Immunoblotting with anti-Gya and anti-Hy to membranes prepared from red cells pre-treated with an Endo F preparation caused a mean reduction in apparent Mr of the glycoprotein by 11 kDa at the leading and trailing edges, when compared with control membranes. These results suggest that the glycoprotein has one or more complex N-glycans that are not completely sensitive to Endo F digestion on intact cells. The majority of Gya/Hy-active molecules are not tightly associated with the red cell membrane skeleton. A gross reduction in reactivity with anti-Gya and anti-Hy by immunoblotting was observed in red cell membranes from patients with paroxysmal nocturnal haemoglobinuria, suggesting a possible membrane linkage via glycosylphosphatidylinositol for the glycoprotein that carries the Gya and Hy antigens. Immunoprecipitation of the glycoprotein by anti-Gya showed that the protein migrates faster under reducing conditions (Mr 45,000-54,000). A putative dimer was also evident in the precipitates. The glycoprotein was demonstrated to be distinct from lymphocyte-function-associated antigen-3 (CD58), the LWab-active glycoprotein, the Fya-active glycoprotein, the Oka-active glycoprotein and the BRIC 125 glycoprotein (CD47).     

Inhibin: studies of stored and secreted forms by biosynthetic labeling and immunodetection in cultured rat granulosa cells

The biosynthesis of inhibin in rat granulosa cells was studied by biosynthetic labeling, immunoblotting, and immunocytochemical techniques. Granulosa cells from immature hypophysectomized estrogen-treated rats were cultured in the presence of [35S]cysteine. Both conditioned media and cell extracts were subjected to immunoprecipitation with an antibody directed against the N-terminal 26 amino acids of the alpha-chain of porcine inhibin (pI alpha 1-26), followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Treatment with FSH (100 ng/ml) and delta 4-androstenedione (10(-7) M) increased the secretion of 35S-labeled inhibin immunoreactivity by 2.6-fold over that in control cultures treated with androstenedione alone. The radiolabeled inhibin had mol wt (Mr) values of 45,000 and 30,000. Upon reduction, the 45,000 Mr polypeptide remained (with increased apparent Mr of 49,000), but the 30,000 Mr species disappeared with the concomitant appearance of two bands with 18,000 and 11,000 Mr. Competition studies with pI alpha 1-26 confirmed that these polypeptides were all related to inhibin. Furthermore, immunoblotting with an antibody directed against the porcine inhibin beta-A chain (pI beta A81-113) indicated that the 11,000 Mr peptide was the inhibin beta-A chain. Extracts of cells treated with FSH contained only a high Mr alpha-related species (Mr, 41,000 nonreduced; 49,200 reduced). The inhibin alpha antibody was also used to immunocytochemically stain cultured granulosa cells. Cells that had been treated with FSH or the adenyl cyclase activator forskolin (3 x 10(-5) M), but not untreated cells, exhibited positive staining. These results indicate that granulosa cells synthesize and store inhibin alpha-chain precursor with 49,000 Mr. Although some of the high Mr alpha-form was secreted, the majority of the alpha-subunit was processed to the 18,000 Mr form and dimerized with the 11,000 Mr beta-chain to form the mature inhibin dimer immediately before secretion. The cultured granulosa cells may provide a model for future studies on the hormonal regulation of inhibin alpha- and beta-gene expression as well as subunit dimerization and secretion.     

Blood group change in a patient with blastic transformation of a myelodysplastic syndrome

Summary Loss of certain red blood cell antigens has been described in patients with acute myelocytic leukemia (AML). This paper describes the loss of blood group A antigen in a patient with AML. The acute leukemia in this patient was preceded by a myelodysplastic syndrome for several months. At the time of diagnosis the patient's red cells showed the 0 Rh(D)⁺ phenotype. After induction of complete remission with two courses of Daunorubicin, Cytosin-arabinosid, and Etoposid his blood group reverted to A₂. Serological studies including saliva analysis revealed that the original blood group was very likely A.     

Patterns of Autologous Blood Use in Elective Orthopedic Surgery: Does the Availability of Autologous Blood Change Transfusion Behavior?

We studied the orthopedic surgery service at our institution to determine whether the mere availability of autologous blood (AB) affected transfusion practice. As a group, patients who had AB available received an average of 1.11 fewer red cell units per hospitalization than did patients with only homologous blood (HB) available. At every transfusion episode, those patients having AB available received fewer red cell units than did patients without AB available. Predeposit of autologous red cells was effective in protecting 77.6% of patients from HB exposure. The availability of autologous red cells resulted in an overall more conservative approach to transfusion.     

A Schistosoma mansoni surface glycoprotein cross-reactive with a T1 antigen of Fasciola hepatica

An antigen recognized by monoclonal antibody 306B3/5 and present on the surface of S. mansoni was expressed by F. hepatica in the pattern typical of T1 antigens. A single polypeptide of molecular weight (Mr) 160,000 was precipitated from metabolically labeled concanavalin A-binding F. hepatica glycoproteins, whereas multiple polypeptides ranging from Mr greater than 200,000 to Mr 45,000 were precipitated from metabolically labeled glycoproteins of male S. mansoni. The polypeptides of both species were also precipitated by sera of infected hosts, and may account for some of the serological cross-reactivity between S. mansoni and F. hepatica in immunodiagnostic assays. These antigens may also represent potentially immunoprophylactic reagents.     

Autoantibodies of U Blood Group Specificity in Autoimmune Haemolytic Anaemia

Autoantibody specificity in acquired haemolytic anaemia of the warm antibody type has been thought to be directed against Rh antigens or the precursors from which they arise. In many cases Rh_null red cells fail to react with these antibodies, providing apparent support for the assumption that they have Rh specificity. Rh_null red cells are also non-reactive with anti-U sera, and in addition have abnormal S and s antigens. We have studied 50 cases of warm antibody autoimmune haemolytic anaemia looking particularly for possible U, S or s blood group specificity of the causative antibodies. In the majority of cases the antibodies were found to be directed against antigens of the Rh complex, but in three cases multi-specific autoantibodies were shown to have complex specificity involving the Rh and U blood groups.     

Functional aspects of red cell antigens

Over 250 blood group determinants are known and most of these are located on integral red cell proteins and glycoproteins. The functions of some of these structures are known: Diego (band 3) is the red cell anion exchanger; Kidd, a urea transporter; Colton (aquaporin 1), a water channel; Cromer (DAF) and Knops (CRI), complement regulators; Diego (band 3) and Gerbich (glycophorin C/D) link the red cell membrane and the membrane skeleton. The Duffy glycoprotein is a chemokine receptor that may act as a scavenger for inflammatory mediators in the peripheral blood, but is also exploited as a receptor by Plasmodium vivax merozoites. The functions of some blood group antigens can be speculated upon because of structural similarity to proteins and glycoproteins of known function. For example, the Lutheran, LW, and Ok glycoproteins are members of the immunoglobulin superfamily of receptors and signal transducers, the Rh proteins and related glycoproteins show homology to ammonium transporters, and the Kell glycoprotein resembles a family of endopeptidases. Yet most blood groups systems contain null phenotypes associated with no apparent pathology. If these blood group antigens have important functions, other structures must be able to carry out those functions in their absence. Almost nothing is known of the biological significance of blood group polymorphism.     

Determination of the N-terminal sequence of human red cell Rh(D) polypeptide and demonstration that the Rh(D), (c), and (E) antigens are carried by distinct polypeptide chains

Human monoclonal antibodies (MoAbs) directed against the blood group Rh(D), (c), and (E) antigens produced by Epstein-Barr virus (EBV)- transformed lymphoblastoid cell lines have been used to characterize the Rh components of human red cell membranes and to determine whether the Rh(D), (c), and (E) epitopes are carried by distinct polypeptides. After immunoprecipitation with the anti-Rh(D) antibody and preparative gel electrophoresis, a homogenous preparation of the Rh(D) protein was obtained from two different erythrocyte samples (Blo and D--/D--), which have an increased density of Rh(D) antigen. Both preparations exhibited the same N-terminal sequence (S)-(S)-K-Y-P-R-S-V-R-R-(L)-L-P- L-X-A, indicating that different Rh(D)-positive red cells are carrying a similar Rh(D) protein. Comparative immunoprecipitation studies with the human monoclonal anti-Rh(D), (c), and (E) antibodies have also shown that Rh components from intact and papain-treated erythrocytes have similar apparent mol wt of 30 to 32 Kd and are buried in the lipid bilayer and are not readily available to the proteolytic enzyme. Further investigations by indirect affinity chromatography and one- dimensional peptide mapping of the Rh(D), (c), and (E) molecules immunopurified from a single red cell sample demonstrate that a common Rh haplotype encodes three distinct polypeptide chains carrying the Rh(D), (c), and (E) epitopes, respectively.     

Expression of Red Cell Antigens C and c on Neonatal and Adult Erythrocytes: A Flow-Cytometric Analysis

Red cell membrane proteins expressing Rh antigens appear to be important for the stabilization of the phospholipid membrane bilayer. Neonatal red cells possess a shorter in vivo life span and different deformability properties as compared to adult red blood cells, differences which could potentially be explained by different cell surface antigen densities of Rh-antigenic determinants. Although conventional serologic techniques have demonstrated Rh antigens on fetal and neonatal red cells, other more sensitive techniques are required when quantitative information is needed. Therefore, in this study, 40 adult and 45 neonatal red blood cell suspensions of differing Rh phenotypes were evaluated for C and c antigen density using flow cytometry. Neonatal and adult red blood cells had the same mean channel fluorescence intensity (expressed in mV) of c antigen in either the homozygous (cc) or heterozygous (cC) state: 485 +/- 44 versus 471 +/- 29 and 416 +/- 31 versus 411 +/- 23, respectively. In addition, neonatal and adult C antigen density was similar in heterozygous (Cc; 415 +/- 49 versus 455 +/- 44) and homozygous (CC; 438 +/- 53 versus 469 +/- 38 C states. As expected, homozygous (double dose) states for either C or c had greater intensities than heterozygous states for both neonates and adults. Although no differences were noted for C or c antigens, studies of other Rh antigens may indicate lower antigenic density in neonatal red cells.     

The 1.3-A resolution structure of Nitrosomonas europaea Rh50 and mechanistic implications for NH3 transport by Rhesus family proteins

The Rhesus (Rh) proteins are a family of integral membrane proteins found throughout the animal kingdom that also occur in a number of lower eukaryotes. The significance of Rh proteins derives from their presence in the human red blood cell membrane, where they constitute the second most important group of antigens used in transfusion medicine after the ABO group. Rh proteins are related to the ammonium transport (Amt) protein family and there is considerable evidence that, like Amt proteins, they function as ammonia channels. We have now solved the structure of a rare bacterial homologue (from Nitrosomonas europaea) of human Rh50 proteins at a resolution of 1.3 A. The protein is a trimer, and analysis of its subunit interface strongly argues that all Rh proteins are likely to be homotrimers and that the human erythrocyte proteins RhAG and RhCE/D are unlikely to form heterooligomers as previously proposed. When compared with structures of bacterial Amt proteins, NeRh50 shows several distinctive features of the substrate conduction pathway that support the concept that Rh proteins have much lower ammonium affinities than Amt proteins and might potentially function bidirectionally.