Current topics in red cell biology: report on the Red Cell Special Interest Group meeting held at NHS Blood and Transplant Bristol on 30 October 2015.

The Red Cell Special Interest Group (SIG) meeting, hosted by the British Blood Transfusion Society, provides an annual forum for the presentation of UK- and European-based red cell research. The 2015 meeting was held on Friday 30 October at the National Health Service Blood & Transplant (NHSBT) facility in Filton, Bristol and provided an exciting and varied programme on the themes of erythropoiesis, malaria biology and pathophysiology and red cells properties in stress and disease. Ten speakers presented on these topics over the course of one day. The meeting was well attended by over 90 delegates. Posters were presented during the lunch break, and abstracts from the posters are published at the end of this issue.

A new blood group system, RHAG: three antigens resulting from amino acid substitutions in the Rh-associated glycoprotein.

BACKGROUND AND OBJECTIVES: Rh-associated glycoprotein (RhAG) is closely associated with the Rh proteins in the red cell membrane. Two high frequency antigens (Duclos and DSLK) and one low frequency antigen (Ol(a)) have serological characteristics suggestive of expression on RhAG. MATERIALS AND METHODS: RHAG was sequenced from the DNA of one Duclos-negative, one DSLK-negative, and two Ol(a+) individuals. Recombinant protein was expressed in HEK 293 cells. Protein models with RhAG subunits were constructed. RESULTS: The original Duclos-negative patient was homozygous for RHAG 316C>G, encoding Gln106Glu. HEK 293 cells expressing Gln106Glu mutant RhAG did not react with anti-Duclos. An individual with DSLK-negative red cells was homozygous for 490A>C, encoding Lys164Gln. Two Ol(a+) members of the original Norwegian family were heterozygous for 680C>T, encoding Ser227Leu. A Japanese donor with Rh(mod) phenotype had Ol(a+) red cells and was homozygous for 680C>T. CONCLUSION: The three red cell antigens encoded by RHAG form the RHAG blood group system: Duclos is RHAG1 (030001); Ol(a) is RHAG2 (030002); and DSLK is provisionally RHAG3 (030003).

South-east asian ovalocytic (SAO) erythrocytes have a cold sensitive cation leak: implications for in vitro studies on stored SAO red cells.

South-east Asian ovalocytosis (SAO) results from the heterozygous presence of an abnormal band 3, which causes several alterations in the properties of the erythrocytes. Although earlier studies suggested that SAO erythrocytes are refractory to invasion in vitro by the malarial parasite Plasmodium falciparum, a more recent study showed that fresh SAO cells were invaded by the parasites, but became resistant to invasion on storage because intracellular ATP was depleted more rapidly than normal. Here we show that SAO red cells are much more leaky to sodium and potassium than normal red cells when stored in the cold. This leak was much less marked when the cells were stored at 25 or 37 degreesC. Incubation for 3.5 h at 37 degreesC of cold-stored SAO red cells did not restore sodium and potassium to normal levels, probably because the depleted ATP level in cold-stored SAO red cells is further reduced with incubation at 37 degreesC. The increased leakiness of SAO red cells is non-specific and extends to calcium ions, taurine, mannitol and sucrose. These results suggest that SAO red cells undergo a structural change on cooling. Since many of the reports describing altered properties of SAO red cells have used cells which have been stored in the cold, these results need re-evaluation using never-chilled SAO red cells to assess whether the cells have the same abnormal properties under in vivo conditions.

The Rhesus (D) polypeptide is linked to the human erythrocyte cytoskeleton.

Cytoskeleton preparations derived from lactoperoxidase-radioiodinated human erythrocytes were found to be enriched in a labelled component with the same apparent molecular mass as the Rhesus (D) (Rh(D] antigen polypeptide. Immune precipitation from the cytoskeleton preparations confirmed that this component is the Rh(D) polypeptide. The results suggest that the Rh(D) polypeptide may be linked to the erythrocyte skeletal matrix. The possibility that the Rh(D) antigen is involved in maintaining the shape and viability of the erythrocyte is discussed.

Effect of endoglycosidase F-peptidyl N-glycosidase F preparations on the surface components of the human erythrocyte.

Endo-N-acetyl-beta-D-glucosaminidase F-Peptidyl N-glycosidase F preparations (abbreviated Endo F) and endo-beta-D-galactosidase were used to study the major human erythrocyte membrane glycoproteins and the components carrying the blood group A, B, Rhesus (D), and Duffy (Fya) antigens. The results are consistent with the known presence of an N-glycosyl-linked oligosaccharide on sialoglycoprotein alpha and the absence of such an oligosaccharide from sialoglycoprotein delta. Under the conditions used, only a portion of the N-glycosyl-linked oligosaccharides on band 3 molecules were cleaved by Endo F alone or by Endo F in combination with endo-beta-D-galactosidase. Immunoblotting experiments showed that treatment of red cells with Endo F alone had little effect on the components carrying blood group A and B antigen activity. However, Endo F used in combination with endo-beta-D-galactosidase caused a substantial reduction in the binding of monoclonal anti-A and anti-B antibodies. The results clearly show that sialoglycoproteins alpha and delta carry little or no blood group A or B activity. Endo F alone, or in combination with endo-beta-D-galactosidase, had no effect on the electrophoretic mobility of the Rh(D) polypeptide, supporting previous suggestions that this membrane polypeptide is unusual in not being glycosylated. Endo F had a dramatic effect on the electrophoretic mobility of the component(s) carrying blood group Fya activity. The diffuse Fya component of Mr 38,500-90,000 was sharpened to a band of Mr 26,000. Either endo-beta-D-galactosidase or neuraminidase treatment reduced the Mr of the Fya component(s) but did not significantly sharpen the bands, suggesting that the Fya component contains between 40-50% by mass of N-glycosyl-linked oligosaccharides.

Production of soluble recombinant proteins with Kell, Duffy and Lutheran blood group antigen activity, and their use in screening human sera for Kell, Duffy and Lutheran antibodies.

The aim of this study was to show that soluble recombinant (sr) proteins can mimic blood group antigens and be used to screen human sera for blood-group-specific antibodies. The blood of all pregnant women and pretransfusion patients should be screened for blood-group-specific antibodies to identify and monitor pregnancies at risk of haemolytic disease of the foetus and newborn (HDFN), and to prevent haemolytic transfusion reactions. Current antibody screening and identification methods use human red blood cell panels, which can complicate antibody identification if more than one antibody specificity is present. COS-7 cells were transfected to produce sr forms of the extracellular domains of the red blood cell membrane proteins that express Kell, Duffy or Lutheran blood group antigens. These sr proteins were used to screen for and identify anti-Kell, anti-Duffy or anti-Lutheran blood-group-specific allo-antibodies in human sera by haemagglutination inhibition and in solid-phase enzyme-linked immunosorbent assays (ELISAs). There is a positive correlation (correlation coefficient 0.605, P value 0.002) between antibody titre by standard indirect antiglobulin test (IAT) and signal intensity in the ELISA test. This work shows that sr proteins can mimic blood group antigens and react with human allogeneic antibodies, and that such proteins could be used to develop solid-phase, high-throughput blood group antibody screening and identification platforms.

The Wrb antigen, a receptor for Plasmodium falciparum malaria, is located on a helical region of the major membrane sialoglycoprotein of human red blood cells.

1. Immunoprecipitation of periodate/NaB3H4-labelled human erythrocytes using anti-Wrightb (Wrb) monoclonal antibodies showed that these antibodies specifically react with the major erythrocyte sialoglycoprotein alpha (glycophorin A). 2. Similar experiments on erythrocytes from the only known individual lacking the Wrb antigen but with otherwise normal sialoglycoproteins did not result in the immunoprecipitation of any sialoglycoprotein. 3. We suggest that the Wrb antigen is located on an alpha-helical region between residues 55 and 70 of sialoglycoprotein alpha.

The Wrb antigen in Sta-positive and Dantu-positive human erythrocytes.

Immunoprecipitation using a monoclonal antibody showed that the Wrb antigen is present on the abnormal (delta-alpha) hybrid sialoglycoprotein of Sta-positive human erythrocytes but not on the abnormal (delta-alpha) hybrid sialoglycoprotein of Dantu-positive erythrocytes. These results provide further information regarding the nature and location of the Wrb antigen on the normal erythrocyte sialoglycoprotein alpha.

cDNA cloning of a 30 kDa erythrocyte membrane protein associated with Rh (Rhesus)-blood-group-antigen expression.

The Rh-blood-group antigens (often described as Rhesus antigens) are associated with erythrocyte membrane proteins of approx. 30 kDa. We have determined the N-terminal 54 amino acid residues of the 30 kDa Rh D polypeptide (D30 polypeptide). We used primers based on these sequence data and the polymerase chain reaction (PCR) on human reticulocyte cDNA and genomic DNA to clone two types of PCR product of identical size. The two PCR products had related translated amino acid sequences between the 3' ends of the primers, one of which was identical with that found for the D30 polypeptide. We designate the two related mRNA species which gave rise to the PCR products as Rh30A and Rh30B, the latter corresponding to the D30 polypeptide. We have isolated cDNA clones for the Rh30A protein which encode a hydrophobic membrane protein of 417 amino acids. The Rh30A protein has the same N-terminal 41 amino acids as the D30 polypeptide, but beyond this point the sequence differs, but is clearly related. The Rh30A protein probably corresponds to the R6A32 polypeptide, another member of the Rh 30 kDa family of proteins, which may carry the C/c and/or E/e antigens. Hydropathy analysis suggests that the Rh30A protein has up to 12 transmembrane domains. Three of these domains are bordered by a novel cysteine-containing motif, which might signal substitutions at these cysteine residues. Information which supplements this paper (amino-acid-sequence-analysis histograms) is reported in Supplementary Publication SUP 50160 (4 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1990) 265, 5.

Topology and organization of human Rh (rhesus) blood group-related polypeptides.

The Rh blood group antigens are associated with nonglycosylated human erythrocyte membrane proteins of molecular mass 30 kDa (the Rh30 polypeptides) and a glycoprotein of 40-100 kDa (the Rh glycoprotein). We have studied the topology of this family of proteins in the erythrocyte membrane. We confirmed the predicted cytosolic localization of the C and N termini of the Rh protein family. We located Lys-196 and Arg-323 of the Rh glycoprotein to the cytosol, and Glu-34 to the extracellular side of the plasma membrane in erythrocytes, by N-terminal sequencing of Rh glycoprotein peptides produced by proteolysis at the cytoplasmic or extracellular side of the membrane. We also show that a glycan chain is present on only one (Asn-37) of the three potential N-glycan addition sites in the Rh glycoprotein. Studies of the Rh glycoprotein fragments that co-immunoprecipitated with the Rh30 polypeptides suggest there is an interaction between the Rh30 polypeptides and amino acids 35-196 of the Rh glycoprotein. A model for the organization of the components of the Rh complex in the red cell membrane is proposed.

Structural relationships between human erythrocyte sialoglycoproteins beta and gamma and abnormal sialoglycoproteins found in certain rare human erythrocyte variants lacking the Gerbich blood-group antigen(s).

The human erythrocyte membrane sialoglycoproteins beta and gamma are important for the maintenance of the discoid shape of the normal erythrocyte. In this paper we show that the human erythrocyte sialoglycoproteins beta and gamma (hereafter called beta and gamma) are structurally related. Rabbit antisera produced against purified beta and beta 1 and rendered specific to the cytoplasmic portion of these proteins also react with the cytoplasmic portion of gamma. Some human anti-Gerbich (Ge) sera react with the extracellular portion of both beta and gamma. This reactivity is shown to be directed towards a common epitope on beta and gamma. However, most anti-Ge sera do not react with beta, but react with an extracellular epitope only present on gamma. All individuals who lack the Ge antigens lack beta and gamma. In some cases abnormal sialoglycoproteins are present in the erythrocytes, and these are shown to be structurally related to beta and gamma. Rabbit antisera raised against the purified abnormal sialoglycoprotein from a Ge-negative erythrocyte type reacted with the cytoplasmic portion of both beta and gamma. Unlike normal beta and gamma, the abnormal sialoglycoproteins found in Ge-negative erythrocytes migrate as a diffuse band on SDS/polyacrylamide-gel electrophoresis. Studies using endoglycosidases suggest that the diffuse nature of these bands results from carbohydrate heterogeneity and that the abnormal sialoglycoproteins contain N-glycosidically linked oligosaccharides with repeating lactosamine units. Such polylactosamine chains are not present on normal beta or gamma.

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.

The low-incidence blood group antigen, Wda, is associated with the substitution Val557-->Met in human erythrocyte band 3 (AE1).

The Waldner blood group antigen (Wda) was first identified in members of a Hutterite kindred. Evidence that the gene governing the Waldner polymorphism is located on chromosome 17, and the observation that the antigen is inactivated by chymotrypsin prompted the investigation of a possible association between Wda and band 3. Single Stranded Conformational Polymorphism (SSCP) analysis and DNA sequence analysis of the AE1 gene, from subjects of known Waldner phenotypes, showed a heterozygous mutation leading to the substitution Val557-->Met in the presumptive Wd(a+) heterozygotes. Therefore the Wda blood group antigen is associated with the presence of Met557 on band 3. the Waldner antigen has been assigned to the Diego blood group system with the International Society of Blood Transfusion number D15.

Absence of two membrane proteins containing extracellular thiol groups in Rhnull human erythrocytes.

Rhnull human erythrocytes lack all the antigens of the Rhesus blood-group system and are associated with mild chronic haemolytic anaemia. These erythrocytes have an abnormal shape and increased osmotic fragility. Labelling studies with the impermeant maleimide N-maleoylmethionine [35S]sulphone show that Rhnull erythrocytes lack two extracellular thiol-group-containing membrane components of apparent mol.wts. 32 000 and 34 000. Immunoprecipitation with mouse monoclonal antibody R6A (which reacts with all normal erythrocytes, but fails to react with Rhnull erythrocytes) specifically precipitates the 34 000-mol.wt. component from normal erythrocytes. Similar studies with human anti-Rh(D) serum shows that this antibody reacts with the 32 000-mol.wt. component. The results suggest that the R6A-binding polypeptide and the Rh(D) polypeptide may be involved in the maintenance of the shape and viability of the human erythrocyte.

Microvillar membrane neutral endopeptidases.

Recent developments on neutral endopeptidase (NEP, EC 3.4.24.11) are described. These include (1) the development of a novel colorimetric assay with a chromogenic substrate (Glutaryl-Gly-Gly-Phe-2-naphthylamide) coupled with aminopeptidase M (EC 3.4.11.2). (2) A detergent form of the pig kidney enzyme has been purified by immuno-adsorbent chromatography and its molecular properties compared with other forms of the enzyme from rabbit kidney and pig intestine. (3) Rat kidney microvilli contain two endopeptidases of about equal activity when assayed with [125I]iodo-insulin B chain as substrate. One is similar to the rabbit and pig endopeptidases in being sensitive to inhibition by phosphoamidon. The other is insensitive to the inhibitor, though susceptible to chelating agents. The two enzymes are resolvable and have been partially characterized. (4) Endopeptidases of the phosphoramidon-sensitive type are present in various tissues in addition to the principal locations in brush borders of kidney and intestine.

Individuals lacking the Gerbich blood-group antigen have alterations in the human erythrocyte membrane sialoglycoproteins beta and gamma.

Membranes from erythrocytes with a new Gerbich (Ge)-negative phenotype (Leach phenotype), as well as those from two other Ge-negative phenotypes, were examined. Whereas cells of the Leach phenotype apparently lack three minor sialoglycoproteins (beta, beta 1 and gamma), the membranes of Ge- Yus- and Ge- Yus+ erythrocytes apparently lack beta- and gamma-sialoglycoproteins but contain additional diffusely migrating components of apparent Mr 30 500-34 500 and 32 500-36 500 respectively. Immunoprecipitation experiments showed that the abnormal components of both Ge- Yus- and Ge- Yus+ erythrocytes reacted with two monoclonal antibodies, BRIC 4 and BRIC 10. These antibodies have been shown to react with sialoglycoproteins beta and beta 1 in normal erythrocytes. Cytoskeletal preparations from Ge- Yus- and Ge- Yus+ erythrocyte membranes contained the abnormal components. In contrast with cells of the Leach phenotype, which are elliptocytic, Ge- Yus- and Ge- Yus+ were of normal shape, despite their apparent lack of beta- and gamma-sialoglycoproteins. It seems likely that the abnormal components in these cells contribute to their normal shape. Ovalocytic erythrocytes were shown to incorporate more radioactivity in the sialoglycoprotein-beta 1 region than normal erythrocytes after labelling by the periodate/NaB3H4 technique. It is suggested that abnormal components in Ge- Yus- and Ge- Yus+ erythrocytes result from chromosomal misalignment with unequal crossing-over at meiosis between the genes giving rise to beta-, beta 1- and gamma-sialoglycoproteins.

Protein-sequence studies on Rh-related polypeptides suggest the presence of at least two groups of proteins which associate in the human red-cell membrane.

The Rh D blood-group antigen forms part of a complex, involving several other polypeptides, that is deficient in the red cells of individuals who lack all the antigens of the Rh blood-group system (Rhnull red cells). These include components recognized by anti-(Rh D) antibodies and the murine monoclonal antibodies R6A and BRIC 125. We have carried out protein-sequence studies on the components immunoprecipitated by these antibodies. Anti-(Rh D) antibodies immunoprecipitate an Mr-30,000-32,000 polypeptide (the D30 polypeptide) and an Mr-45,000-100,000 glycoprotein (D50 polypeptide). Antibody R6A immunoprecipitates two glycoproteins of Mr 31,000-34,000 (R6A32 polypeptide) and Mr 35,000-52,000 (R6A45 polypeptide). The D30 and R6A32 polypeptides were found to have the same N-terminal amino acid sequences, showing that they are closely related proteins. The D50 polypeptide and the R6A45 polypeptide also had indistinguishable N-terminal amino acid sequences that differed from that of the D30 and R6A32 polypeptides. The putative N-terminal membrane-spanning segments of the two groups of proteins showed homology in their amino acid sequence, which may account for the association of each of the pairs of proteins during co-precipitation by the antibodies. Supplementary data related to the protein sequence have been deposited as Supplementary Publication SUP 50417 (6 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1988) 249, 5.

Assignment of the chromosomal locus of the human 30-kDal Rh (rhesus) blood group-antigen-related protein (Rh30A) to chromosome region 1p36.13----p34.

Using a panel of somatic cell hybrids, we have localised the 30-kDal Rhesus blood group-antigen-related protein to human chromosome 1 in the region p36.13----p34. This confirms the localisation of this protein described previously using cytogenetic and linkage analyses.

Isolation of cDNA clones for a 50 kDa glycoprotein of the human erythrocyte membrane associated with Rh (rhesus) blood-group antigen expression.

The Rh blood-group antigens are associated with human erythrocyte membrane proteins of approx. 30 kDa (the Rh30 polypeptides). Heterogeneously glycosylated membrane proteins of 50 and 45 kDa (the Rh50 glycoproteins) are coprecipitated with the Rh30 polypeptides on immunoprecipitation with anti-Rh-specific mono- and poly-clonal antibodies. We have isolated cDNA clones representing a member of the Rh50 glycoprotein family (the Rh50A glycoprotein). We used PCR with degenerate primers based on the N-terminal amino acid sequence of the Rh50 glycoproteins and human genomic DNA as a template and cloned and sequenced three types of PCR product of the expected size. Two of these products, Rh50A and Rh50B, gave the same translated amino acid sequence which corresponded to the expected Rh50 glycoprotein sequence but had only 75% DNA sequence similarity. The third product (Rh50C) contained a single base deletion, and the translated amino acid sequence contained an in-frame stop codon. We have isolated cDNA clones containing the full coding sequence of the Rh50A glycoprotein. This sequence predicts that it is a 409-amino acid N-glycosylated membrane protein with up to 12 transmembrane domains. The Rh50A glycoprotein shows clear similarity to the Rh30A protein in both amino acid sequence and predicted topology. Our results are consistent with the Rh30 and Rh50 groups of proteins being different subunits of an oligomeric complex which is likely to have a transport or channel function in the erythrocyte membrane. We mapped the Rh50A gene to human chromosome 6p21-qter, showing that genetic differences in the Rh30 rather than the Rh50 genes specify the major polymorphic forms of the Rh antigens.

Murine monoclonal antibody MB-2D10 recognizes Rh-related glycoproteins in the human red cell membrane.

The human red cell membrane components reacting with monoclonal antibody MB-2D10 were examined by immunoblotting. The antibody bound to a diffusely staining band extending from Mr 30,000 up to the high-molecular-weight region of the gel in normal membranes and in Rhnull U + membranes, but not in Rhnull U - membranes. Treatment of normal red cells with an endoglycosidase F-containing preparation destroyed the epitope recognized by MB-2D10. The reactivity of the antibody with purified preparations of Rh-related glycoproteins D30 polypeptide, D50 polypeptide, R6A32 polypeptide, and R6A45 polypeptide was also examined. Only the purified R6A45 and D50 components reacted with MB-2D10. These results show that MB-2D10 recognizes a carbohydrate-dependent epitope on the R6A45 and D50 group of Rh-related polypeptides. The results also suggest the possibility that the U antigen arises from interaction between glycophorin B and the Rh-related components D50 and R6A45.