The functional importance of blood group-active molecules in human red blood cells.

Antigens of 23 of the 30 human blood group systems are defined by the amino acid sequence of red cell membrane proteins. The antigens of DI, RH, RHAG, MNS, GE and CO systems are carried on blood group-active proteins (Band 3, D and CE polypeptides, RhAG, Glycophorins A and B, Glycophorins C and D and Aquaporin 1, respectively) which are expressed at high levels (>200,000 copies/red cell). These major proteins contribute to essential red cell functions either directly as membrane transporters and by providing linkage to the underlying red cell skeleton or by facilitating the membrane assembly of the protein complexes involved in these processes. The proteins expressing antigens of the remaining 17 blood group systems are much less abundant (<20,000 copies/red cell) and their functional importance for the circulating red cell is largely unknown. Human gene knock-outs (null phenotypes) have been described for many of these minor blood group-active proteins, but only absence of Kx glycoprotein has been clearly linked with pathology directly related to the function of circulating red cells. Recent evidence suggesting the normal quality control system for glycoprotein synthesis is altered during the latter stages of red cell production raises the possibility that many of these low abundance blood group-active proteins are vestigial. In sickle cell disease and polycythaemia vera, elevated Lutheran glycoprotein expression may contribute to pathology. Dyserythropoiesis with reduced antigen expression can result from mutations in the erythroid transcription factors GATA-1 and EKLF.

Human red cell aquaporin CHIP. I. Molecular characterization of ABH and Colton blood group antigens.

Blood group antigens are structural variants in surface carbohydrate or amino acid polymorphisms on extracellular domains of membrane proteins. The red cell water channel-forming integral protein (Aquaporin CHIP) is a homotetramer with only one N-glycosylated subunit, however no CHIP-associated blood group antigens have yet been identified. Immunoblotting, monosaccharide composition analysis, and selective glycosidase digestions revealed that the CHIP-associated oligosaccharide contains ABH determinants and resembles a band 3-type glycan that cannot be cleaved from intact membranes by Peptide:N-glycosidase F. The molecular structure of the Colton antigens was previously unknown, but CHIP was selectively immunoprecipitated with anti-Coa or anti-Co(b). The DNA sequence from Colton-typed individuals predicted that residue 45 is alanine in the Co(a+b-) phenotype and valine in the Co(a-b+) phenotype. The nucleotide polymorphism corresponds to a PflMI endonuclease digestion site in the DNA from Co(a-b+) individuals. These studies have defined antigens within two blood group systems on CHIP: (a) an ABH-bearing polylactosaminoglycan attached to a poorly accessible site in the native membrane; and (b) the Colton antigen polymorphism which may permit the identification of rare individuals with defective water channel expression.

Human red cell Aquaporin CHIP. II. Expression during normal fetal development and in a novel form of congenital dyserythropoietic anemia.

Channel-forming integral protein (CHIP) is the archetypal member of the Aquaporin family of water channels. Delayed CHIP expression was shown recently in perinatal rat (Smith, B. L., R. Baumgarten, S. Nielsen, D. Raben, M. L. Zeidel, and P. Agre. 1993. J. Clin. Invest. 92:2035-2041); here we delineate the human patterns. Compared with adult, second and third trimester human fetal red cells had lower CHIP/spectrin ratios (0.72 +/- 0.12, 0.94 +/- 0.22 vs 1.18 +/- 0.11) and reduced osmotic water permeability (0.029, 0.026 vs 0.037 cm/s); CHIP was already present in human renal tubules by the second trimester. A patient with a novel form of congenital dyserythropoietic anemia (CDA) with persistent embryonic and fetal globins and absent red cell CD44 protein was studied because of reduced CHIP-associated Colton antigens. Novel CDA red cells contained < 10% of the normal level of CHIP and had remarkably low osmotic water permeability (< 0.01 cm/s), but no mutation was identified in Aquaporin-1, the gene encoding CHIP. These studies demonstrate: (a) unlike rat, human CHIP expression occurs early in fetal development; (b) red cell water channels are greatly reduced in a rare phenotype; and (c) disrupted expression of red cell CHIP and CD44 suggests an approach to the molecular defect in a novel form of CDA.

Freeze-fracture electron microscopy of human erythrocytes lacking the major membrane sialoglycoprotein.

Human erythrocytes of blood group En (a-), a rare homozygous condition involving a complete lack of the major sialoglycoprotein of the cell membrane (glycophorin A), were compared with erythrocytes from normal (En (a+)) individuals by freeze-fracture electron microscopy. No decrease in number, or variation in morphology, of the intramembranal particles of En (a-) cells was detectable. The results show that the erythrocyte sialoglycoprotein is not essential for the maintenance of the integrity of the intramembranal particles of the human erythrocyte membrane.

Blood group antigen deficiencies associated with abnormal red cell shape.

Rare individuals are known with erythrocytes which show an inherited deficiency of certain blood group antigens and also have abnormal red cell shape. Studies of these cells can give an insight into the functional role of blood group active components in maintaining the shape and membrane properties of the normal erythrocyte. The biochemical characterisation of the red cell membrane alterations occurring in two such rare erythrocyte phenotypes--the Leach phenotype and the Rhnull phenotype are reviewed here.

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.

The phospholipid organisation in the membranes of McLeod and Leach phenotype erythrocytes.

The phospholipid composition, the distribution of phospholipids over the two membrane layers as well as the phosphatidylcholine-specific transfer protein-mediated exchangeability of phosphatidylcholine from the membrane, has been investigated in two types of abnormal erythrocytes--the McLeod phenotype and the Leach phenotype. The acanthocytic McLeod cells appeared to have a normal phospholipid composition and distribution, but the exchangeability of phosphatidylcholine was found to be markedly enhanced. Unlike control erythrocytes, in which 75% of all of the phosphatidylcholine can be exchanged during an 8 h incubation, the McLeod cell showed a complete exchange of this phospholipid within the same time period. This obviously indicates an enhanced transbilayer mobility of phosphatidylcholine in the membrane of McLeod cells. Erythrocytes of the Leach phenotype showed an elliptocytic shape and increased osmotic fragility, but no abnormalities were observed as to the composition and organisation of the phospholipid complement of their membranes.

Mechanism of regulation of malarial invasion by extraerythrocytic ligands.

Invasion of red cells by Plasmodium falciparum in vitro was inhibited by a range of extracellular ligands, none of which block the major receptors for merozoites. Most effective, in terms of dose response, were two monoclonal antibodies against the Wrb antigen on glycophorin A; wheat germ agglutinin which also binds to glycophorin, and an anti-band 3 monoclonal antibody, caused inhibition of invasion at higher levels of saturation, while concanavalin A, which binds to band 3, was without effect. All the ligands except concanavalin A, increased the rigidity of the host cell membrane. The anti-Wrb antibodies generated the highest dose response effect, but no correlation between invasion and shear elastic modulus of the membrane could be established. All ligands, with the exception of concanavalin A, caused a reduction in the translationally mobile fractions of band 3 and glycophorin, as revealed by fluorescence recovery after photobleaching (FRAP). Invasion diminished with loss of mobile band 3, engendered by bound wheat germ agglutinin or anti-band 3, falling precipitately when the mobile fraction fell below 40% of that in unperturbed membranes. Both anti-Wrb antibodies suppressed invasion completely at concentrations insufficient to affect significantly either membrane rigidity or intramembrane protein diffusion. A univalent anti-glycophorin A (Fab) fragment, the parent antibody of which was previously shown to inhibit invasion strongly, had only a modest effect on invasion and induced a correspondingly small change in the mobile fraction of band 3.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of functional and quantitative assays to study glycoprotein Ib in platelets stored under various in vitro conditions.

There is much evidence to suggest that platelet membrane glycoprotein Ib is involved in the haemostatic function of platelets and it has been suggested that loss of this glycoprotein may occur during in vitro storage of platelet concentrates. In this study two quantitative radioimmunoassays were developed to measure the content of glycoprotein Ib in platelets stored under a range of conditions used in blood banks. One assay involved the use of iodinated Maclura pomifera lectin and the other the binding of a monoclonal antibody (AN51) specific for glycoprotein Ib. The results showed that there was no significant reduction in the glycoprotein Ib content of platelets under the storage conditions used. These results suggest that any loss of haemostatic effectiveness which occurs on in vitro storage of platelet concentrates is not attributable to a selective loss of glycoprotein Ib from the platelet surface.

Screening of blood donors for IgA deficiency: a study of the donor population of south-west England.

Altogether 29 745 English blood donors were screened for IgA deficiency by double diffusion analysis; 57 had apparent absence of IgA, a frequency of 1:522. Further examination by the more sensitive haemagglutination inhibition assay revealed 34 samples having no detectable IgA, a frequency of 1:875. All donors negative by double diffusion analysis were tested for the presence of antibodies to IgA. Six class specific anti IgA antibodies and four anti IgA antibodies of limited specificity were detected. Three of these had the specificity anti alpha2 and one anti A2m(2). The 34 IgA deficient donors detected provide a source of IgA deficient blood for transfusion to patients with anti IgA antibodies.

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 erythroid cells from human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPS).

Erythroid progenitors can be generated ex vivo from human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPS). Development of laboratory scale culture conditions capable of generating mature functional erythrocytes from human embryonic stem cells or human induced pluripotent stem cells would open the possibility for manufacture of therapeutic quantities of red cells and thereby new clinical transfusion products. Current attempts to produce erythrocytes from human embryonic stem cells reveal the need for greater understanding of the process whereby primitive erythropoiesis switches to definitive fetal and adult erythropoiesis and the factors driving erythrocyte maturation. Studies with human embryonic stem cells have already yielded encouraging results but functional mature biconcave erythrocytes have yet to be generated from these cells.

Blood group antigens defined by the amino acid sequences of red cell surface proteins.

The antigens of 18 blood group systems are expressed on proteins that are intrinsic to the red cell. The proteins which carry the antigens of these systems have been identified and primary sequence information is available for all but two (SC, DO). Several different functional groups are evident. Antigens of the DI, CO, RH, XK and JK systems are located on proteins which have the structure of membrane transport proteins. The FY antigens mark a cytokine receptor. The IN, LW, XG antigens are associated with molecules which have adhesion functions and the LU glycoprotein also has a structure which suggests a role in adhesion. YT and KEL antigens are located on cell surface enzymes and the CR and KN antigen on molecules involved in complement regulation. Finally, the MN and GE antigens are located on sialic acid-rich glycoproteins (glycophorins A, B and C/D respectively), a group of molecules which do not, as yet, have a clearly defined function. The molecular basis of antigens in several blood group systems have been defined and shown to depend upon the amino acid sequence.

Blood group MNSs-active sialoglycoproteins of the human erythrocyte membrane.

The human erythrocyte membrane contains at least four different PAS-staining sialic acid-rich glycoproteins. The major sialoglycoprotein, which carries blood group M or N antigen activity, has been extensively characterized. The Ss antigens are located on a minor sialoglycoprotein, which also has "N' activity. The amino acid sequence at positions 1 and 5 of these glycoproteins correlates with the presence of M or N antigen activity. Little is known about the other minor sialoglycoproteins (beta and gamma). Membranes from erythrocytes of type (En(a-)Fin lack the major MN-active sialoglycoprotein; those from S-s-erythrocytes lack normal Ss-active sialoglycoproteins, although they contain an abnormal component that may be an altered Ss glycoprotein. Mk Mk cells lack both the MN- and Ss-active glycoproteins. These sialoglycoprotein-deficient cells are found in apparently healthy individuals. The sera of individuals with sialoglycoprotein-deficient cells may contain antisialoglycoprotein antibody, which has properties similar to those of auto-anti-Pr. Miltenberger Class III, IV, and VI erythrocytes have abnormal Ss-active sialoglycoproteins. Component beta appears altered in Miltenberger Classes I and II. These abnormalities may account for the unique serological properties of Class I, II, III, IV, and VI erythrocytes. Membranes from erythrocytes of type EnU K/Mk, Miltenberger Class V, and Ph contain abnormal sialogylcoproteins that may result from fusion of the genes that give rise to the Mn-and Ss-active sialoglycoproteins. If this is so, then the genes giving rise to the MN and Ss glycoproteins must be adjacent on the same chromosome.

Blood group-active surface molecules of the human red blood cell.

The surface of the human red blood cell is dominated by a small number of abundant blood group active proteins. The major proteins are the anion transport protein (band 3) which has AB(H) activity, and Glycophorin A which has MN activity. Band 3 and Glycophorin A are of equal abundance in the normal red cell membrane (approximately 10(6) copies of each) and the two proteins may associate together as a complex. The glucose transporter (band 4.5) had AB(H) activity and there are about 5 x 10(5) copies/red cell. Several polypeptides associate together to form the Rh complex. The major components of this complex (abundance 1-2 x 10(5) copies/red cell) are polypeptides of Mr 30,000, polypeptides of Mr 45,000-100,000 and Glycophorin B. The antigens of the Rh blood group system appear to be associated with the polypeptides of Mr 30,000 and those of Mr 45,000-100,000 (the latter also express AB(H) activity). Glycophorin B expresses the blood group 'N' antigen and the Ss antigens. Glycophorins C and D carry the Gerbich antigens and, together, these polypeptides comprise approximately 10(5) copies/red cell. The complete protein sequence of all the above-mentioned proteins is known, except for the Mr 30,000 and Mr 45,000-100,000 polypeptides of the Rh complex for which only partial sequences are available, and Glycophorin D, the sequence of which can be inferred from that of Glycophorin C. Several of the minor blood group active proteins at the red cell surface (abundance less than 1.2 x 10(4)/red cell) have been the subject of recent studies. The polypeptide expressing Cromer-related blood group antigens has been identified as decay-accelerating factor and that carrying the Ina/Inb antigens as CD44. The protein sequence of both of these proteins has been deduced form nucleotide sequencing. The polypeptides expressing Kell antigens, Lutheran antigens, Fy antigens, and LW antigens have also been identified and partially characterised.