Carbohydrate content of human erythrocyte membrane. Variations with ABO-blood group.

The study of the carbohydrates of human erythrocyte membranes has been mainly focused on their glycopeptidic and glycolipidic complexes. Modifications of these carbohydrates have been described in subjects with various pathological states. In order to characterize possible changes of the glycopeptides, or glycolipids obtained from erythrocyte membrane in various pathological situations, the determination of the carbohydrate content of the whole membrane appeared a necessary preliminary. This study concerns the determination of the normal values of the main carbohydrates of whole human erythrocyte membranes, with respect to their blood group. Erythrocyte membranes were prepared from donors of the four ABO blood groups. After acidic hydrolysis, the contents of fucose, mannose, galactose, glucose, glucosamine, galactosamine and N-acetylneuraminic acid in each blood group were determined and compared with one another. The galactosamine content of A, B and AB erythrocyte membranes is significantly higher than that of the O-erythrocyte. For galactose, the differences are significant for the following pairs: A/O; B/O; AB/O; A/B; A/AB. Significant differences in the mannose contents of O-erythrocytes and A, B and AB erythrocytes have also been found. This result suggests that a basic difference, in the core of the oligosaccharide chains, may exist between O and A, B, AB erythrocyte membranes.     

The transfer of bovine J blood group activity to erythrocytes: chemical nature of transferable and of non-transferable J1

The bovine J blood group substance exists as a glycosphingolipid (ceramide deca-hexoside as well as ceramide dodecahexoside) and as a glycoprotein. The lipidic form occurs in erythrocyte membranes, both forms are found in serum. The lipidic J substances were isolated from erythrocytes and from serum, and identified by thin-layer chromatography with lipidic J substances isolated from spleen. The glycoprotein nature of the non-lipidic J of serum was evident by pronase-catalysed hydrolysis yielding J-active glycopeptides of lower molecular weights. The lipidic J was completely extracted from lyophilized stroma with chloroform/methanol. From lyophilized serum, however. it was completely extracted only in the presence of water, indicating different binding partners in serum and in erythrocyte membranes. The J lipid was incorporated as intact molecule into the erythrocyte membrane by a simple incubation technique. The incorporation was inhibited by various glyc-erophospholipids (called blockers). The J glycoprotein could not be transferred to the erythrocyte membrane. Three methods are descrjbed which are suitable for the preparation of a blocker-free fraction enriched with J lipids from J-positive serum.     

Level of the A, B and H blood group glycosyltransferases in red cell membranes from patients with malignant hemopathies

Eight patients (4 suffering from acute myeloid leukemia) exhibiting a loss of ABO red cell antigens, as seen by a mixed-field reaction pattern in agglutination tests, were selected and examined for the level of the A, -B, -H blood group glycosyltransferases within membranes prepared from erythrocyte subpopulations (A or B positive and A or B negative red cells). A or B enzyme activities were largely decreased in membranes which had lost A or B antigens (A or B negative subpopulations) but were within normal level in membrane from cells which had not lost A or B antigens (A or B positive subpopulations). The H enzyme level which was frequently low in the serum was within normal limits in the membrane preparations examined. Since A or B negative subpopulations were normally glycosylated in vitro into A or B reactive structures, the results demonstrate that loss of A or B antigens is related to some alteration of the blood group gene products rather than to significant abnormalities of the membrane precursors.     

Genetic mechanism of blood group (ABO)-expression

It has generally been believed that human blood group ABO is controlled by allelic ABO genes. However, this hypothesis has not yet been experimentally proven, and other possibilities such as the non-allelic gene model and the regulatory gene model for ABO locus have also been proposed. The genetic mechanisms of many unusual blood group expressions remain unanswered. Purification of human blood group N-acetylgalactosyltransferase (A-enzyme) which synthesizes A-substance, and blood group galactosyltransferase which is responsible for synthesis of B-substance, allows us to resolve these problems from an immuno-biochemical approach. It was found that rabbit antibody against-A-enzyme completely neutralized not only A-enzyme but also B-enzyme activity. Moreover, plasma from blood type O subjects contained an enzymatically inactive but immunologically cross-reactive material (CRM). Plasma from heterozygous AO and BO subjects also contained CRM, but plasma from homozygous AA and BB subjects did not contain CRM. These facts led us to conclude that the ABO genes are allelic in the strict sense, refuting other genetic models for ABO locus. Genotypes of phenotype A and B subjects can be unequivocally determined by examining the presence or absence of CRM in their plasma. Mechanism of the unusual blood group inheritance of Cis-AB (i.e., AB and/or O childbirth from AB X O parent) was elucidated by examining properties of the A and B enzymes, CRM in their plasma, and separation of active enzymes and CRM by affinity chromatography. It became clear that Cis-AB expressions in one family was due to unequal chromosomal crossing-over producing a single chromosome with the genes for A and B enzymes. In contrast, in the other two unrelated families, the Cis-AB expression was due to a structural mutation in A or B gene producing a single abnormal enzyme which was capable of transferring both GalNAc and Gal to H-substance. Mechanism of very weak B expression in a family with A1Bm character was studied. Plasma enzyme activity and kinetic characteristics of B-enzyme from the subjects was not different from that of normal. However, the A1Bm red cells contained a large amount of unoccupied H-sites which can be galactosylated in vitro and become B active. Examination of membrane components by isoelectric focussing revealed that blood group components of the A1Bm membranes were distinctively different from that of the usual membranes. Consequently, the weak B expression is not due to direct mutation of ABO locus, but due to a secondary consequence of genetic abnormality of a membrane component (or components) associated with blood group substances.     

Isolation and partial characterization of two minor glycoproteins from human erythrocyte membranes

Two minor glycoproteins GP-II and GP-III, were isolated from human erythrocyte membranes and characterized chemically and immunologically. The chemical composition of GP-II and GP-III was similar: GP-II consisted of 81% protein and 19 % carbohydrate of which 4.9 % was hexose. 5.4 % hexosamine and 7.8 % sialic acid. GP-III consisted of 76 % protein and 24 % carbohydrate of which 7.6 % was hexose, 7.2 % hexosamine and 8.1 % sialic acid. The amino acid composition of GP-II and GP-III was also similar. GP-II and GP-III, however, differed in chemical composition from the MN glycoprotein. GP-II and GP-III were associated with the blood group activities Ss, I and A, but not with the MN antigens. GP-III had higher blood group activities per μg of protein than did GP-II. The specific activities for the Ss blood group antigens were increased 3–10-fold by purification of GP-III from the aqueous phase of chloroform methanol extracts.     

Miltenberger Class I and II erythrocytes carry a variant of glycophorin A.

The membranes from Miltenberger Class I (Mi I) and II (Mi II) erythrocytes, two rare variants at the blood group MNSs locus, exhibited an abnormal glycoprotein of 32 kDa apparent molecular mass sharply stained by the periodic acid/Schiff procedure and a decreased content of glycoprotein alpha (synonym glycophorin A, glycoprotein MN) as seen on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Purified 125I-labelled Vicia graminea lectin binds to the unusual 32 kDa glycoprotein separated from Mi I and Mi II erythrocyte membrane of blood group NN or MN, but no significant labelling of this band was observed with Mi samples typed MM. On the basis of such lectin-labelling experiments we have described two heterozygous MN, Mi I individuals that carry one copy of an M gene producing a normal alpha-glycoprotein with M-specificity and one copy of a MiI gene producing a 32 kDa glycoprotein with N-specificity. Further investigations have shown that the 32 kDa glycoprotein was immunoprecipitated by two mouse monoclonal antibodies (R18 and R10) reacting specifically with the external domain of glycoprotein alpha. These results demonstrate that Mi I and Mi II erythrocytes carry an unusual variant of glycoprotein alpha.     

Association of human erythrocyte membrane glycoproteins with blood-group Cad specificity.

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of erythrocyte membranes from a blood-group-B individual with the rare Cad phenotype indicates a lower-than-normal mobility of the main sialoglycoproteins, suggesting an increase in apparent molecular mass of 3kDa and 2kDa respectively for glycoprotein alpha (synonym glycophorin A) and glycoprotein delta (synonym glycophorin B). Since the chief structural determinant of Cad specificity is N-acetylgalactosamine, the membrane receptors have been isolated by affinity binding on immobilized Dolichos biflorus (horse gram) lectin. The predominant species eluted from the gel was the abnormal glycoprotein alpha, whereas in control experiments no material could be recovered from the adsorbent incubated with group-B Cad-negative erythrocyte membranes. After partition of the membranes with organic solvents, the blood-group-Cad activity was found in aqueous phases containing the sialoglycoproteins, but not in the organic phases containing simple or complex glycolipids, which, however, retained the blood-group-B activity. The carbohydrate composition of highly purified lipid-free glycoprotein alpha molecules prepared from Cad and control erythrocytes was determined. Interestingly the molar ratio of N-acetylneuraminic acid to N-acetylgalactosamine was equal to 2:1 in the case of controls and equal to 1:1 in the case of Cad erythrocytes. Taken together these results suggest that Cad specificity is defined by N-acetylgalactosamine residues carried by the alkali-labile oligosaccharide chains attached to the erythrocyte membrane sialo-glycoproteins.     

Structure of the Ss blood group antigens, II: a methionine/threonine polymorphism within the N-terminal sequence of the Ss glycoprotein

The N-terminal amino acid sequence (residues 1--35) of the Ss sialoglycoprotein (or glycophorin B) from human erythrocyte membranes of defined Ss blood group activity was determined by manual sequencing methods, using N-terminal tryptic or chymotryptic glycopeptides and various secondary peptides. The proposed structure differs considerably from that suggested on the basis of work with glucopeptides of unknown Ss blood group activity (Furthmayr, Nature 271, 519--523, 1978). Only one difference between glycopeptides from Ss and ss erythrocytes was found, i.e. a methionine/threonine polymorphism at position 29. On the basis of previous work (Dahr et al., Hoppe-Seyler's Z. Physiol. Chem. 361, 145--152, 1980), it is concluded that this amino acid heterogeneity represents the Ss polymorphism rather than the UX or UZ polymorphisms, which are in strong genetic linkage disequilibrium with the Ss antigens. A part of the sequence (residues 9--30) of the major (MN) red cell membrane sialoglycoprotein (glycophorin A) was re-investigated and revised at positions 11 and 17. As judged from the present data, the first 26 residues of the Ss and the blood group N-specific MN glycoprotein are identical. The sequence 27--35 of the Ss glycoprotein shows a homology with the residues 56--64 and 59--67 of the MN glycoprotein. Data on the partial N-terminal sequence of glycopeptides from a third erythrocyte membrane sialoglycoprotein (component D or glycophorin C) indicate that its structure is different from those of the two other glycoproteins.     

Serological and molecular analysis of ABO and Rh blood group chimeras

The serological examination, blood transfusion strategies and the molecular analysis to blood group chimera were conducted to demonstrate existent of chimera in blood group. The blood grouping of ABO or/and RhD, newborn red blood cells separated by capillary centrifugation. Aabsorption tests and DTT treated agglutination erythrocyte tests were implemented in four patients. Further molecular biological research was conducted on one patient''s sample. The results showed that for patient 1: ABO blood group was AB/B chimera, Rh blood cells contained the RhCE chimera gene; Patient 2: Rh blood cells contained the RhD chimera gene; Patient 3: ABO blood group was AB/B chimera, Rh blood cells contained the RhD chimera gene; Patient 4: ABO blood group was O/B chimera, Rh blood cells contained the RhCE chimera gene. The study suggests that the individuals categorized as chimeras are likely to be more common than existing literature reports. According to the serological tests, in the absence of a history of recent blood transfusion or disease to cause reduced antigen, the phenomena of hybrid aggregation of the ABO and Rh blood system were the main feature. In terms of transfusion strategy, the selection of ABO and Rh blood groups should be depended on the group of cells with more antigens.     

The Mz variety of the St(a+) phenotype--a variant of glycophorin A exhibiting a deletion

The NeuNAc level of erythrocyte membranes from two related donors exhibiting the Mz variety of St(a+) phenotype within the MNSs blood group system was found to be decreased by about 16%. The quantity of glycophorin A was decreased by about 38%, whereas that of glycophorin B was not significantly different from normal. Mz erythrocyte membranes were also found to contain an abnormal component (molar ratio to glycophorin A about 0.89:1.0) with an apparent molecular mass of about 24,000 Da. Immunoblotting experiments and amino-acid sequence analysis revealed that the novel component (and glycophorin A in one of the donors) carries blood group M activity. Blood group N activity was demonstrable for glycophorin A and glycophorin B from both donors. Amino-acid sequence analysis of chymotryptic, tryptic and cyanogen bromide peptides demonstrated that the novel molecule exhibits the typical structure of a Sta-active molecule. However, since it exhibits blood group M activity, it appears to represent a variant of glycophorin A lacking the residues 27-58 (encoded by exon three of the glycophorin A gene) rather than a glycophorin B-glycophorin A-hybrid molecule of the anti-Lepore type. Since one of the Mz heterozygotes was found to exhibit both M- and N activity on glycophorin A, the Mz gene complex appears to encode a blood group N-active glycophorin A apart from the novel component and a blood group s-active glycophorin B, although the level of glycophorin A in the erythrocyte membranes is decreased by about half.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blood-group ABH-specific macroglycolipids of human erythrocytes: isolation in high yield from a crude membrane glycoprotein fraction

Highly glycosylated, water-soluble ABH-specific sphingolipids, designated macroglycolipids, were isolated in high yield, up to 5 mg per unit of blood, from the crude human-erythrocyte-membrane glycoprotein fraction which is obtained by extraction of the membranes with chloroform/methanol/water. Both serological tests and radioactive labelling experiments indicated that these substances, rather than the glycoproteins, are the principal ABH-components in this fraction. The activities of A-specific, B-specific and H-specific macroglycolipids were very high, approximately 0.1 microgram inhibiting four hemagglutinating doses of the respective agglutinating reagents, and were thus comparable to those of secreted blood-group ABH-specific glycoproteins. The substances were stable to mild alkaline conditions. They contained fucose, galactose, glucosamine, glucose, sialic acid, sphingosine and fatty acids; blood-group-A-specific substances contained, in addition, galactosamine. No amino acids were detected. Assuming one glycosyl residue per molecule, the average number of sugars in A and B macroglycolipids was 31, and their molecular weights approximately 6100. The presence of beta-D-galactosidase-labile and sialic acid residues indicated that these substances contain nonreducing termini additional to the ABH immunodeterminants. In the B macroglycolipid, the ratio between nonreducing terminal alpha-D-galactopyranosyl and beta-D-galactopyranosyl residues was 1.7:1.0. The macroglycolipids formed clear aqueous solutions at concentrations as high as 30 mg/ml, were insoluble in 60--70% aqueous ethanol, and did not migrate on thin-layer chromatography unless they were acetylated. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed the macroglycolipids to be a heterogeneous mixture migrating throughout most of the region in which the periodic acid/Schiff-positive membrane glycoproteins are found. On the basis of the evidence presented, it is concluded that macroglycolipids are the predominant ABH-specific component in human erythrocyte membranes, and that they most likely account for previous observations of ABH activity in membrane glycoprotein fractions.     

Altered cell surface antigen expression in bladder carcinoma detected by a new hemagglutinating monoclonal antibody

A hemagglutinating monoclonal IgM antibody (MoAb145) was produced against a high incidence red blood cell membrane antigen. By the specific red cell adherence test, the antibody also reacted with human bladder epithelium; in addition, expression of the MoAb145 antigen was lost in some cases of transitional cell carcinoma of the bladder, in a manner similar to the ABH blood group. Hemagglutination studies with a panel of erythrocytes lacking specific high incidence red blood cell membrane antigens indicated that MoAb145 did not recognize ABH specificity but rather a determinant absent from rare MN variant erythrocytes, including En(a-) erythrocytes, which lack glycophorin-alpha. Failure of MoAb145 to stain, by indirect immunofluorescence, the erythroleukemia cell line K562, which expresses glycophorin-alpha and the MN blood group, and failure to inhibit MoAb145 hemagglutination with an erythrocyte sialoglycoprotein fraction that contained MN blood group activity suggests that MoAb145 does not recognize either glycophorin-alpha or the MN blood group, but rather another membrane determinant, which is altered in En(a-) erythrocytes. This study demonstrates a new epitope detected by MoAb145 that is shared between human erythrocyte membranes and bladder epithelia, and is affected by neoplastic transformation in transitional cell carcinoma of the bladder.     

人ABO血型定型试剂(单克隆抗体)临床试验

通过与１１家单位协作,对中国药品生物制品检定所分发的ＡＢＯ血型单克隆抗体定型试剂进行临床考核,检测１～１２个月婴儿、５９岁以上老人、１～５９岁成人、健康献血员、癌症患者及其它疾病患者共计７０９６８例,单抗试剂检测结果与反定型结果完全相符,特别是对红细胞表面抗原位点少、亚型抗原性弱的新生儿和红细胞表面抗原成份可能发生变化的癌症患者,均呈现较好的凝集模式。     

Protein blot analysis of virus receptors: identification and characterization of the Sendai virus receptor

Receptors for Sendai virions in human erythrocyte ghost membranes were identified by virus overlay of protein blots. Among the various erythrocyte polypeptides, only glycophorin was able to bind Sendai virions effectively. The detection of Sendai virions bound to glycophorin was accomplished either by employing anti-Sendai virus antibodies or by autoradiography, when 125I-labeled Sendai virions were used. The binding activity was associated with the viral hemagglutinin/neuraminidase (HN) glycoprotein, as inferred from the observation that the binding pattern of purified HN glycoprotein to human erythrocyte membranes was identical to that of intact Sendai virions. No binding was observed when blots, containing either human erythrocyte membranes or purified glycophorin, were probed with the viral fusion factor (F glycoprotein). Active virions competed effectively with the binding of 125I-labeled Sendai virions (or purified HN glycoprotein), whereas no competition was observed with inactivated Sendai virus. The results of the present work clearly show that protein blotting can be used to identify virus receptors in cell membrane preparations.     

The ABO, Lewis and related blood group antigens; a review of structure and biosynthesis

Numerous studies have shown that the antigenic determinants of the ABO blood group system are closely related in biochemical terms to the antigenic determinants of the Hh, P, Lewis and Ii blood group systems. The blood group antigens of each of these systems are formed by the addition of specific sugars to an oligosaccharide precursor chain which may be bound through sphingosine to fatty acids (glycolipid) or through serine or threonine to a peptide chain (glycoproteins). The direct gene products of each of these blood group systems are the glycosyltransferase enzymes which catalyse the addition of the specific sugar thus conferring the specified blood group activity to the glycolipid or glycoprotein molecule. The antigenic determinants of the ABO and Lewis systems in addition to red cells also exist in the body secretions in soluble form when the relevant genes are expressed in the phenotype. The antigens expressed on both the red cells and in the secretions are determined by the interaction of Hh, Sese, ABO and Lele genes.     

The ABO, Lewis and related blood group antigens; a review of structure and biosynthesis

Abstract Numerous studies have shown that the antigenic determinants of the ABO blood group system are closely related in biochemical terms to the antigenic determinants of the Hh, P, Lewis and Ii blood group systems. The blood group antigens of each of these systems are formed by the addition of specific sugars to an oligosaccharide precursor chain which may be bound through sphingosine to fatty acids (glycolipid) or through serine or threonine to a peptide chain (glycoproteins). The direct gene products of each of these blood group systems are the glycosyltransferase enzymes which catalyse the addition of the specific sugar thus conferring the specified blood group activity to the glycolipid or glycoprotein molecule. The antigenic determinants of the ABO and Lewis systems in addition to red cells also exist in the body secretions in soluble form when the relevant genes are expressed in the phenotype. The antigens expressed on both the red cells and in the secretions are determined by the interaction of Hh, Sese, ABO and Lele genes.     

A monoclonal antibody to swine erythrocytes recognizes the B blood group on the major glycophorin

Recently monoclonal antibodies (mAbs) to swine red blood cells have been described. One of them (1AC11) was specific for the major swine glycoprotein with a molecular weight of 45 kDa and another mAb, 2G2, recognized the B ^a allele in the B system of swine blood groups. Immunoblotting experiments to characterize the mAb 2G2 indicated that it reacts with an antigen of 45 kDa, present on the aqueous phase, glycophorin fraction, of swine red blood cells with the B ^a allele and does not react with B ^bB^b homozygous cells. The antigen recognized by 2G2 has the same characteristics as the major glycophorin recognized by 1AC11, so we can conclude that the B system of the swine blood group is on the major glycophorin of swine erythrocyte membranes.     

A hemagglutinin specific for sialic acids in a rat brain synaptic vesicle-enriched fraction

A hemagglutinating activity was detected in a synaptic vesicle-enriched fraction prepared from adult rat brain, using trypsinized glutaraldehyde-fixed rabbit erythrocytes. The specific activity of the fraction, in two series of experiments, was 7.5 and 16-fold higher than in the other subcellular fractions. The activity was absent from the synaptosome cytosol. In a study using twenty-five different carbohydrates and glycoproteins, best inhibitors were N-acetylneuraminic acid and N-glycolylneuraminic acid, together with bovine submaxillary mucin and a glycopeptide fraction prepared from rabbit erythrocyte membranes. The activity was thermolabile and very sensitive to proteolytic enzymes (but insensitive to neuraminidase) indicating that a protein (agglutinin) is responsible for the activity. Experiments using detergents and high ionic strength showed that the agglutinin is tightly bound to membranes, inactivated by the so-called non denaturing detergents, and stable in diluted sodium dodecyl sulphate. Hypotheses are discussed on the possible function of the agglutinin.     

Temperature-dependent abnormalities of the erythrocyte membrane in porcine malignant hyperthermia

The temperature dependence of ATPase activities and stearic acid spin label motion in red blood cells of normal and MH-susceptible pigs have been examined. Arrhenius plots of red blood cell ghost Ca-ATPase and calmodulin-stimulable Ca-ATPase activities were identical for both normal and MH erythrocyte ghosts. Arrhenius plots of Mg-ATPase activity exhibited a break (defined as a change in slope) at 24 degrees C in both MH and normal erythrocyte ghosts. However, below 24 degrees C the apparent activation energy for this activity was less in MH than normal ghosts. To determine whether breaks in ATPase Arrhenius plots could be correlated with changes in the physical state of the red blood cell membrane, the spin label 16-doxyl-stearate was introduced into the bilayer of both erythrocyte ghosts and red blood cells. With both ghosts and intact cells, at each temperature examined, the mobility of the probe in the lipid bilayer, as measured by electron paramagnetic resonance, was greater in normal than in MH membranes. While there were no breaks in Arrhenius plots for probe motion in the erythrocyte ghosts, the apparent activation energy for probe motion was significantly greater in normal than in MH ghost membranes. While there was no break in the Arrhenius plot of probe motion in normal intact red blood cell membranes, there were breaks in the Arrhenius plot of probe motion at both 24 and 33 degrees C in intact MH red blood cell membranes. Based on the altered temperature dependence of Mg-ATPase activity and spin probe motion in membranes derived from MH red blood cells, we conclude that there may be a generalized membrane defect in MH pigs which is reflected in the red blood cell as an altered membrane composition or organization.