Erythrocyte Glycophorin Research Articles

Flow cytometric analysis of erythrocyte populations in tn syndrome blood using monoclonal antibodies to glycophorin A and the Tn antigen

Flow cytometric analysis employing monoclonal antibodies to the Tn antigen and glycophorin A was used to characterize the erythrocyte populations present in blood samples from individuals with Tn syndrome. Four monoclonal antibodies specific for the Tn antigen, Gal-NAc monosaccharide, on human erythrocytes were obtained from a fusion of splenocytes from a Biozzi mouse immunized with red cells from a Tn individual. These monoclonal antibodies specifically recognize GalNAc monosaccharide sites located on the erythrocyte cell surface sialoglycoproteins, glycophorin A and glycophorin B, and do not bind to fixed normal red cells presenting the Neu-NAc alpha 2-3Gal beta 1-3(NeuNAc alpha 2-6)GalNAc alpha 1-O-Ser(Thr) tetrasaccharide or to fixed neuraminidase-digested cells presenting the Gal-GalNAc disaccharide. The percentages of Tn-positive red cells in samples from six unrelated Tn donors ranged from 28 to 99%. Binding of the glycophorin A-specific monoclonal antibodies showed that the erythrocytes composing the Tn-negative fraction presented normal amounts of the M and N epitopes on glycophorin A. The presumed somatic mutational origin of Tn-positive cells was tested in blood samples from five normal donors; three possible Tn cells were observed after analysis of a total of 1.1 x 10(7) erythrocytes, suggesting that the frequency of such cells in normal individuals is less than 1 x 10(-6).

Ontogenetic expression of the murine erythrocyte glycophorins.

The major sialoglycoproteins or glycophorins of the murine erythrocyte membrane, gp2 (Mr 44,000) and gp3 (Mr 29,000), are expressed as identical or closely related antigens by the three erythroid cell lines that succeed each other in normal mouse development. The embryonic forms of gp2 and gp3 differ from the adult forms by their mobility on SDS-PAGE. The apparent Mr values are increased by 1000-2000 for gp2 and 500 for gp3. An increase in the microheterogeneity of both embryonic and fetal forms of gp2 is also detected. Variations in glycosylation account in part for the apparent differences in Mr. Sizing of O-glycosidically linked oligosaccharide chains reveals that fetal glycophorins contain predominantly trisaccharide units while their adult counterparts are mostly tetrasaccharides. The kinetics of gp2 and gp3 biosynthesis in fetal liver are comparable to those established for the splenic erythroblasts of adult anemic mice. Like their adult counterparts, fetal glycophorins incorporate [3H]palmitate and [3H]galactose. The results indicate that, in murine ontogeny, distinct but antigenically related sialoglycoproteins are produced at each erythropoietic stage.

Phosphorus nuclear magnetic resonance studies of lipid-protein interactions: human erythrocyte glycophorin and phospholipids.

Human erythrocyte glycophorin containing four molecules of phospholipid tightly bound to the protein was isolated from human red cell ghosts. This protein preparation was reconstituted into a digalactosyl diglyceride bilayer. The 31P NMR spectrum of this reconstituted membrane produced an axially symmetric powder pattern arising exclusively from the phospholipids bound to glycophorin. The width of the powder pattern, about 90 ppm, is about twice as broad as that normally exhibited by a phospholipid bilayer. The chemical shift tensor is perturbed relative to phospholipids in a bilayer. The spin-lattice relaxation rate of these protein-bound phospholipids is found to be nearly an order of magnitude faster than phospholipids in a bilayer. The results are consistent with phospholipids tightly bound to the membrane protein and undergoing rotational diffusion, perhaps as a complex of phospholipid and protein.

Human erythrocyte glycophorin C: Gene structure and rearrangement in genetic variants

We have previously shown that a deletion of approximately 3 kilobases in the unique glycophorin C (GPC) gene, which encodes for the human erythrocyte glycophorins C and D, is associated with the Gerbich (Ge) blood group deficiency (Ge-2,-3 and Ge-2,+3 types) (Le van Kim, C., Colin, Y., Blanchard, D., Dahr, W., London, J. & Cartron, J.P. (1987) Eur. J. Biochem. 165, 571-579). We have now isolated and characterized the structure of the GPC gene from the common Ge+2,+3 donors and from a Ge-2,-3 variant (Ge-2,-3 gene). The GPC gene is organized in four exons distributed over 13.5-kilobase pairs (kbp) DNA and contains two directly repeated domains of 3.4 kbp in length which are likely derived from the recent duplication of a unique ancestral domain. Restriction mapping and sequence analysis indicate that a 3.4-kbp deletion within this gene, arising probably by unequal crossing over between the two repeated domains, is responsible for the formation of the Ge-2,-3 gene. The breakpoints of the deletion are located within introns 2 and 3, and therefore exon 3 is removed. The defective gene is transcribed as a mRNA with a continuous open reading frame extending over 300 nucleotides which is translated into an unusual sialoglycoprotein present on Ge-2,-3 red cells. The primary structure of this new glycoprotein has been deduced from nucleotide sequencing. It is proposed in addition, that another 3.4-kb deletion within the GPC gene eliminates exon 2 only by a similar mechanism and generates a defective gene encoding for the abnormal glycoprotein present on Ge-2,+3 erythrocytes. Interestingly, the same deletion which lead to the rare Ge-2,-3 genetic condition, occurred spontaneously and frequently in the cloned GPC gene during the propagation of the recombinant phages in Escherichia coli. From these observations we suggest that the Ge-2,-3 and Ge-2,+3 genes might represent the two allelic forms of a unique ancestral form of the GPC gene, following successive internal duplication and deletion events.

Structure of the majorO-glycosidic oligosaccharide of monkey erythrocyte glycophorin

Sialic acids and the major O-glycosidic oligosaccharide of glycophorin MK from monkey (Japanese monkey, Macaca fuscata) erythrocyte membranes were characterized. N-Glycolylneuraminic acid (Neu5Gc) was found as the major sialic acid, which was confirmed by gas-liquid chromatography-mass spectrometry as the trimethylsilyl methyl ester. Three O-glycosidic oligosaccharide units were obtained from a tryptic glycopeptide that contained all of the carbohydrate units in glycophorin MK by mild alkaline borohydride/borotritide treatment. Carbohydrate analyses of the oligosaccharides revealed that they were composed of Neu5Gc, galactose and N-acetylgalactosaminitol in the molar ratios of 1:1:1 (trisaccharide), 2:1:1 (tetrasaccharide) and 3:1:1 (pentasaccharide). The content of oligosaccharide units was estimated to be 1:12:5 for penta-, tetra- and trisaccharide, respectively, based on the yields, the molecular weight, and the number of oligosaccharide attachment sites in the amino-acid sequence. The tetrasaccharide was the major oligosaccharide and its structure was proposed to be Neu5Gc alpha 2-3Gal beta 1-3[Neu5Gc alpha 2-6]GalNAcol.

Hydrogen exchange from the transbilayer hydrophobic peptide of glycophorin reconstituted in lipid bilayers

The hydrophobic transbilayer peptide of erythrocyte glycophorin has been purified following exchange of tritium into the backbone amides, and reconstituted in egg phosphatidylcholine micelles. Analysis of tritium exchange from the backbone amides of the membrane-reconstituted peptide shows that about two of the amides are virtually non-exchangeable, about 10 are slowed by factors of 10 7 relative to free amides in unstructured water soluble peptides and the remainder of the amides (about 20) have slowing factors of less than 1000. These classes of amides are proposed to reflect the stability of the peptide with respect to hydrogen bond breaking fluctuations and the accessibility of the amides to exchange catalysts in different regions of the bilayer.

Antigenicity of rat erythrocyte glycophorins

The relationship between rat red blood cell (RBC) glycophorins and the antigens recognised by anti-rat RBC antibodies was examined. Initially, murine monoclonal antibodies specific for surface epitopes on whole rat RBCs were tested for their reactivity with RBC membranes on Western blots and two were found which reacted with blotted antigens. These antibodies recognised two bands corresponding to the major PAS-stainable bands of rat RBC membranes (i.e., the glycophorins) and a number of minor bands, thus demonstrating that the bands are antigenically related. This band-pattern was remarkably similar to that obtained with mouse anti-rat RBC serum. Digestion with neuraminidase altered the electrophoretic mobility of most of the bands, providing additional evidence that they are sialoglycoproteins, although sialic acid was shown not to contribute to their antigenicity. The glycophorin nature of the major antigens was verified by reelectrophoresis and blotting of bands excised from SDS gels, which showed that they were interconvertible monomeric and dimeric forms of the same polypeptide chain. It is suggested that rat RBC glycophorins are a related family of sialoglycoproteins with the high molecular weight members being formed by dimerization of five lower molecular weight poly-peptide chains in various combinations.

Monoclonal antibodies against glycophorins

Glycophorins of human erythrocytes have been extensively studied and the structure of three of them is fully (glycophorins A and C) or almost fully (glycophorin B) known [1, 2]. Glycophorins span the erythrocyte membrane and their NH 2 -terminal domains exposed at the cell surface are heavily glycosylated. Glycophorin A occurs in two genetically determined forms carrying at NH 2 -terminal end blood group M and N antigenic determinants. Glycophorin B (blood group Ss glycoprotein) has the structure of NH 2 -terminal region (a.a. residues 1–26) identical to glycophorin A of blood type N, and also shows a high degree of homology with glycophorin A in the internal portion of the molecule, whereas glycophorin C has a different amino acid sequence. The knowledge of structure and orientation in the membrane and genetic differentiation of glycophorins facilitate elucidation of the fine specificity of anti-glycophorin antibodies. The 30 anti-glycophorin-antibodies obtained were tested by agglutination of untreated and modified erythrocytes, immunoblotting, and binding to glycophorin A in microtiter plate ELISA. Moreover, inhibition of antibodies by untreated and modified glycophorin A preparations was studied. The methods used were described in detail in our recent publications [3, 5]. The antibodies could be divided into groups (Table I) , depending on specificity. The 19 antibodies recognized epitopes located at the NH 2 -terminal end of glycophorin A that could be easily shown by specific or distinctly preferable reactivity with blood group M (8 MoAbs) or N (11 MoAbs) antigen. The antibodies with anti-N specificity also reacted with glycophorin B. Among the remaining blood group MN-unrelated antibodies, 4 were specific for glycophorin A, 3 recognized epitopes common for glycophorins A and B, 2 reacted to glycophorin C, and the specificity of 2 antibodies could not be clearly established. The antibodies in each group differed in sub-specificity and antigen-binding properties.

The structure of glycophorins of animal erythrocytes

Glycophorins are red cell membrane sialoglycoproteins, which contain multipleO-linked oligosacchride chains and carry most of the cell surface sialic acid. Due to this high content of sialic acid the glycophorins are strongly stained with periodic acid-Schiff (PAS) reagent after sodium sodicylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The term “glycophorin” was proposed initially for human red cell sialoglycoproteins [1,2] and now it is also used for sialoglycoproteins in animal red cell membranes. Furthermore, similar glycoproteins of non-erythrocyte origin have also been identified and given the same name [3], although the terms “leukosialin” and “sialophorin” were proposed for a major sialoglycoproteins of human leukocytes [4,5]. In this article the term “glycophorin” will be used only for sialoglycoproteins existing in the erythrocyte membrane. Glycophorins of human erythrocytes, carrying blood group MN, Ss and other determinants, have been thoroughly studied and their properties described in several review articles [3,6,7,8]. The aim of this article is to summarize studies carriedout on the structure of non-human glycophorins, although some data concerning human glycophorins are included for comparative purposes.

Biogenesis of glycophorin A in K562 human erythroleukemia cells.

A monoclonal antibody (mAb-233) directed against an epitope in the nonglycosylated carboxyl-terminal region of human erythrocyte glycophorin A (GPA) was used in combination with metabolic labeling, the modification of N- and O-linked oligosaccharide processing by tunicamycin and monensin, and digestions with neuraminidase and O-glycanase to elucidate the pathway of GPA biogenesis in K562 human erythroleukemia cells. Cell-surface GPA is derived from two obligatory precursors in a stepwise manner. The initial GPA precursor has a Mr of 27,000 and appears to contain one N-linked high mannose oligosaccharide chain. In tunicamycin-treated cells, the initial precursor is similar in size (Mr = 24,000) to deglycosylated GPA from human erythrocytes. The 27-kDa initial precursor is rapidly converted to a transient 31-kDa intermediate by the addition of N-acetylgalactosamine residues to serine/threonine hydroxyl groups. Subsequent maturation involves the conversion of the high mannose chain to a complex-type oligosaccharide and the concomitant addition of galactose and sialic acid to internal N-acetylgalactosamine residues to extend the O-linked chains. These results define a single, stepwise processing pathway for the generation of all cell-surface GPA molecules and document for the first time the occurrence of both a unique initial precursor that contains a high mannose N-linked oligosaccharide chain but no O-linked sugars and a transient intermediate that appears to contain the same N-linked group and N-acetylgalactosamine at multiple serine/threonine residues. The properties of the intracellular GPA precursors and the relatively simple nature of the processing pathway reported herein contrast markedly with the characteristics of three intermediates and the complexity of two independent pathways in previously postulated schemes for GPA biogenesis (Gahmberg, C. G., Jokinen, M., Karhi, K. K., Kampe, O., Peterson, P. A., and Andersson, L. C. (1983) Methods Enzymol. 96, 281-298; Jokinen, M., Andersson, L. C., and Gahmberg, C. G. (1985) J. Biol. Chem. 260, 11314-11321).

Structures of novel sialylated O-linked oligosaccharides isolated from human erythrocyte glycophorins.

The O-linked oligosaccharides attached to human erythrocyte glycophorins were extensively characterized. In addition to the previously described disialylated tetrasaccharide, NeuNAc alpha 2----3Gal beta 1----3 (Neu-NAc alpha 2----6)GalNAcOH and monosialylated trisaccharide, NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, novel trisialylated oligosaccharides were isolated. Methylation analysis, fast atom bombardment-mass spectrometry, and enzymatic degradation were used to elucidate the following novel structures: formula; see text: These results suggest that O-linked oligosaccharides with a disialosyl group, NeuNAc alpha 2----8NeuNAc alpha 2----, may be present in various tissues.

Membrane glycophorins of Dantu blood group erythrocytes.

Glycophorins of erythrocytes of two unrelated individuals who exhibit the Dantu blood group phenotype were studied. Immunoblots indicated that erythrocytes of each individual contained a complement of a normal alpha-glycophorin (glycophorin A) and a variant N-glycophorin. delta-Glycophorin (glycophorin B) was present in one donor's cells but not the other's; the s and N phenotypes of the latter's erythrocytes may derive from the variant glycophorin. The variant glycophorin is of a smaller size, does not bind to Lens culinaris lectin agarose, and lacks residues approximately 40-60 of alpha-glycophorin and its single asparagine-linked carbohydrate; it contains approximately 2 less O-glycosidically bound units whose structures are identical to those found in alpha-glycophorins. All these properties are characteristic of delta-glycophorin. The variant is related to alpha-glycophorin in the carboxyl-terminal region as shown by reaction with a specific antiserum. Sequence analyses of a mixture of chymotryptic peptides of a CNBr fragment of the variant glycophorin identified the sequence Val-His-Arg-Phe-Thr-Val-Pro-Glu-Ile-Thr-Leu-Ile-Ile that contains the junction point of delta- and alpha-glycophorins spanning residues 33-38/39 of delta-glycophorin and residues 71/72-77 of alpha-glycophorin. Sequence analysis of a mixture of CNBr fragments allowed us to conclude that the variant originates from delta-s- rather than delta-S-glycophorin. The quantity of the variant Dantu glycophorin when compared to alpha-glycophorin differed in the two individuals, the ratio being 2/1 in one individual's cells and 0.5/1 in the other's. This may reflect that the two donors belong to different varieties of Dantu phenotypes. Together, the evidence indicates that both donors' erythrocytes contain a (delta-alpha) variant glycophorin, whose amino terminus originates from delta-s-glycophorin and the carboxyl end from alpha-glycophorin with a junction point around residues 39 of delta- and 71 of alpha-glycophorins. The results suggest that the unique junction region may be characteristic of the Dantu phenotype.

Human erythrocyte antigens. III. Characterization of a panel of murine monoclonal antibodies that react with human erythrocyte and erythroid precursor membranes.

Human erythrocyte membrane proteins express antigens which serve as markers for erythroid differentiation as well as targets for human blood group alloantibodies. We have produced and characterized a new panel of five monoclonal antibodies to erythrocyte membrane proteins. Three monoclonal antibodies (E3, E4, E5) were specific for erythrocyte glycophorins. One antibody (E3) identified the sialoglycoprotein alpha and beta homologous regions proximal to the plasma membrane, a second antibody (E4) was specific for sialoglycoprotein alpha, while a third (E5) was a sialoglycoprotein-beta-specific antibody. Two antibodies (E6 and TE10) to the 65,000-dalton chymotrypsin cleavage product of band 3 were also produced. These antibodies constitute a new panel of probes for investigation of normal erythroid differentiation, erythroleukemia, and the expression of normal and anomalous blood group antigens.

Human Erythrocyte Antigens

Human erythrocyte membrane proteins express antigens which serve as markers for erythroid differentiation as well as targets for human blood group alloantibodies. We have produced and characterized a new panel of five monoclonal antibodies to erythrocyte membrane proteins. Three monoclonal antibodies (E3, E4, E5) were specific for erythrocyte glycophorins. One antibody (E3) identified the sialoglycoprotein α and β homologous regions proximal to the plasma membrane, a second antibody (E4) was specific for sialoglycoprotein a, while a third (E5) was a sialogylcoprotein-β-specific antibody. Two antibodies (E6 and TE10) to the 65,000-dalton Chymotrypsin cleavage product of band 3 were also produced. These antibodies constitute a new panel of probes for investigation of normal erythroid differentiation, erythroleukemia, and the expression of normal and anomalous blood group antigens.

Structure of human erythrocyte glycophorin C deduced from cDNA analysis

Structure of human erythrocyte glycophorin C deduced from cDNA analysis

Isolation of cDNA clones and complete amino acid sequence of human erythrocyte glycophorin C.

Two cDNA clones for glycophorin C, a transmembrane glycoprotein of the human erythrocyte which carries the blood group Gerbich antigens, have been isolated from a human reticulocyte cDNA library. The clones were identified with a mixture of 32 oligonucleotide probes (14-mer) which have been synthetized according to the amino acid sequence Asp-Pro-Gly-Met-Ala present in the N-terminal tryptic peptide of the molecule. The primary structure of glycophorin C deduced from the nucleotide sequence of the 460 base-pair insert of the pGCW5 clone indicates that the complete protein is a single polypeptide chain of 128 amino acids clearly organized in three distinct domains. The N-terminal part (residues 1-57, approximately) which is N- and O-glycosylated is connected to a hydrophilic C-terminal domain (residues 82-128, approximately) containing 4 tyrosine residues by a hydrophobic stretch of nonpolar amino acids (residues 58-81, approximately) probably interacting with the membrane lipids and permitting the whole molecule to span the lipid bilayer. Northern blot analysis using a 265-base-pair restriction fragment obtained by DdeI digestion of the inserted DNA shows that the glycophorin C mRNA from human erythroblasts is approximately 1.4 kilobases long and is present in the human fetal liver and the human K562 and HEL cell lines which exhibit erythroid features. The glycophorin C mRNA, however, is absent from adult liver and lymphocytes, indicating that this protein represents a new erythrocyte-specific probe which might be useful to study erythroid differentiation.

Cross polarization P-31 nuclear magnetic resonance of phospholipids

P-31 single-pulse and cross-polarization (CP) nuclear magnetic resonance spectra were obtained of aqueous dispersions of pure phospholipids. Dimyristoyl phosphatidylcholine, dipalmitoylphosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, egg phosphatidylcholine, bovine brain sphingomyelin, and transphosphatidylated (from egg phosphatidylcholine) phosphatidylethanolamine were studied. The spectra from all the phospholipids, taken in the usual single-pulse mode, showed the pseudo-axially symmetric powder pattern typical of phospholipids in a hydrated lamellar form. P-31 CP spectra of all the phosphatidylcholines and phosphatidylethanolamine revealed a decrease in intensity in the vicinity of the isotropic chemical shift as long as the lipid was above the gel-to-liquid crystalline phase transition temperature. This intensity pattern has been observed previously for C-13 CP spectra of molecules rotating rapidly about a single well-defined axis (e.g., solid benzene) (Pines, A., M.G. Gibby, and J.S. Waugh, 1973, J. Chem. Phys., 59:569-590). Pure lipid dispersions below their gel-to-liquid crystalline phase transition temperature, including dipalmitoylphosphatidylcholine and sphingomyelin, do not exhibit a local minimum in the CP spectrum at the position of the isotropic chemical shift. Thus, below the phase transition temperature, there is not the same rapid rotation of the headgroup about a well-defined axis. A dramatic change in the rate of headgroup rotation is shown to take place at the pretransition of dipalmitoylphosphatidylcholine. P-31 CP spectra were also obtained for bovine rod outer segment disk membranes, rabbit muscle sarcoplasmic reticulum membranes, a total lipid extract of the latter, and a recombined membrane containing human erythrocyte glycophorin. The CP spectra were similar to the single-pulse spectra, indicating a substantial difference in behavior from pure phospholipid dispersions. This is interpreted in terms of a slower headgroup rotation.

Structural studies of O-glycosidic oligosaccharide units of dog erythrocyte glycophorin

Dog glycophorin, the major sialoglycoprotein of dog erythrocyte membranes, contains either N-acetyl- or N-glycolylneuraminic acid, depending upon the strain of dog. Glycolipids also contained the same sialic acid as those found in glycophorin when both materials are prepared from erythrocyte membranes of individual dogs. The O-glycosidic oligosaccharides were released from glycophorin, prepared from individual dogs, by alkaline borohydride treatment, and purified by gel filtration and ion-exchange chromatography. The structures of the reduced oligosaccharides were determined by methylation analysis and gas-liquid chromatography-mass spectrometry. The O-glycosidic oligoscharides identified were one tetrasaccharide - Neu5Ac(2→3)Gal)1→3)[Neu5Ac(2→6)[GalNAcol - two trisaccharides - Neu5Ac(2→3)Gal1→3)GaINAcol and Gal(1→3)[Neu5Ac(2→6)]GalNAcol.

Plasmodium falciparum malaria: band 3 as a possible receptor during invasion of human erythrocytes.

Human erythrocyte band 3, a major membrane-spanning protein, was purified and incorporated into liposomes. These liposomes, at nanomolar concentrations of protein, inhibited invasion of human erythrocytes in vitro by the malaria parasite Plasmodium falciparum. Liposomes containing human band 3 were ten times more effective in inhibiting invasion than those with pig band 3 and six times more effective than liposomes containing human erythrocyte glycophorin. Liposomes alone or liposomes containing erythrocyte glycolipids did not inhibit invasion. These results suggest that band 3 participates in the invasion process in a step involving a specific, high-affinity interaction between band 3 and some component of the parasite.

Purification and characterization of four isolectins of mushroom (Agaricus bisporus).

Four lectins were purified from a mushroom (Agaricus bisporus) by ammonium sulfate fractionation, anion-exchange chromatography, affinity chromatography on bovine submaxillary mucin-Sepharose 4B and preparative isoelectric focusing. They were designated as ABA-I (pI 6.70), II (pI 5.98), III (pI 5.69) and IV (pI 5.53). Polyacrylamide gel electrophoresis of each lectin in the presence of sodium dodecyl sulfate gave a single band with an apparent molecular mass of 16 000 Da. Sedimentation equilibrium analysis suggested that each lectin is a tetramer of subunits. The four lectins were found to have quite similar carbohydrate-binding specificities. The hemagglutination activities of the lectins were effectively inhibited by bovine and porcine submaxillary mucins (BSM and PSM), and NH2-terminal glyco-octapeptides obtained by cyanogen bromide cleavage of human erythrocyte glycophorin A. In addition, desialylated PSM-glycopeptides were more potent inhibitors than untreated PSM-glycopeptides. Among monosaccharides and their glycosides, only methyl N-acetyl-alpha-galactosaminide inhibited lectin binding at a high concentration, but a synthetic oligosaccharide, O-beta-galactopyranosyl-(1----3)-O-(2-acetamido-2-deoxy-alpha-D- galactopyranosyl)-N-tosyl-L-serine, was a strong inhibitor.