Isolation of cDNA clones for human erythrocyte membrane sialoglycoproteins alpha and delta.

We have isolated almost full-length cDNA clones corresponding to human erythrocyte membrane sialoglycoproteins alpha (glycophorin A) and delta (glycophorin B). The predicted amino acid sequence of delta differs at two amino acid residues from the sequence determined by peptide sequencing. The sialoglycoprotein delta clone we have isolated contains an interrupting sequence within the region that gives rise to the cleaved N-terminal leader sequence for the protein and represents a product that is unlikely to be inserted into the erythrocyte membrane. Comparison of the cDNA sequences of alpha and delta shows very strong homology at the DNA level within the coding regions. The two mRNA sequences are closely related and differ by a number of clearly defined insertions and deletions.     

Characterization of O-glycosidically linked oligosaccharides of rat erythrocyte membrane sialoglycoproteins

The carbohydrate units of the rat erythrocyte membrane sialoglycoprotein rSGP-4 [Edge, A. S. B., & Weber, P. (1981) Arch. Biochem. Biophys. 209, 697-705] have been characterized. All of the carbohydrate of this Mr 19,000 glycoprotein occurs in O-glycosidic linkage to the peptide; following alkaline borohydride treatment and chromatography on Bio-Gel P-2, sialic acid containing oligosaccharides terminating in N-acetylgalactosaminitol were obtained. Their structures were determined by compositional analysis, exoglycosidase digestions, alkaline sulfite degradation, and periodate oxidation. The oligosaccharides were characterized for molecular weight and linkage by direct chemical ionization and gas-liquid chromatography/mass spectrometry, respectively. The structures are proposed to be NeuAc alpha 2----3Gal beta 1----3GalNAc-ol, Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, and NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)GalNAc-ol. Two of the N-acetylglucosamine-containing hexasaccharides were present per molecule of rSGP-4 along with two trisaccharides and seven tetrasaccharides.     

Purification and characterization of the major sialoglycoproteins of the rat erythrocyte membrane

The major sialoglycoproteins of the rat erythrocyte membrane were purified by hot phenol partitioning followed by cation-exchange chromatography on SP-Sephadex. Further purification was obtained by extraction with n-butanol and anion-exchange chromatography on DEAE-cellulose. The resulting sialoglycoprotein fraction was free of lipids and nonsialylated glycoproteins and gave rise to four major periodic acid-Schiff staining bands when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fastest migrating protein on these gels with an apparent molecular weight of 19,000 was purified to homogeneity by gel filtration. The amino acid and sugar compositions of these materials are reported. The protein moiety is rich in serine, threonine, and hydrophobic amino acids and the carbohydrate moiety is high in sialic acid and N-acetylgalactosamine. Most of the carbohydrate is linked O-glycosidically to serine and threonine residues, as shown by susceptibility to base-catalyzed β-elimination and concomitant reduction of serine and threonine to alanine and α-aminobutyric acid and of N-acetylgalactosamine to N-acetylgalactosaminitol in the presence of reducing agents. The significance of these data in light of the known role of the rat erythrocyte membrane sialoglycoproteins in erythropoiesis is discussed. The properties of the rat erythrocyte membrane sialoglycoproteins are compared to those of other species.     

The biosynthesis and fatty acid acylation of the murine erythrocyte sialoglycoproteins

The biosynthesis and post-translocational processing of the murine erythrocyte sialoglycoproteins gp2 and gp3 have been studied on splenic erythroblasts obtained from mice rendered anemic by treatment with phenylhydrazine. A putative precursor-product relationship has been established between gp3 and a peptide (gp3pr) of apparent Mr = 22,000. No precursor to gp2 has been found. gp3pr was selectively and efficiently converted to gp3 in pulse-chase experiments after a 45-60-min chase. [3H]Palmitate labeled a series of splenic cell proteins, including gp2, gp3, and gp3pr. Chemical analyses indicated that the fatty acid is covalently linked to protein by an ester bond. Splenic cells incorporated [3H]galactose in both gp2 and gp3 but not in gp3pr. The results indicate that the murine sialoglycoproteins are modified in succession by fatty acid acylation and terminal glycosylation. [3H]Palmitate labeling appears to be an early modification that affects concomitantly gp3pr and gp3, suggesting that fatty acid acylation is a cytosolic event not obligatorily coupled to translocation.     

Two individuals with elliptocytic red cells apparently lack three minor erythrocyte membrane sialoglycoproteins.

We have studied the erythrocytes of two individuals (P. L. and K. W.) who lack the Gerbich (Ge) blood-group antigen. The erythrocytes of P. L. and K. W. were not reactive with two monoclonal antibodies (NBTS/BRIC 4 and NBTS/BRIC 10) which reacted with normal erythrocytes. The membranes of P. L. and K. W. erythrocytes appeared to lack three minor sialoglycoproteins (beta, beta 1 and gamma). These three minor sialoglycoproteins were found to be associated with the cytoskeletons of normal erythrocytes. Approx. 10% of the erythrocytes of P. L. and K. W. were frankly elliptocytic. We suggest that one or more of the minor sialoglycoproteins may play a part in maintaining the discoid shape of the human erythrocyte.     

Aggregates of human erythrocyte membrane sialoglycoproteins in the presence of deoxycholate and dodecyl sulfate

Gel electrophoresis in the presence of deoxycholate of human erythrocyte membranes solubilized with deoxycholate resolves four glycoprotein zones. Electrophoresis in dodecyl sulfate in a second dimension reveals several components, three of which migrate in the region of PAS-2. One of the zones in deoxycholate gel electrophoresis contains component PAS-3, and this glycoprotein seems to exist as a monomer in deoxycholate, but aggregates partially upon addition of dodecyl sulfate. The major sialoglycoprotein migrates as a diffuse zone in dodecyl sulfate. The major sialoglycoprotein migrates as a diffuse zone in deoxycholate gel electrophoresis, indicating association and dissociation during the electrophoresis. The use of deoxycholate followed by dodecyl sulfate in two-dimentional electrophoresis gave high resolution of membrane proteins and can be used for detection of complexes in one of the detergents.     

Betaine prevents loss of sialic acid residues and peroxidative injury of erythrocyte membrane in ethanol-given rats

To evaluate chronic ethanol toxicity on erythrocyte membrane and preventive action of betaine as a methyl donor, 24 male Wistar albino rats were divided into three groups: control, ethanol and ethanol plus betaine group. Animals were fed 60 ml diet per day for two months. Rats in the ethanol group were fed ethanol 8 g/kg/day. The ethanol + betaine groups were fed ethanol plus betaine (0.5% w/v). After two months, all animals were killed. Malondialdehyde (MDA) and sialic acid (SA) levels were determined in plasma samples. Osmotic fragility tests were performed on whole blood samples and erythrocyte membrane thiol contents were determined using membrane suspensions. Plasma MDA levels in ethanol-given rats were increased significantly compared to the control group of rats (p < 0.05). MDA in the betaine group was significantly lower than that in the ethanol group (p < 0.05). Erythrocyte membrane thiol contents in ethanol group were decreased compared with those of the control group (p < 0.05). Thiol contents were increased slightly after betaine therapy, but this increase was not statistically significant (p > 0.05). Plasma sialic acid levels in the ethanol group were significantly higher than in the control group (p < 0.05). Sialic acid was decreased in the betaine group compared to the ethanol group (p < 0.05). In the osmotic fragility test, we observed that chronic ethanol consumption increased erythrocyte hemolysis. Betaine protected against ethanol-induced hemolysis. Our findings show that chronic ethanol administration affects erythrocyte membrane properties and this may be related to oxidative stress. Betaine protects erythrocyte membrane alterations against chronic ethanol toxicity. Therefore betaine as a nutritional agent, may protect ethanol induced clinical problems associated with membrane abnormalities.     

High-frequency antigens of human erythrocyte membrane sialoglycoproteins, IV. Molecular properties of the U antigen

The nature of the common erythrocyte antigen U, that is absent from S-s-U-cells, which lack glycophorin B (Ss sialoglycoprotein), was investigated using six different antisera. The molecular features of a U-like antigen (Duclos), detected by a hitherto unique serum, were also studied. The U and Duclos antigens are complex in that they exhibit relationships with the MNSs and Rh blood group systems. Various fractionation, cleavage, or modification products of normal erythrocyte membranes were used in hemagglutination inhibition assays. Both, the U and Duclos antigens were found to represent labile structures that require lipids, at least for optimum expression of antigen activity. The antigens could be solubilized using conditions of Triton X-100 extraction that release glycophorin B, but solubilize the Rh antigens only to a small extent. Anti-U and anti-Duclos were also inhibited, albeit weakly, by glycophorin B-containing fractions obtained by chromatographic separation of Triton X-100 extracts. The residues approx. 33-39 of glycophorin B represent essential parts of the U antigen, as judged from proteolytic digestion and chemical modification. Conversely, the expression of Duclos activity seems to require a region of glycophorin B (C-terminal of the positions approx. 34-36) that could not be cleaved by various proteinases. Data obtained with anti-Duclos have to be interpreted with caution, since there is evidence that this serum might contain a mixture of antibodies.     

Protein and lipid components of the pigeon erythrocyte membrane.

The plasma membrane of the nucleated pigeon erythrocyte was isolated by a method that is simple, reproducible and minimally disruptive, the final preparation consisting of whole cell 'ghosts', recovered at over 40% yield. Alternative methods, which yield membrane fragments, were also tested and some of their possible disadvantages demonstrated. Analysis of the protein components of the isolated membranes by gel elctrophoresis in the presence of sodium dodecyl sulphate revealed that their composition is very similar to that of the proteins of human erythrocyte membranes. However, two major proteins are unique to the nucleated cell membrane; these have apparent mol.wts. of 97000 and 57000. Also, the bands designated 4.2 (74500 mol.wt.) and 6 (35000 mol wt.) by Steck [(1974) J. Cell Biol. 62, 1-19] for the human cell membrane are absent from pigon cell membrane. Glycosylated membrane proteins could not be detected in gels stained with the periodate-Schiff-base procedure. Analysis of membrane phospholipids revealed the same components known to be present in mammalian erythrocytes, though in different proportions. These findings are discussed in the light of known physiological and biochemical differences between avian and mature mammalian erythrocytes.     

Heterogeneity of human cell membrane sialoglycoproteins.

Discontinuous sodium dodecysulfate polyacrylamide gel electrophoresis (disc SDS-PAGE) followed by periodic acid/Schiff staining reveals the presence of six sialoglycoprotein bands in human red cell membranes or glycoprotein preparations therefrom. In agreement with previous investigations it is shown that PAS-1 and PAS-2 (mol. weight 37 000) are different forms of the same molecule (MN glycoprotein). Using separation of glycoproteins by the system of Weber and Osborn and reelectrophoresis of gel slices by disc SDS-PAGE it is demonstrated that the minor component C (mol. weight 41 000) represents the dimeric form of PAS-3 (Ss glycoprotein). Band B corresponds to an aggregate of PAS-3 and PAS-2 and/or the trimer of PAS-3 with possible differences between extracted glycoproteins and those present in the membrane. The minor component D (mol. weight 35 000) is, as far as could be elucidated, not involved in aggregation phenomena. Some technical problems of glycoprotein fractionation by SDS-PAGE and the remarkable effect of phosphate buffers on the glycoprotein pattern are discussed.     

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.     

Group C rotavirus requires sialic acid for erythrocyte and cell receptor binding.

Current immunological and biochemical information regarding the hemagglutinin and virus-cell interactions of rotavirus is obtained exclusively from studies with group A rotaviruses. In this study, I report that the immunologically and genetically distinct group C rotavirus also possesses a hemagglutinin. The viral hemagglutinin was identified on a cultivable porcine group C rotavirus strain (strain AmC-1) by using agglutinated human and guinea pig erythrocytes. Neuraminidase treatment of fresh human erythrocytes or blocking with glycophorin A or fetuin prevented hemagglutination. Infection of swine testicular cells with group C AmC-1 virus was also prevented by glycophorin A, fetuin, and neuraminidase treatment, suggesting that sialic acid constitutes an essential part of the cell receptor.     

Reduced diversity of the human erythrocyte membrane sialic acids in polycythemia vera. Absence of N-glycolylneuraminic acid and characterisation of N-acetylneuraminic acid 1,7 lactone

Sialic acids from the erythrocyte (RBC) membrane of a patient suffering from polycythemia vera, a malignant orphan disorder of hematopoietic cells, was studied using GC/MS. We found that the sialic acid diversity of these membranes was drastically reduced since only four entities were identified: Neu5Ac (91.5%) and its 1,7 lactone Neu5Ac1,7L (7.5%) which is absent in normal RBC, Neu4,5Ac(2) (0.50%) and Neu4,5Ac(2) 9Lt (0.50%); in normal RBC, Neu5,7Ac(2), Neu5,9Ac(2), Neu5Ac9Lt, Neu5Ac8S and Neu, as well as traces of Kdn, were also present. Neu5Gc and its O-alkylated or O-acetylated derivatives, which are considered by various authors as cancer markers, were not detected.     

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.     

Uptake of sialic acid by human erythrocyte. Characterization of a transport system

Upon incubation of human red blood cells (RBC) with [4-9-14C] N-acetylneuraminic acid, the cells incorporated this sugar, as demonstrated by the identification of labelled N-acetylmannosamine in the cytosol, as a result of the action of the sialic acid pyruvate-lyase we discovered previously (Biochimie 84 (2002) 655). The mechanism is saturable and indicates the presence of a limited number of transporter molecules in the RBC membrane. This transport process may have relevance to the desialylation of membrane glycoconjugates which occurs during ageing of erythrocytes.