Expression of human glycophorin A in wild type and glycosylation-deficient Chinese hamster ovary cells. Role of N- and O-linked glycosylation in cell surface expression (original) (raw)
Related papers
Glycoconjugate Journal, 1994
Alterations in N-and O-linked glycosylation affect cell surface expression and antigenicity of recombinant glycophorin A expressed in transfected Chinese hamster ovary (CHO) cells. To understand these effects further, glycophorin A was purified by immunoaffinity chromatography from transfected wild type and glycosylation deficient CHO cells. The O-glycans were characterized both biochemically, using gel filtration and high performance anion exchange chromatography, and immunologically, using carbohydrate specific monoctonal antibodies to probe Western blots. The O-glycans of human erythrocyte glycophorin A consist mainly of short oligosaccharides with one, two, or three sialic acid residues linked to a common disaccharide core, Gat/~l-3GalNAccd-Ser/Thr, with the disialylated structure being the most abundant. With the exception of the trisialylated derivative, the same structures were found on recombinant glycophorin A expressed by wild type CHO cells. However, in contrast to human erythrocyte glycophorin A, the monosialylated oligosaccharide was the most abundant structure on the recombinant protein. Furthermore, recombinant glycophorin A was shown to express a small amount of the Tn antigen (GalNAccO-Ser/Thr). Recombinant glycophorin A had the same O-glycan composition, whether purified from clones expressing high or moderate levels of the recombinant glycoprotein. This indicates that the level of expression of the transfected glycoprotein did not affect its O-glycan composition. Deletion of the N-linked glycosylation site at Asn26, by introducing the Mi.I mutation (Thr~8 -~ Met) by site-directed mutagenesis, did not markedly affect the O-glycan composition of the resulting recombinant glycoprotein expressed in wild type CHO cells. This demonstrates that the presence or absence of the N-glycan did not influence O-linked glycosylation of the recombinant glycoprotein. Finally, the O-gtycans on recombinant glycophorin A expressed in the Lec 2 and Lec 8 glycosylation deficient CHO cells were characterized. The O-glycans on Lec 2 cell glycophorin A were predominantly Gal/~l-3GalNAc~l-Ser/Thr (T antigen), while those on Lec 8 glycophorin A were exclusively GalNAcel-Ser/Thr (Tn antigen). These results will lead to a better understanding of the cell biology and immunology of this important human erythrocyte glycoprotein.
Blood, 1993
Glycophorin A is a heavily glycosylated glycoprotein (1 N-linked and 15 O-linked oligosaccharides) and is highly expressed on the surface of human red blood cells. It is important in transfusion medicine because it carries several clinically relevant human blood group antigens. To study further the role of glycosylation in surface expression of this protein, four mutations were separately introduced into glycophorin A cDNA by site-directed mutagenesis. Each of these mutations blocks N-linked glycosylation at Asn26 of this glycoprotein by affecting the Asn-X-Ser/Thr acceptor sequence. Two of these mutations are identical to the amino acid polymorphisms found at position 28 in the Mi.I and Mi.II Miltenberger blood group antigens. The mutated recombinant glycoproteins were expressed in transfected wild-type and glycosylation-deficient Chinese hamster ovary (CHO) cells. When expressed in wild-type CHO cells and analyzed on Western blots, each of the four mutants had a faster electrophor...
Monoclonal antibodies specific for the M- and N-forms of human glycophorin A
Molecular Immunology, 1983
Four mouse monoclonal antibodies directed against the red cell membrane protein glycophorin A have been isolated and characterized. They are produced by hybridomas derived from SP2/0 myeloma cells and spleen cells from Biozzi mice immunized with a mixture of human erythrocytes from homozygous blood group M and N individuals. These antibodies recognize and bind to purified glycophorin A and to glycophorin on the red cell surface. All are of the IgGl, kappa light chain subclass and bind to determinants presented on the 39 amino acid, trypsin-sensitive, N-terminal peptide of glycophorin A. Three display differential specificities for the two allelic forms of glycophorin A; two are exquisitely specific for the M-form and one preferentially binds the N-form. Treatment of red cells with neuraminidase, which removes N-acetylneuraminic acid from glycophorin A, abolishes the binding of these three antibodies. The binding of the N-specific antibody is also sensitive to modification of the amino-terminal residue of the antigen. The fourth antibody binds equally well to both the M-and N-forms as well as to neuraminidase-treated red cells; thus it recognizes a public, N-acetylneuraminic acid independent glycophorin A determinant.
The binding of human alloantibodies to recombinant glycophorin A
Transfusion, 1992
Chinese hamster ova (CHO) cells were transfected with the wild-type, M allele of glycophorin A cDNA. f i e binding of human alloantibodies to recombinant glycophorin A was assessed with a modified hemagglutination-inhibition assay. Patient sera were incubated with acetone powders derived from CHO cells, and the adsorbed supernatants were tested in standard hemagglutination assays. Five M antibodies and one sample containing anti-En' bound to transfected CHO cells expressing glycophorin A but did not bind to untransfected CHO cells. Three N antibodies as well as 21 other alloantibodies (representing other major red cell blood group specificities) bound to neither CHO cell line. The M allele specificity of recombinant gl cophorin A was further verified by the demonstration that a high-titer D alloantibodYy maintained the same titer of agglutination after incubation with recombinant glycophorin A. Transfected CHO cells thus express an M blood group antigen that appears to be serologically equivalent to that found on human red cells. A panel of cell lines expressing mutant glycophorin A molecules with defined variations in amino acid sequence and carbohydrate composition will be useful in studies of the fine specificity of human glycophorin alloantibodies. This approach .may also provide an abundant source of artificial antigens for clinical use in blood group serology. TRANSFUSION 1992;32:62S 632.
Species specificity in mouse glycophorin
Biochemical and Biophysical Research Communications, 1991
89, 65, 46 and 29 Kd mouse glycophorin proteins identified during polyacrylamide gel electrophoresis of mouse erythrocytes have been further characterized. These proteins (1) sta.ilagd positive with Periodic Acid Schiff reagent after sodium hydroxide treatment; (2) labeled using [lz3I] in intact cells; (3) co-isolated along with integral membrane proteins in the pellet fraction of sodium hydroxide treated ghosts; and (4) demonstrated a molecular weight downshift after neuraminidase treatment during electrophoresis. We have called them mouse Sialoglycoproteins 1,2,3 and 4. Immuno-blot analysis revealed distinct species specificity between human and mouse erythrocyte ghosts, and some cross-reactivity between rat and mouse erythrocyte ghosts. ® 1991 Academic Press, Inc. Glycophorin represents the major family of sialoglycoproteins (SGP) on the surface of mature human red blood cells and has been studied extensively as a model for transmembrane glycoproteins (1 for review). Glycophorin has been shown to be present on the surface of mature erythrocytes of several species (2,3,4,5,6,7,8,9,10). Both Hudson et al. (11) and Hamada (personal communication, Showa University, Japan) have made comparisons of the composition of red cell membrane glycoproteins from these species and have found significant differences in their amino acid and carbohydrate composition. This report demonstrates the presence of four distinct major murine sialoglycoproteins whose antisera demonstrates species specificity between human and murine glycophorin. While the function of glycophorin in normal red blood cells has not yet been specifically delineated, glycophorin is believed to play a role in the recognition and/or invasion of erythrocytes by malarial parasites and other microorganisms (1,12,13,14). Characterization of murine glycophorin may be used in the formation of a model mammalian system for the study of hostparasite interaction. MATERIALS AND METHODS Red cell ghost preparation and treatment. RBC ghost for all experiments conducted were prepared from packed RBC of fre]l~lcv collected and washed heparinized blood (30 units heparin/ml). Packed cells were labeled with [ ~aI] using lactoperoxidase/glucose oxidase (a modification of the Hubbard and Cohn (15) procedure), or hemolyzed unlabeled in phosphate buffer (16), The yield was approximately 3.5 ug protein/ul of ghost. Whole packed red cells were treated with 1.1 unit/ml of neuraminidase (N'ase) at 37°C for 10 and 90 minutes, with and without subsequent treatment of the ghost preparation with 0.1N NaOH, where indicated.
Transfusion clinique et biologique : journal de la Société française de transfusion sanguine, 1997
Recombinant wild-type and variant forms of glycophorin A (GPA) were expressed in wild-type and glycosylation-defective Chinese hamster ovary cells. The binding to recombinant GPA of antibodies submitted to the Third International Workshop was assessed by immunoprecipitation and indirect immunofluorescence. This is a powerful approach for determining the fine specificity of antibodies to blood group antigens. The advantages and limitations of this approach are discussed.
Antigenicity of rat erythrocyte glycophorins
Immunology Letters, 1988
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 polypeptide chains in various combinations.
Isolation of cDNA clones and complete amino acid sequence of human erythrocyte glycophorin C
The Journal of biological chemistry, 1986
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...
Molecular Genetics of Human Erythrocyte Sialoglycoproteins Glycophorins A, B, C, and D
Blood Cell Biochemistry, 1990
Nomenclature and Properties of the Major Red Cell Membrane Glycoproteins Red cell membrane glycophorinsa Properties Synonyms Fairbanks et al. (1971) Dahr et al. (1975) Anstee et al. (1979) Blood group antigens PAS-positive material(%) Apparent molecular mass (kDa)h Copies/cell (X J0-3)c Polypeptide chain (residues) 0-linked sugar chains N-linked sugar chains Percent solubilized by Triton X