Abstract
We have investigated the oligosaccharides in the human transferrin receptor from three different cell lines. During our studies on the structures of the N-linked oligosaccharides of the receptor, we discovered that the receptor contains O-linked oligosaccharides. This report describes the isolation and characterization of these O-linked oligosaccharides. Three different human cell lines--K562, A431, and BeWo--were grown in media containing either [2-3H] mannose or [6-3H]glucosamine. The newly synthesized and radiolabeled transferrin receptors were purified by immunoprecipitation from cell extracts and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor was proteolytically digested or treated directly with mild base/borohydride. The released radiolabeled glycopeptides and oligosaccharides were separated by a variety of chromatographic techniques, and their structures were analyzed. The transferrin receptor from all three cell types contains O-linked oligosaccharides that are released from peptide by mild base/borohydride treatment. The receptor from K562 cells contains at least one O-linked oligosaccharide having two sialic acid residues and a core structure of the disaccharide galactose-N-acetyl-galactosamine. In contrast, the O-linked oligosaccharides in the transferring receptors from both A431 and BeWo cell lines are not as highly sialylated and were identified as both the neutral disaccharide galactose-N-acetylgalactosamine and the neutral monosaccharide N-acetylgalactosamine. In addition, the receptors from all three cell lines contain both complex-type and high mannose-type N-linked oligosaccharides. The complex-type chains in the receptor from A431 cells have properties of blood group A antigens, whereas oligosaccharides in receptors from both BeWo and K562 cells lack these properties. These results are interesting since both A431 and BeWo cells, but not K562 cells, are positive for blood group A antigens. Thus, our results demonstrate that the human transferrin receptor contains O-linked oligosaccharides and that there are differences in the structures of both the O-linked and complex-type N-linked oligosaccharides on the receptors synthesized by different cell types.
Highlights
We have investigated the oligosaccharides in the human transferrin receptor from three different cell lines
We have found that the human transferrin receptor contains O-linked oligosaccharides linked through N-acetylgalactosamine to peptide
Both cell lines are agglutinated by antisera to human blood group A, but they are not agglutinated by antisera to human blood group B
Summary
Materials for SDS-polyacrylamide gel electrophoresis and fluorography were purchased from Bio-Rad and Du Pont-New England Nuclear, respectively. Monoclonal antibody against the human transferrin receptor (B3/25) and the goat anti-mouse IgG antibody were purchased from Hybri-Tech. The subconfluent (50-70%) cells were incubated for 24 h in media containing either [2-3H]mannose (1 mCi/ml) or [6-3H]glucosamine (1 mCi/ml). Receptor and SDS-Polyacrylamide GeE Electrophoresis-Metabolically radiolabeled cells were washed with PBS (6.7 mM KH2P04, 150 mM NaCl, pH 7.4) twice and were primarily solubilized with 1.0 ml of 1% Nonidet P-40 in PBS containing the protease inhibitors pepstatin (O.Ol%), aprotinin (O.l%), and phenylmethylsulfonyl fluoride (0.1 mM) at 4 “C for 20 min. The supernatant was mixed with 10 ~1 of B3/25 monoclonal antibody and incubated on ice for 15 min. 15 ~1 of goat anti-mouse IgG was added and incubated for another 15 on ice
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