Presence of O-linked oligosaccharide on a threonine residue in the human transferrin receptor.
We have previously demonstrated that the human transferrin receptor (TfR) of approximately 90 kDa contains Ser/Thr-linked (O-linked) oligosaccharides. In the present study, we report our identification of the site of attachment of the O-linked oligosaccharides in the receptor. A 70 kDa fragment from the external domain of the TfR was generated by trypsin treatment of the [3H]glucosamine-labelled receptor purified from human K562 cells. The beta-elimination of the intact TfR, but not the 70 kDa fragment, released Gal-[3H]Gal-NAcitol, indicating that the 70 kDa fragment lacks O-linked oligosaccharides. In the remaining 20 kDa fragment there are three potential sites (Thr96, Thr104 and Ser106) for O-glycosylation in the extracellular domain. To identify which of these residues are O-glycosylated, both the [3H]Thr- and [3H]Ser-labelled TfR were directly treated with mild base to effect beta-elimination, and the radiolabelled amino acids and their derivatives were analysed. Approximately 2% of the total radiolabelled Thr, but no radiolabelled Ser, was converted to expected beta-elimination products by this treatment. These and other results demonstrate that only one O-linked oligosaccharide is present in the TfR and that it occurs on either Thr96 or Thr104. From human serum we purified the cleaved, soluble form of the TfR (s-TfR), which contains Thr104, but lacks Thr96. The s-TfR was sensitive to O-glycanase and bound to Jacalin lectin, indicating that the s-TfR contains an O-linked oligosaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Biochemistry, University of Georgia, Athens 30602.
Cummings
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques; D004915:Leukemia, Erythroblastic, Acute; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D011990:Receptors, Transferrin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D013912:Threonine / Q000032:analysis*; D014357:Trypsin; D014407:Tumor Cells, Cultured
D000596:Amino Acids; D009844:Oligosaccharides; D010446:Peptide Fragments; D011990:Receptors, Transferrin; D013912:Threonine; D014357:Trypsin