The hamster transferrin receptor contains Ser/Thr-linked oligosaccharides: use of a lectin-resistant CHO cell line to identify glycoproteins containing these linkages.
We recently reported that the human transferrin receptor (TfR) contains O-linked GalNAc residues [1]. To investigate whether this modification is shared by transferrin receptors in other mammals, we investigated the glycosylation of TfR in hamster cells. To facilitate our analysis the lectin-resistant Chinese hamster ovary (CHO) cell line Lec8 was used. These cells are unable to galactosylate glycoproteins, resulting in truncation of the Ser/Thr-linked oligosaccharides to a single residue of terminal alpha-linked GalNAc. This structure is bound with high affinity by the lectin Helix pomatia agglutinin (HPA). The TfR was affinity purified from Lec8 cells metabolically radiolabeled with [3H]glucosamine and the receptor was found to bind tightly to HPA-Sepharose. Treatment of the purified TfR with mild alkaline/borohydride released [3H]GalNAcitol, demonstrating the presence of O-linked GalNAc. We also found that many other unidentified [3H]glucosamine-labeled glycoproteins from Lec8 cells were bound by HPA-Sepharose. The bound and unbound glycoproteins were separated by SDS/PAGE and individual species were selected for treatment with mild base/borohydride. Treatment of glycoproteins bound by HPA, but not those unbound, resulted in the release of [3H]GalNAcitol. These studies demonstrate both that the hamster TfR contains O-linked oligosaccharides and that this approach may have general utility for identifying the presence of these oligosaccharides in other glycoproteins.
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Department of Biochemistry, University of Georgia, Athens 30602.
Cummings
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D000818:Animals; D016466:CHO Cells / Q000187:drug effects; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D006224:Cricetinae; D004351:Drug Resistance / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry; D037102:Lectins; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis*; D011990:Receptors, Transferrin / Q000737:chemistry*; D012694:Serine / Q000737:chemistry*; D013912:Threonine / Q000737:chemistry*
D006023:Glycoproteins; D037102:Lectins; D009844:Oligosaccharides; D011990:Receptors, Transferrin; D013912:Threonine; D012694:Serine