O-linked fucose in glycoproteins from Chinese hamster ovary cells.
We report our discovery that many glycoproteins synthesized by Chinese hamster ovary (CHO) cells contain fucose in O-glycosidic linkage to polypeptide. To enrich for the possible presence of O-linked fucose, we studied the lectin-resistant mutant of CHO cells known as Lec1. Lec1 cells lack N-acetylglucosaminyltransferase I and are therefore unable to synthesize complex-type N-linked oligosaccharides. Lec1 cells were metabolically radiolabelled with [6-3H]fucose and total glycoproteins were isolated. Glycopeptides were prepared by proteolysis and fractionated by chromatography on a column of concanavalin A (Con A)-Sepharose. The sets of fractionated glycopeptides were treated with mild base/borohydride to effect the beta-elimination reaction and release potential O-linked fucosyl residues. The beta-elimination produced [3H]fucitol quantitatively from [3H]fucose-labelled glycopeptides not bound by Con A-Sepharose, whereas none was generated by treatment of glycopeptides bound by the lectin. The total [3H]fucose-labelled glycoproteins from Lec1 cells were separated by SDS-PAGE and detected by fluorography. Treatment of selected bands of detectable glycoproteins with mild base/borohydride quantitatively generated [3H]fucitol. Pretreatment of the glycoproteins with N-glycanase prior to the SDS-PAGE method of analysis caused an enrichment in the percentage of radioactivity recovered as [3H]fucitol. Trypsin treatment of [3H]fucose-labelled intact CHO cells released glycopeptides that contained O-linked fucose, indicating that it is present in surface glycoproteins. These findings demonstrate that many glycoproteins from CHO cells contain O-linked fucosyl residues and raise new questions about its biosynthesis and possible function.
Stults
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University of Georgia, Department of Biochemistry, Athens 30602.
Cummings
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RD
D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity; D002854:Chromatography, Paper; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005643:Fucose / Q000032:analysis* / Q000096:biosynthesis; D006020:Glycopeptides / Q000737:chemistry; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry*; D037102:Lectins
D006020:Glycopeptides; D006023:Glycoproteins; D037102:Lectins; D005643:Fucose