A method for [3H]mannose labeling of Asn-linked oligosaccharides on recombinant glycoproteins synthesized in Xenopus oocytes.
We have developed an efficient method for labeling the Asn-linked oligosaccharides of recombinant glycoproteins synthesized in Xenopus laevis oocytes. By coinjecting GDP-[3,4-(3)H]mannose with mRNA for human cathepsin D, it was possible to incorporate as much as 1800 cpm per oocyte into each of the two Asn-linked oligosaccharides of this glycoprotein. Overall, about 50% of the microinjected GDP-[3,4-(3)H]mannose was incorporated into Asn-linked oligosaccharides, a 10-fold greater value than that obtained when [2-(3)H]mannose was microinjected. Less than 10% of the injected GDP-[3,4-(3)H]mannose was metabolized to water or converted to amino acids. This technique should facilitate studies of Asn-linked oligosaccharide biosynthesis, processing, and structure in recombinant proteins synthesized in Xenopus oocytes.
Cantor
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Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Kornfeld
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D000818:Animals; D002240:Carbohydrate Sequence; D002402:Cathepsin D / Q000008:administration & dosage; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008358:Mannose / Q000737:chemistry*; D008845:Microinjections; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D009865:Oocytes; D011994:Recombinant Proteins / Q000737:chemistry; D014316:Tritium; D014981:Xenopus
D006023:Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014316:Tritium; D002402:Cathepsin D; D008358:Mannose