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. 1978 Apr;75(4):1768–1772. doi: 10.1073/pnas.75.4.1768

mRNA-dependent synthesis of a glycosylated subunit of human chorionic gonadotropin in cell-free extracts derived from ascites tumor cells

Malgorzata Bielinska 1,2,3, Irving Boime 1,2,3
PMCID: PMC392421  PMID: 273908

Abstract

Protein synthesized in ascites cell-free extracts in response to first trimester placental mRNA was immunoprecipitated with antisera directed against the α subunit of human chorionic gonadotropin. The immunoprecipitable proteins were resolved on sodium dodecyl sulfate/polyacrylamide slab gels. In membrane-depleted extracts placental mRNA directed the synthesis of the “preprotein” form of the α subunit. However, when membranes were added to the cell-free extracts a protein migrating more slowly than pre-α subunit was observed. This protein was specifically adsorbed to a concanavalin A column, and its migration on sodium dodecyl sulfate gels was enhanced after treatment with α-mannosidase (EC 3.2.1.24) or endo-β-N-acetylglucosaminidase CII or H. Similar results were obtained when mRNA was translated in lysates derived from first trimester placenta instead of in ascites cell extracts. Kinetic studies of the glycosylation reaction revealed that sugar attachment can occur just prior to release of the protein. These data show that the apoprotein of the α subunit can be glycosylated in vitro. The data also suggest that the glycosylated protein contains a sugar core consisting of di-N-acetylchitobiose and at least four mannose residues, some of which are α-linked. In addition, it appears that this carbohydrate unit is present in homologous placental membranes as well as in the ascites tumor membranes.

Keywords: glycoprotein hormone, secretory protein, in vitro protein synthesis, membrane-dependent sugar transfer

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