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. 1979 Mar;76(3):1208–1212. doi: 10.1073/pnas.76.3.1208

Glycosylation of human chorionic gonadotropin in mRNA-dependent cell-free extracts: Post-translational processing of an asparagine-linked mannose-rich oligosaccharide

Malgorzata Bielinska 1,2,3, Irving Boime 1,2,3
PMCID: PMC383219  PMID: 286306

Abstract

Placental RNA has previously been shown to direct the synthesis of an asparagine-linked mannose-rich glycosylated form of the α subunit of human chorionic gonadotropin (hCG-α) in lysates derived from mouse ascites tumor cells. Glycosylation was dependent on the presence of homologous microsomal membranes, and the glycosylated protein was sequestered into the microsomal vesicles. Here we show that when Triton X-100 is added after 60 min of translation and the incubation is continued, there is a shift of this glycosylated form to new discrete lower molecular weight proteins. The formation of these new proteins was not the apparent result of proteolysis because (i) treatment of the fully glycosylated protein or the proteins formed in the presence of Triton with endoglycosidase H resulted in the formation of a single protein migrating at the same rate on sodium dodecyl sulfate gels; (ii) the migration of nonglycosylated hCG-α synthesized in the presence of membranes isolated from tunicamycin-pretreated ascites tumor cells was not changed upon Triton addition; and (iii) the Triton-induced change was inhibited by mannonolactone, yeast mannan, and purified mannose oligosaccharides. It was also shown that little processing of the mannose-rich glycoprotein occurred in the presence of microsomal membranes alone. However, addition of the ribosome-free supernatant fraction to the glycoprotein resulted in processing. These data suggest that processing of the oligosaccharide core is a compartmentalized process in which removal of sugar, presumably mannose, requires a transfer of the glycoprotein from the endoplasmic reticulum to another component of the secretory cascade.

Keywords: carbohydrate processing in vitro, glycoprotein hormone, cell-free protein synthesis

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Selected References

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