Glycosylation of recombinant prorenin in insect cells: the insect cell line Sf9 does not express the mannose 6-phosphate recognition signal.
Sf9 cells infected with a recombinant baculovirus containing the gene for human prorenin were cultured in the presence of [3H]mannose. In vivo labeled prorenin was isolated by immunoprecipitation from the culture medium and digested with Pronase. The oligosaccharide structures on the resulting glycopeptides were analyzed by a combination of lectin, ion-exchange, paper, and high-pressure liquid chromatography. Of the N-linked oligosaccharides isolated from the Sf9-produced prorenin, 98% were of a truncated (trimannosyl) high-mannose type, approximately two-thirds of which contained a fucose residue linked to the reducing N-acetylglucosamine. The remaining 2% constituted a mixture of high-mannose-type structures containing six, seven, or eight mannose residues; none of these structures were core-fucosylated. None of the oligosaccharide structures recovered from recombinant prorenin synthesized by Sf9 cells were phosphorylated or contained any other form of charge. Furthermore, assays for UDP-GlcNAc-lysosomal-enzyme N-acetylglucosamine phosphotransferase demonstrated no activity above background in lysates prepared from Sf9 cells. Blotting of Sf9 cell lysates with an 125I-labeled, soluble form of the cation-independent mannose 6-phosphate receptor failed to detect any proteins carrying the mannose 6-phosphate recognition signal. Taken together, the data suggest that Sf9 cells do not synthesize high-mannose-type oligosaccharides containing mannose 6-phosphate, and consequently it appears unlikely that these cells utilize the mannose 6-phosphate receptor mediated pathway for targeting of lysosomal enzymes.
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Department of Biochemistry, Upjohn Company, Kalamazoo, Michigan 49001.
Elhammer
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D000818:Animals; D002240:Carbohydrate Sequence; D002460:Cell Line; D004792:Enzyme Precursors / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D008247:Lysosomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology*; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D012083:Renin / Q000378:metabolism*; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism
D004792:Enzyme Precursors; D011498:Protein Precursors; D017527:Receptor, IGF Type 2; D011994:Recombinant Proteins; D017855:Transferases (Other Substituted Phosphate Groups); C030012:UDP-N-acetylglucosamine-lysosomal-enzyme-N-acetylglucosaminephosphotransferase; D012083:Renin