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 [H-3]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 I-125-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 B-phosphate, and consequently it appears unlikely that these cells utilize the mannose 6-phosphate receptor mediated pathway for targeting of lysosomal enzymes.