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 v ivo labeled prorenin was isolated by immunoprecipitation from the cult ure medium and digested with Pronase. The oligosaccharide structures o n 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 t wo-thirds of which contained a fucose residue linked to the reducing N -acetylglucosamine. The remaining 2% constituted a mixture of high-man nose-type structures containing six, seven, or eight mannose residues; none of these structures were core-fucosylated, None of the oligosacc haride structures recovered from recombinant prorenin synthesized by S f9 cells were phosphorylated or contained any other form of charge. Fu rthermore, assays for UDP-GlcNAc-lysosomal-enzyme N-acetylglucosamine phosphotransferase demonstrated no activity above background in lysate s prepared from Sf9 cells. Blotting of Sf9 cell lysates with an I-125- labeled, soluble form of the cation-independent mannose 6-phosphate re ceptor 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 uti lize the mannose 6-phosphate receptor mediated pathway for targeting o f lysosomal enzymes.