Use of inhibitors to characterize intermediates in the processing of N-glycans synthesized by insect cells: a metabolic study with Sf9 cell line

The most frequent tape of N-glycan synthesized by lepidopteran Sf9 cells ap pears to be fucosylated Man(3)GlcNAc(2), and this has been a limitation for a large scale production and utilization of therapeutic glycoproteins in c ultured insect cells. The current knowledge of the protein glycosylation pa thway derived from structural studies on recombinant glycoproteins expresse d by using baculovirus vectors. In this work we provide more direct evidenc e for the sequential events occurring in the processing of endogenous N-gly coproteins of noninfected Sf9 cells. By metabolic labeling with radioactive mannose, we characterized the glycan structures which accumulated in the p resence of processing inhibitors (castanospermine and swainsonine) and in t he presence of an intracellular trafficking inhibitor (monensin). We thus d emonstrated that from the glycan precursor Glc(3)Man(9)GlcNAc(2) to GlcNAc- Man(5)(Fuc)GlcNAc(2) intermediate, the processing pathway in Sf9 cells para lleled the one demonstrated in mammalian cells. By using monensin, we demon strated the formation of Man(3)(Fuc)GlcNAc(2) from GlcNAcMan(3)(Fuc)GlcNAc( 2), a reaction which has not been described in mammalian cells. Our results support the idea that the hexosaminidase activity is of physiological rele vance to the glycosylation pathway and is Golgi located.