N-LINKED GLYCOSYLATION OF A BACULOVIRUS-EXPRESSED RECOMBINANT GLYCOPROTEIN IN INSECT LARVAE AND TISSUE-CULTURE CELLS

The potential of insect cell cultures and larvae infected with recombi nant baculoviruses to produce authentic recombinant glycoproteins clon ed from mammalian sources was investigated. A comparison was made of t he N-linked glycans attached to secreted alkaline phosphatase (SEAP) p roduced in four species of insect larvae and their derived cell lines plus one additional insect cell line and larvae of one additional spec ies. These data survey N-linked oligosaccharides produced in four fami lies and six genera of the order Lepidoptera, Recombinant SEAP express ed by recombinant isolates of Autographa californica and Bombyx mori n ucleopolyhedroviruses was purified from cell culture medium, larval he molymph or larval homogenates by phosphate affinity chromatography. Th e N-linked oligosaccharides were released with PNGase-F, labeled with 8-aminonaphthalene-13-6-trisulfonic acid, fractionated by polyacrylami de gel electrophoresis, and analyzed by fluorescence imaging. The olig osaccharide structures were confirmed with exoglycosidase digestions. Recombinant SEAP produced in cell lines of Lymantria dispar (IPLB-LdEI ta), Heliothis virescens (IPLB-HVT1), and Bombyx mori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni, H.virescens, B,moli, and D anaus plexippus contained oligosaccharides that were structurally iden tical to the 10 oligosaccharides attached to SEAP produced in T,ni cel l lines. The oligosaccharide structures were all mannose-terminated. S tructures containing two or three mannose residues, with and without c ore fucosylation, constituted more than 75% of the oligosaccharides fr om the cell culture and larval samples.