title: N-Linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells.
abstract: The potential of insect cell cultures and larvae infected with recombinant baculoviruses to produce authentic recombinant glycoproteins cloned from mammalian sources was investigated. A comparison was made of the N-linked glycans attached to secreted alkaline phosphatase (SEAP) produced in four species of insect larvae and their derived cell lines plus one additional insect cell line and larvae of one additional species. These data survey N-linked oligosaccharides produced in four families and six genera of the order Lepidoptera. Recombinant SEAP expressed by recombinant isolates of Autographa californica and Bombyx mori nucleopolyhedroviruses was purified from cell culture medium, larval hemolymph or larval homogenates by phosphate affinity chromatography. The N-linked oligosaccharides were released with PNGase-F, labeled with 8-aminonaphthalene-1-3-6-trisulfonic acid, fractionated by polyacrylamide gel electrophoresis, and analyzed by fluorescence imaging. The oligosaccharide structures were confirmed with exoglycosidase digestions. Recombinant SEAP produced in cell lines of Lymantria dispar (IPLB-LdEIta), Heliothis virescens (IPLB-HvT1), and Bombyx mori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni , H.virescens , B.mori , and Danaus plexippus contained oligosaccharides that were structurally identical to the 10 oligosaccharides attached to SEAP produced in T.ni cell lines. The oligosaccharide structures were all mannose-terminated. Structures containing two or three mannose residues, with and without core fucosylation, constituted more than 75% of the oligosaccharides from the cell culture and larval samples.
authors.affiliation: Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, NY 14853-1801, USA.
authors.forename: P C
authors.initials: PC
authors.lastname: Kulakosky
authors.forename: P R
authors.initials: PR
authors.lastname: Hughes
authors.forename: H A
authors.initials: HA
authors.lastname: Wood
mesh_terms: D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D012831:Bombyx; D002240:Carbohydrate Sequence; D002460:Cell Line; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D007814:Larva; D007915:Lepidoptera; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
chemical_list: D006023:Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase