Structures of the asparagine-289-linked oligosaccharides assembled on recombinant human plasminogen expressed in a Mamestra brassicae cell line (IZD-MBO503).
In this report, we have fortified and extended a previous investigation [Davidson, D. J., Fraser, M. J., & Castellino, F. J. (1990) Biochemistry 29, 5584-5590] in which we demonstrated for the first time that lepidopteran insect (Spodoptera frugiperda) cells (IPLB-SF-21AE) were capable of assembling N-linked complex oligosaccharide on a human protein (plasminogen), the cDNA of which had been inserted into these cells via recombinant DNA technology with a baculovirus vector. In order to investigate whether a more general capability of lepidopteran insect cells to produce complex oligosaccharide existed, and to identify the chemical nature of the types of oligosaccharides that such insect cells were able to assemble, we have infected Mamestra brassicae (IZD-MBO503) cells for 48 h with a recombinant (r) baculovirus containing the [R561E]human plasminogen (HPg) cDNA and characterized the nature of the glycopeptidase F (GF) released N-linked oligosaccharides contained on Asn289 of the r-HPg expressed by these cells. We found that approximately 63% of the total N-linked oligosaccharides were of the complex type, with bisialo-biantennary (28%), asialo-biantennary (7%), fucosylated bisialo-biantennary (25%), and fucosylated asialo-biantennary (3%) oligosaccharides representing the major complex-type carbohydrate species. The remainder of the oligosaccharides were of the high-mannose type, with (mannose)9(N-acetylglucosamine)2 (22%), (mannose)5(N-acetylglucosamine)2 (13%), and (mannose)3(N-acetylglucosamine)2 (2%) representing the major oligosaccharides observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Davidson
D J
DJ
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Castellino
F J
FJ
D000818:Animals; D001216:Asparagine / Q000737:chemistry*; D016367:Baculoviridae / Q000235:genetics; D002240:Carbohydrate Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005814:Genes, Viral; D006031:Glycosylation; D006801:Humans; D007915:Lepidoptera / Q000166:cytology; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D010958:Plasminogen / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D009844:Oligosaccharides; D011994:Recombinant Proteins; D001216:Asparagine; D010958:Plasminogen; D004247:DNA