Oligosaccharide processing in the expression of human plasminogen cDNA by lepidopteran insect (Spodoptera frugiperda) cells.
A comparison has been made between the Asn289-linked oligosaccharide structures of human plasma plasminogen and a recombinant human plasminogen, expressed in lepidopteran insect (Spodoptera frugiperda) cells, after infection of these cells with a recombinant baculovirus containing the entire human plasminogen cDNA. Using anion-exchange liquid chromatography mapping of the oligosaccharide units cleaved from the proteins by glycopeptidase F, compared with elution positions of standard oligosaccharide structures, coupled with monosaccharide compositional analysis, we find that the human plasma protein contained only bisialo-biantennary complex-type carbohydrate and asialo-biantennary complex carbohydrate, confirming earlier work published by this laboratory. The glycosylation pattern of the insect cell expressed recombinant human plasminogen showed considerable microheterogeneity, with identifiable high-mannose carbohydrate (Man9GlcNAc2) and truncated high-mannose oligosaccharide (Man5GlcNAc2, Man4GlcNAc2, and Man3GlcNAc2). Of major importance, approximately 40% of the oligosaccharide population consisted of complex carbohydrate (bisialo-biantennary), identical in structure with that of the human plasma protein. This is the first direct identification of complex carbohydrate in proteins produced in insect cells and demonstrates that trimming and processing of high-mannose carbohydrate into complex-type oligosaccharide can occur. Our data indicate that both normal and alternate pathways exist in these cells for incorporation and trimming of high-mannose oligosaccharides and that mannosidases, as well as galactosyl-, hexosaminidasyl-, and sialyltransferases are present, and/or can be induced, in these cells. From these observations, we conclude that amino acid sequences and/or protein conformational properties can control oligosaccharide processing events.
Davidson
D J
DJ
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Fraser
M J
MJ
Castellino
F J
FJ
D000818:Animals; D001483:Base Sequence; D002240:Carbohydrate Sequence; D004247:DNA / Q000235:genetics*; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths / Q000235:genetics; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism*; D010958:Plasminogen / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics
D009844:Oligosaccharides; D011994:Recombinant Proteins; D010958:Plasminogen; D004247:DNA