Comparison of oligosaccharide processing among various insect cell lines expressing a secreted glycoprotein.
The processing of the N-linked oligosaccharide modifying a secreted alkaline phosphatase glycoprotein (SEAP) expressed with a recombinant Autographa californica nuclear polyhedrosis virus was evaluated in insect cell lines established from Spodoptera frugiperda, Trichoplusia ni, and Mamestra brassicae. Studies with Endoglycosidase H (Endo H), which removes high-mannose oligosaccharides, revealed that 79% of the intracellular SEAP produced in the M. brassicae-derived MB0503 cell line was Endo H resistant. The commonly used S. frugiperda Sf21 and Sf9 cell lines produced 44 and 21% Endo H-resistant intracellular SEAP, respectively. Detection of oligosaccharide moieties with lectins, which selectively recognize terminal sugars, identified only mannose residues on SEAP expressed in the six insect cell lines. However, the oligosaccharide moiety of SEAP expressed in a Chinese hamster ovary cell line contained sialic acid. Therefore, when expressed in mammalian cells, the oligosaccharide present on SEAP is processed into complex oligosaccharide, but in insect cells it is of the high-mannose type. Studies with inhibitors of the initial oligosaccharide processing steps demonstrated that all six cell lines possessed glycosidase I/II and mannosidase I activity and that glycosylation was required for secretion.
Davis
T R
TR
Boyce Thompson Institute for Plant Research, Ithaca, NY 14853.
Schuler
M L
ML
Granados
R R
RR
Wood
H A
HA
D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism*; D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D007313:Insecta; D037102:Lectins / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008970:Molecular Weight; D017924:Nucleopolyhedroviruses / Q000235:genetics; D009844:Oligosaccharides / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011994:Recombinant Proteins / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
D037102:Lectins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014415:Tunicamycin; D017485:1-Deoxynojirimycin; D000469:Alkaline Phosphatase; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase