Evidence for a sialic acid salvaging pathway in lepidopteran insect cells.
We previously described a transgenic insect cell line, Sfbeta4GalT/ST6, that expresses mammalian beta-1,4-galactosyltransferase and alpha2,6-sialyltransferase genes and produces glycoproteins with terminally sialylated N-glycans. The ability of these cells to produce sialylated N-glycans was surprising because insect cells contain only small amounts of sialic acid and no detectable CMP-sialic acid. Thus, it was of interest to investigate potential sources of sialic acids for sialoglycoprotein synthesis by these cells. We found that Sfbeta4GalT/ST6 cells can produce sialylated N-glycans when cultured in the presence but not in the absence of fetal bovine serum. The serum component(s) supporting N-glycan sialylation by Sfbeta4GalT/ST6 cells is relatively large-it was not removed by dialysis in a 50,000-molecular-weight cutoff membrane. Serum-free media supplemented with purified fetuin but not asialofetuin supported N-glycan sialylation by Sfbeta4GalT/ST6 cells. The terminally sialylated N-glycans isolated from fetuin also supported glycoprotein sialylation by Sfbeta4GalT/ST6 cells. Finally, serum-free medium supplemented with N-acetylneuraminic acid or N-acetylmannosamine supported glycoprotein sialylation by Sfbeta4GalT/ST6 cells but to a much lower degree than serum or fetuin. These results provide the first evidence of a sialic acid salvaging pathway in insect cells, which begins to explain how Sfbeta4GalT/ST6 and other transgenic insect cell lines can sialylate recombinant glycoproteins in the absence of a more obvious source of CMP-sialic acid.
Hollister
Jason
J
Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA.
Conradt
Harald
H
Jarvis
Donald L
DL
D000818:Animals; D016367:Baculoviridae; D001685:Biological Factors / Q000737:chemistry / Q000302:isolation & purification*; D002461:Cell Line, Transformed; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D007915:Lepidoptera / Q000166:cytology* / Q000378:metabolism*; D011134:Polysaccharides / Q000032:analysis; D044967:Serum / Q000737:chemistry; D012794:Sialic Acids / Q000378:metabolism*; D012795:Sialoglycoproteins / Q000737:chemistry / Q000302:isolation & purification
D001685:Biological Factors; D016895:Culture Media, Serum-Free; D011134:Polysaccharides; D012794:Sialic Acids; D012795:Sialoglycoproteins