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Evidence for a sialic acid salvaging pathway in lepidopteran insect cells.

ABSTRACT: 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.

SUBMITTER: Hollister J

PROVIDER: S-EPMC3632433 | biostudies-other | 2003 Jun

REPOSITORIES: biostudies-other

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Evidence for a sialic acid salvaging pathway in lepidopteran insect cells.

Hollister Jason J Conradt Harald H Jarvis Donald L DL

Glycobiology 20030220 6

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 sy ...[more]

PMID: 12626401

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Project description:Epoxyoctadecamonoenoic acids (EpOMEs) are epoxide derivatives of linoleic acid (9,12-octadecadienoic acid) and include 9,10-EpOME and 12,13-EpOME. They are synthesized by cytochrome P450 monooxygenases (CYPs) and degraded by soluble epoxide hydrolase (sEH). Although EpOMEs are well known to play crucial roles in mediating various physiological processes in mammals, their role is not well understood in insects. This study chemically identified their presence in insect tissues: 941.8 pg/g of 9,10-EpOME and 2,198.3 pg/g of 12,13-EpOME in fat body of a lepidopteran insect, Spodoptera exigua. Injection of 9,10-EpOME or 12,13-EpOME into larvae suppressed the cellular immune responses induced by bacterial challenge. EpOME treatment also suppressed the expression of antimicrobial peptide (AMP) genes. Among 139 S. exigua CYPs, an ortholog (SE51385) to human EpOME synthase was predicted and its expression was highly inducible upon bacterial challenge. RNA interference (RNAi) of SE51385 prevented down-regulation of immune responses at a late stage (> 24 h) following bacterial challenge. A soluble epoxide hydrolase (Se-sEH) of S. exigua was predicted and showed specific expression in all development stages and in different larval tissues. Furthermore, its expression levels were highly enhanced by bacterial challenge in different tissues. RNAi reduction of Se-sEH interfered with hemocyte-spreading behavior, nodule formation, and AMP expression. To support the immune association of EpOMEs, urea-based sEH inhibitors were screened to assess their inhibitory activities against cellular and humoral immune responses of S. exigua. 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) was highly potent in suppressing the immune responses. The addition of AUDA to a pathogenic bacterium significantly increased bacterial pathogenicity by suppressing host immune defense. In sum, this study demonstrated that EpOMEs play a crucial role in facilitating anti-inflammatory responses in S. exigua.

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Evidence for a sialic acid salvaging pathway in lepidopteran insect cells.

Publications

Evidence for a sialic acid salvaging pathway in lepidopteran insect cells.

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