Abstract
9-O-Acetylation is a common sialic acid modification, expressed in a developmentally regulated and tissue/cell type-specific manner. The relevant 9-O-acetyltransferase(s) have not been isolated or cloned; nor have mechanisms for their regulation been elucidated. We previously showed that transfection of the GD3 synthase (ST8Sia-I) gene into Chinese hamster ovary (CHO)-K1 cells gave expression of not only the disialoganglioside GD3 but also 9-O-acetyl-GD3. We now use differential display PCR between wild type CHO-K1 cells and clones stably expressing GD3 synthase (CHO-GD3 cells) to detect any increased expression of other genes and explore the possible induction of a 9-O-acetyltransferase. The four CHO mRNAs showing major up-regulation were homologous to VCAM-1, Tis21, the KC-protein-like protein, and a functionally unknown type II transmembrane protein. A moderate increase in expression of the FxC1 and SPR-1 genes was also seen. Interestingly, these are different from genes observed by others to be up-regulated after transfection of GD3 synthase into a neuroblastoma cell line. We also isolated a CHO-GD3 mutant lacking 9-O-acetyl-GD3 following chemical mutagenesis (CHO-GD3-OAc(-)). Analysis of the above differential display PCR-derived genes in these cells showed that expression of Tis21 was selectively reduced. Transfection of a mouse Tis21 cDNA into the CHO-GD3-OAc(-) mutant cells restored 9-O-acetyl-GD3 expression. Since the only major gangliosides expressed by CHO-GD3 cells are GD3 and 9-O-acetyl-GD3 (in addition to GM3, the predominant ganglioside type in wild-type CHO-K1 cells), we conclude that GD3 enhances its own 9-O-acetylation via induction of Tis21. This is the first known nuclear inducible factor for 9-O-acetylation and also the first proof that 9-O-acetylation can be directly regulated by GD3 synthase. Finally, transfection of CHO-GD3-OAc(-) mutant cells with ST6Gal-I induced 9-O-acetylation specifically on sialylated N-glycans, in a manner similar to wild-type cells. This indicates separate machineries for 9-O-acetylation on alpha2-8-linked sialic acids of gangliosides and on alpha2-6-linked sialic acids on N-glycans.
Highlights
9-O-Acetylation is a common sialic acid modification, expressed in a developmentally regulated and tissue/ cell type-specific manner
We previously showed that transfection of the GD3 synthase (ST8Sia-I) gene into Chinese hamster ovary (CHO)-K1 cells gave expression of the disialoganglioside GD3 and 9-O-acetyl-GD3
Isolation of a Clone of CHO-K1 Cells Stably Transfected with the GD3 Synthase Gene—Wild type CHO cells express predominantly the monosialoganglioside GM3 (64 – 66)
Summary
9-O-Acetylation is a common sialic acid modification, expressed in a developmentally regulated and tissue/ cell type-specific manner. We previously showed that transfection of the GD3 synthase (ST8Sia-I) gene into Chinese hamster ovary (CHO)-K1 cells gave expression of the disialoganglioside GD3 and 9-O-acetyl-GD3.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have