Genes Modulated by Expression of GD3 Synthase in Chinese Hamster Ovary Cells

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 upregulation were homologous to VCAM-1, Tis21, the KCprotein-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 wildtype 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 ␣2-8-linked sialic acids of gangliosides and on ␣2-6-linked sialic acids on N-glycans.Sialic acids are a family of 9-carbon carboxylated monosaccharides typically located at the termini of mammalian cell surface sugar chains on both glycoproteins and glycolipids. N-acetylneuraminic acid, the most common sialic acid, is subjected to various modifications in vivo (1-5). One of the most prevalent modifications is O-acetylation of the hydroxyl group at the 9-carbon position. This modification is known to reduce or abolish the recognition of sialic acid residues by sialidases (2, 4, 6, 7), by certain sialic acid-binding lectins like Siglecs (4, 5, 8 -10), and by several viral recognition proteins (4,5,11,12). Conversely, other viruses require 9-O-acetylation for recognition of their target cells (4,5,(13)(14)(15)(16). It is also known that 9-O-acetylation is regulated during development and aberrantly expressed in melanomas and basal cell carcinomas (17)(18)(19)(20). These findings ind...

O-acetylated N-acetylneuraminic acid as a novel target for therapy in human pre-B acute lymphoblastic leukemia

“…Interestingly, our experiments showed that the ALL cells fail to 9- O -acetylate exogenously added GD3. In CHO cells, exposure to exogenous GD3 was shown to result in increased levels of 9- O -Ac GD3, and the authors concluded that in these cells, GD3 induces sialyl O -acetyltransferase (SOAT) production or activation (Satake et al, 2003). Because ALL cells contain endogenous 9- O -Ac–GD3, it is reasonable to assume that the SOAT enzyme, which is directly or indirectly responsible for 9- O -Ac–GD synthesis, must be expressed in pre-B ALL cells.…”

Functions and Biosynthesis of O-Acetylated Sialic Acids

“…To speculate further, induction of apoptosis would then just require activation of the cellular SIAE to generate the proapoptotic ganglioside GD3. Another molecule possibly involved in O-acetylation of GD3 was identified as Tis21, a cell cycle regulator and cell death molecule [179]. It is possible that Tis21 may be a mediator of O-acetylation, but it appears unlikely that Tis21 represents the elusive SOAT.…”

9-O-Acetylation of Exogenously Added Ganglioside GD3

“…CHO-K1 cells were grown in ␣-minimum essential medium supplemented with 10% (v/v) fetal bovine serum. CHO-GD3, a cell line established previously by stable transfection of mouse GD3 synthase into parental CHO-K1 cells (31) were grown in the same medium containing 0.8 mg/ml G418, to assure continued expression of GD3 synthase. Fibroblast cells were grown in ␣-minimum essential medium supplemented with non-heat-inactivated 15% (v/v) fetal bovine serum, and 293T cells were grown in Dulbecco's modified Eagle's Medium supplemented with 10% (v/v) fetal bovine serum.…”