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J. Biol. Chem., Vol. 278, Issue 10, 7942-7948, March 7, 2003

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Genes Modulated by Expression of GD3 Synthase in Chinese Hamster Ovary Cells
EVIDENCE THAT THE Tis21 GENE IS INVOLVED IN THE INDUCTION OF GD3 9-O-ACETYLATION^*

Honoo Satake^§, Helen Y. Chen, and Ajit Varki^¶

From the Glycobiology Research and Training Center, Departments of Medicine and Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093-0687

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 2-8-linked sialic acids of gangliosides and on 2-6-linked sialic acids on N-glycans.

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				H. Y. Chen, A. K. Challa, and A. Varki 9-O-Acetylation of Exogenously Added Ganglioside GD3: THE GD3 MOLECULE INDUCES ITS OWN O-ACETYLATION MACHINERY J. Biol. Chem., March 24, 2006; 281(12): 7825 - 7833. [Abstract] [Full Text] [PDF]