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.
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.
Satake
Honoo
H
Glycobiology Research and Training Center, Department of Medicine, University of California, San Diego, La Jolla 92093-0687, USA.
Chen
Helen Y
HY
Varki
Ajit
A
D000107:Acetylation; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002999:Clone Cells; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers; D018076:DNA, Complementary; D005786:Gene Expression Regulation / Q000235:genetics*; D016147:Genes, Tumor Suppressor; D017874:Immediate-Early Proteins / Q000235:genetics*; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D025521:Tumor Suppressor Proteins; D015854:Up-Regulation
C489435:Btg2 protein, mouse; D017931:DNA Primers; D018076:DNA, Complementary; D017874:Immediate-Early Proteins; D011134:Polysaccharides; D025521:Tumor Suppressor Proteins; D012799:Sialyltransferases; C043662:alpha-N-acetylneuraminate alpha-2,8-sialyltransferase; D019158:N-Acetylneuraminic Acid