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J. Biol. Chem., Vol. 278, Issue 10, 7942-7948, March 7, 2003
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§,
, and
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.
These two authors contributed equally to this work.
§
Supported by the Suntory Institute for Bioorganic Research (Osaka, Japan).
¶
Supported by United States Public Health Service Grant
R01-GM32373. To whom correspondence should be addressed: CMM-E, Rm. 1065, Mail Code 0687, University of California, San Diego, La Jolla, CA
92093-0687. Tel.: 858-534-3296; Fax: 858-534-5611; E-mail: avarki@ucsd.edu.
This article has been cited by other articles:
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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] |
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