Induction of sialic acid 9-O-acetylation by diverse gene products: implications for the expression cloning of sialic acid O- acetyltransferases |
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Authors: | Shi WX; Chammas R; Varki A |
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Institution: | Glycobiology Program, UCSD Cancer Center, Division of Cellular and Molecular Medicine, University of California San Diego, La Jolla 92093, USA. |
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Abstract: | Sialic acids can be modified by O-acetyl esters at the 7- and/or 9-
position, altering recognition by antibodies, lectins and viruses. 9(7)-
O-acetylation is mediated by a sialic acid-specific O- acetyltransferase,
which has proven difficult to purify. Two groups have recently isolated
cDNAs possibly encoding this enzyme, by expression cloning of human
melanoma libraries in COS cells expressing the substrate ganglioside GD3.
Pursuing a similar approach, we have isolated additional clones that can
induce 9-O-acetylation. One clone present in a melanoma library encodes a
fusion protein between a bacterial tetracycline resistance gene repressor
and a sequence reported to be part of the P3 plasmid. Expression of the
open reading frame is necessary for inducing 9-O-acetylation, indicating
that this is not a reaction to the introduction of bacterial DNA. Another
clone from a rat liver cDNA library induced 9-O-acetylation on COS cells
expressing alpha2-6-linked sialic acids, and encodes an open reading frame
identical to the Vitamin D binding protein. However, truncation at the 5'
end eliminates the amino-terminal hydrophobic signal sequence, predicting
cytosolic hyperexpression of a truncated protein. Thus, diverse types of
cDNAs can indirectly induce sialic acid 9-O- acetylation in the COS cell
system, raising the possibility that the real enzyme may be composed of
multiple subunits which would not be amenable to expression cloning.
Importantly, the cDNAs we isolated are highly specific in their ability to
induce 9-O-acetylation either on alpha2-6-linked sialic acids of
glycoproteins (truncated vitamin D binding protein) or on the
alpha2-8-linked sialic acids of gangliosides (Tetrfusion protein). These
data confirm our prior suggestion that a family of O-acetyltransferases
with distinctive substrate specificities exists in mammalian systems.
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