The two rat α2,6-sialyltransferase (ST6Gal I) isoforms: evaluation of catalytic activity and intra-Golgi localization

α2,6-Sialyltransferase (ST6Gal I) functions in the Golgi to terminally sialylate the N-linked oligosaccharides of glycoproteins. Interestingly, rat ST6Gal I is expressed as two isoforms, STtyr and STcys, that differ by a single amino acid in their catalytic domains. In this article, our goal was to...

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Published in:Glycobiology (Oxford) 2003-02, Vol.13 (2), p.109-117
Main Authors: Chen, Tung-ling L., Chen, Chun, Bergeron, Nyahne Q., Close, Brett E., Bohrer, Tracy J., Vertel, Barbara M., Colley, Karen J.
Format: Article
Language:English
Subjects:
6-sialyltransferase
catalytic activity
cellular localization
Chinese hamster ovary
CHO
endoplasmic reticulum
ER-Golgi-intermediate compartment
ERGIC
FBS
fetal bovine serum
Golgi
isoform
PBS
phosphate buffered saline
Sambucas nigra agglutinin
SDS–PAGE
sialyltransferase
SNA
sodium dodecyl sulfate–polyacrylamide gel electrophoresis
ST6GalI
TBS
TGN
trans-Golgi network
Tris-buffered saline
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Summary:α2,6-Sialyltransferase (ST6Gal I) functions in the Golgi to terminally sialylate the N-linked oligosaccharides of glycoproteins. Interestingly, rat ST6Gal I is expressed as two isoforms, STtyr and STcys, that differ by a single amino acid in their catalytic domains. In this article, our goal was to evaluate more carefully possible differences in the catalytic activity and intra-Golgi localization of the two isoforms that had been suggested by earlier work. Using soluble recombinant STtyr and STcys enzymes and three asialoglycoprotein substrates for in vitro analysis, we found that the STcys isoform was somewhat more active than the STtyr isoform. However, we found no differences in isoform substrate choice when these proteins were expressed in Chinese hamster ovary cells, and sialylated substrates were detected by lectin blotting. Immuno-fluorescence and immunoelectron microscopy revealed differences in the relative levels of the isoforms found in the endoplasmic reticulum (ER) and Golgi of transiently expressing cells but similar intra-Golgi localization. STtyr was restricted to the Golgi in most cells, and STcys was found in both the ER and Golgi. The ER localization of STcys was especially pronounced with a C-terminal V5 epitope tag. Ultrastructural and deconvolution studies of immunostained HeLa cells expressing STtyr or STcys showed that within the Golgi both isoforms are found in medial-trans regions. The similar catalytic activities and intra-Golgi localization of the two ST6Gal I isoforms suggest that the particular isoform expressed in specific cells and tissues is not likely to have significant functional consequences.
ISSN:0959-6658
1460-2423
DOI:10.1093/glycob/cwg015