Glycobiology Advance Access originally published online on January 9, 2009
Glycobiology 2009 19(4):418-427; doi:10.1093/glycob/cwn156
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Enhanced expression of β3-galactosyltransferase 5 activity is sufficient to induce in vivo synthesis of extended type 1 chains on lactosylceramides of selected human colonic carcinoma cell lines
2 Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan
3 Institute of Bioinformatics and Structural Biology, National Tsing-Hua University, Hsinchu 300, Taiwan
4 Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan
5 Taiwan and Institute of Biochemical Science, National Taiwan University, Taipei 106, Taiwan
1 To whom correspondence should be addressed: Tel: +886-2-2785-5696; Fax: +886-2-2788-9759; e-mail: kkhoo{at}gate.sinica.edu.tw
Received on March 28, 2008; revised on December 24, 2008; accepted on December 27, 2008
In general, an elevated expression of β3-galactosyltrans- ferase (β3GalT) activity contributed by β3GalT5 correlates well with increased biosynthesis and expression of type 1 chain (Galβ1-3GlcNAcβ1-) derivatives such as Lewis A and sialyl Lewis A, which are mostly recognized as terminal epitopes and not further extended. Most known β3-N-acetylglucosaminyltransferases show a higher activity toward extending type 2 chain (Galβ1-4GlcNAcβ1-), and an over-expression of β3GalT5 could suppress the formation of the type 2 chain poly-N-acetyllactosaminoglycans. The potential of extending instead the predominant type 1 chain termini synthesized under such circumstances was, however, not investigated, partly due to technical difficulty in unambiguous identification of extended type 1 chains. Using an advanced mass spectrometry-based glycomic mapping and glycan sequencing approach, we show here that type 1 chains carried on the lacto-series glycosphingolipids of colonic carcinoma cells can be extended when the endogenous β3GalT activity relative to competing β4GalT activity, as defined against a common GlcNAcβ1-3Galβ1-4Glc acceptor, is sufficiently high, as found in Colo205 and SW1116, but not in DLD-1 cells. In support of this positive correlation, the lacto-series glycosphingolipids isolated from stably transfected DLD-1 clones over-expressing β3GalT5 were shown to comprise fucosylated dimeric type 1 chains, whereas a mock transfectant and the DLD-1 parent carried only fucosylated dimeric type 2 chains on their lactosylceramides. It suggests that while the natural expression of extended type 1 chain is likely to be determined by many contributing factors including the relative amounts of competing glycosyltransferases and the UDP-Gal level, the enhanced expression of β3GalT5 is sufficient to promote in vivo extension of type 1 chains by furnishing a significantly higher amount of type 1 chain precursors relative to competing type 2 chains.
Key words: β3-galactosyltransferase / β3GalT5 / extended type 1 chain / glycomics / mass spectrometry