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

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.