Enhanced expression of  3-galactosyltransferase 5 activity is sufficient to induce in vivo synthesis of extended type 1 chains on lactosylceramides of selected human colonic carcinoma cell lines

Abstract

In general, an elevated expression of beta 3-galactosyltransferase (beta 3GalT) activity contributed by beta 3GalT5 correlates well with increased biosynthesis and expression of type 1 chain (Gal beta 1-3GlcNAc beta 1-) derivatives such as Lewis A and sialyl Lewis A, which are mostly recognized as terminal epitopes and not further extended. Most known beta 3-N-acetylglucosaminyltransferases show a higher activity toward extending type 2 chain (Gal beta 1-4GlcNAc beta 1-), and an over-expression of beta 3GalT5 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 beta 3GalT activity relative to competing beta 4GalT activity, as defined against a common GlcNAc beta 1-3Gal beta 1-4Glc 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 beta 3GalT5 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 beta 3GalT5 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.

Ceramide Glycosylation by Glucosylceramide Synthase Selectively Maintains the Properties of Breast Cancer Stem Cells

“…3C). To verify these findings, GSLs further profiled using MALDI-MS and MS/MS analysis (43). The Gb3 levels in MCF-/Dox cells was 250% higher than in MCF-7 cells (10,179 versus 4,139 in absolute height; p Ͻ 0.001) and 347% higher than in MCF-12A cells (10,179 versus 2,833 in absolute height; p Ͻ 0.001) (Fig.…”

“…profiling of permethylated GSLs was performed as described previously (43). Briefly, the extracted GSLs (from 7.5 ϫ 10 5 cells) were incubated with leech ceramide glycanase (50 milliunits/100 l) (44) in 50 mM sodium acetate, pH 5.5, containing 0.1% sodium cholate at 37°C for 16 h. The released glycans were separated from the ceramides and detergent by passing through a Sep-Pak C18 cartridge (Waters, Milford, MA), and further desalted by a porous graphitized carbon solid phase extraction cartridge.…”

Helicobacter pylori chronic infection and mucosal inflammation switches the human gastric glycosylation pathways

“…2A, B). Enhanced expression of β1,3-galactosyltransferase 5 (β3GalT5) has been reported to result in increased biosynthesis of extended type 1 chains on lactosylceramides and glycoproteins in colon carcinoma cells [42,43], while suppression of β3GalT5 in pancreas adenocarcinoma cells reduced the expression of sialyl-Le a [44]. In line with these observations, the induction of β3GalT5, in response to H. pylori infection, can drive the extension of precursor chains towards the biosynthesis of type 1 sequences, therefore contributing for the increased expression of sialyl-Le a (Fig.…”

Specificity of β1,4-galactosyltransferase inhibition by 2-naphthyl 2-butanamido-2-deoxy-1-thio-β-D-glucopyranoside

“…β3GalT5 is the key enzyme involved in the synthesis of type 1 chains [16][17][18][19][20]. The activity and transcript levels of β3GalT5 were shown to be decreased in tissue samples of human colon adenocarcinoma, relative to the surrounding normal mucosa [15][16][17].…”