α-N-Acetylgalactosamine-capping of chondroitin sulfate core region oligosaccharides primed on xylosides

Abstract

We previously reported that cultured mammalian cells incubated with 4-methylumbelliferyl (MU) or p-nitrophenyl (pNP) β-xyloside synthesize an α-GalNAc-terminated pentasaccharide resembling the glycosaminoglycan-core protein linkage region. Here we show that human melanoma M21 cells and human neuroblastoma cells incubated with Xylβ-MU/pNP also make an α-GalNAc-terminated heptasaccharide containing one chondroitin disaccharide repeat. High performance liquid chromatography and matrix-assisted laser desorption ionization mass spectrometry analysis of intact or glycosidase-digested xyloside showed the structure as: GalNAcαGlcAβ1,3GalNAcβ1,4GlcAβ1,3Galβ1,3Galβ1,4Xylβ-MU/pNP. The α-GalNAc-terminated xylosides can account for ~10% of the total Xylβ-MU/pNP products (~1.5 nmol/h/mg). These results show that GalNAcαGlcAβ-modification is relatively abundant, but not unique to the GAG-linkage tetrasaccharide, α-GalNAc addition to the GlcA residue does not appear to be an extension of general phase II detoxification of xenobiotics that involve glucuronidation, since M21 cells incubated with MU synthesize only 0.3 pmol GlcAβ-MU/h/mg protein, and undetectable amount of GalNAcαGlcAβ-MU (<40 fmol/h/mg). Further, subcellular fractionation shows that the α-N-acetylgalactosaminyltransferase activity colocalizes in the Golgi with other glycosyl transferases and not in the ER, where xenobiotic detoxification glucuronosyltransferases are found. Although GalNAcαGlcAβ-terminal modification has not been detected on naturally occurring GAG chains, the substantial amount of α-GalNAc transferase activity suggests that the α-GalNAc transferase could utilize other GlcA-containing glycoconjugates as acceptors.