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

We previously reported that cultured mammalian cells incubated with 4-methylumbelliferyl (MU) or p-nitrophenyl (pNP) alpha-xyloside synthesize an alpha-GalNAc-terminated pentasaccharide resembling the glycosaminoglycan-core protein linkage region. Here me show that human melanoma M21 cells and human neuroblastoma cells incubated with Xyl beta-MU/pNP also make an a-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: GalNAc alpha GlcA-beta 1,3GalNAc beta 1,4GlcA beta 1,3Gal beta 1,3Gal beta 1,4Xyl beta-MU/pNP. The alpha-GalNAc-terminated xylosides can account for similar to 10% of the total Xyl beta-MU/pNP products (similar to 1.5 nmol/h/mg). These results show that GalNAc alpha GlcA beta-modification is relatively abundant, but not unique to the GAG-linkage tetrasaccharide, alpha-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 GlcA beta-MU/h/mg protein, and undetectable amount of GalNAc alpha GlcA beta-MU (<40 fmol/h/mg), Further, subcellular fractionation shows that the alpha-N-acetylgalactosaminyltransferase activity colocalizes in the Golgi with other glycosyl transferases and not in the ER, where xenobiotic detoxification glucuronosyltransferases are found, Although GalNAc alpha GlcA beta-terminal modification has not been detected on naturally occurring GAG chains, the substantial amount of alpha-GalNAc transferase activity suggests that the a-GalNAc transferase could utilize other GlcA-containing glycoconjugates as accepters.