The accompanying article (Manzi, A., Salimath, P. V., Spiro, R. C., Keifer, P. A., and Freeze, H. H. (1995) J. Biol. Chem. 270, 9154-9163) reported the complete structure of a novel molecule made by human
melanoma cells incubated with 1 mM 4-methylumbelliferyl-beta Xyl (Xyl beta MU). The product resembles a common pentasaccharide core region found in
chondroitin/
dermatan sulfate glycosaminoglycans, except that a terminal alpha-Gal-NAc residue is found in a location normally occupied by beta-GalNAc in these chains or alpha-GlcNAc in
heparan sulfate chains. In this paper we show that several other human
cancer cell lines and Chinese hamster ovary cells also make alpha-GalNAc-capped
xylosides. The [6-3H]
galactose-labeled Xyl beta MU product binds to immobilized alpha-GalNAc-specific
lectin from Helix pomatia and the binding is competed by GalNAc, but not by Glc. Binding to the
lectin is destroyed by digestion with
alpha-N-acetylgalactosaminidase, but not
beta-hexosaminidase. The nature of the aglycone influences the amount and relative proportion of this material made, with p-nitrophenyl-beta-xyloside being a better promoter of alpha-GalNAc-terminated product than Xyl beta MU. This novel
oligosaccharide accounts for 45-65% of xyloside-based products made by both human
melanoma and Chinese hamster ovary cells when they are incubated with 30 microM Xyl beta MU, but at 1 mM both the total amount and the proportion decreases to only 5-10%. In both cell lines this product is replaced by a corresponding amount of Sia alpha 2,3Gal beta 4Xyl beta MU. Preferential synthesis of the alpha-GalNAc-capped material at very low xyloside concentration argues that it is a normal biosynthetic product and not an experimental artifact. This pentasaccharide may be a previously unrecognized intermediate in
glycosaminoglycan chain biosynthesis. Since this alpha-GalNAc residue occurs at a position that determines whether
chondroitin or heparan chains are added to the acceptor, it may influence the timing, type, and extent of further chain elongation.