IDENTIFICATION OF A NOVEL GLYCOSAMINOGLYCAN CORE-LIKE MOLECULE .2. ALPHA-GALNAC-CAPPED XYLOSIDES CAN BE MADE BY MANY CELL-TYPES

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-GalNAc 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-H-3]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 ac counts for 45-65% of xyloside-based products made by both human melanoma and Chinese hamster ovary cells when they are incubated with 30 mu M 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.