title: Identification of a novel glycosaminoglycan core-like molecule. II. Alpha-GalNAc-capped xylosides can be made by many cell types.
abstract: 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.
authors.affiliation: La Jolla Cancer Research Foundation, California 92037, USA.
authors.forename: P V
authors.initials: PV
authors.lastname: Salimath
authors.forename: R C
authors.initials: RC
authors.lastname: Spiro
authors.forename: H H
authors.initials: HH
authors.lastname: Freeze
mesh_terms: D000116:Acetylgalactosamine / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D006025:Glycosaminoglycans / Q000032:analysis*; D006027:Glycosides / Q000032:analysis / Q000096:biosynthesis*; D006596:Hexosaminidases / Q000494:pharmacology; D006801:Humans; D048809:alpha-N-Acetylgalactosaminidase
chemical_list: D006025:Glycosaminoglycans; D006027:Glycosides; C010807:xylosides; D006596:Hexosaminidases; C482722:NAGA protein, human; D048809:alpha-N-Acetylgalactosaminidase; D000116:Acetylgalactosamine