Identification of a novel glycosaminoglycan core-like molecule. II. 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-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.
Salimath
P V
PV
La Jolla Cancer Research Foundation, California 92037, USA.
Spiro
R C
RC
Freeze
H H
HH
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
D006025:Glycosaminoglycans; D006027:Glycosides; C010807:xylosides; D006596:Hexosaminidases; C482722:NAGA protein, human; D048809:alpha-N-Acetylgalactosaminidase; D000116:Acetylgalactosamine