Structure of the heparan sulfate-protein linkage region. Demonstration of the sequence galactosyl-galactosyl-xylose-2-phosphate.
We have treated bovine lung heparan sulfate with alkaline [3H]borohydride to end label the chains with [3H]xylitol. After subsequent periodate oxidation-alkaline elimination products were separated by gel permeation and ion exchange chromatography. The linkage region fragment expected to have 2 galactoses and 1 [3H]xylitol residue appeared in the tetra-/trisaccharide region after gel filtration and was bound to the anion exchange resin. A similar negatively charged fragment, expected to have 2 galactoses, 1 xylose and 1 serine, was isolated after periodate oxidation-alkaline elimination of unlabeled heparan sulfate. The negative charge was due to the presence of alkaline phosphatase-labile phosphate ester. The molar ratio of galactose:phosphate:xylose was 2.17:1.19:1.00. The phosphate ester was associated with the xylose/[3H] xylitol moiety as indicated by the formation of phosphoxylose/-xylitol by beta-galactosidase digestion of the phosphorylated trisaccharide. Furthermore, orcinol reactivity disappeared after periodate oxidation of the dephosphorylated trisaccharide. The phosphate ester must be located to C-2 of xylose/xylitol as the 1-3H radioactivity could be released by periodate oxidation when it was preceded by alkaline phosphatase treatment. It is estimated that almost every chain of heparan sulfate carries 2-phosphoxylose. It would be of interest to know if glycosaminoglycan chains that are artificially initiated onto exogeneous beta-D-xylosides also acquire the 2-phosphoxylose moiety.
Fransson
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LA
Silverberg
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Carlstedt
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D000818:Animals; D001894:Borohydrides; D002240:Carbohydrate Sequence; D002417:Cattle; D011508:Chondroitin Sulfate Proteoglycans / Q000032:analysis*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006025:Glycosaminoglycans / Q000032:analysis*; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000032:analysis*; D008168:Lung / Q000378:metabolism*; D010428:Pentosephosphates / Q000032:analysis*; D011509:Proteoglycans / Q000032:analysis*; D013403:Sugar Phosphates / Q000032:analysis*; D014312:Trisaccharides / Q000032:analysis*
D001894:Borohydrides; D011508:Chondroitin Sulfate Proteoglycans; D006025:Glycosaminoglycans; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D010428:Pentosephosphates; D011509:Proteoglycans; D013403:Sugar Phosphates; D014312:Trisaccharides