Biosynthesis of heparin. O-sulfation of the antithrombin-binding region.
The antithrombin-binding region in heparin is a pentasaccharide sequence with the predominant structure GlcNAc(6-OSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-IdoA -(2-OSO3)-GlcNSO3(6-OSO3) (where GlcA and IdoA represent D-glucuronic and L-iduronic acid, respectively), in which the 3-O-sulfate residue on the internal glucosaminyl unit is a marker group for this particular region of the polysaccharide molecule. A heparin octasaccharide which contained the above pentasaccharide sequence was N/O-desulfated and re-N-sulfated and was then incubated with adenosine 3'-phosphate 5'-phospho[35S]sulfate in the presence of a microsomal fraction from mouse mastocytoma tissue. Fractionation of the resulting 35S-labeled octasaccharide on antithrombin-Sepharose yielded a high affinity fraction that accounted for approximately 2% of the total incorporated label. Structural analysis of this fraction indicated that the internal glucosamine unit of the pentasaccharide sequence was 3-O-35S-sulfated, whereas both adjacent glucosamine units carried 6-O-[35S]sulfate groups. In contrast, the fractions with low affinity for antithrombin (approximately 98% of incorporated 35S) showed no consistent O-35S sulfation pattern and essentially lacked glucosaminyl 3-O-[35S]sulfate groups. It is suggested that the 3-O-sulfation reaction concludes the formation of the antithrombin-binding region. This proposal was corroborated in a similar experiment using a synthetic pentasaccharide with the structure GlcNSO3(6-OSO3)-GlcA-GlcNSO3(6-OSO3)-Id oA (2-OSO3)-GlcNSO3(6-OSO3) as sulfate acceptor. This molecule corresponds to a functional antithrombin-binding region but for the lack of a 3-O-sulfate group at the internal glucosamine unit. The 35S-labeled pentasaccharide recovered after incubation bound with high affinity to antithrombin-Sepharose and contained a 3-O-[35S]sulfate group at the internal glucosamine residue as the only detectable labeled component. The use of this pentasaccharide substrate along with the affinity matrix provides a highly specific assay for the 3-O-sulfotransferase.
D000818:Animals; D000991:Antithrombins / Q000378:metabolism*; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002460:Cell Line; D006493:Heparin / Q000096:biosynthesis*; D012515:Mast-Cell Sarcoma / Q000201:enzymology; D051379:Mice; D008861:Microsomes / Q000201:enzymology; D009844:Oligosaccharides / Q000378:metabolism; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism*; D013466:Sulfurtransferases / Q000378:metabolism
D000991:Antithrombins; D009844:Oligosaccharides; D013431:Sulfates; D010724:Phosphoadenosine Phosphosulfate; D006493:Heparin; D013466:Sulfurtransferases