Occurrence of sulfate in the asparagine-linked complex carbohydrate units of thyroglobulin. Identification and localization of galactose 3-sulfate and N-acetylglucosamine 6-sulfate residues in the human and calf proteins.
Human thyroglobulin glycopeptides representing the multiple asparagine-linked complex (unit B) carbohydrate units of this protein were found to contain substantial amounts of sulfate (ranging from 0.5 to 2.5 mol/mol of oligosaccharide); this substituent was shown to occur primarily in the form of terminal beta-linked Gal-3-SO4 residues which represent novel capping groups occurring alternatively to sialic acid and in comparable amounts. Upon hydrazine/nitrous acid fragmentation and radiolabeling with NaB3H4, all human unit B DEAE-resolved glycopeptide fractions yielded an acidic disaccharide which was characterized as Gal-3-SO4 beta 1----4-anhydromannitol. Studies on glycopeptides modified by desialylation, desulfation, and beta-galactosidase treatment indicated that the majority (approximately 70%) of the complex carbohydrate units contain sulfate groups and that Gal-3-SO4 and sialic acid residues can coexist in terminal positions on the same N-linked oligosaccharide. In addition to Gal-3-SO4, the most acidic unit B variants were found to contain GlcNAc-6-SO4 which was recovered as Gal beta 1----4-anhydromannitol-6-SO4 after hydrazine/nitrous acid treatment and NaB3H4 reduction. On the basis of chromatography on immobilized concanavalin A, it was determined that whereas the Gal-3-SO4 groups occur on biantennary as well as more highly branched carbohydrate units, GlcNAc-6-SO4 is exclusively present in the latter oligosaccharides. In contrast to the N-linked carbohydrate units, the previously described O-linked glycosaminoglycan chain of human thyroglobulin yielded GlcA beta 1----3-anhydrotalitol-6-SO4 upon hydrazine/nitrous acid/NaB3H4 treatment, indicating that it is a chrondroitin 6-sulfate-like polymer. The distribution of sulfate in the complex oligosaccharides of calf thyroglobulin was quite different from that in the human protein; sulfate was not detectable in most of the glycopeptides and was sequestered in a single multibranched complex-type glycopeptide fraction (1.6 mol of sulfate/mol of oligosaccharide) which contained about equal amounts of Gal-3-SO4 and GlcNAc-6-SO4. The difference in galactose sulfation between human and calf thyroglobulins may be related to the substitution in the latter protein of some of the galactose residues by alpha-D-Gal capping groups.
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Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts.
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D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000032:analysis; D000818:Animals; D001216:Asparagine; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle; D005690:Galactose / Q000032:analysis; D005944:Glucosamine / Q000031:analogs & derivatives*; D006020:Glycopeptides / Q000302:isolation & purification*; D006801:Humans; D007202:Indicators and Reagents; D009844:Oligosaccharides / Q000302:isolation & purification; D012794:Sialic Acids / Q000032:analysis; D013045:Species Specificity; D013464:Sulfuric Acids / Q000032:analysis*; D013954:Thyroglobulin / Q000302:isolation & purification; D013961:Thyroid Gland / Q000032:analysis
D006020:Glycopeptides; D007202:Indicators and Reagents; D009844:Oligosaccharides; D012794:Sialic Acids; D013464:Sulfuric Acids; C033137:N-acetylglucosamine-6-sulfate; D001216:Asparagine; D013954:Thyroglobulin; D005944:Glucosamine; D000117:Acetylglucosamine; D005690:Galactose