Abstract
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.
Highlights
From the Denartments of Biological Chemistry and Medicine, Harvard Medico1School and the Elliott P
Upon hydrazine/nitrous acid hydrate, predominantlyin the form of asparagine-linked fragmentation and radiolabeling withNaBSH4,all hu- polymannose and complex oligosaccharides man unitB DEAE-resolved glycopeptide fractions (9, lo), distinguishing species differences have been noted yielded an acidic disaccharide which was characterized (IO-lZ), among which is the occurrence in the human protein as Gal-3-S04/31+4-anhydromannitol.Studies on gly- of a sulfated 0-linked glycosaminoglycan chain [13]
We found that hydrazine treatment did not bring about detectable cleavage of 0-sulfate saccharide substituents [15], we observed, using [3H]GlcH2-6-Pas a model, that extensive dephosphorylation did take place with this reagent
Summary
Clearly distinct from the product whichwas produced by Presence of Sulfate in Unit B Glycopeptides-Sulfate anal- hydrazine/nitrous acid treatment of the0-linked sulfated yses of DEAE-cellulose-fractionatedglycopeptidescontaining glycosaminoglycan chain of human thyroglobulin [13] which the complex (unit B) carbohydrate of human thyroglobulin wouldbe expected to elute in Dowex 1 fraction 4. Indicated the presence of substantial amountsof this substit- treatment of unfractionated human thyroglobulin glycopepuent in all of the column peaks (Table I). the sulfate tides, as well as the purified glycosaminoglycan-peptide, by content varied among the glycopeptide fractions, increasing hydrazine/nitrous acid yielded, after NaB3H4 reduction, a as the elution progressed, in general, the ratio of the sum of component present in this Dowex 1fraction which migrated sulfate and sialic acid residues to galactose remained close to (RhcH, = 0.52) tothe position of standard GlcAPl+. Because of the symmetry of the reduced anhydromannose residue, Gal@l+3GlcNAc andGalB14GlcNAc sequences would be expected to yield the identical Gal@l+AnManHZ disaccharide after hydrazine/nitrous acid/NaB3H4treatment To circumvent this difficulty, acidic asialoglycopeptidesfrom human thyroglobulin were desulfated and radiolabeled with galactose oxidase NaB3H4 prior to hydrazine/nitrous acid degradation. After NaBH4 reduction, as anticipated, the [3H]galactoselabeled disaccharide standards as well as the thyroglobulin
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