Abstract
Treatment of thyroglobulins from several mammalian sources (calf, sheep, pig, dog, rat, rabbit, guinea pig, and man) with alpha-galactosidase demonstrated a species-dependent occurrence of terminal alpha-D-galactosyl residues which ranged from 11 mol/mol of protein (23% of total galactose) in calf to a complete absence in man. The presence of the alpha-D-galactosyl groups resulted in a partial binding of the thyroglobulins (greater than 70% in calf and sheep) to Bandeiraea simplicifolia I-agarose, and this lectin-thyroglobulin interaction could be quantitated by a solid-phase assay utilizing 125I-labeled B. simplicifolia I. Sequential glycosidase digestions of calf thyroglobulin glycopeptides containing the complex carbohydrate unit (unit B) and characterization of oligosaccharide obtained by partial acid hydrolysis indicated that the alpha-D-galactosyl residues are located on oligosaccharide branches with an alpha-D-Gal-(1----3)-beta-D-Gal-(1----4)-D-GlcNAc sequence. While mild acid treatment of calf thyroglobulin glycopeptides yielded a disaccharide, alpha-D-Gal-(1----3)-D-Gal, and a trisaccharide, alpha-D-Gal-(1----3)-beta-D-Gal-(1----4)-D-GlcNAc, which could be resolved by B. simplicifolia I-agarose or thin-layer chromatography, similar hydrolysis of the human unit B-containing glycopeptides did not produce such components. A study of various glycopeptides indicated that the alpha-D-galactosyl residues are unevenly distributed among the multiple complex carbohydrate units of calf thyroglobulin and are preferentially located in units with a relatively low sialic acid content. During affinity chromatography on B. simplicifolia I-agarose, glycopeptides with multiple alpha-D-galactosyl groups bound more firmly to the lectin than those which contained only a single residue. In contrast to the alpha-D-galactosyl residues, beta-linked galactose of calf thyroglobulin was primarily bound in penultimate locations being susceptible to enzymatic release only after prior removal of capping sialyl and alpha-D-galactosyl groups. The isolation of N-acetyllactosamine and a beta-D-Gal----beta-D-GlcNAc----D-Man trisaccharide from partial acid hydrolysates helped to position the beta-D-galactosyl residues in the oligosaccharide branches of the complex carbohydrate units.
Highlights
From the Departments of Biological Chemistry and Medicine, Harvard Medical School and the Elliott P
A study of various glycopeptides indicated that the a-D-galactosyl residues are unevenldyistributed among the multiple complex carbohydrate unitsof calf Preparation of Thyroglobulins-Thyroids from calves, sheep, and thyroglobulin and are preferentially located in units pigswere obtained from a local slaughterhouse, while those from with a relatively low sialic acid content
The content of terminal a - ~ - tion with B. simplicifoliu I-agarose, this was not a uniform galactosyl residues in thyroglobulin was species dependent (Table I) with the highest values being observed in the calf, sheep, dog, and pig proteins; the human thyroglobulin was notable by the complete absence of a-galactose-releasable finding as the dog, pig, and rat proteins bound to the lectin to a lesser degreethan might have been anticipated (Table I)
Summary
From the Departments of Biological Chemistry and Medicine, Harvard Medical School and the Elliott P. The presenceof the a-D-gdaCtOSyl groups re- the linkage and location of the galactose residues, was undersulted ina partial bindingof the thyroglobulins(>70% takento explain thepartial @-galactosidaseresistance of in calf and sheep) to Bandeiraea simplicifoliaI-aga- desialylated glycopeptides from calf thyroglobulin as well as rose, and this lectin-thyroglobulin interacticoonuld be earlier findings [1, 2] which indicated that the complex carquantitated bya solid-phase assay utilizing1261-labeled bohydrate units of this protein contain a greater amount of. Ride obtainedby partial acid hydrolysis indicatedthat of the galactose residues in calf thyroglobulin occur in athe a-D-galactosyl residuesare located on oligosaccha- linked terminal positions and are constituentsof an a-D-Galride branches with an a-~-Gal-(1+3)-8-~-Ga1-(1+4)(1-+3)-/3-~-Gal-1( 4 ) - ~ - GlcNA cs’equence. A study of various glycopeptides indicated that the a-D-galactosyl residues are unevenldyistributed
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