Metabolic Labeling of Human Thyroglobulin with [³⁵S] Sulfate: Incorporation into Chondroitin 6-Sulfate and Endoglycosidase-F-Susceptible Carbohydrate Units*

Abstract

To study the incorporation of sulfate into thyroglobulin, human thyroid tissue was incubated with [35S]sulfate. Labeled thyroglobulin was purified by ammonium sulfate precipitation, gel exclusion chromatography, and equilibrium density gradient centrifugation, the last of these specifically to remove proteoglycans. [35S]Sulfate was found in thyroglobulin with low density, indicating that sulfate was incorporated into newly synthesized molecules before their iodination. Chondroitin ABC lyase and chondroitin AC lyase released equal amounts of [35S]sulfate, demonstrating the presence of chondroitin units, and TLC showed this to be predominantly chondroitin 6-sulfate. Additional [35S]sulfate was released by endoglycosidase-F (Endo-F), but not by Endo-H. The Endo-F-sensitive sulfate-labeled complex carbohydrate units were heterogeneous, one fraction passing through a Concanavalin-A-Sepharose column without delay and another fraction showing weak affinity for the lectin. More than 90% of the incorporated [35S]sulfate was accounted for by the chondroitin ABC lyase and Endo-F-sensitive fractions. Thus, human thyroglobulin contains sulfate in at least three types of carbohydrate units, 1) chondroitin 6-sulfate units, 2) complex units with no affinity for Concanavalin-A (tri- or tetraantennary forms), and 3) complex units with weak affinity for Concanavalin-A (biantennary forms).

Thyrotropin regulates tyrosine sulfation of thyroglobulin

“…Similar data have been reported for porcine, calf, and human thyroglobulin (16)(17)(18). We observed that, with TSH, oligosaccharide chains of porcine thyroglobulin bore 29 sulfate residues per mol thyroglobulin, probably bound to galactose and N-acetylglucosamine residues.…”

“…Sulfate residues are bound essentially to complex carbohydrate units of calf (16,17), human (16,18), and porcine (19,20) thyroglobulin. Chondroitins are also sulfated, but are present only in human thyroglobulin (18).…”

“…Sulfate residues are bound essentially to complex carbohydrate units of calf (16,17), human (16,18), and porcine (19,20) thyroglobulin. Chondroitins are also sulfated, but are present only in human thyroglobulin (18). Contradictory results from studies of porcine thyroglobulin suggested that a large proportion of the sulfate residues are bound to tyrosine molecules, while the rest are linked to polymannose moieties (15,21).…”

Role of Asparagine-linked Oligosaccharides in Protein Folding, Membrane Targeting, and Thyrotropin and Autoantibody Binding of the Human Thyrotropin Receptor

“…These cells express Nacetylgalactosaminyl-transferase and N-acetylgalactosaminyl-N-acetylglucosaminyl-mannosyl-sulfotransferase and produce N-linked oligosaccharides terminating in sulfate in Ͼ70% of the chains (41). Of interest is that some glycoproteins synthesized in the thyroid gland have been reported to be sulfated (42,43). TSHR expressed in the thyroid gland may also be, at least in part, sulfated.…”

Insights into the posttranslational structural heterogeneity of thyroglobulin and its role in the development, diagnosis, and management of benign and malignant thyroid diseases

“…Sulfate residues are present in carbohydrate units and in some amino acids, especially tyrosine (28). The sulfate residues of Tg are essentially present in complex carbohydrates, particularly in chondroitin sulfate chains, an event that only occurs in human Tg (29).…”