Anionic carbohydrate groups of human thyroglobulin containing both phosphate and sulfate

We showed previously that human thyroglobulin (hTG) contains anionic complex carbohydrate units with up to four sulfate groups, some containing both sulfate and sialic acid. Recent reports indicate that the carbohydrate units of hTG may also contain phosphate, but these reports are not all in accord. The purpose of this study was to confirm the presence of phosphate on the carbohydrate units of hTG and to determine whether phosphate coexists with other acidic moieties, such as sulfate and sialic acid, on the same carbohydrate units. Alkaline phosphatase and acid hydrolysis were used to detect phosphate on the sulfated carbohydrate units of hTG derived from normal and neoplastic tissues. Thyroid fragments from two patients were incubated for 16 h in [35S]sulfate-containing medium, and hTG was purified. Complex carbohydrates were released from hTG with endoglycosidase-F and analyzed at pH 2.2 on a HPLC ion exchange column. Sulfate-containing peaks were monitored by radioactivity, and sialic acid-containing ones were identified by their reduced charge after neuraminidase or acid treatment. None of the sulfate-labeled carbohydrate peaks shifted after alkaline phosphatase treatment alone, indicating that none of them contained phosphomonoesters. Several of the sulfate-labeled peaks shifted after acid hydrolysis, some to positions of decreased charge, due to removal of sialic acid, and some to positions of increased charge, suggesting the presence of phosphodiesters. The latter was confirmed by the observation that some of the newly formed peaks were susceptible to alkaline phosphatase digestion. Thus, acid hydrolysis converted phosphodiesters into alkaline phosphatase-susceptible phosphomonoesters, most likely mannose-6-phosphate. We conclude that some anionic complex carbohydrate units of hTG contain exclusively sulfate, while others contain combinations of sulfate, sialic acid, and phosphodiesters. Phosphodiesters are present in the sulfated carbohydrate units of hTG from normal as well as neoplastic thyroid tissue.