Metabolic labeling of human thyroglobulin with [35S]sulfate: incorporation into chondroitin 6-sulfate and endoglycosidase-F-susceptible carbohydrate units.
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).
D000645:Ammonium Sulfate; D050260:Carbohydrate Metabolism; D011232:Chemical Precipitation; D002807:Chondroitin / Q000031:analogs & derivatives*; D002808:Chondroitin Lyases / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006801:Humans; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D013431:Sulfates / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D013954:Thyroglobulin / Q000302:isolation & purification / Q000378:metabolism*; D013961:Thyroid Gland / Q000032:analysis
D013431:Sulfates; D013462:Sulfur Radioisotopes; D002807:Chondroitin; D002809:Chondroitin Sulfates; D013954:Thyroglobulin; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D002808:Chondroitin Lyases; D000645:Ammonium Sulfate