Immunochemical evidence for a role of complex carbohydrate chains in thyroglobulin antigenicity.
Thyroglobulin secreted by porcine thyroid cells in serum-free culture was previously found (Ronin, C., Fenouillet, E., Hovsepian, S., Fayet, G., and Fournet, B. (1986) J. Biol. Chem. 261, 7287-7293) to contain more highly branched complex carbohydrate chains than the thyroid-derived molecules. When assayed for their ability to react with polyclonal antibodies directed against the natural prohormone in competitive radioimmunoassays, using the porcine antigen as iodinated tracer and standard competitor, in vitro thyroglobulin appeared to be 4-fold less immunoreactive than the in vivo molecule. However, both types of thyroglobulin exhibited superimposable incomplete displacement curves after peripheral deglycosylation using a mixture of neuraminidase, alpha-, and beta-galactosidases while they behave as good competitors as the native antigen after removal of the majority of the carbohydrate chains by Endo-beta-N-acetylglucosaminidase F. Solid-phase binding assays revealed that in vitro thyroglobulin was able to bind less antibodies than its thyroid-derived counterpart before as well as after treatment with exo- or endoglycosidases. Furthermore, only 20% of thyroglobulin biosynthetically labeled with glucosamine could be precipitated with specific antibodies followed by addition of staphylococcal-protein A, whereas up to 61% of the labeling was specifically bound when the antibodies were preincubated with protein A. After pronase digestion, both thyroglobulin-like material displayed different carbohydrate structures as judged by concanavalin A-Sepharose analysis. Thus, substituting multiantennary complex carbohydrate chains to the usual biantennary and high mannose structures present on thyroid-derived thyroglobulin had a profound effect on the prohormone immunoreactivity.
D000818:Animals; D002241:Carbohydrates / Q000032:analysis*; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D000939:Epitopes / Q000032:analysis*; D006020:Glycopeptides / Q000302:isolation & purification; D011863:Radioimmunoassay; D013552:Swine; D013954:Thyroglobulin / Q000096:biosynthesis / Q000276:immunology*; D013961:Thyroid Gland / Q000378:metabolism
D002241:Carbohydrates; D000939:Epitopes; D006020:Glycopeptides; D013954:Thyroglobulin