Glycosylation of human thyroglobulin and characterization by lectin affinity electrophoresis.
Thyroglobulin, a 660 kDa glycoprotein, is the major product of protein synthesis in the thyroid gland. It has been suggested that modifications of thyroglobulin glycosylation occur in various thyroid disorders. In order to study possible changes in glycosylation of tissue thyroglobulin associated with thyroid disease, we have developed a lectin affinity electrophoresis system which allows characterization of small (less than 1 microgram) quantities of thyroglobulin. Human thyroglobulin was extracted and purified. Agarose gels were cast containing concanavalin A, Ricinus communis agglutinin, L-phytohaemagglutinin and pokeweed mitogen at various concentrations. Purified human thyroglobulin was serially diluted, loaded onto lectin gels and electrophoresed. Concanavalin A, R. communis agglutinin and phytohaemagglutinin all bound thyroglobulin in a concentration-dependent manner. Pokeweed mitogen did not bind thyroglobulin. Purified thyroglobulin was treated with neuraminidase and endoglycosidase H. Two-dimensional immunoelectrophoresis revealed the migration of thyroglobulin to be modified by neuraminidase but not by endoglycosidase H. Lectin affinity electrophoresis of purified human thyroglobulin with and without enzyme treatment indicated the presence of: oligomannose structures as shown by concanavalin A reactivity and modification by endoglycosidase H, and complex oligosaccharides as shown by affinity for R. communis agglutinin and modification by neuraminidase. These structures are in keeping with the proposed patterns of glycosylation of human thyroglobulin and indicate suitability of the method for characterizing the glycosylation of small quantities of thyroglobulin.
D000118:Acetylglucosaminidase; D055598:Chemical Phenomena; D002621:Chemistry; D004586:Electrophoresis / Q000379:methods; D006031:Glycosylation; D006801:Humans; D007122:Immunoelectrophoresis / Q000379:methods; D037102:Lectins; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D009439:Neuraminidase; D013954:Thyroglobulin / Q000378:metabolism*
D037102:Lectins; D013954:Thyroglobulin; D009439:Neuraminidase; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase