Hanham C A, Chapman A J, Sheppard M C, Black E G, Ramsden D B
Biochim Biophys Acta. 1986 Oct 29;884(1):158-65. doi: 10.1016/0304-4165(86)90239-4.
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.
甲状腺球蛋白是一种660 kDa的糖蛋白，是甲状腺中蛋白质合成的主要产物。有人提出，甲状腺球蛋白糖基化修饰在各种甲状腺疾病中都会发生。为了研究与甲状腺疾病相关的组织甲状腺球蛋白糖基化可能发生的变化，我们开发了一种凝集素亲和电泳系统，该系统可以对少量（小于1微克）的甲状腺球蛋白进行表征。提取并纯化了人甲状腺球蛋白。制备含有不同浓度伴刀豆球蛋白A、蓖麻凝集素、L-植物血凝素和商陆有丝分裂原的琼脂糖凝胶。将纯化的人甲状腺球蛋白进行系列稀释，加样到凝集素凝胶上并进行电泳。伴刀豆球蛋白A、蓖麻凝集素和植物血凝素均以浓度依赖的方式结合甲状腺球蛋白。商陆有丝分裂原不结合甲状腺球蛋白。用神经氨酸酶和内切糖苷酶H处理纯化的甲状腺球蛋白。二维免疫电泳显示，甲状腺球蛋白的迁移被神经氨酸酶修饰，但未被内切糖苷酶H修饰。对经过和未经过酶处理的纯化人甲状腺球蛋白进行凝集素亲和电泳，结果表明存在：如伴刀豆球蛋白A反应性和内切糖苷酶H修饰所示的寡甘露糖结构，以及如对蓖麻凝集素的亲和力和神经氨酸酶修饰所示的复合寡糖。这些结构与所提出的人甲状腺球蛋白糖基化模式一致，并表明该方法适用于表征少量甲状腺球蛋白的糖基化。
