Sakurai S, Fogelfeld L, Schneider A B
Michael Reese Hospital and Medical Center, Department of Medicine, University of Illinois, Chicago 60616.
Endocrinology. 1991 Aug;129(2):915-20. doi: 10.1210/endo-129-2-915.
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.
我们之前曾表明，人甲状腺球蛋白（hTG）含有带有多达四个硫酸基团的阴离子复合碳水化合物单元，其中一些同时含有硫酸和唾液酸。最近的报告表明，hTG的碳水化合物单元可能还含有磷酸盐，但这些报告并不完全一致。本研究的目的是确认hTG碳水化合物单元上磷酸盐的存在，并确定磷酸盐是否与其他酸性部分（如硫酸和唾液酸）在同一碳水化合物单元上共存。使用碱性磷酸酶和酸水解来检测源自正常和肿瘤组织的hTG硫酸化碳水化合物单元上的磷酸盐。将两名患者的甲状腺碎片在含[35S]硫酸盐的培养基中孵育16小时，然后纯化hTG。用内切糖苷酶-F从hTG中释放复合碳水化合物，并在pH 2.2的HPLC离子交换柱上进行分析。通过放射性监测含硫酸的峰，通过神经氨酸酶或酸处理后电荷减少来鉴定含唾液酸的峰。仅用碱性磷酸酶处理后，没有一个硫酸标记的碳水化合物峰发生移动，这表明它们都不含有磷酸单酯。几个硫酸标记的峰在酸水解后发生了移动，一些移向电荷减少的位置，这是由于唾液酸的去除，一些移向电荷增加的位置，这表明存在磷酸二酯。通过观察到一些新形成的峰易受碱性磷酸酶消化，证实了后者。因此，酸水解将磷酸二酯转化为易受碱性磷酸酶作用的磷酸单酯，最有可能是6-磷酸甘露糖。我们得出结论，hTG的一些阴离子复合碳水化合物单元仅含有硫酸，而其他单元则含有硫酸、唾液酸和磷酸二酯的组合。磷酸二酯存在于正常和肿瘤甲状腺组织的hTG硫酸化碳水化合物单元中。
