Giraud A, Franc J L, Long Y, Ruf J
INSERM U 38, Faculté de Médecine, Marseille, France.
J Endocrinol. 1992 Feb;132(2):317-23. doi: 10.1677/joe.0.1320317.
Thyroid peroxidase (TPO) is a glycoprotein enzyme which catalyses the iodination of thyroglobulin and the coupling of iodinated tyrosines. Human TPO (hTPO) is the microsomal antigen recognized by the autoantibodies in the serum of patients with autoimmune thyroid disease. An active detergent-solubilized immunoaffinity-purified hTPO was deglycosylated, either by peptide N-glycosidase F (PNGase F) or by endo-beta-N-acetylglucosaminidase H (endo H), and the enzymatic activity and immunoreactivity of the native and deglycosylated forms were compared. Electrophoretic controls and affinoblotting with concanavalin A showed that deglycosylation was not total and that it was more pronounced with endo H than with PNGase F. The enzymatic activity of hTPO was inhibited by endo H deglycosylation, but not by PNGase F deglycosylation; this inhibition was not due to aggregation and/or insolubilization of the molecule subsequent to deglycosylation. Immunoreactivity was monitored by enzyme-linked immunosorbent assay (ELISA) with 13 mouse monoclonal antibodies, rabbit polyclonal antibodies and antibodies from serum of patients with Hashimoto's thyroiditis. In contrast with enzymatic activity, immunoreactivity was not modified or was slightly enhanced (with four monoclonal antibodies) by deglycosylation. The results indicate that strong, if not total, deglycosylation induces a modification of the tertiary structure of hTPO, which affects the enzymatic site but does not modify markedly the epitopes implicated in the recognition of the molecule by the antibodies tested.
甲状腺过氧化物酶（TPO）是一种糖蛋白酶，它催化甲状腺球蛋白的碘化以及碘化酪氨酸的偶联。人TPO（hTPO）是自身免疫性甲状腺疾病患者血清中自身抗体所识别的微粒体抗原。通过肽N - 糖苷酶F（PNGase F）或内切β - N - 乙酰氨基葡糖苷酶H（内切H）对一种活性去污剂增溶的免疫亲和纯化的hTPO进行去糖基化，并比较天然形式和去糖基化形式的酶活性和免疫反应性。电泳对照和伴刀豆球蛋白A亲和印迹表明去糖基化并不完全，且内切H处理比PNGase F处理更明显。hTPO的酶活性被内切H去糖基化抑制，但不被PNGase F去糖基化抑制；这种抑制不是由于去糖基化后分子的聚集和/或不溶性。通过酶联免疫吸附测定（ELISA），用13种小鼠单克隆抗体、兔多克隆抗体以及桥本甲状腺炎患者血清中的抗体监测免疫反应性。与酶活性相反，去糖基化未改变免疫反应性或使其略有增强（四种单克隆抗体的情况）。结果表明，即使不是完全的强去糖基化也会诱导hTPO三级结构的改变，这影响了酶活性位点，但未显著改变所测试抗体识别该分子所涉及的表位。
