Glycosylation of recombinant human thyroid peroxidase ectodomain of insect cell origin has little effect on recognition by serum thyroid peroxidase antibody

• Published in: Chinese medical journal Vol. 126; no. 15; pp. 2907 - 2911
• Main Authors: Liu, Ming-ming, Li, Qing, Zhao, Lan-lan, Gao, Ying, Huang, You-yuan, Lu, Gui-zhi, Gao, Yan-ming, Guo, Xiao-hui, Shi, Bing-yin
• Format: Journal Article
• Language: English
• Published: China Department of Endocrinology, Peking University First Hospital,Beijing 100034, China%CAS Key Laboratory of Pathogenic Microbiology and Immunology,Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China 2013
• Subjects: Animals; Antibodies, Monoclonal - immunology; Baculoviridae; Enzyme-Linked Immunosorbent Assay; Epitopes; Glycosylation; Humans; Insecta - cytology; Iodide Peroxidase - immunology; Recombinant Proteins; 单克隆抗体; 去糖基化; 昆虫; 甲状腺过氧化物酶; 细胞来源; 细胞重组; 胞外区; 血清; fucose; thyroid peroxidase; galactose; sialic acid; baculovirus expression vector system
• ISSN: 0366-6999; 2542-5641
• DOI: 10.3760/cma.j.issn.0366-6999.20130339

Background Thyroid peroxidase （TPO） is an important autoantigen in Hashimoto＇s thyroiditis （HT）, and almost all epitopes are located in TPO ectodomain. The glycosylation of TPO might contribute to breaking self-tolerance, therefore, pudfied glycosylated recombinant TPO ectodomain is prerequisite of elucidating its role in the pathogenesis of HT. The aim of our study was to investigate whether the glycosylation has influence on the antigenic determinants of recombinant TPO. Methods Bac-to-Bac baculovirus expression system was used to generate recombinant human TPO ectodomain. The antigenicity was analyzed by antigen specific enzyme-linked immunosorbant assays （ELISAs）. The glycosylation of recombinant human TPO ectodomain of High Five insect cell origin was detected by lectin-ELISAs. Results TPO ectodomain was recovered from the culture media as a soluble protein, and it was fused with a hexahistidine tag which allowed purification by nickel-affinity chromatography. The recombinant TPO ectodomain could be recognized by all the 54 HT patients and three TPO monoclonal antibodies. Fucose, sialic acid and galactose were all detected on the recombinant TPO ectodomain. Sera TPOAb binding decreased slightly after non-specific deglycosylation of TPO by periodic acid. Conclusions High Five insect cells derived recombinant human TPO ectodomain had N-glycosylation sites, which might have little effect on recognition by serum TPOAb.