Glycosylation of recombinant human thyroid peroxidase ectodomain of insect cell origin has little effect on recognition by serum thyroid peroxidase antibody.
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, purified 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.
Department of Endocrinology, Peking University First Hospital, Beijing 100034, China.
Ming-ming
MM
Liu
Qing
Q
Li
Lan-lan
LL
Zhao
Ying
Y
Gao
You-yuan
YY
Huang
Gui-zhi
GZ
Lu
Yan-ming
YM
Gao
Xiao-hui
XH
Guo
Bing-yin
BY
Shi
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D016367:Baculoviridae; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006031:Glycosylation; D006801:Humans; D007313:Insecta / Q000166:cytology; D007453:Iodide Peroxidase / Q000276:immunology*; D011994:Recombinant Proteins
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; D007453:Iodide Peroxidase