A redundant role of human thyroid peroxidase propeptide for cellular, enzymatic, and immunological activity

Marlena Godlewska, Monika Gora, Ashley Maurice Buckle, Benjamin T Porebski, Erin Helen Kemp, Brian J Sutton, Barbara Czarnocka, J Paul Banga

Research output: Contribution to journalArticleResearchpeer-review

20 Citations (Scopus)

Abstract

Background: Thyroid peroxidase (TPO) is a dimeric membrane-bound enzyme of thyroid follicular cells, responsible for thyroid hormone biosynthesis. TPO is also a common target antigen in autoimmune thyroid disease (AITD). TPO is an unusual enzyme, comprising of two active sites and thus there is much interest in understanding its structure and role in AITD. Homology modeling has shown TPO to be comprised of different structural modules, as well as a propeptide sequence. During the course of studies to obtain homogeneous preparations of recombinant TPO for structural studies, we investigated the role of the large propeptide sequence in TPO. Methods: An engineered recombinant human TPO preparation expressed in Chinese hamster ovary (CHO) cells lacking the propeptide (TPODeltapro) (amino acid residues 21-108) was characterized and compared to its properties with wild-type (wt) TPO. Plasma membrane localization was determined by cell surface protein biotinylation, and biochemical studies for evaluating enzymatic activity and the effect of deglycosylation were performed. Immunological investigations using autoantibodies from AITD patients and other epitope-specific antibodies that recognize conformational determinants on TPO were evaluated for binding to TPODeltapro by flow cytometry, immunocytochemistry and capture ELISA. Molecular modeling and dynamics simulation of TPODeltapro comprising a dimer of MPO-like domains was performed in order to investigate the impact of propeptide removal and the role of glycosylation. Results: The TPODeltapro was expressed on the cell surface at comparable levels to TPOwt. The TPODeltapro was enzymatically active and recognized by patients autoantibodies and a panel of epitope specific antibodies, confirming structural integrity of the two major conformational determinants recognized by autoantibodies. Faithful intracellular trafficking and N-glycosylation of TPODeltapro was also maintained. Molecular modeling and dynamics simulations we
Original languageEnglish
Pages (from-to)371 - 382
Number of pages12
JournalThyroid
Volume24
Issue number2
DOIs
Publication statusPublished - 2014

Cite this