title: A redundant role of human thyroid peroxidase propeptide for cellular, enzymatic, and immunological activity.
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). With two active sites, TPO is an unusual enzyme, and thus there is much interest in understanding its structure and role in AITD. Homology modeling has shown TPO to be composed 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 (TPOΔpro; amino acid residues 21-108) was characterized and its properties compared to wild-type TPO. Plasma membrane localization was determined by cell surface protein biotinylation, and biochemical studies were performed to evaluate enzymatic activity and the effect of deglycosylation. Immunological investigations using autoantibodies from AITD patients and other epitope-specific antibodies that recognize conformational determinants on TPO were evaluated for binding to TPOΔpro by flow cytometry, immunocytochemistry, and capture enzyme-linked immunosorbent assay. Molecular modeling and dynamics simulation of TPOΔpro comprising a dimer of myeloperoxidase-like domains was performed in order to investigate the impact of propeptide removal and the role of glycosylation.
  
  
  RESULTS
  The TPOΔpro was expressed on the cell surface at comparable levels to wild-type TPO. The TPOΔpro 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 TPOΔpro was also maintained. Molecular modeling and dynamics simulations were consistent with these observations.
  
  
  CONCLUSIONS
  Our results point to a redundant role for the propeptide sequence in TPO. The successful expression of TPOΔpro in a membrane-anchored, enzymatically active form that is insensitive to intramolecular proteolysis, and importantly is recognized by patients' autoantibodies, is a key advance for purification of substantial quantities of homogeneous preparation of TPO for crystallization, structural, and immunological studies.
authors.affiliation: 1 Department of Biochemistry and Molecular Biology, Medical Center of Postgraduate Education , Warsaw, Poland .
authors.forename: Marlena
authors.initials: M
authors.lastname: Godlewska
authors.forename: Monika
authors.initials: M
authors.lastname: Góra
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Buckle
authors.forename: Benjamin T
authors.initials: BT
authors.lastname: Porebski
authors.forename: E Helen
authors.initials: EH
authors.lastname: Kemp
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Sutton
authors.forename: Barbara
authors.initials: B
authors.lastname: Czarnocka
authors.forename: J Paul
authors.initials: JP
authors.lastname: Banga
mesh_terms: D000818:Animals; D001323:Autoantibodies / Q000276:immunology; D016466:CHO Cells; D020134:Catalytic Domain / Q000502:physiology; D006224:Cricetinae; D003412:Cricetulus; D004792:Enzyme Precursors / Q000276:immunology* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007453:Iodide Peroxidase / Q000276:immunology* / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D009195:Peroxidase / Q000737:chemistry; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D013961:Thyroid Gland / Q000378:metabolism; D013967:Thyroiditis, Autoimmune / Q000201:enzymology* / Q000276:immunology
chemical_list: D001323:Autoantibodies; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D009195:Peroxidase; D007453:Iodide Peroxidase