J Korean Soc Endocrinol.  2003 Aug;18(4):371-378.

Search for "Proximal Histidine" of Thyroperoxidase Using Site Directed Mutagenesis

• Kim WB
• Shong YK

Abstract

BACKGROUND: Thyroperoxidase (TPO), a transmembrane heme containing glycoprotein, catalyzes iodide organification and thyroid hormone synthesis. It is a single peptide making a loop with more than one disulfide bond. The tertiary conformational structure is essential for its enzymatic activity and immunogenicity. The proximal histidine is thought to play a major role in enzymatic activity since it is linked to the iron center of the heme. The crystal structure of TPO has not yet been reported, but some have suggested histidine 407 be a putative proximal histidine based on comparison of a.a. sequence for TPO and that for myeloperoxidase.
METHODS
The putative histidine 407 and nearby histidine 414 were mutated to arginine to verify their role as the proximal histidine. Using site directed mutagenesis of wild type, human TPO cDNA, mutants H407R and H414R were made. Mutant cDNAs were transiently transfected into COS-7 cells, and the TPO enzyme activities were measured by guaiacol assay. Four cysteine residues around the putative proximal histidines were mutated to serine and their enzymatic activities were measured to check if they were involved in the formation of intra-molecular disulfide bonds.
RESULTS
TPO protein expression of H407R- and H414R- transfected cells was confirmed by Western blot, using Hashimoto's IgG as primary antibody. Both the mutants H407R and H414R showed significant peroxidase enzymatic activity, although lower than those of the wild type. None of the cysteine mutants, C375S, C389S, C598S, and C655S, were detected by Hashimoto's IgG ordisplayed any enzymatic activity.
CONCLUSION
These data suggest that neither histidine 407 nor histidine 414 functions as the "proximal histidine" in human TPO. All the cysteine residues checked (375, 389, 598, 655) might be involved in the formation of disulfide bonds in TPO molecules, but this hypothesis could not be confirmed. A further search for the other putative histidine residues using the same strategy is needed to define the structure-function relationship in the human TPO molecule.

MeSH Terms

Aging
Animals
Arginine
Blotting, Western
COS Cells
Cysteine
DNA, Complementary
Glycoproteins
Guaiacol
Heme
Histidine
Humans
Immunoglobulin G
Insulin Resistance
Iron
Mesenchymal Stromal Cells
Mutagenesis, Site-Directed*
Peroxidase
Serine
Thyroid Gland
Arginine
Cysteine
DNA, Complementary
Glycoproteins
Guaiacol
Heme
Histidine
Immunoglobulin G
Iron
Peroxidase
Serine