A conformational B-cell epitope on the C-terminal end of the extracellular part of human thyroid peroxidase.
To investigate the B-cell autoimmune epitopes on human thyroid peroxidase (TPO), we generated proteolytic peptides by enzymatic hydrolysis of TPO in nondenaturing and nonreducing conditions. The hydrolysate was chromatographed on a reverse phase column. We eluted a material immunoreactive with both a TPO monoclonal antibody recognizing a linear epitope (mAb47, amino acid 713-721) and TPO autoantibodies (aAb) from patients. The aAb immunoreactivity, but not that of mAb47, was lost after reduction. Western blots after electrophoresis without reduction showed that the aAb and mAb47 were immunoreactive with a 66-kDa band and that aAb identified a doublet at 20 kDa. For electrophoresis under reducing conditions, the 66-kDa band resolved into two peptides of 40 and 26 kDa, whereas the doublet at 20 kDa remained unchanged. None of these reduced peptides was immunoreactive with aAb, whereas the 40-kDa peptide was immunoreactive with mAb47. The 40-kDa peptide extends from amino acid 549 to 933 of TPO, and its last 192 amino acids overlap the autoimmune 20-kDa peptide. After iodine labeling, the 20-kDa peptide lost its immunoreactivity. We conclude that the C-terminal end of the extracellular part of TPO, which includes all the tyrosine residues of the 20-kDa peptide, contains at least one conformational B-cell epitope involved in autoimmune thyroid diseases.
Estienne
V
V
Unit 38 of INSERM and the Laboratoire de Biochimie Endocrinienne et Métabolique, Faculté de Médecine, 27, boulevard Jean Moulin, F-13385 Marseille Cedex 5, France.
Duthoit
C
C
Vinet
L
L
Durand-Gorde
J M
JM
Carayon
P
P
Ruf
J
J
D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000276:immunology*; D002851:Chromatography, High Pressure Liquid; D000939:Epitopes / Q000737:chemistry*; D006801:Humans; D007453:Iodide Peroxidase / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011487:Protein Conformation
D000939:Epitopes; D007453:Iodide Peroxidase