MAPPING THYROID PEROXIDASE EPITOPES USING RECOMBINANT PROTEIN-FRAGMENTS

The identification of autoantibody epitopes is important to the unders tanding of autoimmune thyroid diseases. In the case of thyroid peroxid ase antibodies (TPO-ab), recent reports have disagreed about the numbe r and type of autoantibody epitopes found in human TPO. In order to cl arify the nature of these epitopes, we used an approach that provides recombinant human TPO produced by bacterial cells. The cDNA of four sl ightly overlapping fragments of human TPO-TPO 1(Glu 17-Ser 227), TPO 2 (Tyr 226-Thr 476), TPO 3(Glu 471-Ser 720) and TPO 4(Phe 709-Leu 993)-w ere amplified by polymerase chain reaction and subcloned into the expr ession vector pMAL. In addition, a TPO 3 species for an alternatively spliced form of TPO of 876 amino acids was constructed (TPO 3M). Each of these constructs encodes a fusion protein, in which the amino termi nal portion is maltose-binding protein, followed by the sequence of th e fragment of human TPO. The plasmid constructs were transformed in Es cherichia call and, after growth, bacterial cells were harvested, lyse d and the lysate was passed over an amylose affinity column and eluted with maltose. Western blots were performed using 33 sera from patient s with autoimmune thyroid disease (group 1) and 17 sera from patients with nodular goiter and focal thyroiditis (group 2), all positive for TPO-ab measured by radioimmunoassay; sera from 10 healthy people with no clinical evidence of thyroiditis and positive for TPO-ab measured b y radioimmunoassay (group 3) and sera from 30 patients with antigastri c parietal cell antibodies without signs or symptoms of thyroiditis, 1 6 negative for TPO-ab (group 4a) and 14 positive for TPO-ab (group 4b) , were included in the study. The four TPO fragments and the alternati vely spliced form of TPO were used as antigens. Our results show that in the first group 91% of sera recognized TPO 3, 33% recognized TPO 4 while 21% and 18% reacted with TPO 1 and TPO 2, respectively, In the s econd group of patients 76% of sera recognized TPO 3, 35% recognized T PO 4, 29% reacted with TPO 1 and 29% with TPO 2. In group 3 (normal he althy people) the individual peptides were recognized at a similar fre quency compared to the other groups. In group 4a, 75% and in group 4b, 93% of sera reacted with one of the fusion recombinant proteins. Immu noreactivity with TPO 3M was the same as that of TPO 3. In conclusion: (i) we have optimized the production of TPO peptides in bacterial cel ls; (ii) TPO-ab are able to bind many amino acid sequences of the prot ein with a hot spot in TPO 3 (known to contain the linear epitopes C2( 590-622) and C21(709-721); (iii) TPO-ab recognize other epitopes in th e amino and carboxyl portions of the protein; (iv) no difference is ob served when comparing the levels of antibodies and the number or type of peptide fragments recognized: (v) there are no disease-specific epi topes, as sera from normal healthy subjects with TPO-ab recognize the same epitopes in a similar percentage to those recognized by patients; (vi) the immunoreactivity of TPO 3 is not changed when the fragment e xpressed is the alternatively spliced form; (vii) we confirm that anti gastric parietal cell antibodies recognize epitopes on the TPO molecul e even when TPO-ab are negative by radioimmunoassay, suggesting a shar ed epitope between TPO and the gastric parietal cell.