Human thyroid peroxidase-myeloperoxidase chimeric molecules: tools for the study of antigen recognition by thyroid peroxidase autoantibodies.
We constructed seven chimeric molecules in which sequential segments in the cDNA for thyroid peroxidase (TPO) were replaced with the homologous regions of myeloperoxidase (MPO) cDNA. The sizes of the translated cDNA segments A through G ranged from 23-175 amino acid residues in length. The TPO-MPO cDNA chimeras, inserted into an eukaryotic expression vector, were stably transfected into Chinese hamster ovary cells. Protein expression was examined by immunoblotting under reduced/denaturing conditions with a murine monoclonal antibody to denatured wild-type TPO. Expression (at a low level) was confirmed for TPO-MPO chimeras A, B, F, and G. The amino acid substitutions in TPO-MPO-C eliminate the monoclonal antibody epitope, and this chimera, therefore, provides a negative control. TPO-MPO-D and TPO-MPO-E did not generate detectable levels of protein. To study TPO autoantibody interaction with native protein, we performed fluorescence-activated cell sorter analysis using intact Chinese hamster ovary cells stably transfected with the wild-type and TPO-MPO chimeric cDNAs. Of the chimeras, only cells transfected with TPO-MPO-A (N-terminal 146 amino acids of MPO substituted for the N-terminal 121 amino acids of TPO) were recognized by TPO autoantibodies, although to a lesser degree than cells expressing wild-type TPO. In conclusion, the present data indicate that TPO autoantibodies can interact with TPO molecules in which the amino-terminus is replaced with the homologous MPO prosequence region, not normally present in mature MPO. Our study provides a foundation for designing future TPO mutants that may be of value for characterizing disease-associated B-cell epitopes in autoimmune thyroid disease.
Nishikawa
T
T
Thyroid Molecular Biology Unit, Veterans Administration Medical Center, San Francisco, California.
Nagayama
Y
Y
Seto
P
P
Rapoport
B
B
D000818:Animals; D000941:Antigens / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology*; D001483:Base Sequence; D016466:CHO Cells; D002469:Cell Separation; D002678:Chimera; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D005434:Flow Cytometry; D006801:Humans; D007453:Iodide Peroxidase / Q000276:immunology*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D009195:Peroxidase; D014162:Transfection
D000941:Antigens; D001323:Autoantibodies; D018076:DNA, Complementary; D015345:Oligonucleotide Probes; D009195:Peroxidase; D007453:Iodide Peroxidase