Characterization of recombinant monoclonal antithyrotropin receptor antibodies (TSHRAbs) derived from lymphocytes of patients with Graves' disease: epitope and binding study of two stimulatory TSHRAbs.
Anti-TSH receptor autoantibodies (TSHRAbs) are known to be involved in Graves' disease. To elucidate the molecular mechanism of the pathogenesis of Graves' disease, we previously isolated and reconstituted the Ig genes of two B cell clones (101-2 and B6B7) producing a monoclonal thyroid-stimulating antibody (TSAb), a stimulating type of TSHRAb, obtained from patients with Graves' disease. In the present study, we produced a large amount of recombinant monoclonal TSAbs in eukariotic cells using these genes and characterized them. First, we tried to identify their epitopes in the TSHR, by using a panel of mutants of the extracellular domain of the TSH receptor (TSHR). Substantial cell surface expression level of each mutant was confirmed by fluorescence-activated cell sorter analysis using a TSHRAb. Mutations in the N-terminal (but not C-terminal) region of the extracellular domain of TSHR abrogated or reduced TSAb activities of both antibodies, whereas they had opposite effects on TSH activity; cAMP generation by 101-2 significantly decreased in the receptors mutated in amino acids 52-56 and 58-61, and that by B6B7 decreased in amino acids 34-37 and 58-61. Secondly, purified antibodies were radiolabeled and tested for binding to cells expressing high levels of TSHR. Although their affinities were lower than that of TSH, their binding was not displaced by TSH. The antibody binding was not mutually competitive. These findings suggest that these antibodies interact with the N-terminal region of the receptor and transduce a signal through binding sites different from TSH. We believe that this is the first report of the characterization of human monoclonal TSHRAbs on their epitopes and bindings, confirming previous reports using patient sera or murine monoclonal antibodies.
Akamizu
T
T
Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Japan. akataka@kuhp.kyoto-u.ac.jp
Moriyama
K
K
Miura
M
M
Saijo
M
M
Matsuda
F
F
Nakao
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D001323:Autoantibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D001666:Binding Sites, Antibody; D019556:COS Cells; D000939:Epitopes / Q000276:immunology*; D005434:Flow Cytometry; D017353:Gene Deletion; D006111:Graves Disease / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D018828:Immunoglobulins, Thyroid-Stimulating; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016296:Mutagenesis; D051381:Rats; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D000939:Epitopes; D007074:Immunoglobulin G; D018828:Immunoglobulins, Thyroid-Stimulating; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; C094372:thyrotropin-binding inhibitory immunoglobulin