Monoclonal antibodies to the human TSH receptor: epitope mapping and binding to the native receptor on the basolateral plasma membrane of thyroid follicular cells

Abstract

We have characterized four murine monoclonal antibodies (mAbs) to the extracellular domain of the human TSH receptor (TSH-R.E), the target autoantigen of Graves' disease. Recombinant TSH-R.E used as immunogen, was produced in E. coli as a fusion protein with glutathione-S-transferase or in a baculovirus-insect cell system, as a non-fusion glycoprotein. To increase the epitope specificity of the mAbs, two different strains of mice (H-2(b) and H-2(d)) were immunized. The epitopes recognized by the mAbs were characterized by immunoblotting with various recombinant constructs of TSH-R.E and by binding to overlapping synthetic peptides of the receptor. The four IgG mAbs characterized recognized epitopes localized to different regions on the TSH-R.E; amino acids 22-35 (A1O and A11, both IgG2b from H-2(b) animals), amino acids 402-415 (A7, IgG2b from H-2(b) animals) and amino acids 147-228 (A9, IgG1 from H-2(d) animals). Immunolocalization studies showed that mAb A9 recognized TSH-R.E on unfixed cryostat sections, where binding was localized to the basolateral plasma membrane of thyroid follicular cells, suggesting that this antibody reacts with the native receptor on thyroid cells. The binding of the mAbs A7, A10 and A11 was also restricted to the basal surface of thyroid cells, but only after acetone fixation of the sections, implying that the epitopes recognized on the amino and carboxyl terminus of the extracellular region of the receptor are not accessible on the native molecule. None of the mAbs stimulated cyclic AMP responses in COS-7 cells transiently transfected with full-length functioning TSH-R.E, whilst weak inhibition of binding of radiolabelled TSH to porcine membranes in a radioreceptor assay was apparent with mAb A10 and A11, but only at high concentrations of IgG. The ability of mAb A9 to bind to the native receptor without stimulating activity or inhibition of TSH binding suggests that antibody can bind to the central region of the TSH-R.E without perturbing receptor function. The availability of mAbs that recognize epitopes on different regions of the extracellular domain of TSH-R will lead to a better understanding of the autoantigenic regions on TSH-R implicated in disease activity.

Yersinia enterocolitica Provides the Link between Thyroid-Stimulating Antibodies and Their Germline Counterparts in Graves’ Disease

“…In another recent study using a neutral mAb to TSHR generated in our laboratory, mAb A9 (27), cross-reactivity with Y. enterocolitica OmpF was reported (29). Reactivity of Graves' disease patient serum with Y. enterocolitica OmpF also was demonstrated.…”

“…Purified human TSHR protein (Novus Biologicals) was used to evaluate binding of rFab germline by sensitive ECL detection. Membranes were blocked overnight with PBS-5% milk powder/ 0.05% Tween 20 at room temperature and probed with purified rFab preparations (1 mg/ml) or mouse anti-TSHR mAbs KSAb1, KSAb2, or mAb A9 (specific for amino acid residues 214-222 of TSHR) (27,28) (1 mg/ml) as primary Abs, followed by detection with HRP-goat anti-mouse whole IgG conjugate (NA931; GE Healthcare) (diluted 1:1000 in PBS-5% milk powder/0.05% Tween 20). The blots probed with the TSAb M22 (RSR, Cardiff, U.K.) were developed with HRP-sheep anti-human IgG conjugate (NA933; GE Healthcare) (diluted 1:10,000 in PBS-5% milk powder/0.05% Tween 20).…”

“…Bioinformatics searches have reported on regions of sequence homology between Y. enterocolitica outer membrane porin (Omp) M and TSHR (26). Recently, a murine neutral mAb to TSHR (mAb A9) (27), whose linear epitope has been mapped to the TSHR A-subunit (28), was shown to bind Y. enterocolitica OmpF (29).…”

Cytokines, IgG subclasses and costimulation in a mouse model of thyroid autoimmunity induced by injection of fibroblasts co-expressing MHC class II and thyroid autoantigens

“…1a). The presence of TSHR antigen in both preparations was confirmed by the binding of murine MoAb A9, which recognizes an epitope in the amino terminal region of the TSHR [17], and not by normal mouse serum.…”

Monoclonal Pathogenic Antibodies to the Thyroid-Stimulating Hormone Receptor in Graves’ Disease with Potent Thyroid-Stimulating Activity but Differential Blocking Activity Activate Multiple Signaling Pathways

“…Displacement studies using labeled KSAb1 and KSAb2 IgG as tracers were also performed with another panel of anti-TSHR IgG mAbs that are specific for linear determinants on the receptor and that show negligible thyroid stimulatory activity (37). Neither KSAb1 or KSAb2 IgG showed any competition with the mAbs A10, A9, and A7, which are specific for residues located in the amino-terminal, middle, and carboxyl-terminal regions of the receptor, respectively (37) (not shown).…”