Antibody Protection Reveals Extended Epitopes on the Human TSH Receptor

Abstract

Stimulating, and some blocking, antibodies to the TSH receptor (TSHR) have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD). However, successful crystallization of TSHR residues 22–260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs) have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1–412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity.

Antigenic “Hot- Spots” on the TSH Receptor Hinge Region

“…Autoantibodies of the stimulating, blocking, and neutral varieties also develop in TSHR immunization models (32, 33) so it is likely that the native tripartite structure of the TSHR (ECD, hinge, and TMD) helps define its antigenic character and repertoire of autoantibodies in animal models and patients with AITD. The development of stimulating monoclonal antibodies (34–36) along with the crystallization of human stimulating and blocking antibodies bound to a partial TSHR ECD (8) together with antibody protection analysis (10) have all provided deeper insight into the binding epitopes and mode of action of TSHR antibodies (8, 9). The large ECD consisting of residues 1–280 is a major pocket for TSH, stimulating and blocking antibody binding (7, 37).…”

“…Crystallization of the TSHR ECD bound to human stimulating and blocking monoclonal antibodies to the TSHR has provided insight into antibody contact residues showing the LRD as the major binding region (8, 9). Although the crystal structures did not include the full hinge region we have provided evidence that TSHR-Abs also bind to this area (10). The crystal structure of the hinge region of the FSHR (11) has provided evidence of the structural features of this region which likely applies to all glycoprotein hormone receptors including the TSHR and has allowed homology modeling of the entire ectodomain (12) and thus extended our understanding on ligand displacement of specific hinge fragments.…”

Characterizing the Interplay of Lymphocytes in Graves’ Disease

“…Their binding also prevents TSH from binding to and stimulating TSHR, which can lead to hypothyroidism [ 18 ]. The blocking antibodies bind to more varied regions of TSHR, with some binding to the LRR without fitting in the grooves enough to cause a conformational change in TSHR and others binding to the linear epitopes [ 19 , 22 ]. Neutral antibodies also bind to various epitopes of TSHR, though they appear to bind more to the linear epitopes of TSHR [ 19 ].…”

Graves’ Disease Mechanisms: The Role of Stimulating, Blocking, and Cleavage Region TSH Receptor Antibodies

“…Review concave surface of the leucine-rich repeat region (LRR) of the TSHR ectodomain were found to be important for antibody binding. Recent studies from our laboratory looking at conformational epitopes using mass spectrometry [28] have indicated that epitopes also exist outside the LRR for blocking as well as stimulating monoclonal antibodies. In addition, the importance of the N terminal region of the extracellular domain (ECD) has been well illustrated as well as residues in the ''hinge'' region [2].…”