The crystal structures of the leucine-rich repeat domain (LRD) of the FSH receptor (FSHR) in complex with FSH and the TSH receptor (TSHR) LRD in complex with the thyroid-stimulating autoantibody (M22) provide opportunities to assess the molecular basis of the specificity of glycoprotein hormone–receptor binding. A comparative model of the TSH–TSHR complex was built using the two solved crystal structures and verified using studies on receptor affinity and activation. Analysis of the FSH–FSHR and TSH–TSHR complexes allowed identification of receptor residues that may be important in hormone-binding specificity. These residues are in leucine-rich repeats at the two ends of the FSHR and the TSHR LRD structures but not in their central repeats. Interactions in the interfaces are consistent with a higher FSH-binding affinity for the FSHR compared with the binding affinity of TSH for the TSHR. The higher binding affinity of porcine (p)TSH and bovine (b)TSH for human (h)TSHR compared with hTSH appears not to be dependent on interactions with the TSHR LRD as none of the residues that differ among hTSH, pTSH or bTSH interact with the LRD. This suggests that TSHs are likely to interact with other parts of the receptors in addition to the LRD with these non-LRD interactions being responsible for affinity differences. Analysis of interactions in the FSH–FSHR and TSH–TSHR complexes suggests that the α-chains of both hormones tend to be involved in the receptor activation process while the β-chains are more involved in defining binding specificity.
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R Núñez Miguel, J Sanders, D Y Chirgadze, T L Blundell, J Furmaniak, and B Rees Smith
R Núñez Miguel, J Sanders, D Y Chirgadze, J Furmaniak, and B Rees Smith
The TSH receptor (TSHR) ligands M22 (a thyroid stimulating human monoclonal antibody) and TSH, bind to the concave surface of the leucine rich repeats domain (LRD) of the TSHR and here, we show that M22 mimics closely the binding of TSH. We compared interactions produced by M22 with the TSHR in the M22–TSHR crystal structure (2.55 Å resolution) and produced by TSH with the TSHR in a TSH–TSHR comparative model. The crystal structure of the TSHR and a comparative model of TSH based on the crystal structure of FSH were used as components to build the TSH–TSHR model. This model was built based on the FSH–FSH receptor structure (2.9 Å) and then the structure of the TSHR in the model was replaced by the TSHR crystal structure. The analysis shows that M22 light chain mimics the TSHβ chain in its interaction with TSHR LRD, while M22 heavy chain mimics the interactions of the TSHα chain. The M22–TSHR complex contains a greater number of hydrogen bonds and salt bridges and fewer hydrophobic interactions than the TSH–TSHR complex, consistent with a higher M22 binding affinity. Furthermore, the surface area formed by TSHR residues N208, Q235, R255, and N256 has been identified as a candidate target region for small molecules which might selectively block binding of autoantibodies to the TSHR.
F Grennan Jones, A Wolstenholme, S Fowler, S Smith, K Ziemnicka, J Bradbury, J Furmaniak, and B Rees Smith
Expression of a major thyroid autoantigen, thyroid peroxidase (TPO) was studied using the baculovirus-insect cell expression system. Human TPO cDNA modified so as to code for the extracellular fragment of the protein was placed under the control of the strong polyhedrin promoter in baculovirus transfer vector pBlueBacIII and cotransfected with linearized AcMNPV viral DNA. Expression in two insect cell lines Spodoptera frugiperda (Sf9) and Tricoplusia ni (High Five) was investigated and levels of recombinant TPO (rTPO) monitored by RIA and SDS-PAGE followed by Western blotting. Both insect cell lines expressed rTPO, but higher levels (30 mg/l culture medium) were obtained with High Five cells. Culture medium rTPO was purified and its glycosylation and immunoreactivity analysed. Lectin-affinity blotting and treatment with glycosidases indicated that both high mannose and complex-type sugar residues were associated with the recombinant protein. Studies with an ELISA based on biotin-labelled rTPO and an immunoprecipitation assay based on 125I-labelled rTPO indicated that the rTPO and native TPO showed similar reactivity to TPO autoantibodies (r=0·96, P<0·001, n=50 and r=0·99, P<0·001, n=80 respectively).
In addition, rTPO expressed in High Five cells showed enzyme activity comparable with that of native TPO when the heme biosynthesis precursor δ-aminolevulinic acid was included in the culture medium.
Overall, our studies indicate that the High Five insect cell line provides a useful system for the expression of relatively high levels of rTPO which should be suitable for structural analysis of TPO and TPO—TPO autoantibody complexes.
N Wedlock, J Furmaniak, S Fowler, Y Kiso, J Bednarek, A Baumann-Antczak, C Morteo, P Sudbery, A Hinchcliff, and B Rees Smith
Saccharomyces cerevisiae and the methylotrophic yeast Hansenula polymorpha have been used to express both full-length and a large hydrophilic domain of human thyroid peroxidase (TPO). Expression of TPO in S. cerevisiae, using the natural signal sequence or the yeast α-mating factor (MFα) signal sequence, resulted in undetectable or very low levels of recombinant TPO production. However, TPO was expressed when the natural TPO leader sequence was replaced by the yeast STE2 signal sequence. This recombinant TPO reacted with both rabbit anti-human TPO polyclonal and mouse anti-human TPO monoclonal antibodies on Western blots. In the case of H. polymorpha, TPO expression was achieved when the natural TPO leader sequence was replaced by the MFα leader and the construct placed under the control of the methanol-regulated promoter from the methanol oxidase gene. The recombinant TPO produced in H. polymorpha reacted with both TPO polyclonal and TPO monoclonal antibodies. No TPO was produced when the signal sequence of SUC2 (invertase) or the TPO natural signal sequence was used to direct expression.
Y Oda, J Sanders, S Roberts, M Maruyama, R Kato, M Perez, VB Petersen, N Wedlock, J Furmaniak, and B Rees Smith
We have used fragments of the TSH receptor (TSHR) expressed in E. coli as glutathione S-transferase fusion proteins to produce rabbit polyclonal antibodies and a panel (n=5) of monoclonal antibodies to the extracellular fragment of the TSHR. The binding characteristics of the antibodies to linear, conformational, glycosylated and unglycosylated forms of the receptor in different assay systems have been investigated. The reactivity of these antibodies with the TSHR was assessed by Western blotting with both native and recombinant human TSHR expressed in CHO cells, immunoprecipitation of 35S-labelled full-length TSHR produced in an in vitro transcription/ translation system, immunoprecipitation of 125I-TSH/TSHR complexes, inhibition of 125I-TSH binding to the TSHR and fluorescence activated cell sorter (FACS) analysis of binding to CHO-K1 cells expressing the TSHR on their cell surface. Fab fragments of monoclonal antibodies were isolated, labelled with 125I and used to determine the affinity constants of these antibodies with receptor, bound and free Fab being separated by polyethylene glycol (PEG) precipitation. Rabbit polyclonal and mouse monoclonal antibodies reacted with the TSHR in Western blotting and one monoclonal antibody (3C7) was able to inhibit 125I-TSH binding to native human TSHR (74% inhibition), recombinant human TSHR (84% inhibition) and porcine TSHR (65% inhibition). Affinity constant values for TSHR monoclonal antibody Fab fragments calculated using Scatchard analysis were about 10(7) M(-1). Four out of five monoclonal antibodies reacted in FACS analysis with TSHR expressed on the surface of CHO-K1 cells. The FACS unreactive monoclonal (3C7) bound well to detergent solubilised TSH receptors and this emphasised the importance of using a combination of FACS analysis and radioactively-labelled probes in analysis of the TSH receptor. The monoclonal antibodies produced in this study were found to be of relatively low affinity but proved useful for detection of the receptor by Western blotting and by FACS analysis.