Contribution of Specific Disulphide Bonds to Two Epitopes of Thyrotropin β‐Subunit Associated with Receptor Recognition

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Abstract
In previous studies, we have shown that two epitopes of bovine thyrotropin beta-subunit that are recognised by the monoclonal antibodies designated mAb 279 and mAb 299 are also associated with the receptor-binding site of bovine thyrotropin. The present investigation has examined the role of the six disulphide bonds of bovine thyrotropin beta-subunit in the conformational stabilisation of these two epitopes, and hence assessed the relative contribution that these disulphide bonds make to the stabilisation of the receptor-binding region of the beta-subunit. The experimental procedure involved the production of several bovine thyrotropin beta-subunit-related derivatives in which an increasing number of the disulphide bonds were selectively reduced with dithiothreitol and alkylated with iodoacetic acid. Antibody-binding properties of these derivatives were then evaluated in thyrotropin beta-subunit-specific immunoassays based on the use of the well characterised mAb 279 and mAb 299, to determine the effect of disulphide bond reduction and alkylation on each epitopic specificity. In separate experiments, the residual disulphide bonds that remained intact following these selective partial reductive alkylation procedures were then fully reduced and alkylated with the fluorescent reagent 5-N-[(iodoacetamidoethyl)amino]naphthalene 1-sulphonic acid. The relative contribution of individual disulphide bonds in the stabilisation of each epitope could then be assessed after application of reverse-phase HPLC peptide mapping methods. Epitope recognition by mAb 279 was not dependent on the preservation of the so-called determinant loop Cys88-Cys95 disulphide bond nor directly involved binding interactions via the Cys2-Cys52, Cys27-Cys83, and Cys31-Cys85 disulphide bonds. However, the experimental results indicated that the mAb 279 epitope was stabilised by the Cys19-Cys105 and Cys16-Cys67 disulphide bonds, which is consistent with other data on the role of the C-terminal region of the thyrotropin beta-subunit in this epitope. In contrast, the presence of an intact Cys88-Cys95 disulphide bond was required for the stabilisation of the mAb 299 epitope, although the location of this disulphide bond is distal to the hairpin loop structure that constitutes the mAb 299 epitope. These results on the relative contribution of these disulphide bonds are also discussed in terms of their relationship to the stabilisation of the predicted region of bovine thyrotropin beta-subunit involved in receptor binding.