CONTRIBUTION OF SPECIFIC DISULFIDE BONDS TO 2 EPITOPES OF THYROTROPINBETA-SUBUNIT ASSOCIATED WITH RECEPTOR RECOGNITION

In previous studies, we have shown that two epitopes of bovine thyrotr opin beta-subunit that are recognised by the monoclonal antibodies des ignated mAb 279 and mAb 299 are also associated with the receptor-bind ing site of bovine thyrotropin. The present investigation has examined the role of the six disulphide bonds of bovine thyrotropin beta-subun it in thr conformational stabilisation of these two epitopes, and henc e assessed the relative contribution that these disulphide bonds make to the stabilisation of the receptor-binding region of the beta-subuni t. The experimental procedure involved the production of several bovin e thyrotropin beta-subunit-related derivatives in which an increasing number of the disulphide bonds were selectively reduced with dithiothr eitol and alkylated with iodoacetic acid. Antibody-binding properties of these derivatives were then evaluated in thyrotropin beta-subunit-s pecific immunoassays based on the use of the well characterised mAb 27 9 and mAb 299, to determine the effect of disulphide bond reduction an d alkylation on each epitopic specificity. in separate experiments, th e residual disulphide bonds that remained intact following these selec tive partial reductive alkylation procedures were then fully reduced a nd alkylated with thr fluorescent reagent 5-N-[(iodoacetamidoethyl)ami no]naphthalene 1-sulphonic acid. The relative contribution of individu al disulphide bends in the stabilisation of each epitope could then be assessed after application of reverse-phase HPLC peptide mapping meth ods. Epitope recognition by mAb 279 was not dependent on the preservat ion of the so-called determinant loop Cys888-Cys95 disulphide bond nor directly involved binding interactions via the Cys2-Cys52, Cys27-Cys8 3, 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 di sulphide bond was required for the stabilisation of the mAb 299 epitop e, although the location of this disulphide bond is distal to the hair pin loop structure that constitutes the mAb 299 epitope. These results on the relative contribution of these disulphide bonds are also discu ssed in terms of their relationship to the stabilisation of the predic ted region of bovine thyrotropin beta-subunit involved in receptor bin ding.