Evaluation of subunit truncation and the nature of the spacer for single chain human gonadotropins

Three single chain gonadotropins were designed based on the three-dimensional-structure of human choriogonadotropin and structure/activity relationships of the glycoprotein hormones. In each single chain, the C-terminal end of the human choriogonadotropin beta subunit is connected via Ser-Gly repeats to the N-terminal end of the alpha subunit. In addition, two of the single chains have truncated subunits. The three mutants were expressed in CHO cells. In vitro binding of two of the three mutants to the human lutropin/ choriogonadotropin receptor was found to be comparable to wild-type lutropin. In contrast, both the receptor binding and the in vitro bioactivity of the mutant with truncated alpha and beta subunits in which the beta:26-110 disulphide bond cannot be formed, are lowered relative to wild-type lutropin. The fact that this mutant still displays biological activity shows that the seat-belt arrangement proposed by Isaacs and coworkers [Lapthorn, A. J., Harris, D. C., Littlejohn, A., Lustbader, J. W., Canfield, R. E., Machin, K. J., Morgan, F. J. & Isaacs, N. W (1994) Nature 369, 455-461] is important but not essential for receptor binding and biological activity in the context of single chain gonadotropins. Single chains in which Ser-Gly spacers are combined with truncated subunits, provide an attractive approach towards the design and generation of novel, biologically active gonadotropins.