Expression of an in vitro biologically active equine LH/CG without C-terminal peptide (CTP) and/or beta26-110 disulphide bridge

The C-terminal region of the β subunit of the human chorionic gonadotrophin (hCG) is implied in heterodimer stability (β26-110 disulphide bridge), in vitro LH bioactivity (region β102-110) and in in vivo LH bioactivity (β CTP). Like the hCG β, the equine eLH and eCG β subunits, also possess a C-terminal extension (CTP). But, in contrast to hCG, eLH and eCG bind to both LH and FSH receptors in species other than the horse. This allows investigation of the roles of the β subunit C-terminal region of a eLH/CG recombinant molecule on both LH and FSH activities. To do so, the CTP was deleted and/or the β26-110 disulphide bond was mutated and the resulting mutated β subunits were transiently co-expressed with common α subunit in COS7 cells. These regions were also deleted in a βαeLH/CG single chain also expressed in COS7 cells. The hormones produced were characterized by different ELISAs and in vitro LH and FSH bioassays. Mutation of the 26-110 disulphide bond and deletion of the βCTP led to a decrease in eLH/CG heterodimer production. Double mutation promoted an additive effect on production of the heterodimer and of the corresponding tethered eLH/CG. The elimination of the β26-110 disulphide bond in the βα single chain had no effect on its production However, neither the 26-110 disulphide bond nor the CTP mutations affected dimer stability and bioactivities of the secreted heterodimers and/or single chain molecules. Therefore, in contrast to hCG, the 26-110 S-S bond of the recombinant eLH/CG β subunit does not seem to be essential for eLH/CG dimer stability upon secretion and expressing LH and FSH bioactivities.