The β104–109 sequence is essential for the secretion of correctly folded single-chain βα horse LH/CG and for its FSH activity

Abstract

The dual LH and FSH activity of the equine LH (eLH)/equine chorionic gonadotropin (eCG) in heterologous species makes eLH/CG a good model to study structure/function relationships of gonadotropins. In order to bypass the problem of intracellular association of the heterodimer, a recombinant single-chain βαeLH/CG was used to identify sequences in the β-subunit involved in the secretion and activities of the hormone. The C-terminal region of the β-subunit was progressively truncated. All resulting truncated single-chains were secreted in the media as detected by an anti-βpeptide antibody in reducing conditions. However, using a conformation sensitive ELISA we show that the truncated single-chains were differently recognized: deletion of the last 40 amino acids of the β-subunit (β109αeLH/CG) resulted in a 90% decrease in the recognized correctly folded hormone compared with the full-length βαeLH/CG single-chain and no properly folded hormone was detected in the secretion medium when the last 46 amino acids of the β-subunit were deleted (β103αeLH/CG). We thus focused on the six amino acids sequence 104–109, which belongs to the seat-belt region. Mutation of the 104–109 sequence in alanines in the full-length βαeLH/CG (β104–109Alaα) led to a 50% decrease in the production of properly folded hormone in COS-7 as well as in αT3 pituitary cells. Moreover, the FSH activity of this mutant was decreased by 70% whereas its LH activity remained intact. These data lead us to conclude that the 104–109 region of the β eLH/CG subunit is essential for the secretion of a fully folded βαeLH/CG and for its FSH activity but not for its LH activity.