The beta104-109 sequence is essential for the secretion of correctly folded single-chain beta alpha horse LH/CG and for its FSH activity.
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 beta alpha eLH/CG was used to identify sequences in the beta-subunit involved in the secretion and activities of the hormone. The C-terminal region of the beta-subunit was progressively truncated. All resulting truncated single-chains were secreted in the media as detected by an anti-beta peptide 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 beta-subunit (beta109alpha eLH/CG) resulted in a 90% decrease in the recognized correctly folded hormone compared with the full-length beta alpha eLH/CG single-chain and no properly folded hormone was detected in the secretion medium when the last 46 amino acids of the beta-subunit were deleted (beta103alpha eLH/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 beta alpha eLH/CG (beta104-109Ala alpha) led to a 50% decrease in the production of properly folded hormone in COS-7 as well as in alphaT3 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 beta eLH/CG subunit is essential for the secretion of a fully folded beta alpha eLH/CG and for its FSH activity but not for its LH activity.
Galet
Colette
C
Department of Pharmacology, Roy J and Lucille A Carver College of Medicine, The University of Iowa, 2-319B BSB, 51 Newton Road, Iowa City, Iowa 52242-1109, USA.
Guillou
Florian
F
Foulon-Gauze
Florence
F
Combarnous
Yves
Y
Chopineau
Maryse
M
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006063:Chorionic Gonadotropin / Q000235:genetics / Q000378:metabolism*; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D006736:Horses; D007986:Luteinizing Hormone / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D006063:Chorionic Gonadotropin; D011994:Recombinant Proteins; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone