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 beta subunit of the human chorionic gonadotrophin (hCG) is implied in heterodimer stability (beta26-110 disulphide bridge), in vitro LH bioactivity (region beta102-110) and in in vivo LH bioactivity (beta CTP). Like the hCG beta, the equine eLH and eCG beta 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 beta 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 beta26-110 disulphide bond was mutated and the resulting mutated beta subunits were transiently co-expressed with common alpha subunit in COS7 cells. These regions were also deleted in a betaalphaeLH/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 betaCTP 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 beta26-110 disulphide bond in the betaalpha 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 beta subunit does not seem to be essential for eLH/CG dimer stability upon secretion and expressing LH and FSH bioactivities.
Galet
C
C
Laboratoire de Physiologie de la Reproduction des Mammifères Domestiques, URA INRA-CNRS 1291 37380 Nouzilly, France. galet@tours.inra.fr
Chopineau
M
M
Martinat
N
N
Combarnous
Y
Y
Guillou
F
F
D000818:Animals; D001681:Biological Assay / Q000379:methods; D019556:COS Cells; D002522:Chlorocebus aethiops; D004220:Disulfides / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005640:Follicle Stimulating Hormone / Q000096:biosynthesis; D006064:Gonadotropins, Equine / Q000737:chemistry / Q000378:metabolism*; D006736:Horses / Q000378:metabolism*; D007986:Luteinizing Hormone / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
D004220:Disulfides; D006064:Gonadotropins, Equine; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone