Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked α- and β-subunits. To study the function and signal transduction of tethered recombinant-eCG (rec-eCG), a single chain eCG molecule was constructed, and the rec-eCG protein was prepared. In this study, we constructed 5 mutants (Δ1, Δ2, Δ3, Δ4, and Δ5) of rec-eCG using data about known glycoprotein hormones to analyze the role of specific follicle stimulating homone (FSH)-like activity. Three amino acids of certain specific sites were replaced with alanine. The expression vectors were transfected into CHO cells and subjected to G418 selection for 2～3 weeks. The media were collected and the quantity of secreted tethered rec-eCGs was quantified by ELISA. The LH- and FSH-like activities were assayed in terms of cAMP production by rat LH/CG and rat FSH receptors. Then, the metabolic clearance rate analyzed by the injection of rec-eCG (5 IU) into the tail vein was analyzed. The mutant eCGs (Δ1, Δ4, and Δ5) were transcripted, but not translated into proteins. Rec-eCG Δ2 was secreted in much lower amounts than the wild type. Only the rec-eCG Δ3 (β-subunit: Gln94-Ile95-Lys96→Ala94-Ala95-Ala96) was efficiently secreted. Although activity is low, its LH-like activity was similar to that of tethered eCGβα. However, the FSH-like activity of rec-eCGβαΔ3 was completely flat. The result of the analysis of the metabolic clearance rate shoed the persistence of the mutant in the blood until 4 hours after the injection. After then, it almost disappeared at 8 hours. Taken together, these data suggest that 94～96 amino acid sequences in eCG β-subunit appear to be of utmost importance for signal transduction of the FSH receptor.

ABSTRACT   INTRODUCTION   MATERIALS AND METHODS    Materials    Transient and Stable Transfection of CHO Cell Lines    Hormone Quantitation and Western Blot Analysis ofTethered rec-eCGs    RT-PCR    In Vitro Bioassy for LH- and FSH-like Activities    Metabolic Clearance Rate of Tethered rec-eCGβαand -eCGβαΔ3   RESULTS    Production of Transient and Stably Tethered receCGMutants    RT-PCR of Cell Lines Transfected with Tetheredrec-eCG Mutant Vector    Western Blot Analysis    Biological Activity of Tethered rec-eCG in rLH/CGRand rFSHR    Clearance Rates of Tethered eCGβα and rec-eCGβαΔ3   DISCUSSION   REFERENCES

• Jong-Ju Park(Animal Biotechnology, Graduate School of Bio. & Information Technology, Institute of Genetic Engineering,)
• Naidansuren JarGal(Animal Biotechnology, Graduate School of Bio. & Information Technology, Institute of Genetic Engineering,)
• Jong-Taek Yoon(Animal Biotechnology, Graduate School of Bio. & Information Technology, Institute of Genetic Engineering,)
• Kwan-Sik Min(Animal Biotechnology, Graduate School of Bio. & Information Technology, Institute of Genetic Engineering,) Corresponding author