β-Subunit 94~96 Residues of Tethered Recombinant Equine Chorionic Gonadotropin are Important Sites for Luteinizing Hormone and Follicle Stimulating Hormone like Activities
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서지정보
ㆍ발행기관 : 한국동물번식학회
ㆍ수록지정보 : Reproductive & developmental biology / 34권 / 1호
ㆍ저자명 : Jong-Ju Park, Naidansuren JarGal, Jong-Taek Yoon, Kwan-Sik Min
목차
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
영어 초록
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.
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