A Single-Chain Tetradomain Glycoprotein Hormone Analog Elicits Multiple Hormone Activities In Vivo1

Abstract

We previously demonstrated that genetically linking one or more of the glycoprotein hormone-specific beta subunit genes to the common alpha subunit resulted in single-chain analogues that were bioactive in vitro. The ability of such large structures to bind their cognate receptors with high affinity supported the hypothesis that extensive flexibility exists between the ligand and receptor to establish a functional complex. To further characterize the extent of this conformational flexibility, we engineered a single-chain analogue that consists of sequentially linked thyroid-stimulating hormone (TSH) beta, follicle-stimulating hormone (FSH) beta, and chorionic gonadotropin (CG) beta subunits to the alpha subunit and expressed this chimera in transfected CHO (Chinese hamster ovary) cells. Because the four subunits are genetically linked and expressed as a single-chain, this analogue presumably lacks significant native structural features of the individual heterodimers. However, it exhibited FSH, CG, and TSH activities in vitro. Here, we test whether this nonnative structure would be stable in vivo and thus biologically active. Using a variety of bioassay protocols, we demonstrate that the analogue elicits multihormone activities when injected in vivo. First, treatment with the analogue caused increases in ovarian and uterine weights and resulted in elevated serum estradiol. Second, the analogue-stimulated ovarian follicle growth and pharmacologically rescued in vivo FSH deficiency similar to recombinant human FSH or equine CG (eCG) as confirmed by induction of aromatase in the ovaries of FSHbeta knockout mice. Third, in a superovulation protocol, when primed with eCG, the analogue elicited a dose-dependent ovulatory response comparable with that by native heterodimeric human CG. Finally, the analogue-stimulated thyroxin production in hypothyroid mice similar to the pituitary-derived human TSH standard. Based on these data, we conclude that a single-chain tetradomain glycoprotein hormone analogue, despite its presumed altered conformation, is stable and biologically active in vivo. Our results establish the permissiveness and conformational plasticity with which the glycoprotein hormones are recognized in vivo by their target cell receptors.

Single-Chain, Triple-Domain Gonadotropin Analogs with Disulfide Bond Mutations in the α-Subunit Elicit Dual Follitropin and Lutropin Activities in Vivo

“…6B). The expression in the ovaries of the control medium-injected group is the baseline expression of aromatase RNA, typically observed in immature mice at this age (15,16).…”

“…6, A and B). Ovarian aromatase converts testosterone to estrogen and is exquisitely sensitive to FSH stimulation (15,16). We have previously standardized a semiquantitative RT-PCR assay that measures aromatase expression in mouse ovaries (15).…”

“…7). A typical superovulation protocol includes priming by PMSG of immature female mice that support ovarian follicle growth followed by an ovulatory dose of hCG (LH activity) that results in release of multiple ova into the oviducts (15). PMSG-primed immature female mice were injected with either hCG or different doses of the analogs, and the number of ova in the oviduct was counted the next day.…”

“…In the first step, the triple-domain single chain was digested with BamHI/SalI. The fragment containing BamHI-FSH␤-CTP-CG␤-exon2-SalI sequence was ligated into BamHI/SalI site of pM 2 HA (10,15). This construct was then digested with SalI.…”

“…To examine the synthesis of single-chain chimeras, CHO cells expressing the wild-type, triple-domain analog or the ␣ mutated chimeras were labeled for 16 h in cysteine-free Ham's F-12 medium containing dialyzed FBS and [ 35 S]cysteine (10,15). Aliquots of cell lysate and medium were immunoprecipitated with polyclonal antiserum directed against CG␤-subunit, which was raised in our laboratory.…”