Biological Activities of Tethered Equine Chorionic Gonadotropin (eCG) and Its Deglycosylated Mutants

Abstract

Abstract. Equine chorionic gonadotropin (eCG), which consists of highly glycosylated α-and β-subunits, is a unique member of the gonadotropin family because it elicits response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in species other than the horse. In this study, recombinant tethered-eCG as well as its deglycosylated mutants were produced to determine if α-and β-subunits can be synthesized as a single polypeptide chain (tethered-eCG) and display biological activity. We found that tethered-eCG (T-βα) had both LH-and FSH-like activities comparable to dimeric eCG. Luteinizing hormone-like activity of tethered-eCGs deglycosylated at Asn 56 (T-βα56) was decreased. In contrast, LH-like activity of eCG without O-glycosylated carboxylterminal peptide (CTP) (T-βcα) was slightly decreased but still similar to T-βα. Double mutation at Asn 56 and CTP (T-βcα56) caused marked decrease in the activity, indicating that both glycosylations at Asn 56 and CTP are involved in LH-like activity in the tethered form. Interestingly, FSH-like activity remained in all deglycosylated eCG mutants (T-βα56, T-βcα and T-βcα56) as well as T-βα. The biological roles of oligosaccharides at Asn 56 of eCG α-subunit and O-linked peptide of β-subunit appear to be different in LH-and FSH-like activities in tethered-eCG. Key words: Equine chorionic gonadotropin, Luteinizing hormone, Follicular stimulating hormone, Glycosylation, Tethered hormone (J. Reprod. Dev. 50: [297][298][299][300][301][302][303][304] 2004) quine chorionic gonadotropin (eCG) is a member of the glycoprotein hormone family which includes luteinizing hormone (LH), follicles t i m u l a t i n g h o r m o n e ( F S H ) a n d t h y r o i dstimulating hormone (TSH). This hormone family is characterized by a heterodimeric structure composed of a common α-subunit noncovalently linked to a hormone-specific β-subunit [1]. The β-subunits of eCG and eLH, translated from the same gene, have identical primary structures [2,3]. The difference between eCG and eLH lies in the structure of their carbohydrates, which are both sialylated and sulfated in LH and sialylated in CG [4,5]. Equine chorionic gonadotropin shows both LH-and FSH-like activities in many species but not the horse [6][7][8][9][10][11]. The LH-and FSH-like activities of eCG in vitro observed in heterologous species is of fundamental interest to the study of structurefunction relationships of gonadotropins and their receptors.We previously prepared recombinant eCG in CHO-K1 cells, and found that these recombinant

Expression of aldo-keto reductase family 1 member C1 (AKR1C1)gene in porcine ovary and uterine endometrium during the estrous cycle and pregnancy

“…The cells were incubated at 37°C in 5% CO 2 . Cultured CHO-K1 cells were transfected with the expression vectors by using the liposome transfection method described previously [15]. Briefly, DNA (0.8 μg) was diluted in 50 μL of Opti-MEM I reduced medium without serum and mixed gently.…”

Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants

“…Nevertheless, we can suppose that the three-dimensional conformation of single-chain eLH/CG could be altered when compared with heterodimeric eLH/CGs, as previously shown for hCG (Fralish et al 2003). Unlike previous studies which reported that eLH/CG b-a single chain was fully bioactive in vitro , Min et al 2004 and in vivo ( Jablonka-Shariff et al 2007), we report here that the b-a eLH/CG single-chain secretion by virus-infected insect cells was reduced when compared with heterodimeric eLH/CG and exhibited a slightly reduced in vitro LH bioactivity. Considering all these clues, it is of utmost interest to study the bioactivities of highly produced and fully bioactive heterodimeric glycoforms rather than those of single-chain variants whose conformation is potentially less similar to that of natural eCG and eLH.…”

“…The b-a configuration was widely used to produce recombinant single-chain glycoprotein hormones of many species (Narayan et al 1995, Boime & BenMenahem 1999, Dirnberger et al 2001, Fidler et al 2003, Min et al 2004, Jablonka-Shariff et al 2007, sometimes with heterologous CTP from hCG inserted between fused a-and b-subunits to enhance the secretion of these single-chain protein variants (Sugahara et al 1996, Garcia-Campayo et al 1997, Grossmann et al 1997, Fares et al 1998. These earlier studies showed that the CTP played the role of a flexible hydrophilic spacer allowing genetically fused a-and b-subunits to adopt a functional conformation and that secretion in the presence of CTP was enhanced due to its specific O-glycans.…”

The transcription factor Ap-1 regulates monkey 20α-hydroxysteroid dehydrogenase promoter activity in CHO cells

“…The correct plasmids were digested with Kpn I and Xho I, and then inserted into the same digested sites of the pGL3-Basic luciferase reporter vector (Promega, Madison, WI). Nucleotide inversion mutants (mut-1, mut-2, and mut-3) of the putative Ap-1 site were constructed according to the two-step overlapping PCR method [57] using the primers shown in Table 1. PCR was performed with Ex taq polymerase (TaKaRa, Osaka, Japan), and the mutant fragments were subcloned into the pCR 2.1.…”