Site-specific mutagenesis of human chorionic gonadotrophin (hCG)-β subunit: influence of mutation on hCG production

1990 ◽  
Vol 5 (2) ◽  
pp. 97-102 ◽  
Author(s):  
C. Azuma ◽  
K. Miyai ◽  
F. Saji ◽  
S. Kamiura ◽  
Y. Tokugawa ◽  
...  
Keyword(s):  
In Vitro Transcription ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Amino Acid Sequences ◽  
In Vitro Mutagenesis ◽  
Β Subunit ◽  
Site Specific ◽  
Biological Specificity ◽  
Covalently Linked

ABSTRACT The heterodimeric glycoprotein hormones, human chorionic gonadotrophin (hCG), LH, TSH and FSH, consist of two non-covalently linked subunits, the α and β subunits. The β subunit is specific for each hormone and is responsible for the biological specificity, but the β subunits of different hormones show some degree of structural homology. The CAGY (cysteine-alanine-glycine-tyrosine) region is one of the amino acid sequences that is homologous in different β subunits and is highly conserved between species. In the present study, site-specific in-vitro mutagenesis was used to change three individual nucleotides in the centre of the CAGY region of the hCG-β subunit, and the effects of these mutations on hCG production was determined by in-vitro transcription and then translation in Xenopus laevis oocytes. The results indicate that the CAGY region, particularly the glycine residue at position 36 in the β subunit, is essential for the production of hCG. This finding is consistent with previous studies showing that this region is necessary for the biological activity of human TSH.

A single amino acid residue replacement in the β subunit of human chorionic gonadotrophin results in the loss of biological activity

1992 ◽  
Vol 8 (1) ◽  
pp. 87-89 ◽  
Author(s):  
F. Chen ◽  
D. Puett
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Β Subunit ◽  
Single Amino Acid Residue

ABSTRACT The heterodimer, human chorionic gonadotrophin (hCG), contains an a subunit that is common to the glycoprotein hormones and a hormone-specific β subunit. A comparison of all known β amino acid sequences shows that an aspartic acid at position 99 (with the numbering scheme for hCG-β) is one of the seven non-Cys invariant residues. Using site-directed mutagenesis we have replaced hCG-β Asp99 with Arg. Chinese hamster ovary cells, containing a stably integrated gene for bovine a subunit, were transiently transfected with plasmids containing wild-type and mutant hCG-β cDNAs. The Arg99 β mutant associated with the a subunit, but the resulting heterodimer failed to enhance intracellular cyclic AMP production in a gonadotrophin-responsive transformed murine Leydig cell line. Thus, a single amino acid residue replacement in this glycosylated heterodimer containing 237 amino acid residues is sufficient to abolish activity.

Mutations of Arg68 of the human chorionic gonadotrophin β subunit lead to reduced secretion

1996 ◽  
Vol 17 (3) ◽  
pp. 257-262 ◽  
Author(s):  
L Shen ◽  
H Xia ◽  
N Bhowmick ◽  
P Narayan ◽  
D Puett
Keyword(s):  
Leydig Cells ◽  
Expression Vector ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Site Directed Mutagenesis ◽  
Β Subunit ◽  
Wild Type ◽  
Biological Assays ◽  
Immunologic Activity

ABSTRACT The Arg68-Leu69 sequence is invariant in the β subunits of chorionic gonadotrophin and luteinizing hormone from a variety of species. Using site-directed mutagenesis of the human chorionic gonadotrophin (hCG)-β cDNA, several replacements of Arg68, an Ala replacement of Leu69, and a multiple replacement with Ala-Ala-Ala-Ala of the tetrapeptide sequence, Arg68-Leu69-Pro70-Gly71, were prepared and characterized. The wild-type and mutant cDNAs were subcloned into a pRSV expression vector and transiently transfected into CHO cells containing a stably integrated gene for bovine a. Concentrations of secreted wild-type and mutant hCG-β subunit and holoprotein were determined using radioimmunoassays; potencies, i.e. the ratio of biologic to immunologic activity, of several of the mutant heterodimers were measured in vitro via gonadotrophin-mediated steroidogenesis in transformed murine Leydig cells (MA-10). The Leu69→Ala mutant formed a mutant holoprotein that was essentially equipotent with wild-type hormone in the steroidogenesis assay. The Arg68 replacements with Lys, Ala, and Leu were poorly secreted by the cells, e.g. <10% that of wild-type hCG; however, sufficient quantities of mutant holoproteins containing Lys68 and Ala68 were obtained for biological assays, and both exhibited greater apparent potencies than wild-type hormone. Likewise, a mutant holoprotein containing the Arg68-Leu69-Pro70-Gly71→Ala-Ala-Ala-Ala multiple replacement was apparently more potent than wild-type hormone, but it too was secreted at lower levels than wild-type. These results establish that replacements of Arg68 in hCG-β diminish secretion, but the small amount of holoprotein that is formed and secreted appears to be of somewhat greater potency than wild-type hormone.

STIMULATION BY HUMAN CHORIONIC GONADOTROPHIN OF OESTRADIOL PRODUCTION BY DISPERSED CELLS FROM HUMAN CORPUS LUTEUM: COMPARISON WITH PROGESTERONE PRODUCTION; UTILIZATION OF EXOGENOUS TESTOSTERONE

1981 ◽  
Vol 91 (2) ◽  
pp. 197-203 ◽  
Author(s):  
M. C. RICHARDSON ◽  
G. M. MASSON
Keyword(s):  
Luteal Phase ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Cell Suspensions ◽  
Corpora Lutea ◽  
Progesterone Production ◽  
Late Luteal Phase ◽  
Oestradiol Production ◽  
Dispersed Cells

Cell suspensions were prepared from tissue samples of human corpora lutea obtained during the mid- and late-luteal phase of the menstrual cycle. Both oestradiol and progesterone production by dispersed cells were stimulated by similar concentrations of human chorionic gonadotrophin (hCG). As the degree of stimulation of production by hCG was greater for progesterone than for oestradiol (five- to tenfold compared with two- to threefold higher than basal production), the ratio of progesterone to oestradiol produced varied according to the level of trophic stimulation. A comparison of cell suspensions prepared from mid- and late-luteal phase corpora lutea, exposed to the same concentration of hCG (10 i.u./ml) in vitro, did not reveal a shift to oestradiol production in the late-luteal phase. Provision of additional testosterone during incubation raised the level of oestradiol production by dispersed luteal cells. At an optimum concentration of testosterone (1 μmol/l), oestradiol synthesis was not raised further in the presence of hCG or N6, O2-dibutyryl cyclic AMP, suggesting a lack of induction or activation of the aromatase system by gonadotrophin in short-term cultures. Basal and stimulated levels of progesterone production were not significantly impaired in the presence of testosterone.

scholarly journals Newt satellite 2 transcripts self-cleave by using an extended hammerhead structure.

1991 ◽  
Vol 11 (12) ◽  
pp. 6109-6115 ◽  
Author(s):  
L M Pabón-Peña ◽  
Y Zhang ◽  
L M Epstein
Keyword(s):  
Structural Model ◽  
In Vitro Mutagenesis ◽  
Base Pairs ◽  
Site Specific ◽  
Internal Loops

Synthetic transcripts of satellite 2 DNA from newts undergo self-catalyzed, site-specific cleavage in vitro. Cleavage occurs within a domain that is similar to the hammerhead domain used by a number of self-cleaving, infectious plant RNAs. The newt hammerhead has a potentially unstable structure due to a stem composed of two base pairs and a 2-nucleotide loop, and unlike other hammerheads that have been studied, it cannot cleave as an isolated unit. Here we show that cleavage by a single newt hammerhead requires additional satellite 2 sequences flanking both ends of the hammerhead domain. We also present a structural model of a truncated satellite 2 transcript which is capable of cleavage. The structure includes an internally looped extension to one of the conserved stems of the hammerhead. By in vitro mutagenesis, the identities of each of the five nucleotides composing one of the internal loops were shown to be critical for cleavage. Additional evidence that the extension stimulates self-cleavage in a manner other than by simply stabilizing the hammerhead is presented.

scholarly journals Association Between Serum Oestradiol Level on the Human Chorionic Gonadotrophin Administration Day and Neonatal Birthweight After in Vitro Fertilization-embryo Transfer: A Retrospective Study of 3659 Singleton Live Births

2020 ◽  
Author(s):  
Yu Liu ◽  
Jing Li ◽  
Yihong Guo
Keyword(s):  
Retrospective Study ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Vitro Fertilization ◽  
Group 4 ◽  
Group 3 ◽  
Group 2 ◽  
Ivf Et ◽  
Group 1

Abstract BackgroundOestradiol, an important hormone in follicular development and endometrial receptivity, is closely related to clinical outcomes of fresh in vitro fertilization-embryo transfer (IVF-ET) cycles. A supraphysiologic E2 level is inevitable during controlled ovarian hyper-stimulation (COH), and its effect on the outcome of IVF-ET is controversial. The aim of this retrospective study is to evaluate the association between elevated serum oestradiol (E2) levels on the day of human chorionic gonadotrophin (hCG) administration and neonatal birthweight after IVF-ET cycles.MethodsThe data of 3659 infertile patients with fresh IVF-ET cycles were analysed retrospectively between August 2009 and February 2017 in First Hospital of Zhengzhou University. Patients were categorized by serum E2 levels on the day of hCG administration into six groups: group 1 (serum E2 levels≤1000 pg/mL, n=230), group 2 (serum E2 levels between 1001 and 2000 pg/mL, n=524), group 3 (serum E2 levels between 2001 and 3000 pg/mL, n=783), group 4 (serum E2 levels between 3001 and 4000 pg/mL, n = 721), group 5 (serum E2 levels between 4001 and 5000 pg/mL, n=548 ), and group 6 (serum E2 levels > 5000 pg/mL, n=852). Univariate linear regression was used to evaluate the independent correlation between each factor and outcome index. Multiple logistic regression was used to adjust for confounding factors.ResultsThe LBW rates were as follows: 3.0% (group 1), 2.9% (group 2), 1.9% (group 3), 2.9% (group 4), and 2.0% (group 6) (P =0.629), respectively. There were no statistically significant differences in the incidences of neonatal LBW among the six groups. We did not detect an association between peak serum E2 level during ovarian stimulation and neonatal birthweight after IVF-ET.ConclusionThe results of this retrospective cohort study showed that serum E2 peak levels during ovarian stimulation were not associated with birth weight during IVF cycles. In addition, no association was found between higher E2 levels and increased LBW risk. Our observations suggest that the hyper-oestrogenic milieu during COS does not seem to have adverse effects on the birthweight of offspring after IVF.

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