Probing the frontiers of glycoprotein synthesis: the fully elaborated β-subunit of the human follicle-stimulating hormone.
Ontology highlight
ABSTRACT:
SUBMITTER: Nagorny P
PROVIDER: S-EPMC3285374 | biostudies-literature | 2012 Jan
REPOSITORIES: biostudies-literature
Publications
Probing the frontiers of glycoprotein synthesis: the fully elaborated β-subunit of the human follicle-stimulating hormone.
Nagorny Pavel P Sane Neeraj N Fasching Bernhard B Aussedat Baptiste B Danishefsky Samuel J SJ
Angewandte Chemie (International ed. in English) 20111209 4
Similar Datasets
Project description:Described herein is the first total chemical synthesis of the unique ?-subunit of the human glycoprotein hormone (?-hGPH). Unlike the biologically derived glycoprotein hormones, which are isolated as highly complex mixtures of glycoforms, ?-hGPH obtained by chemical synthesis contains discrete homogeneous glycoforms. Two such systems have been prepared. One contains the disaccharide chitobiose at the natural N-glycosylation sites. The other contains dodecamer oligosaccharides at these same sites. The dodecamer sugar is a consensus sequence incorporating the key features associated with human glycoproteins.
| S-EPMC3288947 | biostudies-literature
Project description:The common gonadotrophic hormone α-subunit (GTHα) has been previously isolated by our research group from A. gigas pituitaries; in the present work the cDNA sequences encoding FSHβ and LHβ subunits have also been isolated from the same species of fish. The FSH β-subunit consists of 126 amino acids with a putative 18 amino acid signal peptide and a 108 amino acid mature peptide, while the LH β-subunit consists of 141 amino acids with a putative 24 amino acid amino acid signal peptide and a 117 amino acid mature peptide. The highest identity, based on the amino acid sequences, was found with the order of Anguilliformes (61%) for FSHβ and of Cypriniformes (76%) for LHβ, followed by Siluriformes, 53% for FSHβ and 75% for LHβ. Interestingly, the identity with the corresponding human amino acid sequences was still remarkable: 45.1% for FSHβ and 51.4% for LHβ. Three dimensional models of ag-FSH and ag-LH, generated by using the crystal structures of h-FSH and h-LH as the respective templates and carried out via comparative modeling and molecular dynamics simulations, suggested the presence of the so-called "seat-belt", favored by a disulfide bond formed between the 3rd and 12th cysteine in both β-subunits. The sequences found will be used for the biotechnological synthesis of A. gigas gonadotrophic hormones (ag-FSH and ag-LH). In a first approach, to ascertain that the cloned transcripts allow the expression of the heterodimeric hormones, ag-FSH has been synthesized in human embryonic kidney 293 (HEK293) cells, preliminarily purified and characterized.
| S-EPMC5573580 | biostudies-literature
Project description:Synthesis of the FSH beta-subunit (FSHbeta) is critical for normal reproduction in mammals, and its expression within the pituitary gonadotrope is tightly regulated by activin. Here we show that Runt-related (RUNX) proteins, transcriptional regulators known to interact with TGFbeta signaling pathways, suppress activin induction of FSHbeta gene expression. Runx2 is expressed within the murine pituitary gland and dramatically represses activin-induced FSHbeta promoter activity, without affecting basal expression in LbetaT2 cells, an immortalized mouse gonadotrope cell line. This repressive effect is specific, because RUNX2 induces LHbeta transcription (with or without activin) and does not interfere with GnRH induction of either gonadotropin beta-subunit gene. Analysis of the murine FSHbeta promoter by transfection and gel shift assays reveals that RUNX2 repression localizes to a Runx-binding element at -159/-153, which is adjacent to a previously recognized region critical for activin induction. Mutation of this -153 activin-response element or, indeed, any of the five activin-responsive regions prevents activin induction and, in fact, RUNX2 suppression, instead converting RUNX2 to an activator of the FSHbeta gene. Although the Runx-binding element is required for RUNX2-mediated repression of FSHbeta induction by either activin or Smad3, confirming a functional role of this novel site, protein interactions in addition to those between RUNX2 and Smads are necessary to account for full repression of activin induction. In summary, the present study provides evidence for Runx2-mediated repression of activin-induced FSHbeta gene expression and reveals the context dependence of Runx2 action in hormonal regulation of the gonadotropin genes.
| S-EPMC2875819 | biostudies-literature
Project description:The goal of this study was to characterize the gonadotropins expressed in pituitary glands of the New World squirrel monkey (Saimiri sp.) and owl monkey (Aotus sp.). The various subunits were amplified from total RNA from squirrel monkey and owl monkey pituitary glands by reverse transcription-polymerase chain reaction and the deduced amino acid sequences compared to those of other species. Mature squirrel monkey and owl monkey glycoprotein hormone alpha-polypeptides (96 amino acids in length) were determined to be 80% homologous to the human sequence. The sequences of mature beta subunits of follicle stimulating hormone (FSHbeta) from squirrel monkey and owl monkey (111 amino acids in length) are 92% homologous to human FSHbeta. New World primate glycoprotein hormone alpha-polypeptides and FSHbeta subunits showed conservation of all cysteine residues and consensus N-linked glycosylation sites. Attempts to amplify the beta-subunit of luteinizing hormone from squirrel monkey and owl monkey pituitary glands were unsuccessful. Rather, the beta-subunit of chorionic gonadotropin (CG) was amplified from pituitaries of both New World primates. Squirrel monkey and owl monkey CGbeta are 143 and 144 amino acids in length and 77% homologous with human CGbeta. The greatest divergence is in the C terminus, where all four sites for O-linked glycosylation in human CGbeta, responsible for delayed metabolic clearance, are predicted to be absent in New World primate CGbetas. It is likely that CG secreted from pituitary of New World primates exhibits a relatively short half-life compared to human CG.
| S-EPMC2277479 | biostudies-literature
Project description:Follicle-stimulating hormone beta subunit (Fshb) expression is regulated by transforming growth factor beta superfamily ligands. Recently, we demonstrated that bone morphogenetic proteins (BMPs) stimulate Fshb transcription alone and in synergy with activins. Also, transfection of the BMP type II receptor (BMPR2) and constitutively active forms of the type I receptors (activin A receptor type I [ACVR1] or BMP receptor type IA [BMPR1A]) in immortalized gonadotroph cells (LbetaT2) stimulated murine Fshb promoter-reporter activity. A third type I receptor (BMP receptor type IB [BMPR1B]) is also expressed in LbetaT2 cells, but we did not previously assess its functional role. A point mutation in BMPR1B (Q249R) is associated with increased ovulation rates and elevated FSH levels in Booroola (FecB) sheep. Herein, we assessed whether BMPR1B can regulate Fshb transcription in LbetaT2 cells and whether its ability to do so is altered by the Q249R mutation. As with ACVR1 and BMPR1A, coexpression of BMPR1B with BMPR2 increased Fshb promoter-reporter activity in BMP2-dependent and BMP2-independent fashions. Unexpectedly, the BMPR1B-Q249R mutant was equivalent to the wild type in its ability to stimulate SMAD1/5 phosphorylation and Fshb transcription. Pharmacological inhibition of ACVR1, BMPR1A, and BMPR1B confirmed that one or more of these receptors are required for BMP2-stimulated SMAD1/5 phosphorylation and Fshb reporter activity. Knockdown of endogenous BMPR1A, but not ACVR1 or BMPR1B, significantly impaired the synergism of BMP2 with activin A. Collectively, these data suggest that BMPR1A is the preferred BMP2 type I receptor in LbetaT2 cells and that neither ACVR1 nor BMPR1B compensates for its loss. The specific mechanism(s) through which the Booroola FecB mutation alters BMPR1B function remains to be determined.
| S-EPMC3093989 | biostudies-literature
Project description:A follicle stimulating hormone (FSH) is widely used in the assisted reproduction and a synthetic peptide corresponding to a receptor binding region of the human (h) FSH-β-(34-37) (TRDL) modulated reproduction. Furthermore, a 13-amino acid sequence corresponding to hFSH-β-(37-49) (LVYKDPARPKIQK) was recently identified as the receptor binding site. We hypothesized that the synthetic peptides corresponding to hFSH-β-(37-49) and hFSH-β-(34-49), created by merging hFSH-β-(34-37) and hFSH-β-(37-49), modulate the reproductive functions, with the longer peptide being more biologically active. In male or female prepubertal mice, a single injection of 200 μg/g BW ip of hFSH-β-(37-49) or hFSH-β-(34-49) hastened (p < 0.05) puberty, whereas the same treatments given daily for 4 d promoted (p < 0.05) the gonadal steroidogenesis and gamete formation. In addition of either peptide to the in vitro cell cultures, promoted (p < 0.05) the proliferation of primary murine granulosa cells and the estradiol production by upregulating the expression of Ccnd2 and Cyp19a1, respectively. In adult female mice, 200 μg/g BW ip of either peptide during diestrus antagonized the FSH-stimulated estradiol increase and uterine weight gain during proestrus. Furthermore, hFSH-β-(34-49) was a more potent (p < 0.05) reproductive modulator than hFSH-β-(37-49), both in vivo and in vitro. We concluded that hFSH-β-(37-49) and especially hFSH-β-(34-49), have the potential for reproductive modulation.
| S-EPMC9570415 | biostudies-literature
Project description:Activin is a major physiological regulator of FSH. We identify FoxL2 as a critical component in activin induction of FSHbeta, both for the mouse gene, induction of which is Sma- and Mad-related protein (Smad) dependent, and for the human gene that is Smad independent. FoxL2 has been shown to regulate gonadotrope gene expression (GnRH receptor, alpha-glycoprotein subunit, porcine FSHbeta, and follistatin), but the mechanisms of action are not well understood. We identify novel sites required for activin action in both the mouse and human FSHbeta promoters, some of which bind FoxL2, and show that the FoxL2-binding element encompasses a larger region (12 bp) than the previously identified forkhead-binding consensus (7 bp). Remarkably, although required for activin induction, FoxL2 sites neither contribute to basal FSHbeta promoter activity nor confer activin response to a heterologous promoter; thus, they are neither classical activin-response elements nor is their role solely to recruit Smads to the promoter. FoxL2 overexpression can potentiate activin induction in gonadotropes and can confer activin responsiveness to FSHbeta in heterologous cells where this promoter is normally refractory to activin induction. Although Smad3 requires the presence of FoxL2 sites to induce mouse FSHbeta, even through its consensus Smad-binding element; the human promoter, which is induced by activin independently of Smad3, also requires FoxL2 sites for its induction by activin; thus the actions of FoxL2 are not exclusively through interactions with the Smad pathway. Thus, FoxL2 plays a key role in activin induction of the FSHbeta gene, by binding to sites conserved across multiple species.
| S-EPMC2870942 | biostudies-literature
Project description:FSH glycosylation varies in two functionally important aspects: microheterogeneity, resulting from oligosaccharide structure variation, and macroheterogeneity, arising from partial FSHβ subunit glycosylation. Although advances in mass spectrometry permit extensive characterization of FSH glycan populations, microheterogeneity remains difficult to illustrate, and comparisons between different studies are challenging because no standard format exists for rendering oligosaccharide structures. FSH microheterogeneity is illustrated using a consistent glycan diagram format to illustrate the large array of structures associated with one hormone. This is extended to commercially available recombinant FSH preparations, which exhibit greatly reduced microheterogeneity at three of four glycosylation sites. Macroheterogeneity is demonstrated by electrophoretic mobility shifts due to the absence of FSHβ glycans that can be assessed by Western blotting of immunopurified FSH. Initially, macroheterogeneity was hoped to matter more than microheterogeneity. However, it now appears that both forms of carbohydrate heterogeneity have to be taken into consideration. FSH glycosylation can reduce its apparent affinity for its cognate receptor by delaying initial interaction with the receptor and limiting access to all of the available binding sites. This is followed by impaired cellular signaling responses that may be related to reduced receptor occupancy or biased signaling. To resolve these alternatives, well-characterized FSH glycoform preparations are necessary.
| S-EPMC6534497 | biostudies-literature
Project description:Differential regulation of gonadotropin hormone production in the pituitary is critical for fertility. Activin and progesterone signaling in gonadotrope cells is important for Fshb gene expression. Previously, we reported that synergy between activin and progestins required the binding of SMAD proteins and the progesterone receptor (PR) to the murine Fshb promoter. In this study, we demonstrate that the FOXL2 transcription factor is also necessary for the full synergistic response between activin and progestins. We show that this synergy occurs in a species-specific manner and that multiple elements in the Fshb promoter that bind forkhead box L2 (FOXL2), SMA/mothers against decapentaplegic homologs (SMAD), and PR are required. Furthermore, we demonstrate that FOXL2 can physically interact with PR and SMAD3. Thus, it is likely that protein-protein interactions among FOXL2, SMAD, and PR recruited to the Fshb promoter play a key role in facilitating Fshb transcription before the secondary FSH surge in rodents.
| S-EPMC3320250 | biostudies-literature
Project description:Follicle-stimulating hormone (FSH) is essential for human reproduction. The unique functions of this hormone are provided by the FSH receptor-binding beta-subunit encoded by the FSHB gene. Resequencing and genotyping of FSHB in three European, two Asian and one African population, as well as in the great apes (chimpanzee, gorilla, orangutan), revealed low diversity and significant excess of polymorphisms with intermediate frequency alleles. Statistical tests for FSHB showed deviations from neutrality in all populations suggesting a possible effect of balancing selection. Two core haplotypes were identified (carried by 76-96.6% of each population's sample), the sequences of which are clearly separated from each other. As fertility most directly affects an organism's fitness, the carriers of these haplotypes have apparently had more success in human history to contribute to the next generation. There is a preliminary observation suggesting that the second most frequent FSHB haplotype may be associated with rapid conception success in females. Interestingly, the same haplotype is related to an ancestral FSHB variant shared with the ancestor of the great apes. The determination of the functional consequence of the two core FSHB variants may have implications for understanding and regulating human fertility, as well as in assisting infertility treatments.
| S-EPMC1859982 | biostudies-literature