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Chemical synthesis of the ?-subunit of human luteinizing (hLH) and chorionic gonadotropin (hCG) glycoprotein hormones.

ABSTRACT: Human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) are human glycoprotein hormones each consisting of two subunits, an identical ?-subunit and a unique ?-subunit, that form noncovalent heterodimers. Structurally, ?-hCG shares a high degree of sequence similarity with ?-hLH, including a common N-glycosylation site at the N-terminus but differs mainly in the presence of an extended C-terminal portion incorporating four closely spaced O-linked glycans. These glycoproteins play important roles in reproduction and are used clinically in the treatment of infertility. In addition, the role of hCG as a tumor marker in a variety of cancers has also attracted significant interest for the development of cancer vaccines. In clinical applications, these hormones are administered as mixtures of glycoforms due to limitations of biological methods in producing homogeneous samples of these glycoproteins. Using the powerful tools of chemical synthesis, the work presented herein focuses on the highly convergent syntheses of homogeneous ?-hLH and ?-hCG bearing model glycans at all native glycosylation sites. Key steps in these syntheses include a successful double Lansbury glycosylation en route to the N-terminal fragment of ?-hCG and the sequential installation of four O-linked glycosyl-amino acid cassettes into closely spaced O-glycosylation sites in a single, high-yielding solid-supported synthesis to access the C-terminal portion of the molecule. The final assembly of the individual glycopeptide fragments involved a stepwise native chemical ligation strategy to provide the longest and most complex human glycoprotein hormone (?-hCG) as well as its closely related homologue (?-hLH) as discrete glycoforms.

SUBMITTER: Fernandez-Tejada A

PROVIDER: S-EPMC4227738 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Chemical synthesis of the β-subunit of human luteinizing (hLH) and chorionic gonadotropin (hCG) glycoprotein hormones.

Fernández-Tejada Alberto A Vadola Paul A PA Danishefsky Samuel J SJ

Journal of the American Chemical Society 20140527 23

Human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) are human glycoprotein hormones each consisting of two subunits, an identical α-subunit and a unique β-subunit, that form noncovalent heterodimers. Structurally, β-hCG shares a high degree of sequence similarity with β-hLH, including a common N-glycosylation site at the N-terminus but differs mainly in the presence of an extended C-terminal portion incorporating four closely spaced O-linked glycans. These glycoproteins play i ...[more]

PMID: 24806200

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Project description:During histiotrophic nutrition of the embryo, maternal platelets may be the first circulating maternal cells that find their way into the placental intervillous space through narrow intertrophoblastic gaps within the plugs of spiral arteries. Activation of platelets at the maternal-fetal interface can influence trophoblast behavior and has been implicated in serious pregnancy pathologies. Here, we show that platelet-derived factors impaired expression and secretion of the human chorionic gonadotropin beta-subunit (?hCG) in human first trimester placental explants and the trophoblast cell line BeWo. Impaired ?hCG synthesis was not the consequence of hampered morphological differentiation, as assessed by analysis of differentiation-associated genes and electron microscopy. Platelet-derived factors did not affect intracellular cAMP levels and phosphorylation of CREB, but activated Smad3 and its downstream-target plasminogen activator inhibitor (PAI)-1 in forskolin-induced BeWo cell differentiation. While TGF-? type I receptor inhibitor SB431542 did not restore impaired ?hCG production in response to platelet-derived factors, Smad3 inhibitor SIS3 interfered with CREB activation, suggesting an interaction of cAMP/CREB and Smad3 signaling. Sequestration of transcription co-activators CBP/p300, known to bind both CREB and Smad3, may limit ?hCG production, since CBP/p300 inhibitor C646 significantly restricted its forskolin-induced upregulation. In conclusion, our study suggests that degranulation of maternal platelets at the early maternal-fetal interface can impair placental ?hCG production, without substantially affecting morphological and biochemical differentiation of villous trophoblasts. KEY MESSAGES: Maternal platelets can be detected on the surface of the placental villi and in intercellular gaps of trophoblast cell columns from gestational week 5 onwards. Platelet-derived factors impair hCG synthesis in human first trimester placenta. Platelet-derived factors activate Smad3 in trophoblasts. Smad3 inhibitor SIS3 interferes with forskolin-induced CREB signaling. Sequestration of CBP/p300 by activated Smad3 may limit placental hCG production.

Project description:LH triggers the biosynthesis of androgens in the theca-interstitial (T-I) cells of ovary through the activation of a cAMP-dependent pathway. We have previously shown that LH/human chorionic gonadotropin (hCG) activates mammalian target of rapamycin complex 1 (mTORC1) signaling network, leading to cell proliferation. In the present study, we provide evidence that the LH/hCG-mediated activation of the mTORC1 signaling cascade is involved in the regulation of steroidogenic enzymes in androgen biosynthesis. Treatment with LH/hCG increased the expression of downstream targets of mTORC1, ribosomal protein S6 kinase 1, and eukaryotic initiation factor 4E as well as steroidogenic enzymes. LH/hCG-mediated stimulation of the steroidogenic enzyme mRNA was blocked by the mTORC1 inhibitor, rapamycin. This inhibitory effect was selective because rapamycin failed to block hCG-mediated increase in the expression of Star mRNA levels. Furthermore, pharmacological targeting of mTORC1 with rapamycin also blocked LH/hCG- or forskolin-induced expression of cAMP response element-binding protein (CREB) and steroidogenic enzymes (P450 side-chain cleavage enzyme, 3?-hydroxysteroid dehydrogenase type 1, and 17?-hydroxylase/17,20 lyase) but produced no effect on steroidogenic acute regulatory protein levels. These results were further confirmed by demonstrating that the knockdown of mTOR using small interfering RNA selectively abrogated the LH/hCG-induced increase in steroidogenic enzyme expression, without affecting steroidogenic acute regulatory protein expression. LH/hCG-stimulated androgen production was also blocked by rapamycin. Furthermore, the pharmacological inhibition of mTORC1 or ribosomal protein S6 kinase 1 signaling prevented the LH/hCG-induced phosphorylation of CREB. Chromatin immunoprecipitation assays revealed the association of CREB with the proximal promoter of the Cyp17a1 gene in response to hCG, and this association was reduced by rapamycin treatment. Taken together, our findings show for the first time that LH/hCG-mediated activation of androgen biosynthesis is regulated by the mTORC1 signaling pathway in T-I cells.

Dataset Information

Chemical synthesis of the ?-subunit of human luteinizing (hLH) and chorionic gonadotropin (hCG) glycoprotein hormones.

Publications

Chemical synthesis of the β-subunit of human luteinizing (hLH) and chorionic gonadotropin (hCG) glycoprotein hormones.

Similar Datasets

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