scholarly journals Alternatively Folded Choriogonadotropin Analogs

2001 ◽  
Vol 276 (50) ◽  
pp. 46953-46960 ◽  
Author(s):  
Yongna Xing ◽  
Win Lin ◽  
Mei Jiang ◽  
Rebecca V. Myers ◽  
Donghui Cao ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Carboxyl Terminus ◽  
Glycoprotein Hormones ◽  
Β Subunit ◽  
Hormone Activity ◽  
Α Subunit ◽  
Receptor Interactions ◽  
Lutropin Receptor ◽  
Carboxyl Terminal ◽  
Loop 2

Most heterodimeric proteins are stabilized by intersubunit contacts or disulfide bonds. In contrast, human chorionic gonadotropin (hCG) and other glycoprotein hormones are secured by a strand of their β-subunits that is wrapped around α-subunit loop 2 “like a seatbelt.” During studies of hCG synthesis in COS-7 cells, we found that, when the seatbelt was prevented from forming the disulfide that normally “latches” it to the β-subunit, its carboxyl-terminal end can “scan” the surface of the heterodimer and become latched by a disulfide to cysteines substituted for residues in the α-subunit. Analogs in which the seatbelt was latched to residues 35, 37, 41–43, and 56 of α-subunit loop 2 had similar lutropin activities to those of hCG; that in which it was latched to residue 92 at the carboxyl terminus had 10–20% the activity of hCG. Attachment of the seatbelt to α-subunit residues 45–51, 86, 88, 90, and 91 reduced lutropin activity substantially. These findings show that the heterodimer can form before the β-subunit has folded completely and support the notions that the carboxyl-terminal end of the seatbelt, portions of α-subunit loop 2, and the end of the α-subunit carboxyl terminus do not participate in lutropin receptor interactions. They suggest also that several different architectures could have been sampled without disrupting hormone activity as the glycoprotein hormones diverged from other cysteine knot proteins.

scholarly journals The Carboxyl-Terminal Region Is a Determinant for the Intracellular Behavior of the Chorionic Gonadotropin β Subunit: Effects on the Processing of the Asn-Linked Oligosaccharides

1998 ◽  
Vol 12 (5) ◽  
pp. 766-772
Author(s):  
Mesut Muyan ◽  
Irving Boime
Keyword(s):  
Intramolecular Interaction ◽  
Chinese Hamster ◽  
Glycoprotein Hormones ◽  
Wild Type ◽  
Cho Cell ◽  
Α Subunit ◽  
Glycosylation Sites ◽  
Carboxyl Terminal ◽  
Complex Forms ◽  
Placental Hormone

Abstract The placental hormone human CG (hCG) consists of two noncovalently linked α- and β-subunits similar to the other glycoprotein hormones LH, FSH, and TSH. These heterodimers share a common α subunit but differ in their structurally distinct β subunits. The CGβ subunit is distinguished among the β subunits by the presence of a C-terminal extension with four serine-linked oligosaccharides (carboxyl terminal peptide or CTP). In previous studies we observed that deleting this sequence decreased assembly of the truncated CGβ subunit (CGβ114) with the α-subunit and increased the heterogeneity of the secreted forms of the uncombined subunit synthesized in transfected Chinese hamster ovary (CHO) cells. The latter result was attributed to alterations in the processing of the two N-linked oligosaccharides. To examine at what step this heterogeneity occurs, the CGβ and CGβ114 genes were transfected into wild-type and mutant CHO cell lines that are defective in the late steps of the N-linked carbohydrate-processing pathway. We show here that removal of the CTP alters the processing of the core mannosyl unit of the subunit to complex forms at both glycosylation sites and that the oligosaccharides contain polylactosamine. Although it has been presumed that there is little intramolecular interaction between the CTP and the proximal domains of the subunit, our data suggest that the CTP sequence participates in the folding of the newly synthesized subunit, which is manifest by the posttranslational changes observed here.

Epitope mapping of human thyrotropin

1989 ◽  
Vol 120 (2) ◽  
pp. 201-209 ◽  
Author(s):  
Yuichi Endo ◽  
Kiyoshi Miyai ◽  
Yasushi Iijima ◽  
Toshihiro Nakajima ◽  
Yasuyuki Eda ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Epitope Mapping ◽  
Antigenic Determinants ◽  
Conformational Structure ◽  
Glycoprotein Hormones ◽  
Two Dimensional ◽  
Β Subunit ◽  
Binding Studies ◽  
Α Subunit ◽  
Affinity Constants

Abstract. Epitope mapping of hTSH was carried out using 19 monoclonal antibodies prepared with hTSH or its β-subunit as antigen. The affinity constants of the monoclonal antibodies ranged from 9.6 × 107 to 5.7 × 109 mol/l for hTSH. The binding activities of monoclonal antibodies were maintained or in some cases rather enhanced after removal of the sugar moeity of the subunits of hTSH, and completely diminished after reduction of intramolecular S-S bonds in the subunits of hTSH. Ten monoclonal antibodies recognized the epitopes on hTSH (α:β subunit combined form) and on free α-subunit form. Eight other antibodies recognized the epitopes on free/or combined form of β-subunit, all of which did not recognize any other human glycoprotein hormones. The monoclonal antibodies directed against the α-subunit could bind also other human glycoprotein hormones to a varying extent. On the basis of results from competitive binding studies, the antibodies directed against α-subunit and those against β-subunit were each classified into five subgroups recognizing different antigenic determinants. The remaining one antibody recognized an epitope expressed only by hTSH and not by the free subunits. In addition, a positive cooperativity on the binding of hTSH was observed between monoclonal antibodies directed towards a particular epitope on the α-subunit and those towards an epitope on the β-subunit. From these data, two-dimensional map of epitopes on hTSH was constructed. The epitopes on each subunit were found to form a cluster with complicated overlapping, suggesting a highly conformational structure.

scholarly journals Unconventional Actions of Glycoprotein Hormone Subunits: A Comprehensive Review

2021 ◽  
Vol 12 ◽  
Author(s):  
Bruno Querat
Keyword(s):  
Biological Effects ◽  
G Protein Coupled Receptors ◽  
Glycoprotein Hormones ◽  
Β Subunit ◽  
Specific Receptor ◽  
Truncated Form ◽  
Glycoprotein Hormone ◽  
Α Subunit ◽  
Evolutionary Relatedness ◽  
G Protein Coupled

The glycoprotein hormones (GPH) are heterodimers composed of a common α subunit and a specific β subunit. They act by activating specific leucine-rich repeat G protein-coupled receptors. However, individual subunits have been shown to elicit responses in cells devoid of the receptor for the dimeric hormones. The α subunit is involved in prolactin production from different tissues. The human chorionic gonadotropin β subunit (βhCG) plays determinant roles in placentation and in cancer development and metastasis. A truncated form of the thyrotropin (TSH) β subunit is also reported to have biological effects. The GPH α- and β subunits are derived from precursor genes (gpa and gpb, respectively), which are expressed in most invertebrate species and are still represented in vertebrates as GPH subunit paralogs (gpa2 and gpb5, respectively). No specific receptor has been found for the vertebrate GPA2 and GPB5 even if their heterodimeric form is able to activate the TSH receptor in mammals. Interestingly, GPA and GPB are phylogenetically and structurally related to cysteine-knot growth factors (CKGF) and particularly to a group of antagonists that act independently on any receptor. This review article summarizes the observed actions of individual GPH subunits and presents the current hypotheses of how these actions might be induced. New approaches are also proposed in light of the evolutionary relatedness with antagonists of the CKGF family of proteins.

Insect cells infected with a recombinant baculovirus express both O- and N-glycosylated forms of the rat glycoprotein hormone α-subunit

1996 ◽  
Vol 16 (2) ◽  
pp. 141-149 ◽  
Author(s):  
R Delahaye ◽  
P Berreur ◽  
R Salesse ◽  
R Counis
Keyword(s):  
Post Infection ◽  
Insect Cells ◽  
Recombinant Baculovirus ◽  
Chorionic Gonadotrophin ◽  
Biologically Active ◽  
Glycoprotein Hormones ◽  
Β Subunit ◽  
Glycoprotein Hormone ◽  
Α Subunit ◽  
Infected Cells

ABSTRACT Glycoprotein hormones LH, FSH, TSH and chorionic gonadotrophin are heterodimers composed of two non-covalently associated subunits, a common α- and a specific β-subunit. A recombinant baculo-virus containing a cDNA encoding the α-subunit of rat glycoprotein hormones was constructed. Viral-infected cells expressed, 48 h post infection, 7–10mg immunoreactive α-glycopolypeptide/6×108 cells, of which 65·6% was able to associate with native LHβ and formed a biologically active heterodimeric hormone that bound to testicular receptors. The treatment with specific glycanases showed that the recombinant α-subunit was produced as two differently glycosylated forms; an Mr 23 000 form which contained exclusively N-linked carbohydrate units and another of Mr 25 000 which appeared to contain additional O-linked carbohydrate. Data demonstrated that the α-subunit was expressed by insect cells in a manner similar to that by mammalian pituitary gonadotropes producing both the N- and O-glycosylated forms although only the N-glycosylated α-subunit is known to be capable of associating with the β-subunit.

scholarly journals Translational Fusion of Two β-Subunits of Human Chorionic Gonadotropin Results in Production of a Novel Antagonist of the Hormone

Endocrinology ◽  
2007 ◽  
Vol 148 (8) ◽  
pp. 3977-3986 ◽  
Author(s):  
Satarupa Roy ◽  
Sunita Setlur ◽  
Rupali A. Gadkari ◽  
H. N. Krishnamurthy ◽  
Rajan R. Dighe
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Expression System ◽  
Biologically Active ◽  
Chain Molecule ◽  
Dependent Manner ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
Lh Receptor

The strategy of translationally fusing the α- and β-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active single-chain analog hCGαβ expressed using Pichia expression system. Using the same expression system, another analog, in which the α-subunit was replaced with the second β-subunit, was expressed (hCGββ) and purified. hCGββ could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose-dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable to inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCGαβ and hCGββ using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCGββ interacts with two LH receptor molecules. These studies demonstrate that the presence of the second β-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of α-subunit, the molecule is unable to elicit response. The strategy of fusing two β-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.

Evidence for Structural Changes in Carboxyl-Terminal Peptides of Transducin α-Subunit upon Binding a Soluble Mimic of Light-Activated Rhodopsin†

Biochemistry ◽  
2003 ◽  
Vol 42 (2) ◽  
pp. 302-311 ◽  
Author(s):  
D. M. Brabazon ◽  
N. G. Abdulaev ◽  
J. P. Marino ◽  
K. D. Ridge
Keyword(s):  
Structural Changes ◽  
Α Subunit ◽  
Carboxyl Terminal

scholarly journals Thyroid hormone regulation of mRNAs encoding thyrotropin β-subunit, glycoprotein α-subunit, and thyroid hormone receptors α and β in brain, pituitary gland, liver, and gonads of an adult teleost, Pimephales promelas

2009 ◽  
Vol 202 (1) ◽  
pp. 43-54 ◽  
Author(s):  
Sean C Lema ◽  
Jon T Dickey ◽  
Irvin R Schultz ◽  
Penny Swanson
Keyword(s):  
Thyroid Hormone ◽  
Pituitary Gland ◽  
Teleost Fish ◽  
Hormone Receptors ◽  
Pimephales Promelas ◽  
Early Gene ◽  
Thyroid Hormone Receptors ◽  
Β Subunit ◽  
Glycoprotein Hormone ◽  
Α Subunit

Thyroid hormones (THs) regulate growth, morphological development, and migratory behaviors in teleost fish, yet little is known about the transcriptional dynamics of gene targets for THs in these taxa. Here, we characterized TH regulation of mRNAs encoding thyrotropin subunits and thyroid hormone receptors (TRs) in an adult teleost fish model, the fathead minnow (Pimephales promelas). Breeding pairs of adult minnows were fed diets containing 3,5,3′-triiodo-l-thyronine (T3) or the goitrogen methimazole for 10 days. In males and females, dietary intake of exogenous T3 elevated circulating total T3, while methimazole depressed plasma levels of total thyroxine (T4). In both sexes, this methimazole-induced reduction in T4 led to elevated mRNA abundance for thyrotropin β-subunit (tshβ) in the pituitary gland. Fish treated with T3 had elevated transcript levels for TR isoforms α and β (trα and trβ) in the liver and brain, but reduced levels of brain mRNA for the immediate-early gene basic transcription factor-binding protein (bteb). In the ovary and testis, exogenous T3 elevated gene transcripts for tshβ, glycoprotein hormone α-subunit (gphα), and trβ, while not affecting trα levels. Taken together, these results demonstrate negative feedback of T4 on pituitary tshβ, identify trα and trβ as T3-autoinduced genes in the brain and liver, and provide new evidence that tshβ, gphα, and trβ are THs regulated in the gonad of teleosts. Adult teleost models are increasingly used to evaluate the endocrine-disrupting effects of chemical contaminants, and our results provide a systemic assessment of TH-responsive genes during that life stage.

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