Biosynthesis of a biologically active single peptide chain containing the human common alpha and chorionic gonadotropin beta subunits in tandem.

T, Sugahara; Pixley, Mary R.; Minami, Sawako; Perlas, Emerald; Ben-Menahem, David; Hsueh, Aaron J. W.; Boime, Irving

doi:10.1073/pnas.92.6.2041

Cited by 105 publications

(84 citation statements)

References 32 publications

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“…Furthermore, the signal transduction capacity of mutant 3 is reduced more than can be explained by the reduction in binding affinity only, which would be in line with the partial dissociation between receptor binding and activation in single chains reported by the groups of Boime [3] and Puett 141.…”

Section: Discussionsupporting

confidence: 71%

“…The three single chain human choriogonadotropins presented in this work have distinct features that differ from the published single chain glycoprotein hormones. The spacer moiety in our single chains does not consist of (parts of) the C-terminal peptide of the subunit [3,4,6, 71 but of Ser-Gly repeats. Since mutants 1 and 2 resemble heterodimeric lutropin in terms of receptor binding and to a lesser extent bioactivity, it can be concluded that the composition of the spacer only has minor effects on the structure and function of the single chain human choriogonadotropins.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Evaluation of Subunit Truncation and the Nature of the Spacer for Single Chain Human Gonadotropins

Heikoop¹,

Vaan²,

Boogaart³

et al. 1997

European Journal of Biochemistry

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Three single chain gonadotropins were designed based on the three-dimensional-structure of human choriogonadotropin and structure/activity relationships of the glycoprotein hormones. In each single chain, the C-terminal end of the human choriogonadotropin /? subunit is connected via Ser-Gly repeats to the N-terminal end of the a subunit. In addition, two of the single chains have truncated subunits. The three mutants were expressed in CHO cells. In vitro binding of two of the three mutants to the human lutropinl choriogonadotropin receptor was found to be comparable to wild-type lutropin. In contrast, both the receptor binding and the in vitro bioactivity of the mutant with truncated (I and /? subunits in which the p:26-110 disulphide bond cannot be formed, are lowered relative to wild-type lutropin. The fact that this mutant still displays biological activity shows that the seat-belt arrangement proposed by Isaacs and coworkers [Lapthorn, A. J., Harris, D. C., Littlejohn, A., Lustbader, J. W., Canfield, R. E., Machin, K. Keywords: gonadotropin ; single chain ; structure-based design; spacer; truncation.The glycoprotein hormones 11 1 form a family of structurally related proteins. Members include choriogonadotropin, follitropin, lutropin and thyrotropin. Follitropin, lutropin and thyrotropin are present in most vertebrate species and are synthesized and secreted by the pituitary. Choriogonadotropin has so far been found only in primates, including humans, and in horses, and is synthesized by placental tissue. The glycoprotein hormones are heterodimers composed of two dissimilar subunits, named 0: and /?, which are associated by non-covalent bonds.Within a species, the a subunit is the same for each member of the gonadotropin family. The , / 3 subunits are different for each member, i.e. choriogonadotropin, follitropin, thyrotropin and lutropin, and confer receptor-binding specificity on the hormones.The association of the (x and the p subunit is a very important step in the biosynthesis of the glycoprotein hormones, since only the intact dimers are biologically active. The correct assembly of the heterodimer is essential for efficient secretion, receptor binding, and signal transduction of the hormone. Free a and / 3 subunits are biologically inactive 121. Numerous mutation studies have shown that the dimerization is a highly specific process and can easily be prohibited by single point mutations. Structure/function analysis of the glycoprotein hormones is often hampered by unwanted secondary effects on the assembly of the subunits.Therefore, we and others [3 -71 have pursued approaches to determine whether the active conformation of the glycoprotein hormones can also be formed when the two subunits are synthesized in tandem on a single polypeptide chain. It has been dem- onstrated that covalently linked u and /j subunits are efficiently secreted and able to fold in a conformation that binds the lutropin/choriogonadotropin receptor with high affinity and stimulates adenylate cyclase. Thus, it can be concluded th...

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Section: Discussionsupporting

confidence: 71%

Section: Discussionmentioning

confidence: 99%

Evaluation of Subunit Truncation and the Nature of the Spacer for Single Chain Human Gonadotropins

Heikoop¹,

Vaan²,

Boogaart³

et al. 1997

European Journal of Biochemistry

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“…To overcome these limitations, the genes encoding the common ␣-subunit and either the hCG ␤-, FSH ␤-, or TSH ␤-subunits have been genetically fused. The resulting polypeptide chains were efficiently secreted and were biologically active (7)(8)(9)(10)(11)(12). These studies presumed that addition of the human CG␤ C-terminal peptide (CTP) as a linker sequence between the subunits would be required for flexibility, hydrophilicity, stability, and successful expression of the single chain forms.…”

Section: Thyrotropin (Tsh)mentioning

confidence: 95%

Engineering a Potential Antagonist of Human Thyrotropin and Thyroid-stimulating Antibody

Fares¹,

Levi²,

Reznick³

et al. 2001

Journal of Biological Chemistry

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Thyrotropin (TSH) and the gonadotropins (FSH, LH, hCG) are a family of heterodimeric glycoprotein hormones composed of two noncovalently linked subunits, ␣ and ␤. We have recently converted the hTSH heterodimer to a biologically active single chain (hTSH␤⅐CTP␣) by fusing the common ␣-subunit to the C-terminal end of the hTSH ␤-subunit in the presence of a ϳ30-amino acid peptide from hCG␤ (CTP) as a linker. The hTSH␤⅐CTP␣ single chain was used to investigate the role of the Nlinked oligosaccharides of ␣-and ␤-subunits in the secretion and function of hTSH. Using overlapping PCR mutagenesis, two deglycosylated variants were prepared: one lacking both oligosaccharide chains on the ␣-subunit (hTSH␤⅐CTP␣ 1؉2 ) and the other lacking the oligosaccharide chain on the ␤-subunit (hTSH␤⅐CTP␣(deg)). The single chain variants were expressed in CHO cells and were secreted into the medium. hTSH variants lacking the oligosaccharide chains were less potent than hTSH␤⅐CTP␣ wild-type with respect to cAMP formation and thyroid hormone secretion in cultured human thyroid follicles. Both deglycosylated variants competed with hTSH in a dose-dependent manner. The hTSH␤⅐CTP␣ 1؉2 variant blocked cAMP formation and thyroid hormone secretion stimulated by hTSH as well as by the antibody, thyroidstimulating immunoglobulins, responsible for the most common cause of hyperthyroidism, Graves disease. Thus, this variant behaves as a potential antagonist, offering a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH-secreting pituitary adenoma. Thyrotropin (TSH)1 is a member of the glycoprotein hormone family, which includes lutropin (LH), follitropin (FSH), and human chorionic gonadotropin (hCG). These are heterodimers composed of two noncovalent-linked subunits, a common ␣-subunit and a hormone-specific ␤-subunit (1, 2). Assembly of glycoprotein subunits is vital to the function of these hormones. The ␣-and ␤-subunits contain one (TSH␤ and LH␤) or two (␣, FSH␤, and hCG␤) asparagine-linked (N-linked) oligosaccharides (1, 2). These residues have been shown to play a role in determining the biological activity of glycoprotein hormones, including the maintenance of intracellular stability, assembly, secretion, signal transduction, and modulation of plasma halflife (1, 3). Deglycosylation of glycoprotein hormones have been utilized using chemical or enzymatic treatments, but these however cannot discriminate between individual sites, are nonspecific, and provide only completely deglycosylated hormones.Site-directed mutagenesis has become an important tool for studying the structure and function of glycoprotein hormones. However, mutations in either ␣-or ␤-subunits can alter the folding and ultimately inhibit subunit assembly and secretion of the hormone (4 -6). To overcome these limitations, the genes encoding the common ␣-subunit and either the hCG ␤-, FSH ␤-, or TSH ␤-subunits have been genetically fused. The resulting polypeptide chains were efficiently secreted and were biologically active (7-12). The...

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“…The transfected CHO cells were maintained in Ham's F-12 medium supplemented with penicillin (100 units/ml), streptomycin (100 g/ml), and glutamine (2 mM), containing 5% fetal calf serum and 0.125 mg/ml of the neomycin analog as described previously (28). For metabolic labeling experiments, transfected CHO cells were plated in multiwell plates and labeled for 18 (LH␤ variants) or 6 h (hCG␤ variants) with Pro-mix (28 Ci/ml) in cysteine and methionine-free media, as described previously (28). Cell lysate and media samples were immunoprecipitated with antisera raised against the ovine LH␤ or human CG␤ subunits and resolved under reduced conditions on 12.5 or 15% SDS-PAGE, as indicated in the figure legends, and autoradiographed.…”

Section: Transfection Of Cho Cells Metabolic Labeling and Immunoprementioning

confidence: 99%

“…As a template, we used the hCG␤ subunit gene that includes a SalI site in the 2nd intron (28), and the boCTP sequence was placed following Asp 117 codon of the hCG␤ gene in two rounds of amplification. A forward primer 11 (5Ј-AGCAGACACTCTTCCCCTCCCTTCCCAATAA-AGACTTCTTGATCTAGATCGGACACCCCGATCCTCCCA-3Ј) and a reverse primer 12 (5Ј-GGTTTCCAGAGTTAGGATGGGCATGGGAGG-TTGAAGTGGGGCATCCTTAGAGGAAGAGGAGTCCTGGAAGC-3Ј) were used in the first reaction, and the forward primer 13 (5Ј-CTCCC-TTCCCAATAAAGACTTCTTGAGAATTCTCGGACACCCCGATCCTC-CCA-3Ј) and reverse primer 14 (5Ј-GGGAAGAG TGTCTGCTGGTTTC-CAGAGTTAGGATGGGCATGGGAGGTTG-3Ј) were used in the second round.…”

Section: Construction Of the Hcg␤-boctp Chimeramentioning

confidence: 99%

The LHβ Gene of Several Mammals Embeds a Carboxyl-terminal Peptide-like Sequence Revealing a Critical Role for Mucin Oligosaccharides in the Evolution of Lutropin to Chorionic Gonadotropin in the Animal Phyla

Nakav

Jablonka‐Shariff

Kaner

et al. 2005

Journal of Biological Chemistry

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The expression of a previously untranslated carboxylterminal sequence is associated with the ancestral lutropin (LH) ␤ to the ␤-subunit gene evolution of choriogonadotropins (CG). The peptide extension (denoted as CTP) is rich in mucin-type O-glycans and confers new hormonal properties on CG relative to the LH. Although the LH␤ gene is conserved among mammals and only a few frameshift mutations account for the extension, it is merely seen in primates and equids. Bioinformatics identified a CTP-like sequence that is encrypted in the LH␤ gene of several mammalian species but not in birds, amphibians, or fish. We then examined whether or not decoding of the cryptic CTP in the bovine LH␤ gene (boCTP) would be sufficient to generate the LH␤ species of a ruminant with properties typical to the CG␤ subunit. The mutated bovine LH␤-boCTP subunit was expressed and N-glycosylated in transfected Chinese hamster ovary cells. However, unlike human (h) CG␤ CTP, the cryptic boCTP was devoid of mucin O-glycans. This deficiency was further confirmed when the boCTP domain was substituted for the natural CTP in the human CG␤ subunit. Moreover, when expressed in polarized Madin-Darby canine kidney cells, this hCG␤-boCTP chimera was secreted basolaterally rather than from the apical compartment, which is the route of the wild type hCG␤ subunit, a sorting function attributed to the Oglycans attached to the CTP. This result shows that the cryptic peptide does not orientate CG to the apical face of the placenta, to the maternal circulation as seen in primates. The absence of this function, which distinguishes CG from LH, provides an explanation as to why the LH␤ to CG␤ evolution did not occur in ruminants. We propose that in primates and equids, further natural mutations in the progenitor LH␤ gene resulted in the efficient O-glycosylation of the CTP, thus favoring the retention of an elongated reading frame.The family of glycoprotein hormones includes lutropin (LH), 1 follitropin, and thyrotropin, as expressed by the pituitary, and chorionic gonadotropin (CG), which is produced by the placenta of primates and equids (1). Each hormone is a noncovalent heterodimer composed of a common ␣-subunit and a unique ␤-subunit that confers receptor specificity. The ␤-subunits have substantial sequence similarities, including conserved location of cysteine residues that suggests an origin from a common ancestral gene (2). The human (h) CG␤ genes have evolved from an ancestral LH␤ gene by frameshift mutations that resulted in a readthrough into a previously untranslated region and the extension of the reading frame (3, 4) (Fig. 1A). As a result, a short carboxyl-terminal sequence of hLH␤ is replaced by a longer carboxyl-terminal peptide (CTP) in hCG␤ (3-6). In the equine (e), both the placental CG␤ and pituitary LH␤ subunits are products of the same gene (eLH/CG␤) and contain a CTP that is presumed to have evolved by a distinct mechanism (7) (for further explanations see "Results"). Except for primates and equids, no other identified animal species b...

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Biosynthesis of a biologically active single peptide chain containing the human common alpha and chorionic gonadotropin beta subunits in tandem.

Cited by 105 publications

References 32 publications

Evaluation of Subunit Truncation and the Nature of the Spacer for Single Chain Human Gonadotropins

Evaluation of Subunit Truncation and the Nature of the Spacer for Single Chain Human Gonadotropins

Engineering a Potential Antagonist of Human Thyrotropin and Thyroid-stimulating Antibody

The LHβ Gene of Several Mammals Embeds a Carboxyl-terminal Peptide-like Sequence Revealing a Critical Role for Mucin Oligosaccharides in the Evolution of Lutropin to Chorionic Gonadotropin in the Animal Phyla

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