Delineation of Subunit and Receptor Contact Sites by Site-Directed Mutagenesis of hCGβ

Puett, David; Huang, Jianing; Xia, Haiying

doi:10.1007/978-1-4613-8386-4_12

Cited by 8 publications

(5 citation statements)

References 39 publications

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“…This indicates that these host cells contain all chaperones and folding enzymes necessary to assemble the A and β subunits of hCG and to perform all post-translational modifications necessary for full biological activity. Furthermore, the use of CHO cells in combination with site-directed mutagenesis has proven to be a valuable tool for elucidating functional determinants in the glycoprotein hormones [6] and for designing potential new therapeutic analogs of these hormones [7].…”

mentioning

confidence: 99%

Expression of a bioactive, single‐chain choriogonadotropin in Dictyostelium discoideum

Heikoop¹,

Grootenhuis

Blaauw³

et al. 1998

European Journal of Biochemistry

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Human choriogonadotropin (hCG) is a highly complex glycoprotein consisting of two non-covalently associated subunits. We aimed for the expression of a single-chain hCG in the soil amoebae Dictyostelium discoideum, a host which, in principle, provides simple genetics in combination with complex protein synthesis. To limit anticipated problems in mRNA translation, the first 30 bases of the coding sequence were altered to conform to the Dictyostelium preferred codon usage. We show that, immunologically, active single-chain hCG is indeed produced by Dictyostelium. Furthermore, this single-chain hCG is able to bind to the human luteinizing hormone/CG receptor and elicit a biological response. Its receptorbinding affinity is comparable to single-chain hCG produced by mammalian cells. We conclude that Dictyostelium is able to express bioactive highly complex heterologous glycoproteins.Keywords : gonadotropin; single chain; Dictyostelium; human choriogonadotropin; follitropin.The placental human choriogonadotropin (hCG) is involved in the maintenance of pregnancy in the early stages after conception and has important therapeutic applications. This gonadotropin belongs to the family of glycoprotein hormones, which also include follitropin, lutropin and thyrotropin. These hormones are heterodimeric proteins of around 30 kDa formed by a non-covalent association of a common A subunit and a hormone-specific β subunit. Both the A and β subunits of hCG contain two Nlinked oligosaccharide side chains that have an important impact on its conformation and biological activity. A unique feature of the hCG β-subunit is the carboxy-terminal peptide (CTP) which bears four serine-linked oligosaccharides. The major role of the glycosylated CTP seems to be the prolongation of the circulatory half-life of hCG [1].The biosynthesis of the glycoprotein hormones is a highly complex process. In the last decade, it has become clear that folding, assembly and secretion of gonadotropins is assisted by a large set of chaperones and folding enzymes, residing in the endoplasmic reticulum and the Golgi apparatus [2]. Since both the A and the β subunits contain a so-called cystine knot, it can be anticipated that protein disulphide isomerase plays a key role in the facilitation of the folding process [3]. In addition, it has been shown that the N-linked oligosaccharide side chains are required for proper folding, disulphide formation and secretion of hCG [4].The gonadotropins have been expressed in Chinese Hamster Ovary (CHO) cells, and their recombinant derivatives have bioCorrespondence to P. D. J.

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mentioning

confidence: 99%

Expression of a bioactive, single‐chain choriogonadotropin in Dictyostelium discoideum

Heikoop¹,

Grootenhuis

Blaauw³

et al. 1998

European Journal of Biochemistry

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“…However, the X‐ray crystal structure of hCG shows this disulfide as a part of the cystine knot motif which is very essential for heterodimer formation. The close association of receptor binding sites with subunit contact sites has been documented earlier also (7,20). The N‐terminal region and determinant loop region of the β‐subunit have been implicated in both receptor binding and subunit association.…”

Section: Discussionmentioning

confidence: 60%

“…Individual subunits have very little hormonal activity but noncovalent combination of the subunits to form the native heterodimeric hormone imparts activity. The concept of multiple binding sites originating from the common α‐subunit and the hormone‐specific β‐subunit has been well accepted and this has led to the belief that these binding sites are close to or overlapping the subunit contact sites (7,20).…”

Section: Discussionmentioning

confidence: 89%

Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its β‐subunit: possible involvement of the disulfide bonds Cys⁹−Cys⁵⁷ and Cys²³−Cys⁷² in receptor binding of the hormone

Mishra

Mahale

Iyer

2001

The Journal of Peptide Research

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Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The alpha- and beta-subunits of hCG are highly cross-linked internally by disulfide bonds which seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. The purpose of this study was to delineate the role of the disulfide bonds of hCGbeta in receptor binding of the hormone. Six disulfide peptides incorporating each of the six disulfide bonds of hCGbeta were synthesized and screened, along with their linear counterparts, for their ability to competitively inhibit the binding of [125I] hCG to sheep ovarian corpora luteal LH/CG receptor. Disulfide peptide Cys (9-57) was found to be approximately 4-fold more potent than the most active of its linear counterparts in inhibiting radiolabeled hCG from binding to its receptor. Similarly, disulfide peptide Cys (23-72) exhibited receptor binding inhibition activity, whereas the constituent linear peptides were found to be inactive. The results suggest the involvement of the disulfide bonds Cys(9)-Cys(57) and Cys(23)-Cys(72) of the beta-subunit of hCG in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hCG.

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“…Clearly, potent hCG-based agonists and antagonists of the LH receptor will require a different protein engineering strategy, probably with the inclusion of different portions of the -subunit rather than just the determinant loop-containing seat belt. Also, both subunits of hCG appear to contribute, either directly or indirectly, to LHR binding , 1992, Bielinski & Boime 1992, Liu et al 1993, Yoo et al 1993, Puett et al 1994; thus, portions of and subunits will probably be required for potent single chain LHR agonists and antagonists.…”

Section: Discussionmentioning

confidence: 99%

“…Using various techniques such as site-directed mutagenesis (Chen & Puett 1991a,b, Chen et al 1991, 1992, Bielinski & Boime 1992, Liu et al 1993, Yoo et al 1993, Puett et al 1994, Puett & Narayan 2000, protein chimeras (Campbell et al 1991, Dias et al 1994, Han et al 1996, Grossmann et al 1997, and synthetic peptides (Salesse et al 1990, Keutmann 1992, many of the regions of hCG important for heterodimer formation and receptor binding and activation have been determined, as well as the suggestion that both subunits are required for full activity. The combined findings from these studies concerning ligand-receptor interactions have indicated that the central portion of the -subunit and the determinant loop (Moore et al 1980) of hCG (residues 93-100), i.e.…”

Section: Introductionmentioning

confidence: 99%

Single-chain human chorionic gonadotropin analogs containing the determinant loop of the beta-subunit linked to the alpha-subunit

Schubert

Puett²

2003

Journal of Molecular Endocrinology

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Human chorionic gonadotropin (hCG) is a member of the family of glycoprotein hormones containing a common α-subunit and distinct β-subunits that confer hormonal specificity. hCG binds to the relatively large ectodomain of the human luteinizing hormone receptor (hLHR), a member of the G protein-coupled receptor superfamily, leading to increased intracellular production of cAMP. Using protein engineering, two miniaturized versions of hCGβ have been separately fused to the N-terminus of the α-subunit to give N-des[1-91]hCGβ-α-C and N-des[1-91,110-114]hCGβ-α-C, i.e. fusion proteins of the hCGβ determinant loop (extended to include the complete seat belt and carboxy-terminal peptide) coupled to the α-subunit. Bioactivity of these single-chain gonadotropin analogs was assessed in two systems following transient transfections into HEK 293 cells and subsequent cAMP measurements. In one, each mini-β-α cDNA was fused to that of hLHR and transfected into cells to create yoked miniaturized hCG-hLHR complexes; in the other, the cDNA of each single chain mini-β-α was co-transfected with that of hLHR in an effort to produce non-covalent miniaturized hCG-hLHR complexes. Using yoked hCG-hLHR and hLHR as positive and negative controls respectively, expression of each mini-hCG-hLHR complex was confirmed using antibody and ligand binding assays. The two mini-hCGs led to minimal activation of hLHR, suggesting weak intrinsic activity of the mini-β-α fusion proteins. These results suggest that potent agonists and antagonists will require the presence of other portions of hCGβ in addition to the determinant loop/seat belt.

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Delineation of Subunit and Receptor Contact Sites by Site-Directed Mutagenesis of hCGβ

Cited by 8 publications

References 39 publications

Expression of a bioactive, single‐chain choriogonadotropin in Dictyostelium discoideum

Expression of a bioactive, single‐chain choriogonadotropin in Dictyostelium discoideum

Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its β‐subunit: possible involvement of the disulfide bonds Cys⁹−Cys⁵⁷ and Cys²³−Cys⁷² in receptor binding of the hormone

Single-chain human chorionic gonadotropin analogs containing the determinant loop of the beta-subunit linked to the alpha-subunit

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Delineation of Subunit and Receptor Contact Sites by Site-Directed Mutagenesis of hCGβ

Cited by 8 publications

References 39 publications

Expression of a bioactive, single‐chain choriogonadotropin in Dictyostelium discoideum

Expression of a bioactive, single‐chain choriogonadotropin in Dictyostelium discoideum

Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its β‐subunit: possible involvement of the disulfide bonds Cys9−Cys57 and Cys23−Cys72 in receptor binding of the hormone

Single-chain human chorionic gonadotropin analogs containing the determinant loop of the beta-subunit linked to the alpha-subunit

Contact Info

Product

Resources

About

Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its β‐subunit: possible involvement of the disulfide bonds Cys⁹−Cys⁵⁷ and Cys²³−Cys⁷² in receptor binding of the hormone