Structure-based design and protein engineering of intersubunit disulfide bonds in gonadotropins

Heikoop, Judith C.; Boogaart, Paul van den; Mulders, John W. M.; Grootenhuis, P. D. J.

doi:10.1038/nbt0797-658

Cited by 43 publications

(31 citation statements)

References 26 publications

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“…Unfortunately, our disulfide approach was not successful. Intermolecular disulfide bonds artificially introduced at positions where natural disulfides are present in homologous proteins (23), including a bacteriophage capsid (3), proved stabilizing in several cases. However, most protein oligomers, including the FMDV capsid, have no adequate disulfide-bonded homologs.…”

Section: Discussionmentioning

confidence: 99%

Engineering Viable Foot-and-Mouth Disease Viruses with Increased Thermostability as a Step in the Development of Improved Vaccines

Mateo

Luna

Rincón

et al. 2008

J Virol

101

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We have rationally engineered foot-and-mouth disease virus to increase its stability against thermal dissociation into subunits without disrupting the many biological functions needed for its infectivity. Amino acid side chains located near the capsid intersubunit interfaces and either predicted or found to be dispensable for infectivity were replaced by others that could establish new disulfide bonds or electrostatic interactions between subunits. Two engineered viruses were normally infectious, genetically stable, and antigenically indistinguishable from the natural virus but showed substantially increased stability against irreversible dissociation. Electrostatic interactions mediated this stabilizing effect. For foot-and-mouth disease virus and other viruses, some evidence had suggested that an increase in virion stability could be linked to an impairment of infectivity. The results of the present study show, in fact, that virion thermostability against dissociation into subunits may not be selectively constrained by functional requirements for infectivity. The thermostable viruses obtained, and others similarly engineered, could be used for the production, using current procedures, of foot-and-mouth disease vaccines that are less dependent on a faultless cold chain. In addition, introduction of those stabilizing mutations in empty (nucleic acid-free) capsids could facilitate the production of infectionrisk-free vaccines against the disease, one of the economically most important animal diseases worldwide.

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Section: Discussionmentioning

confidence: 99%

Engineering Viable Foot-and-Mouth Disease Viruses with Increased Thermostability as a Step in the Development of Improved Vaccines

Mateo

Luna

Rincón

et al. 2008

J Virol

101

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“…Purification of hCG from Dictyostelium was performed with the aid of a programmable FPLC system (Pharmacia) using the control and chromatography supervision system UNICORN (Pharmacia). Purification of single-chain hCG was accomplished using a combination of hydrophobic interaction and immuno chromatography with LH/CG β subunit-specific monoclonal antibodies (optimised protocol for hCG produced by CHO cells, see [9]). For this purpose, 150 ml medium were produced with a single-chain hCG content of 0.372 unit/ml as determined by DELFIA ® .…”

Section: Oligonucleotidementioning

confidence: 99%

“…To overcome this problem, we and others have produced mutants in which the coding regions of the hCG β subunit and the common A subunit are connected via peptide spacers [8] or intersubunit disulphide bonds [9]. It has been shown that these covalently linked A and β subunits of hCG are able to fold into biologically active conformations.…”

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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“…This amount is produced in 4-5 days in a petri dish with ϳ10 6 cells per ml of medium. For comparison, the expression level of wt hCG in CHO cells is 800mU/10 6 cells per 24 h (22).…”

Section: Gonadotropin Expression In Dictyosteliummentioning

confidence: 99%

“…Concentrations of wild-type and mutant hCG were measured using a DELFIA hLH assay (Wallac Oy, Turku, Finland), which has a 100% cross-reactivity with hCG, as described by the manufacturer. FSH was quantified by a sandwich immunoassay as described previously (22). Both assays are based on the enzyme-linked immunoassay principle, with a solid-phase anti-␤ chain antibody and a soluble anti-␣ chain antibody.…”

Section: Expression Of Recombinant Hormonesmentioning

confidence: 99%

Random mutagenesis and screening of complex glycoproteins: expression of human gonadotropins inDictyostelium discoideum

Linskens

Grootenhuis²,

Blaauw

et al. 1999

FASEB j.

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The soil amoeba Dictyostelium discoideum is a host cell that provides simple genetics in combination with complex protein synthesis. We show that the complex human heterodimeric gonadotropins can be produced and secreted by this organism. Furthermore, both follicle stimulation hormone and choriogonadotropin produced by D. dictyostelium bind to their human receptors and elicit a biological response comparable to the wild‐type hormones. We also show that structure‐function analysis using random mutagenesis and screening of recombinant glycoprotein hormones is feasible. Thus, expression of gonadotropins in D. dictyostelium opens the way to the engineering of potential new therapeutic analogues. Linskens, M. H. K., Grootenhuis, P. D. J., Blaauw, M., Huisman‐de Winkel, B., van Ravestein, A., van Haastert, P. J. M., and Heikoop, J. C. Random mutagenesis and screening of complex glycoproteins: expression of human gonadotropins in Dictyostelium discoideum. FASEB J. 13, 639–645 (1999)

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Structure-based design and protein engineering of intersubunit disulfide bonds in gonadotropins

Cited by 43 publications

References 26 publications

Engineering Viable Foot-and-Mouth Disease Viruses with Increased Thermostability as a Step in the Development of Improved Vaccines

Engineering Viable Foot-and-Mouth Disease Viruses with Increased Thermostability as a Step in the Development of Improved Vaccines

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

Random mutagenesis and screening of complex glycoproteins: expression of human gonadotropins inDictyostelium discoideum

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