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

Abstract

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.

GTPγS Regulation of a 12-Transmembrane Guanylyl Cyclase Is Retained after Mutation to an Adenylyl Cyclase

“…The plasmid obtained was used as the template for PCR mutagenesis to introduce three point mutations, E440K, S502Q, and H504D, which have been confirmed by sequencing. The obtained PCR products were cloned in the PstI and HindIII sites of full-length DdGCA, which were subsequently cloned in the expression plasmids MB12N (bsr selection) and MB12Neo (G418 selection) (33) Guanylyl and Adenylyl Cyclase Assays-GC assays were performed as described previously (34). In short, cells (10 8 cells/ml) in lysis buffer (40 mM Hepes/NaOH, 6 mM MgSO 4 , and 6 mM EGTA, pH 7.5) with or without GTP␥S (0.1 mM) were lysed by forced filtration through a Nucleopore filter (pore size, 3 m).…”

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

“…The structure and overall organization of the vectors used for expression of the ␣-and ␤-subunits of the gonadotropins is essentially identical to that of MB12n, which has been described previously (16). In short, each subunit was amplified from cDNA containing plasmids by using the appropriate 5Ј and 3Ј primers to generate a DNA fragment that is cloned in a Dictyostelium extra-chromosomal expression vector (16).…”

“…In short, each subunit was amplified from cDNA containing plasmids by using the appropriate 5Ј and 3Ј primers to generate a DNA fragment that is cloned in a Dictyostelium extra-chromosomal expression vector (16). To facilitate expression of two independent plasmids in Dictyostelium, we required two plasmids with different selection markers.…”

“…For construction of the expression vector for the ␣-subunit, its natural cDNA sequence, including the mammalian secretion signal sequence, was produced by polymerase chain reaction (PCR) by using primers that introduce a BglII restriction site both at the 5Ј and 3Ј end of the fragment, so that it could be cloned in MB12neo. For construction of the expression vector for the ␤-subunit of hCG, MB12n was modified to contain another unique restriction site (SphI) 3Ј of the BglII cloning site (16). About 40% of the codons in the ␤-subunit of CG are infrequently used in Dictyostelium (20).…”

“…About 40% of the codons in the ␤-subunit of CG are infrequently used in Dictyostelium (20). To limit possible problems in mRNA translation at the start of the open reading frame, the first 30 bases of the leader sequence of the ␤-subunit were altered conform to Dictyostelium preferred codon usage (16). The hCG ␤-subunit cDNA was amplified using a 5Ј primer (16), resulting in alteration of the first 30 bases of the coding sequence conform the Dictyostelium preferred codon usage.…”