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

Abstract

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...

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

“…Using primers b20aarev and alphater ( Table 1) a single-chain hCG was amplified by PCR from a plasmid containing hCG mutant 1 [β-(1Ϫ111)-(Ser-Gly) 5 -A(1Ϫ92), [8]]. The resulting fragment was cloned in MB12n after digestion with BglII.…”

“…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.…”

Towards minimized gonadotropins with full bioactivity

“…The primers were chosen in such a way that the PCR fragments contained appropriate restriction sites 3 H and 5 H of the mutation, so that they could be exchanged with the native a-chain sequence in the expression vector described previously [9,14]. Mutations were confirmed using an automated sequencer (Pharmacia).…”

“…Previously, we have shown that a truncated single-chain human choriogonadotropin is able to elicit a biological response on the human lutropin/choriogonadotropin receptor [9]. However, both its receptor-binding affinity and in vitro biological activity are reduced substantially.…”

“…Even more substantial reductions were recently published by Yanofsky and co-workers [7] who obtained a 15-residue peptide antagonist of the interleukin-1 receptor that may mimic the binding motif of its natural 153-residue antagonist. Wrighton and co-workers [8] reported the discovery of a 20-residue peptide dimer that binds and activates the erythropoietin receptor.In an attempt to reduce the structural complexity of gonadotropins we have engineered a mini-choriogonadotropin in which the two subunits are truncated by a total of 17 amino acid residues and which, in addition, lacks the C-terminal peptide of the b-subunit and its four O-glycosylation sites [9]. Although this mini-human choriogonadotropin is able to bind to the human lutropin/choriogonadotropin receptor and elicit a biological response, its biological activity is reduced by a factor of 10.…”