Towards minimized gonadotropins with full bioactivity

Abstract

Gonadotropins are highly complex glycoprotein hormones consisting of two noncovalently associated subunits, which are heavily glycosylated. Using the X-ray structure of human choriogonadotropin and structure/activity relationships we aimed to design`minimized' gonadotropins of reduced complexity. Our results show that it is possible to reduce the size of natural human choriogonadotropin by one-third of its molecular weight while retaining its wild-type biopotency. To our knowledge, such`mini'-human choriogonadotropins represent the smallest gonadotropins described so far with an lutropin/choriogonadotropin receptor affinity and in vitro biological activity comparable with that of natural human choriogonadotropin. It provides an important step towards the structure/ function-based design of small molecule drugs to the human gonadotropin receptors.Keywords: gonadotropin; human choriogonadotropin; protein minimization; single-chain.Choriogonadotropin, lutropin and follitropin form the family of gonadotropins that play a key role in fertility [1]. They are heterodimeric glycoproteins composed of an a-subunit and a b-subunit, which are noncovalently linked. Both subunits are glycosylated and contain N-linked oligosaccharides, and in the case of the choriogonadotropin b-subunit, O-linked oligosaccharides. Assembly of the common a-subunit with one of the three b-subunits results in the unique properties of each hormone leading to its specific biological activity.The elucidation of the crystal structure of human choriogonadotropin [2,3] has been a major step forward in the understanding of gonadotropin structure/function relationships. However, the mechanism of gonadotropin receptor interactions is poorly understood at present. The reduction of the highly complex heterodimeric glycoprotein hormones to small structured polypeptides or`mini-proteins' that retain their biological activity would be a major step towards achieving the structure/ function-based design of small molecule drugs for the treatment of infertility. Reducing the size and complexity of protein drugs may also open the way for other routes of administration, e.g. nasal rather than by injection. Successful examples of protein minimization projects [4] include the minimization of protein A [5] and atrial natriuretic peptide [6], both to peptides of nearly half the size the original proteins. 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 ...

New Frontier in Glycoprotein Hormones and Their Receptors Structure–Function

“…The effect of these substitutions on the in vitro bioactivity was highly correlated with their effects on the receptor binding activity. It was repeatedly demonstrated in media and buffers with various salt concentrations, and confirmed by studies in other laboratories ( 25 , 26 ) as well as by using CHO-TSHR cells with largely depleted pool of the negatively charged cell surface proteoglycans. Notably, mutations to alanine did not alter hormone activity, indicating that only selective substitutions to K or R amino acid residues are causing an enhancement of cAMP and IP 3 production, iodine uptake, proliferation of FRTL-5 cells, thyroxine and progesterone production, respectively ( 1 , 21 , 24 ).…”

“…( 3 , 38 , 42 , 43 )]. Moreover, largely reduced binding activity and potency of single-chain hCG and its minimized variants were restored using αL1 loop substitutions (α4K and α4R) ( 25 , 26 ). Remarkably, also the LH activity of the hTSH/hCG “seat-belt” “determinant loop” chimera was further increased by concomitant introduction of a cluster of K residues (α4K) into a highly distant from “seat-belt” αL1 13–20 domain ( 41 ).…”