Translational Fusion of Two β-Subunits of Human Chorionic Gonadotropin Results in Production of a Novel Antagonist of the Hormone

Abstract

The strategy of translationally fusing the alpha- and beta-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active single-chain analog hCGalphabeta expressed using Pichia expression system. Using the same expression system, another analog, in which the alpha-subunit was replaced with the second beta-subunit, was expressed (hCGbetabeta) and purified. hCGbetabeta could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose-dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable to inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCGalphabeta and hCGbetabeta using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCGbetabeta interacts with two LH receptor molecules. These studies demonstrate that the presence of the second beta-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of alpha-subunit, the molecule is unable to elicit response. The strategy of fusing two beta-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.

Human choriogonadotrophin protein core and sugar branches heterogeneity: basic and clinical insights

“…Stronger heterogeneity of free hCGb is found in the molecules secreted by abnormal trophoblast or other tissues, usually malignant. Besides the free hCGb molecules found in normal pregnancy and non-trophoblast tumors, the other hCGb forms with clinical importance are the immature beta form found in placenta, the hCG beta beta monodimer (hCGbb), and hyperglycosylated hCGb (hCGbh) forms, found in pregnancy or trophoblast tumors (Butler and Iles, 2004;Roy et al, 2007). Practically, all the hCGb molecules in urine are nicked (Birken et al, 1991).…”

G protein‐coupled receptors of the hypothalamic–pituitary–gonadal axis: A case for gnrh, LH, FSH, and GPR54 receptor ligands

“…106 Recently, another hormonal antagonist was identified, which was generated by fusing two hCG b-subunits. 107 Antagonists of the LH receptor could lead to novel contraceptive treatments. However, protein hormones need to be administered by intramuscular or subcutaneous injection.…”

Substituted Terphenyl Compounds as the First Class of Low Molecular Weight Allosteric Inhibitors of the Luteinizing Hormone Receptor

“…This compound was shown to have in vivo efficacy upon oral administration . Recently the first high molecular weight antagonist was reported for the LH receptor, two fused beta-subunits of hCG . However, LMW antagonists have not been reported so far .…”