Single chain human chorionic gonadotropin, hCGαβ: Effects of mutations in the α subunit on structure and bioactivity

Abstract

The strategy of translationally fusing the subunits of heterodimeric proteins into single chain molecules is often used to overcome the mutagenesis-induced defects in subunit interactions. The approach of fusing the alpha and beta subunits of human Chorionic Gonadotropin (hCG) to produce a single chain hormone (phCGalphabeta) was used to investigate roles of critical residues of the alpha subunit in hormone receptor interaction and biological activity. The alpha subunit was mutated using PCR-based site-directed mutagenesis, fused to the wild type beta subunit and the fusion protein was expressed using Pichia pastoris expression system. Following partial purification, the mutant proteins were extensively characterized using immunological probes, receptor assays, and in vitro bioassays. The mutation hCGalpha P38A, which disrupts subunit interaction in the heterodimeric molecule, produced a fusion molecule exhibiting altered subunit interactions as judged by the immunological criteria, but could bind to the receptor with lower affinity and elicit biological response. Mutation of hCGalpha T54A disrupting the glycosylation at Asparagine 52, believed to be important for bioactivity, also yielded a biologically active molecule suggesting that the glycosylation at this site is not as critical for bioactivity as it is in the case of the heterodimer. The fusion protein approach was also used to generate a superagonist of hormone action. Introduction of four lysine residues in the Loop 1 of the alpha subunit led to the generation of a mutant having higher affinity for the receptor and enhanced bioactivity. Immunological characterization of single chain molecules revealed that the interactions between the subunits were not identical to those seen in the heterodimeric hormone, and the subunits appeared to retain their isolated conformations, and also retained the ability to bind to the receptors and elicit response. These data suggest the plasticity of the hormone-receptor interactions.

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 ).…”

Membrane Tethered Bursicon Constructs as Heterodimeric Modulators of the Drosophila G Protein–Coupled Receptor Rickets

“…Our observations with bursicon reveal another parallel with mammalian heterodimeric cystineknot proteins. Fusion of the a and b subunits of mammalian glycohormones including TSH, LH, and FSH as single soluble peptides also results in ligands that can activate their corresponding mammalian GPCR (Sugahara et al, 1996;Fares et al, 1998;Sen Gupta and Dighe, 2000;Park et al, 2005;Setlur and Dighe, 2007).…”

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

“…Several reports have shown that single-chain variants of glycoprotein hormones with C terminus of the ␤-subunit fused to the N terminus of the ␣-subunit display bioactivities comparable with those of the corresponding heterodimers (27)(28)(29)(30)(31). However, the relative position of the two subunit does not appear to be important for generation of a biologically active molecule (13).…”