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Single chain human chorionic gonadotropin, $hCG\alpha \beta$: Effects of mutations in the \alpha subunit on structure and bioactivity

Setlur, Sunita R and Dighe, Rajan R (2007) Single chain human chorionic gonadotropin, $hCG\alpha \beta$: Effects of mutations in the \alpha subunit on structure and bioactivity. In: Glycoconjugate Journal, 24 (1). pp. 97-106.

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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 (phCG\alpha\beta) 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 hCG\alpha 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 hCG \alpha 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 hormonereceptor interactions.

Item Type: Journal Article
Additional Information: Copyright of this article belongs to Springer.
Keywords: HCG;Gonadotropins;Single chain glycoprotein hormones;Pichia pastoris;Site directed mutagenesis
Department/Centre: Division of Biological Sciences > Molecular Reproduction, Development & Genetics (formed by the merger of DBGL and CRBME)
Date Deposited: 25 Aug 2008
Last Modified: 19 Sep 2010 04:34
URI: http://eprints.iisc.ernet.in/id/eprint/9386

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