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

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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 α and β subunits of human Chorionic Gonadotropin (hCG) to produce a single chain hormone (phCGαβ) was used to investigate roles of critical residues of the α subunit in hormone receptor interaction and biological activity. The α subunit was mutated using PCR-based site-directed mutagenesis, fused to the wild type β 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 hCGα 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 hCGα 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 α 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.

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Acknowledgments

The work presented here was supported by grants from the Department of Biotechnology, Government of India, the Indian Council of Medical Research, the University Grant Commission, New Delhi, and the Council for Scientific and Industrial Research, India (fellowship for SRS). We also wish to thank National Hormone Pituitary program, NIH, USA for providing the reference preparations of hormones.

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  1. Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, 560012, India

    Sunita R. Setlur & Rajan R. Dighe

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  1. Sunita R. Setlur
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  2. Rajan R. Dighe
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Correspondence to Rajan R. Dighe.

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Setlur, S.R., Dighe, R.R. Single chain human chorionic gonadotropin, hCGαβ: Effects of mutations in the α subunit on structure and bioactivity. Glycoconj J 24, 97–106 (2007). https://doi.org/10.1007/s10719-006-9016-x

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