Functionality of an absolutely conserved glycine residue in the chimeric relaxin family peptide R3/I5
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The insulin superfamily is a group of homologous proteins that are further divided into the insulin family and relaxin family according to their distinct receptors. All insulin superfamily members contain three absolutely conserved disulfide linkages and a nonchiral Gly residue immediately following the first B-chain cysteine. The functionality of this conserved Gly residue in the insulin family has been studied by replacing it with natural l-amino acids or the corresponding unnatural d-amino acids. However, such analysis has not been conducted on relaxin family members. In the present study, we conducted chiral mutagenesis on the conserved B11Gly of the chimeric relaxin family peptide R3/I5, which is an efficient agonist for receptor RXFP3 and RXFP4. Similar to the effects on insulin family foldability, l-Ala or l-Ser substitution completely abolished the in vitro refolding of a recombinant R3/I5 precursor; whereas, d-Ala or d-Ser substitution had no detrimental effect on refolding of a semi-synthetic R3/I5 precursor, suggesting that the conserved Gly residue controls the foldability of relaxin family members. In contrast to the effect on insulin family activity, d-Ala or d-Ser replacement had no detrimental effect on the binding and activation potencies of the mature R3/I5 towards both RXFP3 and RXFP4, suggesting that the conserved Gly residue is irrelevant to the relaxin family’s activity. The present study revealed functionality of the conserved B-chain Gly residue for a relaxin family peptide for the first time, providing an overview of its contribution to foldability and activity of the insulin superfamily.
KeywordsInsulin superfamily Relaxin family Activity Foldability R3/I5
This work was supported by Grants from the National Natural Science Foundation of China (CN) (31670773, 31470767).
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Conflict of interest
The authors declare that they have no conflict of interest.
Research involving human participants and/or animals
This article does not contain any studies with human participants or animals performed by any of the authors.
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