Chimeric relaxin peptides highlight the role of the A-chain in the function of H2 relaxin

• Published in: Peptides (New York, N.Y. : 1980) N.Y. : 1980), 2012-05, Vol.35 (1), p.102-106
• Main Authors: Hossain, Mohammed Akhter, Wade, John D., Bathgate, Ross A.D.
• Format: Article
• Language: English
• Subjects: Activation; agonists; amino acid composition; Amino acids; Binding; binding capacity; bioactive properties; Biological; chimerism; Cyclic AMP - metabolism; disulfide bonds; GPCR; HEK293 Cells; Homology; Humans; In vitro testing; Insulin; Insulin - pharmacology; Ligands; matrix-assisted laser desorption-ionization mass spectrometry; Peptide; Peptide Fragments - pharmacology; Peptides; Protein Binding; Proteins - pharmacology; Receptors, G-Protein-Coupled - agonists; Receptors, G-Protein-Coupled - metabolism; Receptors, Peptide - agonists; Receptors, Peptide - metabolism; Recombinant Fusion Proteins - pharmacology; Relaxin; Relaxin - pharmacology; reversed-phase high performance liquid chromatography; RXFP1
• ISSN: 0196-9781; 1873-5169
• DOI: 10.1016/j.peptides.2012.02.021

► Chimeric peptides combining H2 relaxin-B/INSL peptides-A have been synthesized. ► The peptides demonstrate reduced activity highlighting the importance of the A-chain. ► A-chain chimeras can be produced with receptor specificity. ► H2 relaxin chimeric peptides may be templates for specific RXFP1 agonist development. Human gene-2 (H2) relaxin is a member of the insulin–relaxin peptide superfamily. Because of the potential clinical applications of H2 relaxin, there is a need for novel analogs that have improved biological activity and receptor specificity. In this respect, we have chemically assembled chimeric peptides consisting of the B-chain of H2 relaxin in combination with A-chains from other insulin/relaxin family members. The peptides were prepared using solid phase peptide synthesis together with regioselective disulfide bond formation and characterized by RP-HPLC, MALDI-TOF MS and amino acid analysis. Their in vitro activity was assessed in RXFP1 or RXFP2 expressing cells. Replacement of the H2 relaxin A-chain resulted in parallel losses of binding affinity and activity on RXFP1. Not surprisingly H1A-H2B demonstrated the highest activity as the H1 A-chain shares high homology with H2 relaxin whereas INSLA-H2B, which shows low homology, had very poor activity. Importantly A-chain replacements had a dramatic effect on RXFP2 activity similar to previous results demonstrating different modes of activation of A-chain variants on RXFP1 and RXFP2. H3A-H2B is particularly interesting as it displays moderate activity at RXFP1 but poor activity at RXFP2 indicating that it may be a template for specific RXFP1 agonist development. Our study confirms that the activity of H2 relaxin at both RXFP1 and RXFP2 relies on interactions with both the B- and A-chains, and also provide new biochemical insights into the mechanism of relaxin action that the A-chain needs to be in native or near-native form for strong RXFP1 or RXFP2 agonist activity.