Johan Rosengren

The chemistry and biology of human relaxin-3

Abstract: A novel member of the human relaxin subclass of the insulin superfamily was recently discovered during a genomics database search and named relaxin‐3. Like human relaxin‐1 and relaxin‐2, relaxin‐3 is predicted to consist of a two‐chain structure and three disulfide bonds in a disposition identical to that of insulin. To undertake detailed biophysical and biological characterization of the peptide, its chemical synthesis was undertaken. In contrast to human relaxin‐1 and relaxin‐2, however, relaxin‐3 could not be successfully prepared by simple combination of the individual chains, thus necessitating recourse to the use of a regioselective disulfide bond formation strategy. Solid phase synthesis of the separate, selectively S‐protected A and B chains followed by their purification and the subsequent stepwise formation of each of the three disulfides led to the successful acquisition of human relaxin‐3. Comprehensive chemical characterization confirmed both the correct chain orientation and the integrity of the synthetic product. Relaxin‐3 was found to bind to and activate native relaxin receptors in vitro and stimulate water drinking through central relaxin receptors in vivo. Recent studies have demonstrated that relaxin‐3 will bind to and activate human LGR7, but not LGR8, in vitro. Secondary structural analysis showed it to adopt a less ordered confirmation than either relaxin‐1 or relaxin‐2, reflecting the presence in the former of a greater percentage of nonhelical forming amino acids. NMR spectroscopy and simulated annealing calculations were used to determine the three‐dimensional structure of relaxin‐3 and to identify key structural differences between the human relaxins.