Solid‐phase synthesis of ovine Leydig cell insulin‐like peptide – a putative ovine relaxin?

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Abstract
The primary structure of ovine Leydig cell insulin‐like peptide (Ley I‐L) was recently deduced from the corresponding cDNA sequence. It consists of two peptide chains and three disulphide bonds in an arrangement similar to both relaxin and insulin. As in relaxin B‐chain, an Arg‐X‐X‐X‐Arg sequence exists within the Ley I‐L B‐chain although it is located four residues towards the C‐terminus from the corresponding position within relaxin. This sequence of amino acids is known to be essential for relaxin biological activity and its presence in Ley I‐L suggested that the peptide might possess a relaxin‐like function. Ovine Ley I‐L was assembled by Fmoc‐solid‐phase synthesis of the separate chains followed by their combination in solution at high pH. The purity and identity of the chain‐combined peptide was confirmed by chemical characterization including mass spectrometry. At physiological concentrations, the peptide was shown not to possess relaxin‐like activity in the rat isolated atrial chronotropic and inotropic assay. This strongly suggests that Ley I‐L is not a relaxin in the sheep. In order to explore further a possible structural relationship between Ley I‐L and relaxin, we prepared a synthetic analogue of ovine Ley I‐L containing a single replacement of B‐chain residue 12, His, with Arg. This was found to possess significant relaxin‐like chronotropic and inotropic activity demonstrating that the tertiary structure of Ley I‐L is similar to that of relaxin and highlighting the key requirement for the five‐residue sequence, Arg‐X‐X‐X‐Arg, to be present in position B12–16 for characteristic relaxin activity.