Solid-phase synthesis of ovine Leydig cell insulin-like peptide--a putative ovine relaxin?
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.
Dawson
N F
NF
Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria, Australia.
Tan
Y Y
YY
Macris
M
M
Otvos
L
L
Summers
R J
RJ
Tregear
G W
GW
Wade
J D
JD
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002316:Cardiotonic Agents / Q000494:pharmacology; D006325:Heart Atria / Q000187:drug effects; D006339:Heart Rate / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007328:Insulin; D007537:Isometric Contraction / Q000187:drug effects; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009200:Myocardial Contraction / Q000187:drug effects; D011506:Proteins / Q000138:chemical synthesis* / Q000494:pharmacology / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012065:Relaxin / Q000494:pharmacology / Q000502:physiology*; D012756:Sheep; D012996:Solutions; D013268:Stimulation, Chemical
D002316:Cardiotonic Agents; D007328:Insulin; C084797:Leydig insulin-like protein; D011506:Proteins; D012996:Solutions; D012065:Relaxin; D007545:Isoproterenol