The chemistry and biology of human relaxin-3.
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.
Tregear
Geoffrey W
GW
Howard Florey Institute of Experimental Physiology and Medicine, Parkville, Victoria, 3010, Australia. g.tregear@hfi.unimelb.edu.au
Bathgate
Ross A
RA
Layfield
Sharon
S
Ferraro
Tania
T
Gundlach
Andrew
A
Ma
Sherie
S
Lin
Feng
F
Hanson
Nicola F
NF
Summers
Roger J
RJ
Rosengren
Johan
J
Craik
David J
DJ
Wade
John D
JD
D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D008969:Molecular Sequence Data; D012065:Relaxin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment
C480238:RLN3 protein, human; D012065:Relaxin