Abstract
Insulin-like peptide 5 (INSL5) was first identified through searches of the expressed sequence tags (EST) databases. Primary sequence analysis showed it to be a prepropeptide that was predicted to be processed in vivo to yield a two-chain sequence (A and B) that contained the insulin-like disulfide cross-links. The high affinity interaction between INSL5 and the receptor RXFP4 (GPCR142) coupled with their apparent coevolution and partially overlapping tissue expression patterns strongly suggest that INSL5 is an endogenous ligand for RXFP4. Given that the primary function of the INSL5–RXFP4 pair remains unknown, an effective means of producing sufficient quantities of this peptide and its analogues is needed to systematically investigate its structural and biological properties. A combination of solid-phase peptide synthesis methods together with regioselective disulfide bond formation were used to obtain INSL5. Both chains were unusually resistant to standard synthesis protocols and required highly optimized conditions for their acquisition. In particular, the use of a strong tertiary amidine, DBU, as Nα-deprotection base was required for the successful assembly of the B chain; this highlights the need to consider incomplete deprotection rather than acylation as a cause of failed synthesis. Following sequential disulfide bond formation and chain combination, the resulting synthetic INSL5, which was obtained in good overall yield, was shown to possess a similar secondary structure to human relaxin-3 (H3 relaxin). The peptide was able to inhibit cAMP activity in SK-N-MC cells that expressed the human RXFP4 receptor with a similar activity to H3 relaxin. In contrast, it had no activity on the human RXFP3 receptor. Synthetic INSL5 demonstrates equivalent activity to the recombinant-derived peptide, and will be an important tool for the determination of its biological function.
Highlights
Insulin-like peptide 5 (INSL5) was first identified through a search of the expressed sequence tags (EST) databases for novel insulin-like sequences.[1]
INSL5 shows the highest sequence similarity to H3 relaxin,[15] and was predicted to bind to relaxin family peptide (RXFP) receptors. Both the INSL5 and RXFP4 genes are dysfunctional in the rat and dog genomes; this suggested that INSL5 is the native ligand of RXFP4.[11]. It was subsequently shown that, in vitro, INSL5 binds to RXFP4 with an affinity equal to that of H3 relaxin.[11]
The most frequently encountered side reaction that affects Asp residues during solid phase peptide synthesis (SPPS) is aspartimide formation; this results from a ring closure between the nitrogen of the acarboxyl amide bond and the b-carboxyl side chain, with the loss of the ester protecting group.[16]
Summary
Insulin-like peptide 5 (INSL5) was first identified through a search of the expressed sequence tags (EST) databases for novel insulin-like sequences.[1]. INSL5 shows the highest sequence similarity to H3 relaxin,[15] and was predicted to bind to relaxin family peptide (RXFP) receptors. Both the INSL5 and RXFP4 genes are dysfunctional in the rat and dog genomes; this suggested that INSL5 is the native ligand of RXFP4.[11] It was subsequently shown that, in vitro, INSL5 binds to RXFP4 with an affinity equal to that of H3 relaxin.[11] Importantly, INSL5 does not activate RXFP3, it does bind to the receptor with low affinity and can act as a weak antagonist. The much cheaper Gly derivative, Fmoc-(Dmb)Gly-OH, which is commercially available, can effectively prevent aspartimide formation and chain aggregation.[22] Use of Fmoc-(Dmb)Gly-OH in the middle of the chain together with one pseudoproline, Fmoc-Leu-SerACHTUNGRE(yMeMePro)-OH, at the C-terminal region resulted in a crude Achain peptide with an excellent HPLC profile (Figure 2)
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