The active form of goat insulin-like peptide 3 (INSL3) is a single-chain structure comprising three domains B-C-A, constitutively expressed and secreted by testicular Leydig cells

Abstract

Relaxin-like factor (RLF), also called insulin-like peptide 3 (INSL3), is a member of the insulin/relaxin gene family and is produced by testicular Leydig cells. While the understanding of its effects is growing, very little is known about the structural and functional properties of native INSL3. Here, we demonstrate that native INSL3 isolated from goat testes is a single-chain structure with full biological activity, and is constitutively expressed and secreted by Leydig cells. Using a series of chromatography steps, native INSL3 was highly purified as a single 12-kDa peak as revealed by SDS-PAGE. MS/MS analysis provided 81% sequence coverage and revealed a distinct single-chain structure consisting of the B-, C-, and A-domains deduced previously from the INSL3 cDNA sequence. Moreover, the N-terminal peptide was six amino acid residues longer than predicted. Native INSL3 exhibited full bioactivity in HEK-293 cells expressing the receptor for INSL3. Immunoelectron microscopy and Western blot analysis revealed that INSL3 was secreted by Leydig cells through the constitutive pathway into blood and body fluids. We conclude, therefore, that goat INSL3 is constitutively secreted from Leydig cells as a B-C-A single-chain structure with full biological activity.

Insulin-Like Factor 3 and the HPG Axis in the Male

“…It has a very similar structure to insulin or relaxin, being made as a prepro-hormone, which after intracellular folding becomes post-translationally processed, to give rise to either an A–B heterodimeric peptide, like insulin, or possibly to an uncleaved B–C–A version, analogous to the IGFs. Why this is unclear is that both forms have been identified in the circulation of male mammals (3–5), and both forms are fully and equally bioactive (4). In the male mammal, the major site of INSL3 synthesis is the interstitial Leydig cells of both the fetal and the adult testis [Ref.…”

Insulin-like peptide 3 expressed in the silkworm possesses intrinsic disulfide bonds and full biological activity

“…The C-domains of insulin and relaxin are processed by Golgi-localized endopeptidases, including prohormone convertase 1/3 (PC1/3) 35 , 36 and furin 36 – 38 . However, native INSL3 extracted from boar testes is a single-chain peptide that likely possesses a C-domain 3 because PC1/3 is not expressed in boar testes at any developmental stages 39 and recombinant pINSL3 does not encode a cleavage motif (Arg-X-Lys/Arg-Arg) for furin in the C-peptide (-His-His-His-His-Arg-Arg-) C-terminus 4 . B .…”

“…Recombinant pINSL3 is likely hydrolyzed in the highly basic region of the C-domain by endopeptidases expressed in the Golgi apparatus of host silkworms in the secretion pathway. INSL3-stimulation of RXFP2 activity requires the α-helical structure of the B- and A-domains; native boar and goat INSL3 possess full bioactivity similar to synthetic hINSL3, which does not have a C-domain 3 , 4 . Although the relaxin/insulin superfamily C-domain has been reported to be required for proper precursor folding in the endoplasmic reticulum 42 , it is not fully understood whether the C-domain has any functions on its own.…”

“…INSL3 is constitutively expressed and secreted as either an A-B heterodimer 2 or an A-B-C single-chain form (Supplementary Fig. 1 ) 3 , 4 and initiates intracellular signaling by binding with high affinity to its specific receptor, relaxin family peptide receptor 2 (RXFP2; originally called leucine-rich G protein-coupled receptor 8 [LGR8]) 5 , 6 . Although INSL3 is essential for fetal testis descent as revealed by mouse models targeting either Insl3 7 , 8 or Rxfp2 9 , 10 , there is compelling but inconclusive evidence that INSL3 contributes to sperm production as a germ cell survival/anti-apoptotic factor in rats 11 , 12 and boars 13 , 14 and as a germ cell proliferation factor in zebrafish 15 .…”

“…Thus, interest in INSL3 for both research and clinical applications is growing rapidly. Because INSL3 isolation methods are very limited, with a yield of approximately 11 μg per 300 to 400 g of pig or goat testes 3 , 4 , the purification of biologically active recombinant INSL3 would be a major step towards producing an uninterrupted supply. INSL3 has been produced either by solid-phase synthesis of the separate A- and B-domains and subsequent chain recombination 20 or by oxidative refolding and enzymatic processing of recombinant single peptides expressed in E .…”