Mass Spectrometry Supports That the Structure of Circulating Human Insulin-Like Factor 3 Is a Heterodimer

Abstract

The structure of the testicular peptide hormone insulin-like factor 3 (INSL3) has been the subject of discussion for more than a decade. Some studies support that the central C-domain of INSL3 is proteolytically removed and that INSL3 is secreted by the testicular Leydig cells into circulation as a small heterodimer consisting of an A- and a B-chain linked by two disulfide bridges. Other studies support that the INSL3 peptide remains uncleaved and that the predominant structure of circulating INSL3 is the larger pro-form. Furthermore, the structure of INSL3 could differ between species, and both structural forms of INSL3 could, in principle, be present in circulation. Recently, we have developed a mass spectrometry (MS)-based method for INSL3 in human serum that provides new information about the structure of circulating INSL3. Based on recent and newly presented data, we argue that in healthy men, the common, and probably the only, form of circulating INSL3 is the smaller AB heterodimer. For the first time, we demonstrate that the same analytical principle, with slight modifications, can also be applied to sera from other species, and we show that the INSL3 AB heterodimer is also present in serum from rodents. Improved understanding of the structure and biochemistry of circulating INSL3 could be valuable for the interpretation of INSL3 as a marker for reproductive and developmental disorders in humans and domesticated animals.

Insulin‐like peptide 3 in domestic animals with normal and abnormal reproductive functions, in comparison to rodents and humans

“… 15 Another research group suggested that human, rat, and mouse INSL3 peptides also undergo the same processing as cattle (Figure 1B ). 16 However, Minagawa et al 17 showed in pigs that the B‐chain‐C‐peptide‐A‐chain pro‐form monomer of INSL3 is mainly secreted into the blood circulation (Figure 1C ), indicating a possible difference in the secreted form of INSL3 peptide among species due to different peptide processing mechanisms.…”

“…The preproforms of bovine, rat and human INSL3 undergo processing by removals of the signal peptide at the cleaving site and connecting (C)‐peptide at the conversion sites, and then the B‐chain and A‐chain heterodimer is produced in the gonadal endocrine cells (B). 15 , 16 The preproform of porcine INSL3 forgoes the removal of C‐peptide, and the resultant B‐chain‐C‐peptide‐A‐chain monomer is secreted into circulation (C). 17 The “Ⓒ” and “S‐S" indicate a cysteine residue and a disulfide bond, respectively.…”

“…15 Another research group suggested that human, rat, and mouse INSL3 peptides also undergo the same processing as cattle (Figure 1B). 16 However, Minagawa et al 17 Figure 2 shows an alignment of deduced amino acid sequences for INSL3 peptides consisting of B-chain and A-chain among various mammals, including some domestic animals and humans. There is seemingly a higher homology of INSL3 peptides for taxonomically closer groups.…”

“…Schematic diagrams of a preproform (A), and bovine, human, rat (B), and porcine (C) mature forms of the insulin‐like peptide 3 (INSL3) peptide. The preproforms of bovine, rat and human INSL3 undergo processing by removals of the signal peptide at the cleaving site and connecting (C)‐peptide at the conversion sites, and then the B‐chain and A‐chain heterodimer is produced in the gonadal endocrine cells (B) 15,16 . The preproform of porcine INSL3 forgoes the removal of C‐peptide, and the resultant B‐chain‐C‐peptide‐A‐chain monomer is secreted into circulation (C) 17 .…”

Physiology and evolution of the INSL3/RXFP2 hormone/receptor system in higher vertebrates

“…Whether or not this cleavage occurs in vivo may depend on species, cell type, and/or level of expression. For example, in the male pig evidence suggests that most circulating immunoreactive INSL3 occurs as the B-A-C pro-form (Minagawa et al, 2012), whereas in the human, the cleaved A-B heterodimer appears to be the prevailing form (Albrethsen et al, 2020a).…”