Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin

<p>Abstract</p> <p>Background</p> <p>Repulsive guidance molecule c (RGMc or hemojuvelin), a glycosylphosphatidylinositol-linked glycoprotein expressed in liver and striated muscle, plays a central role in systemic iron balance. Inactivating mutations in the RGMc gene cause juvenile hemochromatosis (JH), a rapidly progressing iron storage disorder with severe systemic manifestations. RGMc undergoes complex biosynthetic steps leading to membrane-bound and soluble forms of the protein, including both 50 and 40 kDa single-chain species.</p> <p>Results</p> <p>We now show that pro-protein convertases (PC) are responsible for conversion of 50 kDa RGMc to a 40 kDa protein with a truncated COOH-terminus. Unlike related molecules RGMa and RGMb, RGMc encodes a conserved PC recognition and cleavage site, and JH-associated RGMc frame-shift mutants undergo COOH-terminal cleavage only if this site is present. A cell-impermeable peptide PC inhibitor blocks the appearance of 40 kDa RGMc in extra-cellular fluid, as does an engineered mutation in the conserved PC recognition sequence, while the PC furin cleaves 50 kDa RGMc <it>in vitro </it>into a 40 kDa molecule with an intact NH₂-terminus. Iron loading reduces release of RGMc from the cell membrane, and diminishes accumulation of the 40 kDa species in cell culture medium.</p> <p>Conclusion</p> <p>Our results define a role for PCs in the maturation of RGMc that may have implications for the physiological actions of this critical iron-regulatory protein.</p

Diversification of the insulin-like growth factor 1 gene in mammals.

Insulin-like growth factor 1 (IGF1), a small, secreted peptide growth factor, is involved in a variety of physiological and patho-physiological processes, including somatic growth, tissue repair, and metabolism of carbohydrates, proteins, and lipids. IGF1 gene expression appears to be controlled by several different signaling cascades in the few species in which it has been evaluated, with growth hormone playing a major role by activating a pathway involving the Stat5b transcription factor. Here, genes encoding IGF1 have been evaluated in 25 different mammalian species representing 15 different orders and ranging over ~180 million years of evolutionary diversification. Parts of the IGF1 gene have been fairly well conserved. Like rat Igf1 and human IGF1, 21 of 23 other genes are composed of 6 exons and 5 introns, and all 23 also contain recognizable tandem promoters, each with a unique leader exon. Exon and intron lengths are similar in most species, and DNA sequence conservation is moderately high in orthologous exons and proximal promoter regions. In contrast, putative growth hormone-activated Stat5b-binding enhancers found in analogous locations in rodent Igf1 and in human IGF1 loci, have undergone substantial variation in other mammals, and a processed retro-transposed IGF1 pseudogene is found in the sloth locus, but not in other mammalian genomes. Taken together, the fairly high level of organizational and nucleotide sequence similarity in the IGF1 gene among these 25 species supports the contention that some common regulatory pathways had existed prior to the beginning of mammalian speciation

The Zmat2 gene in non-mammalian vertebrates: Organizational simplicity within a divergent locus in fish.

ZMAT2 is among the least-studied of mammalian proteins and genes, even though it is the ortholog of Snu23, a protein involved in pre-mRNA splicing in yeast. Here we have used data from genomic and gene expression repositories to examine the Zmat2 gene and locus in 8 terrestrial vertebrates, 10 ray-finned fish, and 1 lobe-finned fish representing > 500 million years of evolutionary diversification. The analyses revealed that vertebrate Zmat2 genes are similar to their mammalian counterparts, as in 16/19 species studied they contain 6 exons, and in 18/19 encode a single conserved protein. However, unlike in mammals, no Zmat2 pseudogenes were identified in these vertebrates, although an expressed Zmat2 paralog was characterized in flycatcher that resembled a DNA copy of a processed and retro-transposed mRNA, and thus could be a proto-pseudogene captured during its evolutionary journey from active to inert. The Zmat2 locus in terrestrial vertebrates, and in spotted gar and coelacanth, also shares additional genes with its mammalian counterparts, including Histidyl-tRNA synthetase (Hars), Hars2, and others, but these are absent from the Zmat2 locus in teleost fish, in which Stem-loop-binding protein 2 (Slbp2) and Lymphocyte cytosolic protein 2a (Lcp2a) are present instead. Taken together, these observations argue that a recognizable Zmat2 was present in the earliest vertebrate ancestors, and postulate that during chromosomal tetraploidization and subsequent re-diploidization during modern teleost evolution, the duplicated Zmat2 gene was retained and the original lost. This study also highlights how information from genomic resources can be leveraged to reveal new biologically significant insights

Alignments of mammalian IGF1 precursor peptides.

<p><b>A</b>. Amino acid sequences of COOH-terminal common E and E_A peptides. <b>B</b>. Amino acid sequences of COOH-terminal E_B peptides. For <b>A</b> and B, identities are indicated by dots and a dash depicts no residue. The number of amino acids in each E_B segment is listed in parenthesis. The color of rat IGF1 amino acids corresponds with the map in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189642#pone.0189642.g001" target="_blank">Fig 1C</a>.</p

Comparison of Stat5b elements in <i>IGF1/Igf1</i> loci.

<p>Comparison of Stat5b elements in <i>IGF1/Igf1</i> loci.</p

Organization of mammalian IGF1 genes^*.

<p>Organization of mammalian IGF1 genes^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189642#t001fn001" target="_blank">*</a>}.</p

The sloth <i>IGF1</i> locus encodes a processed pseudogene.

<p><b>A</b>. Map of the sloth <i>IGF1</i> gene and locus. Exons are depicted as boxes, and introns and flanking DNA as horizontal lines. The enlargement above the main map illustrates the two <i>Igf1</i> promoters, P1 and P2, and exons 1–3. Red circles represent locations of homologues of sites shown to bind Stat5b in a GH-inducible way in the rat <i>Igf1</i> locus. The enlargement below the main map depicts the location and structure of the putative <i>IGF1</i> pseudogene, which consists of DNA segments that are nearly identical to sloth IGF1 exons 2, 3, and 4 (color coded in black, red, and blue, and marked as exons 2’, 3’, and 4’, respectively). <b>B</b>. Comparison of amino acid sequences of sloth IGF1 with sloth pseudogene-IGF1 in single letter code. Differences are depicted in red; a dash indicates no residue.</p

The insulin-like growth factor 2 gene in mammals: Organizational complexity within a conserved locus.

The secreted protein, insulin-like growth factor 2 (IGF2), plays a central role in fetal and prenatal growth and development, and is regulated at the genetic level by parental imprinting, being expressed predominantly from the paternally derived chromosome in mice and humans. Here, IGF2/Igf2 and its locus has been examined in 19 mammals from 13 orders spanning ~166 million years of evolutionary development. By using human or mouse DNA segments as queries in genome analyses, and by assessing gene expression using RNA-sequencing libraries, more complexity was identified within IGF2/Igf2 than was annotated previously. Multiple potential 5' non-coding exons were mapped in most mammals and are presumably linked to distinct IGF2/Igf2 promoters, as shown for several species by interrogating RNA-sequencing libraries. DNA similarity was highest in IGF2/Igf2 coding exons; yet, even though the mature IGF2 protein was conserved, versions of 67 or 70 residues are produced secondary to species-specific maintenance of alternative RNA splicing at a variable intron-exon junction. Adjacent H19 was more divergent than IGF2/Igf2, as expected in a gene for a noncoding RNA, and was identified in only 10/19 species. These results show that common features, including those defining IGF2/Igf2 coding and several non-coding exons, were likely present at the onset of the mammalian radiation, but that others, such as a putative imprinting control region 5' to H19 and potential enhancer elements 3' to H19, diversified with speciation. This study also demonstrates that careful analysis of genomic and gene expression repositories can provide new insights into gene structure and regulation