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

Hemojuvelin-Neogenin Interaction Is Required for Bone Morphogenic Protein-4-induced Hepcidin Expression

Hemojuvelin (HJV) is a glycosylphosphatidylinositol-linked protein and binds both bone morphogenic proteins (BMPs) and neogenin. Cellular HJV acts as a BMP co-receptor to enhance the transcription of hepcidin, a key iron regulatory hormone secreted predominantly by liver hepatocytes. In this study we characterized the role of neogenin in HJV-regulated hepcidin expression. Both HJV and neogenin were expressed in liver hepatocytes. Knockdown of neogenin decreased BMP4-induced hepcidin mRNA levels by 16-fold in HJV-expressing HepG2 cells but only by about 2-fold in cells transfected with either empty vector or G99V mutant HJV that does not bind BMPs. Further studies indicated that disruption of the HJV-neogenin interaction is responsible for a marked suppression of hepcidin expression. Moreover, in vivo studies showed that hepatic hepcidin mRNA could be significantly suppressed by blocking the interaction of HJV with full-length neogenin with a soluble fragment of neogenin in mice. Together, these results suggest that the HJV-neogenin interaction is required for the BMP-mediated induction of hepcidin expression when HJV is expressed. Combined with our previous studies, our results support that hepatic neogenin possesses two functions, mediation of cellular HJV release, and stimulation of HJV-enhanced hepcidin expression

Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin-4

(FAC) indicated, followed by cell-surface biotin labeling, and detection of RGMc by immunoblotting in cell lysates (left panel), on the membrane after surface biotin labeling and streptavidin pull-down (middle panel), and in the medium (right panel). Similar results were observed with Cos-7 cells. Detection of RGMc by immunoblotting of conditioned medium from transiently transfected Cos-7 cells following incubation for 24 h with the concentrations of FAC or deferoxamine (DFO) indicated. Identical results were seen with Hep3B cells. For – , arrows are as in legend to Fig. 2.<p><b>Copyright information:</b></p><p>Taken from "Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin"</p><p>http://www.biomedcentral.com/1471-2091/9/9</p><p>BMC Biochemistry 2008;9():9-9.</p><p>Published online 2 Apr 2008</p><p>PMCID:PMC2323002.</p><p></p

Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin-0

of processing are unknown. Full-length membrane-linked 50 kDa RGMc may be digested by a phospholipase (PI-PLC) or by an uncharacterized protease, and then by a PC to generate the 40 kDa species. Alternatively, a PC may directly cleave 40 kDa RGMc at the membrane. The starbursts represent N-linked glycosylation sites, and the thin lines, disulfide bonds.<p><b>Copyright information:</b></p><p>Taken from "Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin"</p><p>http://www.biomedcentral.com/1471-2091/9/9</p><p>BMC Biochemistry 2008;9():9-9.</p><p>Published online 2 Apr 2008</p><p>PMCID:PMC2323002.</p><p></p

Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin-1

of processing are unknown. Full-length membrane-linked 50 kDa RGMc may be digested by a phospholipase (PI-PLC) or by an uncharacterized protease, and then by a PC to generate the 40 kDa species. Alternatively, a PC may directly cleave 40 kDa RGMc at the membrane. The starbursts represent N-linked glycosylation sites, and the thin lines, disulfide bonds.<p><b>Copyright information:</b></p><p>Taken from "Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin"</p><p>http://www.biomedcentral.com/1471-2091/9/9</p><p>BMC Biochemistry 2008;9():9-9.</p><p>Published online 2 Apr 2008</p><p>PMCID:PMC2323002.</p><p></p

Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin-2

S with Ad-HA-RGMc ± RVKR. Cell-surface proteins were labeled with non-permeable biotin (EZ-link), followed by incubation ± RVKR, and streptavidin pull-down, as described in 'Methods'. Immunoblot for cadherin measures sample loading. PC inhibition does not prevent acute release of RGMc from the cell surface. Membrane-associated RGMc was labeled with EZ-link, followed by incubation ± RVKR, and detection of soluble RGMc after streptavidin pull-down by immunoblotting. For – , arrows are described in legend to Fig. 1. Similar results were observed with Cos-7 cells.<p><b>Copyright information:</b></p><p>Taken from "Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin"</p><p>http://www.biomedcentral.com/1471-2091/9/9</p><p>BMC Biochemistry 2008;9():9-9.</p><p>Published online 2 Apr 2008</p><p>PMCID:PMC2323002.</p><p></p