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

Superior expansion of long-term hematopoietic stem cells using StemPro™ HSC medium kit

The use of CD34+ hematopoietic stem cells (HSC) for transplantation has been limited due to the low CD34+ cell numbers in tissue sources such as peripheral blood and cord blood. Two strategies have been employed to increase the CD34+ cell dosage. These include mobilization of HSC into peripheral blood via injection of G-CSF, and ex vivo expansion of CD34+ cells. A major limitation of current systems used for the expansion of HSC is that ex vivo culture leads to expansion and differentiation of cells, at the expense of the most primitive pluripotent long-term HSC. This has limited the clinical application of ex vivo expanded HSC, since short-term progenitor cells only provide transient protection, ultimately reducing the positive health outcomes, increasing the duration of hospitalizations, and health care costs per patient. Development of a culture system that expands, both short term progenitor cells and long-term HSC would enable immune protection during the early phase of recovery, and provide a suitable solution for transfusion-independent hematopoiesis. Therefore, we have developed an HSC culture medium that enables the expansion of both long-term HSC and short-term progenitor cells, while maintaining their functional properties. We conducted several iterative rounds of Design of Experiments (DOE) involving multifactorial analysis, and mathematical modeling methods. The DOEs allowed us to identify optimal combinations and concentrations of essential media components, small molecules, and growth factors. The performance of candidate HSC expansion media were evaluated after 7 days of culture, upon which the CD34+ cells and CD34+CD90+CD45RA- cells (long-term HSC) were quantified. We were successful in developing a media system- StemPro™ HSC Medium Kit-which is xeno-free, serum-free medium that expands both long term CD34+CD90+CD45RA- HSC and short term CD34+. The expression of aldehyde dehydrogenase was conducted to identify primitive stem cells, and colony-forming unit assays were performed to assess the in vitro differentiation capacity of expanded cells. We plan to determine whether the expanded cells are engraftable by transplanting the cells into immuno-deficient mice. Taken together, we seek to highlight our design philosophy in HSC culture media development, and we believe our efforts are critical for the successful utilization of hematopoietic stem cell transplants in translational cell therapies

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

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-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-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