A pro-protein convertase inhibitor prevents accumulation of soluble 40 kDa RGMc
RGMc is produced by hepatocytes and striated muscle [8, 13, 15]. We have detected RGMc on muscle cell membranes, and have found that proteins of ~50, 35, and 20 kDa are released into the extracellular fluid after incubation with bacterial PI-PLC [14], illustrating that RGMc is attached to the membrane by a GPI linkage. The two smaller protein bands comprise a disulfide-linked heterodimer, while the larger species is full-length single-chain RGMc [14]. Similar results have been observed for RGMc over-expressed in cell lines [14–16]. RGMc also accumulates in medium conditioned by muscle cells as 50 and 40 kDa single-chain proteins [14], suggesting either direct secretion or release from the plasma membrane.
Here we investigate mechanisms responsible for the appearance of 40 kDa RGMc in extra-cellular fluid. Full-length 50 kDa RGMc is a highly conserved protein, with ~76% homology among the 7 mammalian species shown in Fig. 1A, and many stretches of amino acid identity, including the arginine-rich segment found between residues 318 and 327 in mouse RGMc (Fig. 1A) that is not conserved in RGMa or RGMb (Fig. 1B). This motif resembles a recognition and cleavage site for pro-protein convertases (PC). These enzymes, PC1, PC2, furin, PC4, PC5, PACE4 and PC7 [22], cleave substrates at the COOH-terminal basic residue in the sequence (K/R)-(X)
n
-(K/R), where n = 0, 2, 4, or 6 amino acids and X is any residue except Cys or Pro [22]. To determine if full-length RGMc is a substrate for PCs, we first purified the soluble protein by antibody affinity chromatography from culture medium from differentiating muscle cells, and from mouse and human blood. Both 50 and 40 kDa RGMc species were detected from all three sources (Fig. 1C and 1D). Surprisingly, serum concentrations differed substantially between two healthy humans, and between two healthy male mice of the same age and on the same diet, where in one mostly the 50 kDa form of RGMc was detected (Fig. 1D, lane c vs. d). Further study will be required to assess the mechanisms responsible for this variation. When muscle cells, which produce RGMc [14], were incubated with the peptide PC inhibitor, RVKR, the 50 kDa species became the only RGMc protein in the medium (Fig. 1E), implicating PC activity in processing of endogenous RGMc. Similar results were seen in undifferentiated mouse C2 myoblasts and in human Hep3B cells expressing HA-RGMc (Fig. 1F), although it should be noted that the same dose of RVKR was less effective in Hep3B cells in preventing accumulation of 40 kDa RGMc, implying that there is more PC activity in this cell line.
Mapping the location of PC cleavage by analysis of JH-associated RGMc frame-shift mutations
Several frame-shift mutations in human RGMc are associated with JH [4–7]. We mimicked four of these mutants in NH2-terminal HA-tagged mouse RGMc, and added a 6× His tag to each COOH-terminus (Fig. 2A). The mutant proteins when expressed in Cos-7 cells accumulated in culture medium, but not on the cell membrane, as they lacked a GPI attachment sequence (Fig. 2B, and data not shown). A doublet was seen in the medium of cells producing the R378X and C354X mutants, but only a single immunoreactive band with S321X and Q305X. As all protein species were detected with an antibody to the NH2-terminal HA tag, this result suggests that the smaller mutant proteins (and 40 kDa RGMc derived from RGMcΔGPI) may lack COOH-terminal residues (Fig. 2B). After incubation of cells with RVKR, the larger member of the protein doublet for R378X and C354X increased in abundance in the medium, and was recognized by an antibody to the COOH-terminal His tag, while the smaller of the doublet bands was not (Fig. 2C). Similar results were seen for RGMcΔGPI (Fig. 2C). In contrast, detection of S321X and Q305X with the His antibody was constant and was unaffected by RVKR. Taken together with observations in Fig. 1, the results with truncation mutants demonstrate that PC activity potentially removes a COOH-terminal segment of RGMc, and indicate that the cleavage site is located between amino acids 321 and 354 of the mouse protein, in agreement with identification of a conserved PC motif between residues 318 and 327 (Fig. 1A).
Altered processing of newly synthesized RGMc in the presence of PC inhibitor
To study effects of PC inhibition on RGMc biosynthesis and maturation, we infected Hep3B cells with Ad-tTA and Ad-HA-RGMc. Ad-tTA encodes a tetracycline-repressible transcriptional activator that stimulates the promoter regulating the gene for HA-RGMc. Under these conditions, full-length and heterodimeric RGMc were found on the cell membrane beginning at 8 h after viral infection and increased in abundance at 24 h. More membrane-associated RGMc was detected in RVKR-treated cells than in controls, although there was no change in the pattern of immunoreactive proteins. In culture medium 40 kDa RGMc accumulated starting at 8 h, and the 50 kDa species was only seen after incubation of cells with RVKR (Fig. 3A).
We used cell surface-labeling experiments to study the impact of PC inhibition on acute release of membrane-linked RGMc into the medium. In Hep3B cells expressing HA-RGMc, membrane proteins were labeled for 30 min with cell-impermeable biotin cross-linker followed by addition of RVKR or vehicle. Release of RGMc from the cell surface was monitored by immunoblotting after streptavidin pull-down of culture medium. Biotinylated 40 and 50 kDa RGMc were detected in medium at 2 and 4 h after labeling, and more 50 kDa RGMc was seen with RVKR, although the total amount of RGMc declined by ~35% (Fig. 3B). These results show that both 50 and 40 kDa soluble RGMc species derive from cell-associated 50 kDa RGMc, and demonstrate that inhibition of PC activity diminishes but does not prevent release of membrane-linked RGMc into extracellular fluid.
Furin cleaves 50 kDa RGMc to produce a 40 kDa species
We destroyed putative PC recognition/cleavage sites in mouse RGMc by site-directed mutagenesis (glycine for arginine 318, alanines for arginines 321 and 324). When this mutant was expressed in Cos-7 cells, only 50 kDa RGMc was detected in conditioned medium, while wild type RGMc was processed to the 40 kDa species in the absence of RVKR, but 50 kDa RGMc was seen when the inhibitor was added (Fig. 4A).
PC activity may be found in intracellular compartments, at the membrane, and in the extra-cellular milieu [22]. To determine where cleavage of 50 kDa RGMc may occur, conditioned medium from Cos-7 cells expressing HA-RGMc was collected in the presence of RVKR, and after dialysis to remove the inhibitor, added to Hep3B cells. Following incubation for 24 h, significant conversion to 40 kDa RGMc was observed, but was not seen when RVKR was added (Fig. 4B), indicating that these cells produce PCs that act extra-cellularly.
The R-N-R-R sequence in RGMc (Fig. 1A) represents an optimal furin site [23]. In agreement with this idea, incubation of 50 kDa RGMc with recombinant furin in vitro led to its efficient cleavage into a 40 kDa species with an intact NH2-terminus, as shown by detection with both anti-HA and anti-RGMc antibodies (Fig. 4C). Thus by several criteria, 50 kDa RGMc is a PC substrate.
Effects of iron on processing of RGMc and its release from the cell membrane
Recent work has shown that delivery of iron to cultured cells resulted in a decline in abundance of extra-cellular RGMc 24 h later [15, 24]. To address effects of iron on membrane-bound RGMc, adenoviral-infected Hep3B cells were incubated with ferric ammonium citrate, briefly labeled with cell-impermeable biotin cross-linker, and examined for accumulation of RGMc on the cell membrane and in the medium. As seen in Fig. 5A, iron loading led to a dose-dependent increase in cell surface-associated 50 kDa RGMc, and to a decline in abundance of the 40 kDa species in the medium (~50% decrease at 100 μg/ml ferric ammonium citrate). In addition, the amount of 50 kDa soluble RGMc increased. In agreement with these results, incubation of cells with the iron chelator, deferoxamine, prevented accumulation of 50 kDa RGMc in culture medium, and led to a ~30% rise in the amount of soluble 40 kDa RGMc compared with cells incubated with ferric ammonium citrate (Fig. 5B). Thus, iron delivery appears to negatively regulate release of RGMc from the membrane, and may also inhibit PC activity. In this regard, Silvestri et al have found that deferoxamine enhanced and iron loading decreased the abundance of furin in HeLa cells [25].