Neogenin interacts with matriptase-2 to facilitate hemojuvelin cleavage

Abstract

Hemojuvelin (HJV) and matriptase-2 (MT2) are co-expressed in hepatocytes, and both are essential for systemic iron homeostasis. HJV is a glycosylphosphatidylinositol-linked membrane protein that acts as a co-receptor for bone morphogenetic proteins to induce hepcidin expression. MT2 regulates the levels of membrane-bound HJV in hepatocytes by binding to and cleaving HJV into an inactive soluble form that is released from cells. HJV also interacts with neogenin, a ubiquitously expressed transmembrane protein with multiple functions. In this study, we showed that neogenin interacted with MT2 as well as with HJV and facilitated the cleavage of HJV by MT2. In contrast, neogenin was not cleaved by MT2, indicating some degree of specificity by MT2. Down-regulation of neogenin with siRNA increased the amount of MT2 and HJV on the plasma membrane, suggesting a lack of neogenin involvement in their trafficking to the cell surface. The increase in MT2 and HJV upon neogenin knockdown was likely due to the inhibition of cell surface MT2 and HJV internalization. Analysis of the Asn-linked oligosaccharides showed that MT2 cleavage of cell surface HJV was coupled to a transition from high mannose oligosaccharides to complex oligosaccharides on HJV. These results suggest that neogenin forms a ternary complex with both MT2 and HJV at the plasma membrane. The complex facilitates HJV cleavage by MT2, and release of the cleaved HJV from the cell occurs after a retrograde trafficking through the TGN/Golgi compartments.

FIGURE 1.

Neogenin is required for MT2 cleavage of HJV. A, diagram of the MT2 protein. MT-2 is a type II transmembrane protein that has a short cytoplasmic domain, a transmembrane domain, and a large extracellular domain. The extracellular domain contains a membrane-proximal SEA (sea urchin sperm protein, enteropeptidase, agrin) domain, two CUB (complete protein subcomponents C1r/C1s motif, urchin embryonic growth factor, and BMP1) domains, three LDLRa (low density lipoprotein receptor class A) domains, a predicted activation domain, and a C-terminal catalytic domain. B, diagram of different forms of neogenin used in this study. Full-length neogenin (Neo) is a type I transmembrane protein with a large extracellular domain that contains four immunoglobulin-like domains (Ig 1–4) and six fibronectin III domains (FNIII 1–6). NeoΔCD is a truncated form of neogenin lacking the cytoplasmic domain. Its cDNA construct was generated by adding a stop codon immediately after glutamine 1135, the 9th amino acid after the predicted transmembrane domain of the neogenin sequence. Neo/Ecto is a soluble form of neogenin that lacks both the cytoplasmic domain and transmembrane domains. FNIII 1–6 is a soluble form of neogenin that contains FNIII 1–6 domains. FNIII 5–6 is a soluble form of neogenin that contains FNIII 5–6 domains. C, knockdown of endogenous neogenin blocks the secretion of MT2-cleaved HJV in HepG2-HJV cells. HepG2-HJV cells (HJV) were transfected with a control (Ctrl) or neogenin siRNA twice on day 1 and day 3. At 72 h after the second transfection, neogenin (Neo), HJV, and β-actin in total cell lysate (L) and HJV in 15% CM were immunodetected using the corresponding antibodies. HepG2-Ctrl cells (Ctrl) were included as a negative control for HJV. D, neogenin FNIII 5–6 blocks the release of MT2-cleaved HJV in HEK293 cells. HEK293-HJV cells were transiently transfected with either MT2 cDNA (HJV/MT2) or pcDNA3 empty vector, followed by incubation in the absence or presence of 40 nm FNIII 5–6 in DMEM, 5% FCS for about 16 h. MT2, HJV, and β-actin in total cell lysate (L) and HJV in 15% CM were immunodetected. HEK293-Ctrl cells (Ctrl) are included as a control for HJV. All images are from a single gel. E, neogenin FNIII 1–6 blocks HJV release from HepG2 cells. HepG2-HJV cells were incubated in the absence or presence of 0.25 or 0.5 μm neogenin FNIII 1–6 in MEM, 5% FCS for about 16 h. HJV and β-actin in total cell lysate (L) and HJV in 15% CM were immunodetected. F, Neo/Ecto blocks HJV release from HepG2 cells. The experiments were performed as described in E except that 0.5 μm Neo/Ecto was added. All experiments were repeated at least three times with consistent results.

FIGURE 2.

Neogenin interacts with both HJV and MT2. A, MT2 interacts with neogenin in HEK293 cells. HEK293 cells expressing MT2 alone, HJV alone, neogenin (Neo) alone, MT2/HJV, MT2/Neo, or MT2/NeoΔCD were metabolically labeled with 100 μCi of [³⁵S]Met/Cys/ml for 3 h. Immunoprecipitations (IP) were performed using rabbit anti-HJV antibody 18745 (H; generated against residues 1–401 of HJV), rabbit anti-neogenin 21567 antibody (N; generated using the neogenin FNIII 1–6 domains), or rabbit anti-MT2 antibody 23144 (M; generated against the stem region of MT2). Immunoprecipitated proteins were separated by SDS-PAGE, followed by soaking of the gel in Amplify (GE Healthcare) and drying of the gels prior to exposure to x-ray film. The preimmune serum for HJV (piH) and neogenin (piN) were included as negative controls. All images are from a single gel. B, MT2 interacts with neogenin in HepG2 cells. HepG2 cells stably transfected with neogenin (Neo) were transiently transfected with either pcDNA3 or MT2 cDNA (MT2), followed by a metabolic labeling with 100 μCi of [³⁵S]Met/Cys/ml, as described in A, and immunoprecipitation using anti-neogenin antibody. * indicates the MT2 band. C, HJV interacts with both neogenin and MT2. HEK293 cells expressing HJV/MT2 or HJV/Neo/MT2 were metabolically labeled and immunoprecipitated as described in A using anti-HJV (H), neogenin (N), or MT2 (M) antibody. Images are from a single gel. All experiments were repeated at least three times with similar results.

FIGURE 3.

MT2 does not cleave neogenin. A, neogenin undergoes an active secretion from HEK293 cells. HEK293 cells stably transfected with pcDNA3 empty vector (Ctrl), full-length neogenin cDNA (Neo), or NeoΔCD cDNA were incubated in DMEM, 1% FCS with or without 5 μm FCI for 16 h. Neogenin (Neo) and β-actin in total cell lysate (L) and neogenin in 15% of CM were immunodetected using the corresponding antibodies. B, MT2 does not cleave neogenin in HEK293 cells. HEK293-Ctrl or -HJV/Neo cells were transiently transfected with MT2 cDNA or pcDNA3 empty vector. At 48 h post-transfection, medium was changed to DMEM, 1% FCS containing 5 μm FCI or 100 μm leupeptin. After 16 h of incubation, neogenin (Neo), MT2, HJV, and β-actin in total cell lysate (L) and neogenin and HJV in 15% of CM were immunodetected using the corresponding antibodies. The top image was obtained by a sequential probing with anti-MT2, HJV, neogenin, and β-actin antibodies. One antibody was applied each time. The membrane was not stripped before the following antibody was applied. C, TAPI-2 inhibits the neogenin release in HEK293 cells. HEK293-HJV/Neo cells in 12-well plates were incubated in DMEM, 1% FCS with 0, 1, 5, 10, 25, 50, or 100 μm TAPI-2 (TAPI) for 16 h. Neogenin, HJV, and β-actin in total cell lysate (L) and neogenin and HJV in 15% of CM were immunodetected using the corresponding antibodies. D, TAPI-2 does not affect MT2 cleavage of HJV in HEK293 cells. HEK293-HJV/Neo cells were transiently transfected with MT2 cDNA. At 48 h post-transfection, medium was changed to DMEM, 1% FCS with or without 25 μm TAPI-2 (TAPI). After 16 h of incubation, neogenin, MT2, HJV, and β-actin in total cell lysate (L), and HJV in 15% of CM were immunodetected using the corresponding antibodies. E, TAPI-2 does not affect HJV release from HepG2 cells. HepG2-HJV cells were incubated in MEM, 1% FCS with 0, 25, or 50 μm of TAPI-2 (TAPI). After 16 h of incubation, HJV and β-actin in total cell lysate (L) and HJV in 15% of CM were immunodetected using the corresponding antibodies. All experiments were repeated for at least three times with consistent results.

FIGURE 4.

Depletion of neogenin results in the accumulation of both cellular and cell surface MT2. A, knockdown of endogenous neogenin increases the MT2 levels both in the cell extracts and on the plasma membrane. HepG2 cells stably expressing MT2 or HJV were transfected with a control or neogenin siRNA twice on day 1 and day 3. At about 72 h after the second transfection, cell surface proteins were biotinylated at 4 °C. The biotinylated proteins in the cell lysate were isolated using streptavidin-agarose beads. Neogenin, MT2, TfR1, HJV, and β-actin in about 10% of total cell lysate prior to pulldown (lysate input) and in the total eluates from streptavidin-agarose beads (biotinylation eluate) were immunoblotted (IB) using the corresponding antibodies. Two individual HepG2-MT2 clones were used for the analysis. Image in the 3rd panel was obtained by a sequential probing with anti-HJV and β-actin antibodies. One antibody was applied each time. The membrane was not stripped before the following antibody was applied. The experiments were repeated four times with consistent results. B, quantification of the immunodetection for MT2 in A. MT2 bands as showed in A were quantified using an Alexa Fluor 680 goat anti-rabbit secondary antibody and an Odyssey Infrared Imaging System (Licor). The intensities of MT2 bands in cell lysate were normalized to that of the corresponding β-actin. The relative levels of MT2 in control (Ctrl) siRNA-transfected cells were counted as 1. Pair and two-tailed t test was used to calculate the difference of relative MT2 levels between control and neogenin siRNA transfection. Results are from four individual experiments. C, neogenin mutant mice have an increased MT2 level. About 250 μg of liver membrane proteins from age- and gender-matched wild type (+/+), heterozygous (+/−), or homozygous (−/−) neogenin mutant mice was subjected to SDS-PAGE and immunodetection of neogenin (Neo), MT2, and β-actin. Liver membrane extracts from a pair of age- and gender-matched wild type (+/+) and Tmprss6-null (−/−) mice and cell lysates from HepG2-Ctrl and MT2 (M2) lysates were included as controls. * indicates the neogenin band. The results were repeated twice in two different sets of neogenin mutant mice with consistent results. D, Western blot analysis of neogenin and MT2 in HepG2-Ctrl (Ctrl) and HepG2-MT2 cells (MT2). Two MT2 images (short exposure and longer exposure) were illustrated to show the lack of detectable autocleavage products of MT2. The experiments were repeated four times with consistent results. n.s. denotes the nonspecific band. E, quantitative RT-PCR analysis of Tmprss6 mRNA expression in age and gender-matched wild type (Neo^+/+), heterozygous (Neo^+/−), or homozygous (Neo^−/−) neogenin mutant mice. There are five animals per group. The results are expressed as the amount of mRNA relative to β-actin in each sample. No significant difference was detected between groups.

FIGURE 5.

Disruption of neogenin-MT2 interaction slows down MT2 degradation. A, depletion of endogenous neogenin reduces MT2 degradation. HepG2-MT2 cells were transfected with a control (Ctrl) or neogenin siRNA twice on day 1 and day 3. At about 66 h after the second transfection, cells were incubated with 100 μg/ml cycloheximide (CHX) in complete medium for 0, 3, and 6 h. Cell lysates were then prepared and analyzed by immunoblotting using anti-neogenin (Neo), MT2, and β-actin (Actin) antibodies. HepG2 cells stably transfected with a pcDNA3.1 empty vector (Ctrl) were included as a negative control for MT2. n.s. denotes nonspecific band. B, quantification of the immunodetection for MT2 in A. MT2 bands as shown in A were quantified using an Alexa Fluor 680 goat anti-rabbit secondary antibody and an Odyssey Infrared Imaging System (Licor). The intensities of MT2 bands in cell lysate were normalized to that of the corresponding β-actin. The relative levels of MT2 at 3 and 6 h were then expressed as the percentage relative to 0 h for each group. Pair and two-tailed t tests were used to calculate the difference of relative MT2 levels between control and neogenin siRNA transfection. Error bars represent the standard deviation. Results are from four individual experiments. C, incubation with dynasore results in an accumulation of MT2 in HepG2-MT2 cells. HepG2-MT2 cells were incubated in complete medium with 160 μm dynasore (Sigma) in DMSO or an equal volume of DMSO (−) for 18 h. Cell lysates were prepared for immunodetection of MT2 and β-actin. The experiments were repeated three times with consistent results. D, incubation with soluble neogenin ectodomain results in a mild accumulation of MT2 in HepG2-MT2 cells. HepG2-MT2 cells were incubated with 40 nm neogenin FNIII 5–6 from two different preparations (FN5–6), 0.25 μm neogenin FNIII 1–6 (FN1–6), or 0.5 μm soluble neogenin ectodomain (Ecto) in DMEM, 5% FCS for 18 h. MT2 and β-actin in cell lysate were immunodetected. Two MT2 images (short exposure and longer exposure) were illustrated. The experiments were repeated three times with consistent results.

FIGURE 6.

MT2 and HJV traffic to the plasma membrane through distinct pathways. A, MT2 traffics to the plasma membrane through the traditional biosynthetic pathway. Cell surface proteins in HepG2-MT2 cells were biotinylated at 4 °C. The biotinylated proteins in the cell lysate were isolated using streptavidin-agarose beads. Bound proteins were eluted with NET-Triton, 1% β-mercaptoethanol, 0.5% SDS. The eluates were subjected to digestion with mock, Endo-H, or PNGase F, followed by immunodetection of neogenin (Neo), Na⁺/K⁺-ATPase α1 (a specific plasma membrane marker), MT2, and β-actin. All images are from a single gel. The experiment was repeated four times with consistent results. Ctrl, control. B, MT2-cleaved soluble HJV is resistant to Endo-H digestion in HEK293 cells. HEK293-HJV cells in a 12-well plate were transiently transfected with either pcDNA3 empty vector (HJV) or MT2 cDNA (HJV/MT2). At 48 h post-transfection, culture medium was changed to DMEM, 1% FCS. After 16 h of incubation, CM was collected, and cell lysate was prepared. One-third of cell lysate and ∼15% of CM were subjected to mock, Endo-H, and PNGase F digestion, followed by SDS-PAGE separation and immunodetection of MT2, HJV, and β-actin. The image is a representative of four independent experiments with consistent results. C, cell surface HJV has high mannose oligosaccharides, whereas MT2-cleaved cell surface HJV has complex oligosaccharides in HepG2 cells. Cell surface HJV in HepG2-HJV cells was biotinylated at 4 °C, followed by incubation at 37 °C for 4 h in complete medium. The total biotinylated HJV in cell lysate (Lysate/Pulldown) and the biotinylated HJV released into the medium (CM/Pulldown) were isolated using streptavidin-agarose beads. The eluates were digested with Endo-H and PNGase F. Endo-H and PNGase F digestion of one-third of input cell lysates (Lysate/Input) and one-third of cell lysate after streptavidin pulldown (Lysate/Post) are also included. Lysate from HepG2-Ctrl cells (Lysate/Input/Ctrl) or pulldown from the medium of HepG2-HJV cells without biotinylation (CM/Pulldown/HJV**) were used as negative controls. * indicates the MT2-cleaved HJV products in the CM. Experiments were repeated four times with consistent results.

FIGURE 7.

Models of HJV, MT2, and neogenin trafficking in hepatocytes. A, model of MT2 cleavage of HJV at the plasma membrane after retrograde transport. Nascent HJV traffics from ER to plasma membrane bypassing the Golgi compartment (1), and MT2 and neogenin follow the traditional biosynthetic pathway and undergo post-translational modifications in the TGN/Golgi compartment (2). Upon reaching the plasma membrane, HJV forms a complex with MT2 and neogenin, which triggers the internalization and retrograde trafficking of the complex into the TGN/Golgi compartment for post-translational modifications of HJV (3). Then the complex traffics back to the plasma membrane (4) to allow MT2 cleavage of HJV. The cleaved HJV is released from the cells (5). B, model of MT2 cleavage of HJV during retrograde transport. Nascent HJV, MT2, and neogenin traffic from ER to plasma membrane as described in A (1 and 2). Upon reaching the plasma membrane, HJV forms a complex with MT2 and neogenin, which triggers the internalization and retrograde trafficking of the complex into the TGN/Golgi compartment for cleavage (3). The cleaved HJV by MT2 in this compartment is rapidly secreted from the cells (4).