Neogenin Interacts with Matriptase-2 to Facilitate Hemojuvelin Cleavage

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.

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Processing of hemojuvelin requires retrograde trafficking to the Golgi in HepG2 cells

Blood ◽

10.1182/blood-2008-08-174565 ◽

2009 ◽

Vol 113 (8) ◽

pp. 1786-1793 ◽

Cited By ~ 26

Author(s):

Julia E. Maxson ◽

Caroline A. Enns ◽

An-Sheng Zhang

Keyword(s):

Plasma Membrane ◽

Bone Morphogenetic Proteins ◽

Iron Homeostasis ◽

Soluble Form ◽

Protein Signaling ◽

Bone Morphogenetic Protein Signaling ◽

Glycosylphosphatidylinositol Anchor ◽

Retrograde Trafficking ◽

Hepcidin Expression ◽

Morphogenetic Protein

Abstract Hemojuvelin (HJV) was recently identified as a critical regulator of iron homeostasis. It is either associated with cell membranes through a glycosylphosphatidylinositol anchor or released as a soluble form. Membrane-anchored HJV acts as a coreceptor for bone morphogenetic proteins and activates the transcription of hepcidin, a hormone that regulates iron efflux from cells. Soluble HJV antagonizes bone morphogenetic protein signaling and suppresses hepcidin expression. In this study, we examined the trafficking and processing of HJV. Cellular HJV reached the plasma membrane without obtaining complex oligosaccharides, indicating that HJV avoided Golgi processing. Secreted HJV, in contrast, has complex oligosaccharides and can be derived from HJV with high-mannose oligosaccharides at the plasma membrane. Our results support a model in which retrograde trafficking of HJV before cleavage is the predominant processing pathway. Release of HJV requires it to bind to the transmembrane receptor neogenin. Neogenin does not, however, play a role in HJV trafficking to the cell surface, suggesting that it could be involved either in retrograde trafficking of HJV or in cleavage leading to HJV release.

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Processing of Hemojuvelin Requires Retrograde Trafficking to the Golgi in HepG2 Cells.

Blood ◽

10.1182/blood.v112.11.3845.3845 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3845-3845

Author(s):

An-Sheng Zhang ◽

Julia Julia Maxson ◽

Caroline A Enns

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Hepg2 Cells ◽

Iron Homeostasis ◽

Bmp Signaling ◽

Soluble Form ◽

Gpi Anchor ◽

Retrograde Trafficking ◽

Hepcidin Expression ◽

Bone Morphogenic Proteins

Abstract Hemojuvelin (HJV) was recently identified as a critical regulator of iron homeostasis. It is either associated with the cells through a GPI-anchor or released as a soluble form. The cellular form acts as a co-receptor for bone morphogenic proteins (BMPs) and activates the transcription of hepcidin, a hormone that regulates iron efflux from cells. Soluble HJV antagonizes BMP signaling and suppresses hepcidin expression. Secretion of HJV requires binding to the transmembrane receptor neogenin. In this study we examined the trafficking and processing of HJV. Cellular HJV reached the plasma membrane without obtaining complex oligosaccharides, indicating that HJV avoided Golgi processing. Secreted HJV, in contrast, had complex oligosaccharides and could be derived from the pool of HJV at the plasma membrane. Neogenin did not play a role in HJV trafficking to the cell surface but was necessary for secretion of HJV, suggesting that it could be involved in either retrograde trafficking of HJV or in cleavage leading to secretion.

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Hepatocyte Neogenin Is Required for Hemojuvelin-Mediated Hepcidin Expression and Iron Homeostasis in Mice

Blood ◽

10.1182/blood.2020009485 ◽

2021 ◽

Author(s):

Caroline A. Enns ◽

Shall Jue ◽

An-Sheng Zhang

Keyword(s):

Plasma Membrane ◽

Iron Overload ◽

Serum Iron ◽

Iron Homeostasis ◽

Transmembrane Protein ◽

High Serum ◽

Bmp Signaling ◽

Hepcidin Expression ◽

Iron Utilization

Neogenin (NEO1) is a ubiquitously expressed multi-functional transmembrane protein. It interacts with hemojuvelin (HJV), a BMP co-receptor that plays a pivotal role in hepatic hepcidin expression. Earlier studies suggest that the function of HJV relies on its interaction with NEO1. However, the role of NEO1 in iron homeostasis remains controversial because of the lack of an appropriate animal model. Here, we generated a hepatocyte-specific Neo1 knockout (Neo1fl/fl;Alb-Cre+) mouse model that circumvented the developmental and lethality issues of the global Neo1 mutant. Results show that ablation of hepatocyte Neo1 decreased hepcidin expression and caused iron overload. This iron overload did not result from altered iron utilization by erythropoiesis. Replacement studies revealed that expression of the Neo1L1046E mutant that does not interact with Hjv, was unable to correct the decreased hepcidin expression and high serum iron in Neo1fl/fl;Alb-Cre+ mice. In Hjv-/- mice, expression of HjvA183R mutant that has reduced interaction with Neo1, also displayed a blunted induction of hepcidin expression. These observations indicate that Neo1-Hjv interaction is essential for hepcidin expression. Further analyses suggest that the Hjv binding triggered the cleavage of the Neo1 cytoplasmic domain by a protease, which resulted in accumulation of truncated Neo1 on the plasma membrane. Additional studies did not support that Neo1 functions by inhibiting Hjv shedding as previously proposed. Together, our data favor a model in which Neo1 interaction with Hjv leads to accumulation of cleaved Neo1 on the plasma membrane, where Neo1 acts as a scaffold to induce the Bmp signaling and hepcidin expression.

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Role of glycosylation in transport and enzymic activity of neutral endopeptidase-24.11

Biochemical Journal ◽

10.1042/bj3020451 ◽

1994 ◽

Vol 302 (2) ◽

pp. 451-454 ◽

Cited By ~ 27

Author(s):

M H Lafrance ◽

C Vézina ◽

Q Wang ◽

G Boileau ◽

P Crine ◽

...

Keyword(s):

Enzyme Activity ◽

Cell Surface ◽

Intracellular Transport ◽

Neutral Endopeptidase ◽

Soluble Form ◽

Cell Extracts ◽

Glycosylation Sites ◽

Sugar Addition ◽

Membrane Bound

Neutral endopeptidase (NEP, EC 3.4.24.11) is a major ectoenzyme of the brush-border membrane. The ectodomain of NEP contains five putative N-glycosylation sites. In order to determine the role of the addition of sugar moieties on the activity and intracellular transport of NEP, we have used site-directed mutagenesis to remove all or some of the five potential sites of sugar addition in membrane-bound and secreted forms of the enzyme. Expression of NEP glycosylation mutants in COS-1 cells showed that all five sites are used for sugar addition. Immunoblotting of NEP in COS-1 cell extracts or culture media indicated that total expression of normal membrane-bound NEP was not affected by mutations at glycosylation sites, whereas this expression level appeared to be strictly dependent on the number of glycosylation sites retained on the soluble form. The transport to the cell surface was also reduced by decreased glycosylation, but again the phenomenon appeared more drastic in the case of the soluble form than for the membrane-bound enzyme. Enzyme activity was decreased by deglycosylation. However, the presence of either of two crucial sites (sites 1 and 5; numbered from the N-terminus of the protein) was sufficient to recover close-to-normal enzymic activities. Transport to the cell surface and enzyme activity of NEP are thus both dependent on sugar residues, probably through different conformational constraints. These constraints seem to be local for enzyme activity but more global for transport to the cell surface.

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Membrane-bound Steel factor induces more persistent tyrosine kinase activation and longer life span of c-kit gene-encoded protein than its soluble form

Blood ◽

10.1182/blood.v85.3.641.bloodjournal853641 ◽

1995 ◽

Vol 85 (3) ◽

pp. 641-649 ◽

Cited By ~ 195

Author(s):

K Miyazawa ◽

DA Williams ◽

A Gotoh ◽

J Nishimaki ◽

HE Broxmeyer ◽

...

Keyword(s):

Cell Surface ◽

Tyrosine Kinase ◽

Life Span ◽

Protein Degradation ◽

Cell Line ◽

Soluble Form ◽

Steel Factor ◽

Degradation Rates ◽

Membrane Bound ◽

Kinetics Of

Alternative splicing of exon 6 results in the production of two isoforms of Steel factor (SLF): the membrane-bound and soluble forms. To investigate differences in the kinetics of c-kit tyrosine kinase activated by these two isoforms, we used a stromal cell line (SI/SI4) established from SI/SI homozygous murine embryo fetal liver and its stable transfectants containing either hSCF248 cDNA (including exon 6; secreted form) or hSCF220 cDNA (lacking exon 6; membrane-bound form) as the source of each isoform. Interaction of factor dependent myeloid cell line MO7e with stromal cells producing either isoform resulted in activated c-kit tyrosine kinase and induction of the same series of tyrosine phosphorylated cellular proteins in MO7e cells. However, SI4- h220 (membrane-bound form) induced more persistent activation of c-kit kinase than SI4-h248 (soluble form) did. Flow cytometric analysis and pulse-chase studies using [35S]methionine showed that SI4-h248 induced rapid downmodulation of cell-surface c-kit expression and its protein degradation in MO7e cells, whereas SI4-h220 induced more prolonged life span of c-kit protein. Addition of soluble recombinant human SLF to SI4- h220 cultures enhanced reduction of cell-surface c-kit expression and its protein degradation. Because the kinetics of c-kit inactivation strikingly fits with the protein degradation rates of c-kit under the conditions described above, rapid proteolysis of c-kit protein induced by soluble SLF stimulation may function as a “turn-off switch” for activated c-kit kinase.

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Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV

Biochemical Journal ◽

10.1042/bj2100389 ◽

1983 ◽

Vol 210 (2) ◽

pp. 389-393 ◽

Cited By ~ 23

Author(s):

E M Danielsen ◽

H Sjöström ◽

O Norén

Keyword(s):

Organ Culture ◽

Membrane Fraction ◽

Bound State ◽

Dipeptidyl Peptidase ◽

Dipeptidyl Peptidase Iv ◽

Soluble Form ◽

Membrane Bound ◽

Aminopeptidase A ◽

High Mannose ◽

Small Intestinal

The biogenesis of three intestinal microvillar enzymes, maltase-glucoamylase (EC 3.2.1.20), aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. The earliest detectable forms of the enzymes were polypeptides of Mr 225000, 140000 and 115000 respectively. These were found to represent the enzymes in a ‘high-mannose’ state of glycosylation, as judged by their susceptibility to treatment with endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96). After about 40-60 min of chase, maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV were further modified to yield the mature polypeptides of Mr 245000, 170000 and 137000 respectively, which were expressed at the microvillar membrane after 60-90 min of chase. The fact that the enzymes before reaching the microvillar membrane were found in a Ca2+-precipitated membrane fraction (intracellular and basolateral membranes), but not in soluble form, indicates that during biogenesis maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV are transported and assembled in a membrane-bound state.

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Inhibition of Hamp1 Expression by TMPRSS6, GDF15, PIAS4 and SMAD6 Utilize Several Target Sites in the Hamp1 Promoter

Blood ◽

10.1182/blood.v112.11.3838.3838 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3838-3838

Author(s):

Jaroslav Truksa ◽

Pauline Lee ◽

Ernest Beutler

Keyword(s):

Transcription Factors ◽

Bone Morphogenetic Proteins ◽

Iron Homeostasis ◽

Negative Regulator ◽

Luciferase Reporter ◽

Reporter Expression ◽

Hepcidin Expression ◽

Double Deletion ◽

Bmp Pathway ◽

Transmembrane Serine Protease

Abstract Hepcidin, the key regulator of iron homeostasis, is up-regulated by iron excess, bone morphogenetic proteins (BMPs) and inflammatory cytokines and down-regulated by hypoxia and anemia. Known positive regulators at the level of transcription factors include SMAD1/4, STAT3 and CEBPα. In this study, we focused on negative regulators of hepcidin regulation: Matriptase II/TMPRSS6 (Transmembrane serine protease 6, a recently identified negative regulator in which disruption leads to anemia in mice as well in humans); Protein inhibitor of activated STATs no. 4 (PIAS4); Growth and differentiation factor 15 (GDF15, a potential erythroid negative regulator); and SMAD6 (Mothers against decapentaplegic homolog 6, an inhibitory SMAD blocking the SMAD/BMP pathway). All tested inhibitors significantly decreased expression of the luciferase reporter under the control of 2.5 Kb murine Hamp1 promoter with GDF15 and PIAS4 Hamp1 specific since none of the inhibitors were able to reduce expression of the luciferase reporter under the control of the murine Hamp2 promoter. Inhibition of the luciferase reporter under the control of the 2.5 Kb murine Hamp1 promoter by SMAD6, unlike TMPRSS6, PIAS4 and GDF15, did not require liver specific transcription factors since the inhibition could also be observed in transfected HEK293T cells. GDF15, PIAS4, TMPRSS6 and SMAD6 all reduced basal level expression of the luciferase reporter under the control of the 2.5 Kb murine Hamp1 promoter as well as the total level of reporter expression induced by IL-6 and BMP-4. Nevertheless, GDF15 did not affect responsiveness (fold induction) to IL-6 and BMP-4. PIAS4 and TMPRSS6 inhibited responsiveness to IL-6 but had little effect on responsiveness to BMP-4. In contrast, SMAD6 did not affect responsiveness to IL-6 but reduced responsiveness to BMP-4. Deletion of the −140 bp −260 bp region of the murine Hamp1 or double deletion of the BMP-RE1 and BMP-RE2 motifs severely reduced the ability of all inhibitors to reduce reporter expression. Deletion of the STAT site abrogated PIAS4 inhibition while deletion of either BMP-RE1 or BMP-RE2 motifs alone partially reduced inhibition by TMPRSS6 and SMAD6. We conclude that there are several independent pathways that inhibit hepcidin expression.

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Rat ovarian lutropin receptor is a transmembrane protein. Evidence for an Mr-20,000 cytoplasmic domain

Biochemical Journal ◽

10.1042/bj2390083 ◽

1986 ◽

Vol 239 (1) ◽

pp. 83-87 ◽

Cited By ~ 7

Author(s):

K P Keinänen ◽

H J Rajaniemi

Keyword(s):

Plasma Membrane ◽

Transmembrane Protein ◽

Membrane Vesicles ◽

Cytoplasmic Domain ◽

Proteolytic Digestion ◽

Plasma Membrane Vesicles ◽

Human Choriogonadotropin ◽

Lutropin Receptor ◽

Membrane Bound ◽

Inside Out

Membrane topography of the rat ovarian lutropin receptor was studied by two different approaches. Ovarian membrane preparation, labelled with 125I-labelled human choriogonadotropin in vivo, was subjected to extensive chymotryptic digestion. The soluble and membrane-bound radioactive complexes were cross-linked with glutaraldehyde, and analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. Chymotrypsin solubilized 70-75% of the radioactivity as Mr-96,000, Mr-74,000 and Mr-61,000 complexes, and decreased the size of the membrane-bound 125I-labelled human choriogonadotropin-receptor complex from Mr 130,000 to Mr 110,000. The Mr-110,000 complex was not observed when 0.1% Triton X-100 was present in the proteolytic digestion. Enrichment of inside-out-oriented plasma-membrane vesicles by concanavalin A affinity chromatography increased by 70% the fraction of radioactivity that remained in the membrane fraction after chymotrypsin treatment. Chymotrypsin also diminished the size of the membrane-bound unoccupied receptor from Mr 90,000 to Mr 70,000, as detected by ligand (125I-labelled human choriogonadotropin) blotting. These results suggest that the lutropin receptor is a transmembrane protein with a cytoplasmic domain of Mr 20,000 that is sensitive to proteolytic digestion in the inside-out-oriented plasma-membrane vesicles.

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Human soluble phospholipase A2 receptor is an inhibitor of the integrin-mediated cell migratory response to collagen-I

AJP Cell Physiology ◽

10.1152/ajpcell.00239.2017 ◽

2018 ◽

Vol 315 (3) ◽

pp. C398-C408 ◽

Cited By ~ 3

Author(s):

Kazunori Watanabe ◽

Kazuhiro Watanabe ◽

Yosuke Watanabe ◽

Daisuke Fujioka ◽

Takamitsu Nakamura ◽

...

Keyword(s):

Cell Surface ◽

Human Plasma ◽

Collagen Iv ◽

Collagen I ◽

Hek293 Cells ◽

Soluble Form ◽

Integrin Β1 ◽

Migratory Response ◽

Cell Responses ◽

Membrane Bound

Murine membrane-bound phospholipase A2 receptor 1 (PLA2R) is shed and released into plasma in a soluble form that retains all of the extracellular domains. Relatively little is known about human PLA2R. This study examined whether human soluble PLA2R has biological functions and whether soluble PLA2R exists in human plasma. Here, we showed that human recombinant soluble PLA2R (rsPLA2R) bound to collagen-I and inhibited interaction of collagen-I with the extracellular domain of integrin β1 on the cell surface of human embryonic kidney 293 (HEK293) cells. As a result, rsPLA2R suppressed integrin β1-mediated migratory responses of HEK293 cells to collagen-I in Boyden chamber experiments. Inhibition of phosphorylation of FAK Tyr397 was also observed. Similar results were obtained with experiments using soluble PLA2R released from HEK293 cells transfected with a construct encoding human soluble PLA2R. rsPLA2R lacking the fibronectin-like type II (FNII) domain had no inhibitory effects on cell responses to collagen-I, suggesting an important role of the FNII domain in the interaction of rsPLA2R with collagen-I. In addition, rsPLA2R suppressed the migratory response to collagen-IV and binding of collagen-IV to the cell surface of human podocytes that endogenously express membrane-bound, full-length PLA2R. Immunoprecipitation and Western blotting showed the existence of immunoreactive PLA2R in human plasma. In conclusion, human recombinant soluble PLA2R inhibits integrin β1-mediated cell responses to collagens. Further studies are warranted to elucidate whether immunoreactive PLA2R in human plasma has the same properties as rsPLA2R.

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Direct stimulation of cells expressing receptors for macrophage colony- stimulating factor (CSF-1) by a plasma membrane-bound precursor of human CSF-1

Blood ◽

10.1182/blood.v76.7.1308.bloodjournal7671308 ◽

1990 ◽

Vol 76 (7) ◽

pp. 1308-1314 ◽

Cited By ~ 5

Author(s):

J Stein ◽

GV Borzillo ◽

CW Rettenmier

Keyword(s):

Plasma Membrane ◽

Growth Factor ◽

Cell Surface ◽

Mononuclear Phagocytes ◽

Biologically Active ◽

Colony Stimulating Factor ◽

Membrane Bound ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

Secreted forms of macrophage colony-stimulating factor (M-CSF or CSF-1) are generated by proteolytic cleavage of membrane-bound glycoprotein precursors. Alternatively spliced transcripts of the human CSF-1 gene encode at least two different transmembrane precursors that are differentially processed in mammalian expression systems. The larger precursor rapidly undergoes proteolysis to yield the secreted growth factor and does not give rise to forms of CSF-1 detected on the cell surface. By contrast, the smaller human CSF-1 precursor is stably expressed on the plasma membrane where it is inefficiently cleaved to release a soluble molecule. To determine whether the smaller precursor is biologically active on the cell surface, mouse NIH-3T3 fibroblasts expressing the different forms of human CSF-1 were killed by chemical fixation and tested for their ability to support the proliferation of cells that require this growth factor. Only fixed cells expressing human CSF-1 precursors on their surface stimulated the growth in vitro of a murine macrophage cell line or normal mouse bone marrow-derived mononuclear phagocytes. The ability of these nonviable fibroblasts to induce the proliferation of CSF-1-dependent cells was not mediated by release of soluble growth factor, required direct contact with the target cells, and was blocked by neutralizing antiserum to CSF-1. These results demonstrate that the cell surface form of the human CSF-1 precursor is biologically active and indicate that plasma membrane- bound growth factors can functionally interact with receptor-bearing targets by direct cell-cell contact.

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