Collection 3: serum from a healthy individual. lower levels of circulating hemojuvelin than those in healthy patients. The enzyme-linked immunosorbent assay also showed that soluble p53 and MDM2 proteins-interaction-inhibitor chiral hemojuvelin levels were significantly higher in patients with anemia of chronic disease than in control individuals. == Conclusions == This enzyme-linked immunosorbent assay has a good specificity and sensitivity for the quantification CR2 of soluble hemojuvelin in human serum and could be a useful aid to understanding the physiological role of this protein. Keywords:iron metabolism, soluble hemojuvelin, ELISA, rhRGM-c, anemia, serum == Introduction == Hemojuvelin is usually a member of the repulsive guidance molecule (RGM) family, a family of glycosylphos-phatidylinositol-anchored neuronal and muscle mass membrane glycoproteins. Hemojuvelin (also called RGM-c) is mainly expressed in skeletal muscle mass, p53 and MDM2 proteins-interaction-inhibitor chiral heart and liver, and plays a major role in regulating hepcidin expression, the unfavorable regulator of intestinal iron absorption and macrophage heme iron recycling. Patients withHJVmutations develop early-onset, severe iron overload, called juvenile hemochromatosis, due to a complete lack of hepcidin expression.1Hemojuvelin can be expressed both as a membrane-bound form and as a soluble form which have opposite effects on hepcidin gene expression. Cell-associated hemojuvelin acts as a co-receptor of bone morphogenic proteins to enhance hepcidin expression through the Smad pathway.2,3In contrast, soluble hemojuvelin, by binding bone morphogenic proteins, acts as a competitive antagonist of membrane-bound hemojuvelin, leading to decreased hepcidin expression.24Thus, chronic soluble hemojuvelin injection in mice causes iron overload.5 Several isoforms of the hemojuvelin protein are generated by multiple proteolytic cleavages (Determine 1). Membrane-bound hemojuvelin protein can be represented by both single- and two-chain species. The two-chain species is produced from the single chain by an intra-molecular cleavage and the two chains can remain bound to the plasma membrane through the formation of a p53 and MDM2 proteins-interaction-inhibitor chiral disulfide bridge.6,7Soluble hemojuvelin can be produced by cleavage, mediated by a furin-like protease, at the C-terminus of the protein occurring in the endoplasmic reticulum.8,9This cleavage leads to the release of two soluble components (42 and 33 kDa) into extracellular fluids or into blood. These two fragments are probably produced from single- and two-chain species of hemojuvelin, as suggested by the authors. Nevertheless, the fact that this 42 and 33 kDa fragments originating from furin cleavage are released into the blood stream has not been confirmed. The furin-mediated release of soluble hemojuvelin can be enhanced by hypoxia and iron deficiency to reduce rapidly the amount of membrane-bound hemojuvelin and to inhibit hepcidin production. Recently, it has been exhibited that membrane-bound hemojuvelin, the glycosylphosphatidylinositol-anchored form, is usually cleaved by matriptase 2, a transmembrane serine protease encoded by theTMPRSS6gene, mostly expressed in the liver. Co-transfection experiments with vectors expressing hemojuvelin and matriptase 2 have shown that this serine protease can cleave the membrane-bound hemojuvelin to generate several smaller fragments (2535 kDa)10but these fragments have not yet been recognized in serum samples. The physiological role of these soluble hemojuvelin fragments and their presence in human serum remain to be investigated. However, mutational inactivation of matriptase 2 causes iron-refractory iron-deficient anemia in mice11and humans,10,1214by inappropriately high levels of hepcidin expression. In summary, circulating hemojuvelin is usually represented by several soluble forms that can be produced by the action of at least two proteolytic enzymes, furin p53 and MDM2 proteins-interaction-inhibitor chiral and matriptase 2. == Determine 1. == Schematic representation of hemojuvelin (HJV) protein on the cell surface, and of the secreted form. Membrane HJV (mHJV) and soluble HJV (sHJV) appear to be represented by both single- and two-chain species that can remain bound through the formation of disulfide bridge. The longest isoform is a 426-amino-acid protein. Locations of asparagine-linked glycosylation sites are indicated p53 and MDM2 proteins-interaction-inhibitor chiral by asterisks, while the polybasic RNRR site (a site of intra-molecular proteolytic cleavage to generate two-chains) is usually indicated by a squiggle. Membrane HJV is the glycosylphosphatidylinositol anchored form. Soluble HJV (42 kDa) and its shorter fragment of 33 kDa are found in extracellular fluids and in blood,4,7,9potentially through the action of a furin-like protease in the endoplasmic reticulum. Other shorter soluble forms of HJV (2535 kDa fragments) can be released from membrane-bound HJV by the action of the trans-membrane.