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Elemental iron is required for a variety of normal cellular functions and vital for proper growth and development. However, natural iron is quite insoluble and excess iron is harmful, since it can catalyze the formation of potentially damaging reactive oxygen species. Humans also have very limited capacity to excrete iron. Therefore, cells have developed mechanisms to improve solubility of iron and to control intracellular iron levels at the point of absorption in the intestine and other tissue. The major pool of body iron (~85%; 40-50 mg/kg) is found in circulating hemoglobin and muscle myoglobin. Iron absorption occurs primarily in the intestine (duodenum) and inversely related to body iron reserve. Several proteins including Ferritin, transferrin (Tf), transferrin receptors (TfRs), and iron regulatory proteins (IRPs), iron transporter (NRMAP2/DMT1/DCT1) etc play a key role in iron metabolism. Some genes involved in iron-metabolism are associated with genetic disorders such as Friedreich's Ataxia (Frataxin), genetic hemochromatosis (HFE), and Sex-linked anemia (Hephaestin).

Iron regulatory proteins (IRP-1 and IRP-2) are cytoplasmic mRNA-binding proteins that control intracellular iron levels by regulating the translation of ferritin, Tfrs, and other proteins congaing iron-responsive element (IRE) located at the 5'-UTR. IRP binds to IREs with high affinity in situation of iron starvation. Binding of IRP to IRE of ferritin represses its transcription. However, when cells are iron replete, IRPs lose their capacity to bind IREs, allowing efficient translation of ferritin and reducing Tfr mRNA half-life. Although IRP1 and IRP-2 share significant protein sequence homology, they differ in tissue distribution and mode of regulation. IRP-1, also known as IRE-BP-1/aconitase/citrate hydrolyase, (mouse/rat/human 889 aa, chromosome 9, ~98 kDa) also posses aconitase activity and highly homologous with mitochondrial aconitase. IRP-1 is more abundant than IRP-2 and it is widely expressed. IRP-2/IRE-BP2 (rat/human 963 aa) is relatively less abundant than IRP-1 and lacks aconitase activity. It has a unique 73-aa insertion in the N-terminus. In the presence of high iron levels, IRP-2 is rapidly targeted to proteasome-mediated degradation. Although, both IRPs bind the consensus IREs, it is also shown that IRP-2 can recognize an exclusive IRE subset.

Hereditary hemochromatosis (HHC) is most common autosomal recessive disorder characterized by defective intestinal iron absorption, which lead to iron-overload in many tissues and toxic effects. The candidate gene for HHC encodes the HFE protein (formerly called HLA-H) resembling the major histocompatibility complex MHC class-1 molecule. HFE protein (mouse 359 aa, rat 360 aa, human 348 aa, ~48 kDa) is type I membrane protein. It is found in all tissues except brain. HFE protein binds too Tfr and reduces its affinity for iron-loaded Tf. The HFE Cys282-Tyr (C282Y) is homozygous in 83-100% of HHC subjects in the US and North Europe, and Australia. The C282Y mutation results in the loss of a structural disulfide bond in the alpha-3 domain of the protein, which prevents association with beta-2 microglobulin and proper presentation to the surface. Defects in HFE are also a cause of porphyria cutanes tarda (PCT), a disorder characterized by light-sensitive dermatitis and high levels of uroporphyrin in urine.

Hephaestin (Hp) gene was originally cloned as the gene defective in sex-linked anemia (sla) that is characterized by moderate to severe microcytic hypochronic anemia. Hp protein (mouse/rat 1157 aa, human 1158 aa, ~155 kDa) encodes a single-membrane spanning domain protein with extensive homology (50-60%) with copper containing serum ferrooxidase ceruloplasmin that is involved in release of iron from various tissues. It is mostly expressed throughout the small intestine and colon. It is localized in mature villus enterocytes with little or no expression n the crypts. Hp levels are not affected by iron status.
 

Hepcidin (hepatic bactericidal protein) or LEAP (liver expressed antimicrobial peptide) is small, cysteine-rich peptide, antimicrobial peptide similar to defensins and thionins. Hepcidin (unprocessed, proprotein in mouse 83 aa, rat/human 84 aa) are almost exclusively produced in liver. Human hepcidin is produced from 84-aa precursor, including a putative 24-aa signal peptide. The secreted form of hepcidin in blood and urine is consists of C-terminal 20, 22 or 25-aa residues. The link between hepcidins and iron metabolism is that hepcidin expression is abolished in mice exhibiting iron-overload due to the targeted disruption of USF2 (upstream stimulatory factor 2) gene resembling the situation in hfe-/- mice. The human gene is located at chromosome 19, in close proximity with Usf2 gene. Hepcidin levels are increased in iron loading and in beta-2 microglobulin knockout mice. Hepcidins are devoid of IRE. Like other antimicrobial peptides, hepcidin is upregulated by lipopolysaccharides (LPS).

Friedrich ataxia (FA), a degenerative disease characterized by progressive ataxia and hypertrophic cardiomyopathy leading to premature death, is cause by a deficiency in frataxin (Fxn). Fxn (mouse 207 aa, human 210 aa major isoform A; alternatively transcribed minor forms A1, 196 aa, and 171-aa isoform B) is a nuclear-encoded and highly conserved mitochondrial membrane protein. Fxn is expressed in tissues with highly metabolic activity, such as heart, liver, and brown fat. In FA, Fxn levels are greatly reduced due to inhibition of its transcription by expansion of an intronic GAA repeat. Defects in yeast Fxn produce mitochondria iron-overload. It is suggested that Fxn could play a role in mitochondrial iron storage.

Beta-2-microglobulin (B2M) is found in the serum of normal individuals and in the urine in elevated amounts in patients with Wilson disease, cadmium poisoning, and other conditions leading to renal tubular dysfunction. Like immunoglobulins, prealbumin, and the beta protein found in the amyloid of Alzheimer disease, B2M has a predominantly beta-pleated sheet structure that may adopt the fibrillar configuration of amyloid in certain pathologic states. The protein is a single polypeptide chain of molecular weight 11,600 (human 119 aa, chromosome 15). B2M associates with heavy hcain of class I MHC antigens on the cell surface. A transient complex of MHC-heavy chain and B2M is is known to be assembled into the TAP molecule involving interation with a number of chaperones. Binding of the processed peptide releases the class I-B2M complex to the cell surface. Absence of binding leads to degradation in the proteasome. Progressive hepatic iron overload, indistinguishable from that observed in HFE, was found only in mice homozygous for the mutated B2M gene

Ceruloplasmin (Cp) is a plasma metalloprotein synthesized and secreted by the liver. Cp (also known as ferroxidase; iron (II):oxygen oxidoreductase, EC 1.16.3.1) is a blue alpha-2-glycoprotein that binds 90 to 95% of plasma copper and has 6 or 7 cupric ions per molecule. It is involved in peroxidation of Fe(II) transferrin to form Fe(III) transferrin. Human Cp is a single polypeptide chain (1065 aa, 135 kDa; chromosome 8q21.13-q23.1) structurally similar to factors V and VIII. Cp also has abundant expression in specific populations of glial cells within the brain. Hereditary Cp deficiency have profound iron accumulation in most tissues. Cp -/- mice showed no abnormalities in cellular iron uptake but a striking impairment in the movement of iron out of reticuloendothelial cells and hepatocytes. Cp may play an essential physiologic role in determining the rate of iron efflux from cells with mobilizable iron stores.

 

M= Mouse; R=Rat; H=Human; Ha=Hamster; Rb=Rabbit; B=Bovine; CT= near C-terminus; NT=near N-terminus; Internal=Middle of protein.

** Expected antibody crossreactivity information is mostly based upon high (>70%) sequence conservation of antigenic/control peptides in various species. When antibody crossreactivity has actually been experimentally confirmed in various species, it will be mentioned in the appropriate data sheets.

"Neat Antisera" are the unpurified antiserum and it is suitable for ELISA and Western.
"Affinity pure" IgG may be more suitable for immunohistochemical (IHC) applications and to reduce background in most immunological applications including ELISA and Western.
"Control peptides" can not be used for Western as they are very short peptides. They are intended for ELISA or antibody blocking studies to establish antibody specificity.

 

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