Haem transport to the liver by haemopexin. Receptor-mediated uptake with recycling of the protein

Rat [(59)Fe]haem-(125)I-labelled haemopexin complexes (700pmol/rat) associate rapidly and exclusively with the liver after intravenous injection into anaesthetized rats. The two isotopes exhibit different patterns of accumulation. Liver (125)I-labelled haemopexin is maximum 10min after injection (20+/-4.9pmol/g of liver) and then declines by 2h to the low values (about 3pmol/g of liver) seen after injection of the apoprotein. In contrast, [(59)Fe]haem accumulates in the liver for at least 2h. Haemopexin undergoes no extensive proteolysis during 2h of haem transport as shown by precipitation with acid (98%) and specific antiserum (92%) and by electrophoresis. Moreover, only 1-2% of the dose is located in extrahepatic tissues, and there is no significant urinary excretion of either (125)I or (59)Fe. Hepatic uptake at 10min is saturable, reaching 200pmol of haemopexin/g of liver and 350pmol of haem/g of liver at a dose of 9nmol/rat, whereas uptake of the apoprotein is 3-5% of the dose. This suggests that the interaction of haem-haemopexin with the liver is a specific receptor-mediated process. The complex probably interacts via the protein moiety, since the haem analogues mesohaem and deuterohaem do not affect association of the protein with the liver but the species of haemopexin does. Increasing amounts of protein are associated with the liver 5min after injection in the order: human>rabbit>rat, and haem uptake is consistently increased. For both rat and rabbit haemopexin saturation is reached at the same concentration of protein, i.e. 180-200pmol/g of liver, indicating that the different protein species bind to a common receptor. We propose that haemopexin transports haem to the liver by a specific receptor-mediated process and then returns to the circulation.     

Expression of the haemopexin-transport system in cultured mouse hepatoma cells. Links between haemopexin and iron metabolism.

Minimal deviation hepatoma (Hepa) cells, from the mouse hepatoma B7756, synthesize and secrete haemopexin and express both the haemopexin receptor and the membrane haem-binding protein (MHBP) associated with the receptor, making this cell line the first available for detailed study of both haemopexin metabolism and hepatic transport. The 17.5 kDa MHBP was detected in Triton X-100 extracts of Hepa cells by immunoblotting with goat anti-rabbit MHBP. Scatchard-type analysis of haem-125I-haemopexin binding at 4 degrees C revealed 35,000 receptors per cell of high affinity (Kd 17 nM). Haemopexin-mediated haem transport at 37 degrees C is saturable, having an apparent Km of 160 nM and a Vmax. of 7.5 pmol of haem/10(6) cells per h during exponential growth. Haem-transport capacity is highest in the period just before the cells enter their exponential phase of growth and slowest in stationary phase. Interestingly, haem-haemopexin serves as effectively as iron-transferrin as the sole source of iron for cell growth by Hepa cells. Furthermore, depriving Hepa cells of iron by treatment with desferrioxamine (DF) increases the number of cell-surface haemopexin receptors to 65,000 per cell and consequently increases haemopexin-mediated haem transport. The effects of DF do not appear to require protein synthesis since they are not prevented by cycloheximide. Treatment of Hepa cells with hydroxyurea, an inhibitor of the iron-requiring enzyme ribonucleotide reductase that is obligatory for DNA synthesis, enhanced haemopexin-mediated haem transport. Thus, these studies provide the first evidence for regulation of haem transport by the iron status of cells and suggest a linkage between haemopexin, iron homeostasis and cell growth.     

Emerging strategies in microbial haem capture

Gram-negative pathogenic bacteria have evolved novel strategies to obtain iron from host haem-sequestering proteins. These include the production of specific outer membrane receptors that bind directly to host haem-sequestering proteins, secreted haem-binding proteins (haemophores) that bind haem/haemoglobin/haemopexin and deliver the complex to a bacterial cell surface receptor and bacterial proteases that degrade haem-sequestering proteins. Once removed from haem-sequestering proteins, haem may be transported via the bacterial outer membrane receptor into the cell. Recent studies have begun to define the steps by which haem is removed from bacterial haem proteins and transported into the cell. This review describes recent work on the discovery and characterization of these systems. Reference is also made to the transport of haem in serum (via haemoglobin, haemoglobin/haptoglobin, haemopexin, albumin and lipoproteins) and to mechanisms of iron removal from the haem itself (probably via a haem oxygenase pathway in which the protoporphyrin ring is degraded). Haem protein-receptor interactions are discussed in terms of the criteria that govern protein-protein interactions in general, and connections between haem transport and the emerging field of metal transport via metallochaperones are outlined.     

Galactose-specific receptors on liver cells. II. Characterization of the purified receptor from macrophages reveals no structural relationship to the hepatocyte receptor

We have isolated a galactose-specific receptor protein from rat liver macrophages by three techniques, all using EDTA extraction and subsequent affinity chromatography. The purified receptor has an apparent molecular mass of 30 kDa and exhibits hemagglutinating activity. Monospecific receptor-antisera produce one precipitation line with the macrophage receptor in Ouchterlony double diffusion but show no cross-reaction with the hepatocyte receptor. Sinusoidal cells, but not hepatocytes, are stained with monoclonal antibodies to the macrophage receptor, whereas anti-hepatocyte receptor antibodies stain hepatocyte surfaces but not sinusoidal cells. We conclude that the galactose-specific receptor from liver macrophages is structurally different from the hepatocyte receptor, although the two lectins share a similar binding specificity.     

Haem utilization in Vibrio cholerae involves multiple TonB-dependent haem receptors

Vibrio cholerae has multiple iron transport systems, one of which involves haem uptake through the outer membrane receptor HutA. A hutA mutant had only a slight defect in growth using haemin as the iron source, and we show here that V. cholerae encodes two additional TonB-dependent haem receptors, HutR and HasR. HutR has significant homology to HutA as well as to other outer membrane haem receptors. Membrane fractionation confirmed that HutR is present in the outer membrane. The hutR gene was co-transcribed with the upstream gene ptrB, and expression from the ptrB promoter was negatively regulated by iron. A hutA, hutR mutant was significantly impaired, but not completely defective, in the ability to use haemin as the sole iron source. HasR is most similar to the haemophore-utilizing haem receptors from Pseudomonas aeruginosa and Serratia marcescens. A mutant defective in all three haem receptors was unable to use haemin as an iron source. HutA and HutR functioned with either V. cholerae TonB1 or TonB2, but haemin transport through either receptor was more efficient in strains carrying the tonB1 system genes. In contrast, haemin uptake through HasR was TonB2 dependent. Efficient utilization of haemoglobin as an iron source required HutA and TonB1. The triple haem receptor mutant exhibited no defect in its ability to compete with its Vib- parental strain in an infant mouse model of infection, indicating that additional iron sources are present in vivo. V. cholerae used haem derived from marine invertebrate haemoglobins, suggesting that haem may be available to V. cholerae growing in the marine environment.     

The use of wheat-germ lectin-Sepharose for the purification of human haemopexin.

Haemopexin was prepared in 37% yield from normal human serum by a simple procedure involving fractional poly(ethylene glycol) precipitation and subsequent chromatography on DEAE-Sepharose CL-6B. One peak from the ion exchanger consisted of only haemopexin and transferrin. These proteins were separated by chromatography on wheat-germ lectin-Sepharose 6MB. Haemopexin was selectively bound and was subsequently desorbed by N-acetyl-D-glucosamine. No impurities could be detected in the final preparation by immunoelectrophoresis or by immunodiffusion against a range of antisera. The protein gave two partially separated bands in polyacrylamide-gradient-gel electrophoresis, corresponding to apohaemopexin and haem-haemopexin complex.     

Haemophore-mediated bacterial haem transport: evidence for a common or overlapping site for haem-free and haem-loaded haemophore on its specific outer membrane receptor

Bacterial extracellular haemophores also named HasA for haem acquisition system form an independent family of haemoproteins that take up haem from host haeme carriers and shuttle it to specific receptors (HasR). Haemophore receptors are required for the haemophore-dependent haem acquisition pathway and alone allow free or haemoglobin-bound haem uptake, but the synergy between the haemophore and its receptor greatly facilitates this uptake. The three-dimensional structure of the Serratia marcescens holo-haemophore (HasASM) has been determined previously and revealed that the haem iron atom is ligated by tyrosine 75 and histidine 32. The phenolate of tyrosine 75 is also tightly hydrogen bonded to the Ndelta atom of histidine 83. Alanine mutagenesis of these three HasASM residues was performed, and haem-binding constants of the wild-type protein, the three single mutant proteins, the three double mutant proteins and the triple mutant protein were compared by absorption spectrometry to probe the roles of H32, Y75 and H83 in haem binding. We show that one axial iron ligand is sufficient to ligate haem efficiently and that H83 may become an alternative iron ligand in the absence of Y75 or both H32 and Y75. All the single mutant proteins retained the ability to stimulate haemophore-dependent haem uptake in vivo. Thus, the residues H32, Y75 and H83 are not individually necessary for haem delivery to the receptor. The binding of haem-free and haem-loaded HasASM proteins to HasRSM-producing strains was studied. Both proteins bind to HasRSM with similar apparent Kd. The double mutant H32A-Y75A competitively inhibits binding to the receptor of both holo-HasASM and apo-HasASM, showing that there is a unique or overlapping site on HasRSM for the apo- and holo-haemophores. Thus, we propose a new mechanism for haem uptake, in which haem is exchanged between haem-loaded haemophores and unloaded haemophores bound to the receptor without swapping of haemophores on the receptor.     

Iron acquisition systems in the pathogenic Neisseria

Pathogenic neisseriae have a repertoire of high-affinity iron uptake systems to facilitate acquisition of this essential element in the human host. They possess surface receptor proteins that directly bind the extracellular host iron-binding proteins transferrin and lactoferrin. Alternatively, they have siderophore receptors capable of scavenging iron when exogenous siderophores are present. Released intracellular haem iron present in the form of haemoglobin, haemoglobin-haptoglobin or free haem can be used directly as a source of iron for growth through direct binding by specific surface receptors. Although these receptors may vary in complexity and composition, the key protein involved in the transport of iron (as iron, haem or iron-siderophore) across the outer membrane is a TonB-dependent receptor with an overall structure presumably similar to that determined recently for Escherichia coli FhuA or FepA. The receptors are potentially ideal vaccine targets in view of their critical role in survival in the host. Preliminary pilot studies indicate that transferrin receptor-based vaccines may be protective in humans.     

Mannose receptor-mediated uptake of ricin toxin and ricin A chain by macrophages. Multiple intracellular pathways for a chain translocation

The role of the high mannose carbohydrate chains in the mechanism of action of ricin toxin was investigated. Ricin is taken up by two routes in macrophages, by binding to cell surface mannose receptors, or by binding of the ricin galactose receptor to cell surface glycoproteins. Removal of carbohydrate from ricin by periodate oxidation led to a large loss in toxicity via both routes of uptake by an effect on the B chain not due to a loss of galactose binding affinity. These data suggest that the carbohydrate chains of ricin B chain may be required for full toxicity. The pathway of uptake of ricin by the macrophage mannose receptor was found to differ in several respects from uptake via the galactose-specific pathway. Analysis of intoxication of macrophages by ricin in the presence of ammonium chloride suggested that mannose receptor bound ligand passes through acidic vesicles prior to translocation, unlike galactose bound ligand. Intoxication by ricin via galactose-specific uptake was potentiated by swainsonine but not by castanospermine, suggesting that ricin may be attacked by an endogenous mannosidase within the cell, and that ricin passes through either a lysosomal or a Golgi compartment prior to translocation.     

Quelling the red menace: haem capture by bacteria

Haem is an important bacterial nutrient. As a prosthetic group of several proteins, haem functions as a cofactor mediating oxygen transport, energy generation, and mixed-function oxidation. In addition, the iron chelated in the porphyrin ring may serve as an iron substrate for growth. However, because of its propensity for oxidizing cellular constituents, haem is always associated with proteins. Therefore, the uptake and transit of haem across bacterial membranes requires the participation of protein escorts. Bacteria have evolved a diverse array of surface-exposed receptors dedicated to binding haem and haem-proteins. Following this selective recognition at the bacterial cell surface, haem is transported across the outer membrane via a TonB-dependent process. The control of receptor expression appears to be multifactorial, probably involving a number of global regulators. A model integrating this information is presented.     

Uptake of sialo and asialo transferrins by isolated rat hepatocytes. Comparison of a heterologous and a homologous system

Incubation of isolated rat hepatocytes with different human sialo transferrins shows that interaction with the specific transferrin receptor is insensitive to differences in the carbohydrate composition of the glycans. Asialo transferrins lead to an increased iron uptake, which is dependent on the amount of exposed galactose. This is explained by the presence of the asialo glycoprotein (AsGP) receptor. Experiments with selective saturation of the two receptor systems show that on incubation with human asialo transferrin (AsHTf) transferrin uptake proceeds increasingly via the AsGP receptor on raising the concentration. Homologous rat asialo transferrin (AsRTf) behaves similarly, but less pronounced. Iron is accumulated via both receptor systems in the heterologous system, but only via the transferrin receptors in the homologous system. The difference in interaction with the AsGP-receptor may be caused by the difference in galactose content of the two asialo transferrins. As an explanation for the differences in intracellular metabolism a hitherto unknown recognition system for species specificity is postulated which protects homologous AsRTf from degradation, but directs foreign AsTf to lysosomes.     

Characterization of HasB, a Serratia marcescens TonB-like protein specifically involved in the haemophore-dependent haem acquisition system

In Gram-negative bacteria, the TonB-ExbB-ExbD inner membrane multiprotein complex is required for active transport of diverse molecules through the outer membrane. We present evidence that Serratia marcescens, like several other Gram-negative bacteria, has two TonB proteins: the previously characterized TonBSM, and also HasB, a newly identified component of the has operon that encodes a haemophore-dependent haem acquisition system. This system involves a soluble extracellular protein (the HasA haemophore) that acquires free or haemoprotein-bound haem and presents it to a specific outer membrane haemophore receptor (HasR). TonBSM and HasB are significantly similar and can replace each other for haem acquisition. However, TonBSM, but not HasB, mediates iron acquisition from iron sources other than haem and haemoproteins, showing that HasB and TonBSM only display partial redundancy. The reconstitution in Escherichia coli of the S. marcescens Has system demonstrated that haem uptake is dependent on the E. coli ExbB, ExbD and TonB proteins and that HasB is non-functional in E. coli. Nevertheless, a mutation in the HasB transmembrane anchor domain allows it to replace TonBEC for haem acquisition. As the change affects a domain involved in specific TonBEC-ExbBEC interactions, HasB may be unable to interact with ExbBEC, and the HasB mutation may allow this interaction. In E. coli, the HasB mutant protein was functional for haem uptake but could not complement the other TonBEC-dependent functions, such as iron siderophore acquisition, and phage DNA and colicin uptake. Our findings support the emerging hypothesis that TonB homologues are widespread in bacteria, where they may have specific functions in receptor-ligand uptake systems.     

Preclustered receptor arrangement is a prerequisite for galactose-specific clearance of large particulate ligands in rat liver

We had hypothesized that preclustered arrangement of galactose-specific receptor activity on rat liver macrophages enables these cells to internalize multivalent, particulate ligands in contrast to the clearance of molecules mediated by statistically distributed receptors on hepatocytes. We now took advantage of the nonclustered receptor distribution in newborn rat liver macrophages to study the in vivo clearance of particulate ligands. Gold particles 5, 17, and 50 nm in diameter (Au5, Au17, Au50), coated with lactosylated bovine serum albumin (LacBSA), were injected into the vena cava and livers were perfusion fixed after allowing for binding and uptake for 3 min. In sinusoidal cells from rats 15 days old LacBSA-Au5 and LacBSA-Au17 were taken up by endothelial cells and all sizes by liver macrophages. In newborn rat liver no LacBSA-Au50 or LacBSA-Au17 was retained in liver macrophages. Uptake of LacBSA-Au5 by sinusoidal cells was significant. LacBSA-Au17 was taken up in significant amounts by endothelial cells of newborn rats which correlates to the findings that galactose-specific binding sites on endothelial cells were found to localize as clusters over coated pits irrespective of age. These results demonstrate the crucial role of clustered receptors in binding and uptake of larger particulate ligands via this lectin-like binding activity.     

Elemental iron does repress transferrin, haemopexin and haemoglobin receptor expression in Haemophilusinfluenzae

The iron repressible nature of Haemophilus influenzae transferrin binding proteins suggests a regulatory role for elemental iron in their expression. The existence of a Haemophilus ferric uptake repressor (Fur) binding motif identified in the promoter region of both tbpA and tbpB further supports this hypothesis. However, a recent study using brain heart infusion growth medium suggested that transferrin binding protein synthesis in H. influenzae was haem- rather than iron-regulated. The present study re-investigates this observation and using a chemically defined medium, we demonstrate that elemental iron haem or protoporphyrin IX can each regulate Haemophilus influenzae transferrin, haemopexin and haemoglobin receptor expression.     

Specificity of hepatic iron uptake from plasma transferrin in the rat.

1. The role of specific interaction between transferrin and its receptors in iron uptake by the liver in vivo was investigated using 59Fe-125I-labelled transferrins from several animal species, and adult and 15-day rats. Transferrin-free hepatic uptake of 59Fe was measured 2 or 0.5 hr after intravenous injection of the transferrins. 2. Rat, rabbit and human transferrins gave high and approximately equal levels of hepatic iron uptake while transferrins from a marsupial (Sentonix brachyurus), lizard, crocodile, toad and fish gave very low uptake values. Chicken ovotransferrin resulted in higher uptake than with any other species of transferrin. 3. Iron uptake by the femurs (as a sample of bone marrow erythroid tissue) and, in another group of 19-day pregnant animals by the placentas and fetuses, was also measured, for comparison with the liver results. The pattern of uptake from the different transferrins was found to be similar to that of iron uptake by the liver except that with femurs, placentas and fetuses ovotransferrin gave low values comparable to those of the other non-mammalian species. 4. It is concluded that iron uptake by the liver from plasma transferrin in vivo is largely or completely dependent on specific transferrin-receptor interaction. The high hepatic uptake of iron from ovotransferrin was probably mediated by the asialoglycoprotein receptors on hepatocytes.     

Expression of membrane-associated C-reactive protein by human monocytes: indications for a selectin-like activity participating in adhesion

We have shown previously that rat liver macrophages (Kupffer cells) express a membrane-bound form of C-reactive protein (mCRP) on their surface which is identical to a galactose-specific particle receptor activity. We now establish the presence of mCRP on human monocyte-macrophages using immunocytochemistry with an anti-neoCRP specific monocloncal antibody and RNA-RNAin situ hybridization to demonstrate the presence of CRP-specific mRNA. Concomitant with mCRP expression, cells exhibit galactose-dependent uptake of particles coated with lactosylated bovine serum albumin. Adhesion experiments on fibronectin-coated surfaces that mCRP on human blood monocytes may act as a selectin-like adhesion molecule, mediating initial carbohydrate-specific contacts which are followed by peptide-specific recognition via integrin receptors.     

Biochemistry of nonheme iron in man. I. Iron proteins and cellular iron metabolism

Total plasma iron turnover in man is about 36 mg/day. Transferrin is the iron transport protein of plasma, which can bind 2 atoms of iron per protein molecule, and which interacts with various cell types to provide them with the iron required for their metabolic and proliferative processes. All tissues contain transferrin receptors on their plasma membrane surfaces, which interact preferentially with diferric transferrin. In erythroid cells as well as certain laboratory cell lines, the removal of iron from transferrin apparently proceeds via the receptor-mediated endocytosis process. Transferrin and its receptor are recycled to the cell surface, whereas the iron remains in the cell. The mode of iron uptake in the hepatocyte, the main iron storage tissue, is less certain. The release of iron by hepatocytes, as well as by the reticuloendothelial cells, apparently proceeds nonspecifically. All tissues contain the iron storage protein ferritin, which stores iron in the ferric state, though iron must be in the ferrous state to enter and exit the ferritin molecule. Cellular cytosol also contains a small-molecular-weight ferrous iron pool, which may interact with protoporphyrin to form heme, and which apparently is the form of iron exported by hepatocytes and macrophages. In plasma, the ferrous iron is converted into the ferric form via the action of ceruloplasmin.     

Haematoporphyrin and OO''-diacetylhaematoporphyrin binding by serum and cellular proteins. Implications for the clearance of these photochemotherapeutic agents by cells.

Haematoporphyrin derivative (HpD), a mixture of porphyrins, is currently used as a photochemotherapeutic agent in the treatment of neoplasias. The interaction of purified components of HpD with serum and cellular proteins was investigated using absorption and fluorescence spectroscopy. The interactions of haematoporphyrin and OO'-diacetylhaematoporphyrin with human albumin and with haemopexin, the two major serum porphyrin-binding proteins, show stoichiometries of 1 mol of porphyrin bound per mol of protein. The apparent dissociation constants, Kd, are in the range of 1-2 microM for albumin and 3-4 microM for haemopexin. These two major components of HpD would, after intravenous injection, bind to albumin and circulate in serum as albumin complexes. Free porphyrin rather than porphyrin bound to albumin interacts with Morris hepatoma tissue culture cells. A rapid high-affinity saturable transport system operates at free porphyrin concentrations of less than 2 microM. In addition, fluorescence spectra show that components in rat liver cytosol can bind haematoporphyrin and OO'-diacetylhaematoporphyrin and distinguish these binders from those present in rat serum.     

Haem acquisition is facilitated by a novel receptor Hma and required by uropathogenic Escherichia coli for kidney infection

Iron acquisition, mediated by specific outer membrane receptors, is critical for colonization of the urinary tract by uropathogenic Escherichia coli (UPEC). The role of specific iron sources in vivo , however, remains largely unknown. In this study, we identified a 79 kDa haem receptor, h ae m a cquisition protein Hma, and established that it functions independently of ChuA to mediate haemin uptake by UPEC strain CFT073. We demonstrated that expression of hma promotes TonB-dependent haemin utilization and the Hma protein binds haemin with high affinity ( K _d = 8 μM). Hma, however, lacks conserved His residues shown to mediate haem uptake by other bacterial receptors. In contrast, we identified Tyr-126 as a residue necessary for Hma-mediated haemin utilization. In a murine co-infection model of UTI, an isogenic hma mutant was out-competed by wild-type CFT073 in the kidneys ( P  < 0.001) and spleens ( P  <  0.0001) of infected mice, indicating its expression provided a competitive advantage in these organs. Furthermore, a hma chuA double mutant, which is unable to utilize haemin, was unable to colonize the kidneys to wild-type levels during independent infection ( P  = 0.02). Thus, we demonstrate that UPEC requires haem for kidney colonization and that uptake of this iron source is mediated, in part, by the novel receptor, Hma.