Identification of the receptor scavenging hemopexin-heme complexes by Vibeke Hvidberg, Maciej B. Maniecki, Christian Jacobsen, Peter Højrup, Holger J. Møller, and Søren K. Moestrup Blood Volume 106(7):2572-2579 October 1, 2005 ©2005 by American Society of Hematology

Purification of the Hx-heme receptor from placenta and liver by Hx-heme affinity chromatography. Purification of the Hx-heme receptor from placenta and liver by Hx-heme affinity chromatography. (A) SDS-PAGE of the purified human Hx used for coupling to heme and the Sepharose matrix. (B) SDS-PAGE showing the elution profile of the eluate from the Hx-heme-Sepharose column loaded with solubilized human placenta membranes. (C) SDS-PAGE of the eluate (lane 1, peak fraction) from a Hx-heme-Sepharose column loaded with human liver membranes. Lanes 2 and 3 show Western blotting of liver Hx-heme affinity eluate using the monoclonal antibodies A2MRα-2 and A2MRβ-1, which recognize the LRP/CD91 α- and β-subunits, respectively. Lane 4 shows SDS-PAGE of the eluate from a Hx-heme column after multiple (> 10) loading-elution runs. Notice that the asialoglycoprotein receptor (ASGP-R) now is becoming a predominant band. Vibeke Hvidberg et al. Blood 2005;106:2572-2579 ©2005 by American Society of Hematology

Binding of Hx-heme to immobilized LRP/CD91. Binding of Hx-heme to immobilized LRP/CD91. (A) Inhibition of binding of 125I-labeled Hx-heme by unlabeled Hx-heme complexes and noncomplexed Hx. (B) Effect on binding of 125I-labeled Hx-heme by RAP (100 μg/mL), EDTA (10 mM), rabbit anti-LRP/CD91 IgG (300 μg/mL), nonimmune rabbit IgG (300 μg/mL), and activated α2-macroglobulin (α2M*, 100 μg/mL). All values in panels A and B, representing the measured radioactivity in percentage of the added radioactivity, are the mean ± 1 SD of triplicate determinations. Each 200-μL well was incubated with 3000 cpm 125I-Hx-heme (∼0.5 ng). (C) Surface plasmon resonance analysis of the binding of Hx (500 nM and 5 μM) and heme (500 nM) to immobilized LRP/CD91. (D) Surface plasmon resonance analysis of the binding of Hx-heme (50 nM to 5 μM) to immobilized LRP/CD91. Vibeke Hvidberg et al. Blood 2005;106:2572-2579 ©2005 by American Society of Hematology

LRP/CD91-mediated endocytosis of 125I-labeled Hx-heme and activated α2-macroglobulin (α2M*) in COS-1 cells. LRP/CD91-mediated endocytosis of125I-labeled Hx-heme and activated α2-macroglobulin (α2M*) in COS-1 cells. (A) Time course of cell-associated radioactivity and degradation (measured as increase in TCA-soluble radioactivity) in cells incubated with 125I-labeled Hx-heme. The inset shows Western blot of solubilized COS-1 cells with the monoclonal antibodies (refer to legend for Figure 1) against the LRP/CD91 subunits. (B) Time course of uptake and degradation of 125I-labeled Hx-heme in the presence of the lysosomal inhibitors chloroquine and leupeptin (both 300 μM). (C-D) Similar experiments as in panels A and B using receptor-binding (methylamine-activated) 125I-α2-macroglobulin (α2M*) as radioligand instead. All values, which represent the measured radioactivity in percentage of the added radioactivity, are the mean ± 1 SD of triplicate determinations. Each well containing 300 μL medium was incubated with 3000 cpm 125I-Hx-heme (∼0.5 ng). Vibeke Hvidberg et al. Blood 2005;106:2572-2579 ©2005 by American Society of Hematology

Inhibition of LRP/CD91-mediated endocytosis of 125I-Hx-heme and 125I-Hx. Inhibition of LRP/CD91-mediated endocytosis of 125I-Hx-heme and 125I-Hx. (A) The effect on endocytosis 125I-Hx-heme and 125I-Hx by RAP (100 μg/mL), rabbit anti-LRP/CD91 IgG (300 μg/mL), nonimmune rabbit IgG (300 μg/mL), Hx-heme (100 μg/mL), Hx (100 μg/mL), and heme (50 μg/mL). The experimental conditions were otherwise as described in the legend for Figure 4. (B) The effect of various Hx-heme concentrations on 125I-Hx-heme endocytosis. Values shown in panels A and B represent the mean ± SD of triplicate determinations. Vibeke Hvidberg et al. Blood 2005;106:2572-2579 ©2005 by American Society of Hematology

Confocal microscopy of the uptake of Hx-heme and receptor-binding α2-macroglobulin in COS-1 cells. Confocal microscopy of the uptake of Hx-heme and receptor-binding α2-macroglobulin in COS-1 cells. (A) Immunostaining of cells incubated 2 minutes at 37°C with Alexa 488-labeled Hx-heme (green color) and monoclonal rhodamine-labeled anti-LRP/CD91 antibody (red color). Notice the colocalization of the 2 fluorochromes. (B) Cells incubated 2 minutes at 37°C with Alexa 488-labeled Hx-heme (green color) and immunostaining for EEA1 using Alexa 594-labeled secondary antibody (red color). (C) Cells incubated for 60 minutes with Alexa 488-labeled Hx-heme (green color) and immunostained with LRP/CD91 using Alexa 594-labeled secondary antibody (red color). Notice the distinct staining of the 2 fluorochromes. (D) Vesicular staining of COS-1 cells incubated for 60 minutes with Alexa 488-labeled Hx-heme (green color) and receptor-binding (activated) α2-macroglobulin (α2M*), which was immunostained by an Alexa 594-labeled secondary antibody (red color). Notice the overlapping coloring in the overlay picture (right panel). Optic magnification was × 630 in all the figure displays. Vibeke Hvidberg et al. Blood 2005;106:2572-2579 ©2005 by American Society of Hematology

Pulse-chase of COS-1 cells incubated with 125I-labeled Hx-heme and α2-macroglobulin. Pulse-chase of COS-1 cells incubated with125I-labeled Hx-heme and α2-macroglobulin. The figure shows the time course of the appearance of TCA-precipitable and soluble radioactivity in the medium after pulse with 125I-Hx-heme (A) and methylamine-activated 125I-α2-macroglobulin (α2M*) for 60 minutes at 4°C followed by wash of the cells and incubation at 37°C (B). All values, which represent the measured radioactivity in percentage of the initial cell-associated radioactivity, are the mean ± 1 SD of triplicate determinations. Vibeke Hvidberg et al. Blood 2005;106:2572-2579 ©2005 by American Society of Hematology

Overview of the receptor pathways for endocytosis of extracellular heme and hemoglobin in complex with hemopexin and haptoglobin, respectively. Overview of the receptor pathways for endocytosis of extracellular heme and hemoglobin in complex with hemopexin and haptoglobin, respectively. LRP/CD91 and CD163 represent 2 pathways for uptake of extracellular heme incorporated in Hx-heme and haptoglobin-hemoglobin. Both receptors are highly expressed in phagocytic macrophages, which are known to metabolize heme into bilirubin, Fe, and carbon monoxide. In addition to the expression in macrophages, LRP/CD91 is highly expressed in several other cell types including hepatocytes, neurons, and syncytiotrophoblasts.31 Vibeke Hvidberg et al. Blood 2005;106:2572-2579 ©2005 by American Society of Hematology