Jonas Heilskov Graversen

Identification of the haemoglobin scavenger receptor.

Intravascular haemolysis is a physiological phenomenon as well as a severe pathological complication when accelerated in various autoimmune, infectious (such as malaria) and inherited (such as sickle cell disease) disorders. Haemoglobin released into plasma is captured by the acute phase protein haptoglobin, which is depleted from plasma during elevated haemolysis. Here we report the identification of the acute phase-regulated and signal-inducing macrophage protein, CD163, as a receptor that scavenges haemoglobin by mediating endocytosis of haptoglobin–haemoglobin complexes. CD163 binds only haptoglobin and haemoglobin in complex, which indicates the exposure of a receptor-binding neoepitope. The receptor–ligand interaction is Ca2+-dependent and of high affinity. Complexes of haemoglobin and multimeric haptoglobin (the 2-2 phenotype) exhibit higher functional affinity for CD163 than do complexes of haemoglobin and dimeric haptoglobin (the 1-1 phenotype). Specific CD163-mediated endocytosis of haptoglobin–haemoglobin complexes is measurable in cells transfected with CD163 complementary DNA and in CD163-expressing myelo-monocytic lymphoma cells.

CD163: a signal receptor scavenging haptoglobin-hemoglobin complexes from plasma.

CD163 is a highly expressed macrophage membrane protein belonging to the scavenger receptor cysteine rich (SRCR) domain family. The CD163 expression is induced by interleukin-6, interleukin-10 and glucocorticoids. Its function has remained unknown until recently when CD163 was identified as the endocytic receptor binding hemoglobin (Hb) in complex with the plasma protein haptoglobin (Hp). This specific receptor-ligand interaction leading to removal from plasma of the Hp-Hb complex-but not free Hp or Hb-now explains the depletion of circulating Hp in individuals with increased intravascular hemolysis. Besides having a detoxificating effect by removing Hb from plasma, the CD163-mediated endocytosis of the Hp-Hb complex may represent a major pathway for uptake of iron in the tissue macrophages. The novel functional linkage of CD163 and Hp, which both are induced during inflammation, also reveal some interesting perspectives relating to the suggested anti-inflammatory properties of the receptor and the Hp phenotypes.

Molecular characterization of the haptoglobin.hemoglobin receptor CD163. Ligand binding properties of the scavenger receptor cysteine-rich domain region.

CD163 is the macrophage receptor for endocytosis of haptoglobin·hemoglobin complexes. The extracellular region consisting of nine scavenger receptor cysteinerich (SRCR) domains also circulates in plasma as a soluble protein. By ligand binding analysis of a broad spectrum of soluble CD163 truncation variants, the amino-terminal third of the SRCR region was shown to be crucial for the binding of haptoglobin·hemoglobin complexes. By Western blotting of the CD163 variants, a panel of ten monoclonal antibodies was mapped to SRCR domains 1, 3, 4, 6, 7, and 9, respectively. Only the two antibodies binding to SRCR domain 3 exhibited effective inhibition of ligand binding. Furthermore, analysis of purified native CD163 revealed that proteolytic cleavage in SRCR domain 3 inactivates ligand binding. Calcium protects against cleavage in this domain. Analysis of the calcium sensitivity of ligand binding to CD163 demonstrated that optimal ligand binding requires physiological plasma calcium concentrations, and an immediate ligand release occurs at the low calcium concentrations measured in acidifying endosomes. In conclusion, SRCR domain 3 of CD163 is an exposed domain and a critical determinant for the calcium-sensitive coupling of haptoglobin·hemoglobin complexes.

Crystal structure of tetranectin, a trimeric plasminogen-binding protein with an α-helical coiled coil

Tetranectin is a plasminogen kringle 4‐binding protein. The crystal structure has been determined at 2.8 Å resolution using molecular replacement. Human tetranectin is a homotrimer forming a triple α‐helical coiled coil. Each monomer consists of a carbohydrate recognition domain (CRD) connected to a long α‐helix. Tetranectin has been classified in a distinct group of the C‐type lectin superfamily but has structural similarity to the proteins in the group of collectins. Tetranectin has three intramolecular disulfide bridges. Two of these are conserved in the C‐type lectin superfamily, whereas the third is present only in long‐form CRDs. Tetranectin represents the first structure of a long‐form CRD with intact calcium‐binding sites. In tetranectin, the third disulfide bridge tethers the CRD to the long helix in the coiled coil. The trimerization of tetranectin as well as the fixation of the CRDs relative to the helices in the coiled coil indicate a demand for high specificity in the recognition and binding of ligands.

Targeting the Hemoglobin Scavenger receptor CD163 in Macrophages Highly Increases the Anti-inflammatory Potency of Dexamethasone

Synthetic glucocorticoids are potent anti-inflammatory drugs but serious side effects such as bone mobilization, muscle mass loss, immunosuppression, and metabolic alterations make glucocorticoid therapy a difficult balance. The therapeutic anti-inflammatory effect of glucocorticoids relies largely on the suppressed release of tumor-necrosis factor-α and other cytokines by macrophages at the sites of inflammation. We have now developed a new biodegradable anti-CD163 antibody-drug conjugate that specifically targets the glucocorticoid, dexamethasone to the hemoglobin scavenger receptor CD163 in macrophages. The conjugate, that in average contains four dexamethasone molecules per antibody, exhibits retained high functional affinity for CD163. In vitro studies in rat macrophages and in vivo studies of Lewis rats showed a strong anti-inflammatory effect of the conjugate measured as reduced lipopolysaccharide-induced secretion of tumor-necrosis factor-α. The in vivo potency of conjugated dexamethasone was about 50-fold that of nonconjugated dexamethasone. In contrast to a strong systemic effect of nonconjugated dexamethasone, the equipotent dose of the conjugate had no such effect, measured as thymus lymphocytes apoptosis, body weight loss, and suppression of endogenous cortisol levels. In conclusion, the study shows antibody-drug conjugates as a future approach in anti-inflammatory macrophage-directed therapy. Furthermore, the data demonstrate CD163 as an excellent macrophage target for anti-inflammatory drug delivery.

A Unique Loop Extension in the Serine Protease Domain of Haptoglobin Is Essential for CD163 Recognition of the Haptoglobin-Hemoglobin Complex

Haptoglobin and haptoglobin-related protein are homologous hemoglobin-binding proteins consisting of a complement control repeat (α-chain) and a serine protease domain (β-chain). Haptoglobin-hemoglobin complex formation promotes high affinity binding of hemoglobin to the macrophage scavenger receptor CD163 leading to endocytosis and degradation of the haptoglobin-hemoglobin complex. In contrast, complex formation between haptoglobin-related protein and hemoglobin does not promote high affinity interaction with CD163. To define structural components of haptoglobin important for CD163 recognition, we exploited this functional difference to design and analyze recombinant haptoglobin/haptoglobin-related protein chimeras complexed to hemoglobin. These data revealed that only the β-chain of haptoglobin is involved in receptor recognition. Substitution of 4 closely spaced amino acid residues of the haptoglobin β-chain (valine 259, glutamate 261, lysine 262, and threonine 264) abrogated the high affinity receptor binding. The 4 residues are encompassed by a part of the primary structure not present in other serine protease domain proteins. Structural modeling based on the well characterized serine protease domain fold suggests that this sequence represents a loop extension unique for haptoglobin and haptoglobin-related protein. A synthetic peptide representing the haptoglobin loop sequence exhibited a pronounced inhibitory effect on receptor binding of haptoglobin-hemoglobin.

Haptoglobin and CD163: captor and receptor gating hemoglobin to macrophage lysosomes.

Abstract The plasma protein haptoglobin and the endocytic hemoglobin receptor HbSR/CD163 are key molecules in the process of removing hemoglobin released from ruptured erythrocytes. Hemoglobin in plasma is instantly bound with high affinity to haptoglobin – an interaction leading to the recognition of the complex by HbSR/CD163 and endocytosis in macrophages. The haptoglobin-dependent HbSR/CD163 scavenging system for hemoglobin clearance prevents toxic effects of hemoglobin in plasma and kidney and explains the decrease in the haptoglobin plasma concentration in patients with accelerated hemolysis. The HbSR/CD163 activity may be of quantitative importance for iron uptake in macrophages in general and for some iron-associated pathological processes, e.g. the atherogenesis-promoting oxidation of LDL leading to foam cell formation and apoptosis in the vessel wall.

STRUCTURE OF THE C-TYPE LECTIN CARBOHYDRATE RECOGNITION DOMAIN OF HUMAN TETRANECTIN

Tetranectin (TN) is a C-type lectin involved in fibrinolysis, being the only endogenous ligand known to bind specifically to the kringle 4 domain of plasminogen. TN was originally isolated from plasma, but shows a wide tissue distribution. Furthermore, TN has been found in the extracellular matrix of certain human carcinomas, whereas none or little is present in the corresponding normal tissue. The crystal structure of full-length trimeric TN (2.8 A resolution) has recently been published [Nielsen et al. (1997). FEBS Lett. 412, 388-396]. The crystal structure of the carbohydrate recognition domain (CRD) of human TN (TN3) has been determined separately at 2.0 A resolution in order to obtain detailed information on the two calcium binding sites. This information is essential for the elucidation of the specificity of TN towards oligosaccharides. TN3 crystallizes as a dimer, whereas it appears as a monomer in solution. The overall fold of TN3 is similar to other known CRDs. Each monomer is built of two distinct regions, one region consisting of six beta-strands and two alpha-helices, and the other region is composed of four loops harboring two calcium ions. The calcium ion at site 1 forms an eightfold coordinated complex and has Asp116, Glu120, Gly147, Glu150, Asn151, and one water molecule as ligands. The calcium ion at site 2, which is believed to be involved in recognition and binding of oligosaccharides, is sevenfold coordinated with ligands Gln143, Asp145, Glu150, Asp165, and two water molecules. One sulfate ion has been located at the surface of TN3, forming contacts to Glu120, Lys148, Asn106 of a symmetry-related molecule, and to an ethanol molecule.

The plasminogen binding site of the C-type lectin tetranectin is located in the carbohydrate recognition domain, and binding is sensitive to both calcium and lysine.

Tetranectin, a homotrimeric protein belonging to the family of C-type lectins and structurally highly related to corresponding regions of the mannose-binding proteins, is known specifically to bind the plasminogen kringle 4 protein domain, an interaction sensitive to lysine. Surface plasmon resonance and isothermal calorimetry binding analyses using single-residue and deletion mutant tetranectin derivatives produced in Escherichia coli showed that the kringle 4 binding site resides in the carbohydrate recognition domain and includes residues of the putative carbohydrate binding site. Furthermore, the binding analysis revealed that the interaction is sensitive to calcium in addition to lysine.

A pivotal role of the human kidney in catabolism of HDL protein components apolipoprotein A-I and A-IV but not of A-II.

Renal handling of major HDL components was studied by analyzing urine from patients with Fanconi syndrome, a rare renal proximal tubular reabsorption failure, including dysfunction of the kidney HDL receptor, cubilin. A high urinary excretion of apolipoprotein A-I and A-IV corresponding to a major part of the metabolism of these proteins was measured. In contrast, no urinary excretion of apolipoprotein A-II which is more hydrophobic and tighter bound to HDL was found. Control urines displayed absence of the three apolipoproteins. Urinary excretion of phospholipids, triglycerides, cholesterol and cholesterol esters in patients was as low as in controls. In conclusion, these data indicate that the human kidney is a major site for filtered nascent apolipoprotein A-I and A-IV but not for HDL particles.