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 CD 163 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.

The present report shows the molecular characterization of the rat 460-kDa epithelial glycoprotein that functions as the receptor facilitating uptake of intrinsic factor-vitamin B 12 complexes in the intestine and kidney. The same receptor represents also the yolk sac target for teratogenic antibodies causing fetal malformations in rats. Determination of its primary structure by cDNA cloning identified a novel type of peripheral membrane receptor characterized by a cluster of eight epidermal growth factor type domains followed by a cluster of 27 CUB domains. In accordance with the absence of a hydrophobic segment, the receptor could be released from renal cortex membranes by nonenzymatic and nonsolubilizing procedures. The primary structure has no similarity to known endocytic receptors but displays homology to epidermal growth factor and CUB domain proteins involved in fetal development, e.g. the bone morphogenic proteins. Electron microscopic immunogold double labeling of rat yolk sac and renal proximal tubules demonstrated subcellular colocalization with the endocytic receptor megalin, which is expressed in the same epithelia as the 460-kDa receptor. Furthermore, megalin affinity chromatography and surface plasmon resonance analysis revealed a calcium-dependent high affinity binding of the 460-kDa receptor to megalin, which thereby may mediate its vesicular trafficking. Due to the high number of CUB domains, accounting for 88% of the protein mass, we propose the name cubilin for the novel receptor.

Cubilin is the intestinal receptor for the endocytosis of intrinsic factor-vitamin B12. However, several lines of evidence, including a high expression in kidney and yolk sac, indicate it may have additional functions. We isolated apolipoprotein A-I (apoA-I), the main protein of high-density lipoprotein (HDL), using cubilin affinity chromatography. Surface plasmon resonance analysis demonstrated a high-affinity binding of apoA-I and HDL to cubilin, and cubilin-expressing yolk sac cells showed efficient 125I-HDL endocytosis that could be inhibited by IgG antibodies against apoA-I and cubilin. The physiological relevance of the cubilin-apoA-I interaction was further emphasized by urinary apoA-I loss in some known cases of functional cubilin deficiency. Therefore, cubilin is a receptor in epithelial apoA-I/HDL metabolism.

Cubilin, the receptor for intrinsic factor-vitamin B 12 , is a novel type of high molecular weight receptor consisting of a 27 CUB (complement components C1r/C1s, Uegf, and bone morphogenic protein-1) domain cluster preceded by 8 epidermal growth factor repeats and a short N-terminal sequence. In addition to binding the vitamin B 12 -carrier complex, cubilin also binds receptor-associated protein. 1 depends on gastric intrinsic factor (IF), which undergoes a specific and strong complex formation with the vitamin in the intestinal lumen (1). The vitamin-carrier complex is subsequently internalized after being recognized by the high affinity membrane receptor, cubilin (2, 3). The binding of IF-B 12 is suggested to occur via the N-terminal region of IF (4) and only when B 12 is bound to the carrier protein (1-3, 5). The physiological importance of the specific B 12 carrier and its receptor is evident from the B 12 deficiency, characterized by the megaloblastic anemia and neurological symptoms related to defective functional expression of IF or cubilin (6 -11). In addition to binding IF-B 12 , cubilin also binds receptor-associated protein (RAP) (5), and growing evidence suggests that the receptor has other biological functions. This is in particular indicated by the fact that kidney and yolk sac epithelia exhibit a high cubilin expression (2, 5, 12), albeit IF is synthesized in the gastric epithelium and virtually only is present in the gastrointestinal lumen.The recent determination of the primary structure of the rat and human cubilin molecule (13, 14) revealed an ϳ3600-aa protein with a short N-terminal segment followed by 8 epidermal growth factor (EGF) repeats and a large cluster of 27 CUB 2 domains. No classical transmembrane segment has been predicted, and the overall structure is distinct from known endocytic receptors, including the RAP-binding members of the low density lipoprotein (LDL) receptor family (15). Two mutations, one causing a Pro/Leu substitution in CUB domain 8 and another suggested to activate an intronic cryptic splice site leading to truncation of the receptor in CUB domain 6, have recently been identified in the cubilin gene of patients with inherited B 12 malabsorption (11).To link the structural and functional features of cubilin, we established a comprehensive panel of transfected Chinese hamster ovary (CHO) cells expressing various segments of the receptor. Biochemical analyses of the cells and expression products now delineate the regions important for membrane association and binding of IF-B 12 and RAP. EXPERIMENTAL PROCEDURESLigands and Receptors-Porcine and human IF-B 12 were purified from gastric mucosa extract as described (16). Human RAP was produced as a recombinant protein in Escherichia coli. Rabbit cubilin was purified from renal cortex by IF-B 12 and RAP affinity chromatography, as described previously (5). Construction of Plasmids for Expression of Recombinant Rat CubilinFragments-Cubilin cDNA fragments extended with enzyme restriction sites were amplified by polymerase c...

Uptake of vitamin B12 (cyanocobalamin) is facilitated by the cobalamin-binder gastric intrinsic factor (IF), which recognizes a 460-kD receptor, cubilin, present in the epithelium of intestine and kidney. Surface plasmon resonance analysis of ligand-affinity-purified human cubilin demonstrated a high-affinity calcium- and cobalamin-dependent binding of IF-cobalamin. Complete cDNA cloning of the human receptor showed a 3597 amino acid peripheral membrane protein with 69% identity to rat cubilin. Amino-terminal sequencing of the receptor indicates that the cDNA sequence encodes a precursor protein undergoing proteolytic processing due to cleavage at a recognition site (Arg7-Glu8-Lys9-Arg10) for the trans-Golgi proteinase furin. Using fluorescence in situ hybridization, radiation hybrid mapping, and screening of YAC clones, the human cubilin gene was mapped between the markers D10S1661 and WI-5445 on the short arm of chromosome 10. This is within the autosomal recessive megaloblastic anemia (MGA1) 6-cM region harboring the unknown recessive-gene locus of juvenile megaloblastic anemia caused by intestinal malabsorption of cobalamin (Imerslund-Gräsbeck's disease). In conclusion, the present molecular and genetic information on human cubilin now provides circumstantial evidence that an impaired synthesis, processing, or ligand binding of cubilin is the molecular background of this hereditary form of megaloblastic anemia.

Megaloblastic anemia 1 (MGA1) is an autosomal recessive disorder caused by the selective intestinal malabsorption of intrinsic factor (IF) and vitamin B12/cobalamin (Cbl) in complex. Most Finnish patients with MGA1 carry the disease-specific P1297L mutation (FM1) in the IF-B12 receptor, cubilin. By site-directed mutagenesis, mammalian expression, and functional comparison of the purified wild-type and FM1 mutant forms of the IF–Cbl-binding cubilin region (CUB domains 5-8, amino acid 928-1386), we have investigated the functional implications of the P1297L mutation. Surface plasmon resonance analysis revealed that the P1297L substitution specifically increases the Kd for IF–Cbl binding several-fold, largely by decreasing the association rate constant. In agreement with the binding data, the wild-type protein, but not the FM1 mutant protein, potently inhibits 37°C uptake of iodine 125–IF–Cbl in cubilin-expressing epithelial cells. In conclusion, the data presented show a substantial loss in affinity of the FM1 mutant form of the IF–Cbl binding region of cubilin. This now explains the malabsorption of Cbl and Cbl-dependent anemia in MGA1 patients with the FM1 mutation.

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Uptake of vitamin B12 (cyanocobalamin) is facilitated by the cobalamin-binder gastric intrinsic factor (IF), which recognizes a 460-kD receptor, cubilin, present in the epithelium of intestine and kidney. Surface plasmon resonance analysis of ligand-affinity-purified human cubilin demonstrated a high-affinity calcium- and cobalamin-dependent binding of IF-cobalamin. Complete cDNA cloning of the human receptor showed a 3597 amino acid peripheral membrane protein with 69% identity to rat cubilin. Amino-terminal sequencing of the receptor indicates that the cDNA sequence encodes a precursor protein undergoing proteolytic processing due to cleavage at a recognition site (Arg7-Glu8-Lys9-Arg10) for the trans-Golgi proteinase furin. Using fluorescence in situ hybridization, radiation hybrid mapping, and screening of YAC clones, the human cubilin gene was mapped between the markers D10S1661 and WI-5445 on the short arm of chromosome 10. This is within the autosomal recessive megaloblastic anemia (MGA1) 6-cM region harboring the unknown recessive-gene locus of juvenile megaloblastic anemia caused by intestinal malabsorption of cobalamin (Imerslund-Gräsbeck's disease). In conclusion, the present molecular and genetic information on human cubilin now provides circumstantial evidence that an impaired synthesis, processing, or ligand binding of cubilin is the molecular background of this hereditary form of megaloblastic anemia.