Hemolysis in the spleen drives erythrocyte turnover.

Red pulp macrophages (RPMs) of the spleen mediate turnover of billions of senescent erythrocytes per day. However, the molecular mechanisms involved in sequestration of senescent erythrocytes, their recognition, and their subsequent degradation by RPMs remain unclear. In this study, we provide evidence that the splenic environment is of substantial importance in facilitating erythrocyte turnover through induction of hemolysis. Upon isolating human spleen RPMs, we noted a substantial lack of macrophages that were in the process of phagocytosing intact erythrocytes. Detailed characterization of erythrocyte and macrophage subpopulations from human spleen tissue led to the identification of erythrocytes that are devoid of hemoglobin, so-called erythrocyte ghosts. By using in vivo imaging and transfusion experiments, we further confirmed that senescent erythrocytes that are retained in the spleen are subject to hemolysis. In addition, we showed that erythrocyte adhesion molecules, which are specifically activated on aged erythrocytes, cause senescent erythrocytes to interact with extracellular matrix proteins that are exposed within the splenic architecture. Such adhesion molecule-driven retention of senescent erythrocytes under low shear conditions was found to result in steady shrinkage of the cell and ultimately resulted in hemolysis. In contrast to intact senescent erythrocytes, the remnant erythrocyte ghost shells were prone to recognition and breakdown by RPMs. These data identify hemolysis as a key event in the turnover of senescent erythrocytes, which alters our current understanding of how erythrocyte degradation is regulated.

Mutations in CUBN, encoding the intrinsic factor-vitamin B12 receptor, cubilin, cause hereditary megaloblastic anaemia 1.

Megaloblastic anaemia 1 (MGA1, OMIM 261100) is a rare, autosomal recessive disorder characterized by juvenile megaloblastic anaemia, as well as neurological symptoms that may be the only manifestations. At the cellular level, MGA1 is characterized by selective intestinal vitamin B12 (B12, cobalamin) malabsorption. MGA1 occurs worldwide, but its prevalence is higher in several Middle Eastern countries and Norway, and highest in Finland (0.8/100,000). We previously mapped the MGA1 locus by linkage analysis in Finnish and Norwegian families to a 6-cM region on chromosome 10p12.1 (ref. 8). A functional candidate gene encoding the intrinsic factor (IF)-B12 receptor, cubilin, was recently cloned; the human homologue, CUBN, was mapped to the same region. We have now refined the MGA1 region by linkage disequilibrium (LD) mapping, fine-mapped CUBN and identified two independent disease-specific CUBN mutations in 17 Finnish MGA1 families. Our genetic and molecular data indicate that mutations in CUBN cause MGA1.

Dietary supplementation of cystinotic mice by lysine inhibits the megalin pathway and decreases kidney cystine content.

Megalin/LRP2 is a major receptor supporting apical endocytosis in kidney proximal tubular cells. We have previously reported that kidney-specific perinatal ablation of the megalin gene in cystinotic mice, a model of nephropathic cystinosis, essentially blocks renal cystine accumulation and partially preserves kidney tissue integrity. Here, we examined whether inhibition of the megalin pathway in adult cystinotic mice by dietary supplementation (5x-fold vs control regular diet) with the dibasic amino-acids (dAAs), lysine or arginine, both of which are used to treat patients with other rare metabolic disorders, could also decrease renal cystine accumulation and protect cystinotic kidneys. Using surface plasmon resonance, we first showed that both dAAs compete for protein ligand binding to immobilized megalin in a concentration-dependent manner, with identical inhibition curves by L- and D-stereoisomers. In cystinotic mice, 2-month diets with 5x-L-lysine and 5x-L-arginine were overall well tolerated, while 5x-D-lysine induced strong polyuria but no weight loss. All diets induced a marked increase of dAA urinary excretion, most prominent under 5x-D-lysine, without sign of kidney insufficiency. Renal cystine accumulation was slowed down approx. twofold by L-dAAs, and totally suppressed by D-lysine. We conclude that prolonged dietary manipulation of the megalin pathway in kidneys is feasible, tolerable and can be effective in vivo.

The intrinsic factor-vitamin B12 receptor, cubilin, is a high-affinity apolipoprotein A-I receptor facilitating endocytosis of high-density lipoprotein.

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.

New immunological serum markers in bacteraemia: anti-inflammatory soluble CD163, but not proinflammatory high mobility group-box 1 protein, is related to prognosis.

High mobility group-box 1 protein (HMGB1) is a late-onset proinflammatory cytokine. Soluble haemoglobin scavenger receptor (sCD163) is a specific marker of anti-inflammatory macrophages. The study purpose was to relate the levels of these new markers in bactaeremic patients to levels of well-known pro- and anti-inflammatory markers [procalcitonin, lipopolysaccharide (LPS)-binding protein, interleukin (IL)-6, IL-10] and to evaluate the levels in relation to disease severity and aetiology. A total of 110 patients with bacteraemia were included in a prospective manner from the medical department at a large Danish university hospital. Levels of HMGB1 and sCD163 were higher in patients with bacteraemia compared to controls (P < 0.001). HMGB1 correlated with proinflammatory molecules [procalcitonin (PCT)] and traditional infectious parameters [C-reactive proteins (CRP), white blood cells (WBC) and neutrophils], whereas sCD163 correlated with levels of IL-6, IL-10 but not to lipopolysaccharide-binding protein (LBP), PCT or CRP. Levels of sCD163 and IL-6 were significantly higher among non-survivors compared to survivors (P < 0.05). Neither HMGB1 nor any of the proinflammatory markers were elevated in fatal cases compared to survivors. There was no statistically significant difference in HMGB1 and sCD163 levels in Gram-negative versus Gram-positive bacteraemia. HMGB1 reflects proinflammatory processes, whereas sCD163 reflects anti-inflammatory processes as judged by correlations with traditional marker molecules. sCD163 and IL-6, but not HMGB1, were prognostic markers in this cohort pointing to an anti-inflammatory predominance in patients with fatal disease outcome.

Evidence that epithelial glycoprotein 330/megalin mediates uptake of polybasic drugs.

Glycoprotein 330 (gp330) is an endocytic receptor expressed in the renal proximal tubules and some other absorptive epithelia, e.g., in the inner ear. The present study shows that the antifibrinolytic polypeptide, aprotinin, and the nephro- and ototoxic antibiotics, aminoglycosides, and polymyxin B compete for binding of 125I-urokinase-plasminogen activator inhibitor type-1 complexes to purified rabbit gp330. Half maximal inhibition was measured at 4 microM for aprotinin, 50 microM for gentamicin, and 0.5 microM for polymyxin B. Drug binding to gp330 was validated by equilibrium dialysis of [3H] gentamicin-gp330 incubations and binding/uptake studies in rat proximal tubules and gp330-expressing L2 carcinoma cells. Analyses of mutant aprotinins expressed in Saccharomyces cerevisiae revealed that basic residues are essential for the binding to gp330 and renal uptake. The polybasic drugs also antagonized ligand binding to the human alpha 2-macroglobulin receptor. However, the rapid glomerular filtration of the drugs suggests kidney gp330 to be the quantitatively most important target. In conclusion, a novel role of gp330 as a drug receptor is demonstrated. The new insight into the mechanism of epithelial uptake of polybasic drugs might provide a basis for future design of drugs with reduced toxicity.

Expression of alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein and scavenger receptor in human atherosclerotic lesions.

Macrophage- and smooth muscle cell (SMC)-derived foam cells are typical constituents of human atherosclerotic lesions. At least three receptor systems have been characterized that could be involved in the development of foam cells: alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2 MR/LRP), scavenger receptor, and LDL receptor. We studied the expression of these receptors in human atherosclerotic lesions with in situ hybridization and immunocytochemistry. An abundant expression of alpha 2MR/LRP mRNA and protein was found in SMC and macrophages in both early and advanced lesions in human aortas. alpha 2MR/LRP was also present in SMC in normal aortas. Scavenger receptor mRNA and protein were expressed in lesion macrophages but no expression was found in lesion SMC. LDL receptor was absent from the lesion area but was expressed in some aortas in medial SMC located near the adventitial border. The results demonstrate that (a) alpha 2MR/LRP is, so far, the only lipoprotein receptor expressed in lesions SMC in vivo; (b) scavenger receptors are expressed only in lesion macrophages; and (c) both receptors may play important roles in the development of human atherosclerotic lesions.

beta2-glycoprotein-I (apolipoprotein H) and beta2-glycoprotein-I-phospholipid complex harbor a recognition site for the endocytic receptor megalin.

Screening of serum by using a surface plasmon resonance analysis assay identified beta2-glycoprotein-I/apolipoprotein H as a plasma component binding to the renal epithelial endocytic receptor megalin. A calcium-dependent megalin-mediated beta2-glycoprotein-I endocytosis was subsequently demonstrated by ligand blotting of rabbit renal cortex and uptake analysis in megalin-expressing cells. Immunohistochemical and immunoelectron microscopic examination of kidneys and the presence of high concentrations of beta2-glycoprotein-I in urine of mice with disrupted megalin gene established that megalin is the renal clearance receptor for beta2-glycoprotein-I. A significant increase in functional affinity for purified megalin was observed when beta2-glycoprotein-I was bound to the acidic phospholipids, phosphatidylserine and cardiolipin. The binding of beta2-glycoprotein-I and beta2-glycoprotein-I- phospholipid complexes to megalin was completely blocked by receptor-associated protein. In conclusion, we have demonstrated a novel receptor recognition feature of beta2-glycoprotein-I. In addition to explaining the high urinary excretion of beta2-glycoprotein-I in patients with renal tubule failure, the data provide molecular evidence for the suggested function of beta2-glycoprotein-I as a linking molecule mediating cellular recognition of phosphatidylserine-exposing particles.

Identification of rat yolk sac target protein of teratogenic antibodies, gp280, as intrinsic factor-cobalamin receptor.

Previous studies in the rat have shown that antibodies to gp280, a protein > 200 kD and closely associated with the early endocytic system can induce fetal malformations. Although gp280 is thought to act as a receptor, its ligand(s) is not known. In the current study, we report that purified gp280 from rat kidney, like the intrinsic factor-Cobalamin receptor (IFCR), binds to the intrinsic factor-cobalamin (IFCbl) complex with an association constant of 0.3 x 10(9) M-1 and mediates its internalization. Furthermore, antibodies raised to purified gp280 and IFCR inhibited the binding of IF-[57Co]Cbl complex to intestinal, renal, and yolk sac apical membranes and revealed a single identically sized protein on immunoblotting of the renal membranes. Both antibodies precipitated a single radiolabeled protein > 200 kD from cellular extract from [35S]methionine-labeled yolk sac epithelial cells, and antibody to gp280 inhibited the uptake and internalization of 125IF-Cbl. Immunoelectron microscopy using the two antibodies revealed that in the kidney, both proteins were colocalized. These observations suggest that IF-Cbl complex is a ligand for gp280 and that gp280 and IFCR are identical proteins.

Cubilin is an albumin binding protein important for renal tubular albumin reabsorption.

Using affinity chromatography and surface plasmon resonance analysis, we have identified cubilin, a 460-kDa receptor heavily expressed in kidney proximal tubule epithelial cells, as an albumin binding protein. Dogs with a functional defect in cubilin excrete large amounts of albumin in combination with virtually abolished proximal tubule reabsorption, showing the critical role for cubilin in the uptake of albumin by the proximal tubule. Also, by immunoblotting and immunocytochemistry we show that previously identified low-molecular-weight renal albumin binding proteins are fragments of cubilin. In addition, we find that mice lacking the endocytic receptor megalin show altered urinary excretion, and reduced tubular reabsorption, of albumin. Because cubilin has been shown to colocalize and interact with megalin, we propose a mechanism of albumin reabsorption mediated by both of these proteins. This process may prove important for understanding interstitial renal inflammation and fibrosis caused by proximal tubule uptake of an increased load of filtered albumin.

High Plasma Lipid Levels Reduce Efficacy of Adenovirus-Mediated Gene Therapy.

Adenoviruses are very efficient vectors for delivering therapeutic genes in preclinical and clinical trials. However, randomized controlled human trials have often been lacking clear clinically relevant results. We hypothesized that high lipid levels and specific lipoproteins could significantly decrease adenoviral transduction efficiency in vivo. Here we demonstrate that mice on a high fat diet have lower transgene expression compared to mice on a regular chow. In addition, on a high fat diet, ApoE-/- mice have much higher plasma transgene levels compared to LDLR-deficient mice. We also found that specific lipoprotein receptors play an important role in adenoviral transduction. These findings suggest that high plasma lipid levels, especially apoE-containing lipoproteins, reduce efficacy of adenoviral transduction in mice, which implies that high cholesterol levels in humans could be protective against viral infections and also lead to insufficient transgene expression in clinical trials using adenoviral vectors.

Characterization, size estimation and solubilization of alpha-macroglobulin complex receptors in liver membranes.

Receptors for alpha 2-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of 125I-labelled alpha 2-macroglobulin.trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8-9.0. The half-time for association was about 5 min at 37 degrees C in contrast to about 5 h at 4 degrees C. The half-saturation constant was about 100 pM at 4 degrees C and 1 nM at 37 degrees C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 +/- 71 kDa (S.D., n = 7) for alpha 2-macroglobulin.trypsin binding activity. Affinity cross-linking to rat and human membranes of 125I-labelled rat alpha 1-inhibitor-3.chymotrypsin, a 210 kDa analogue which binds to the alpha 2-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55-60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked alpha 2-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound 125I-alpha 1-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]propane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400-500 kDa alpha 2-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.

Binding and receptor-mediated endocytosis of pregnancy zone protein-proteinase complex in rat macrophages.

125I-labelled pregnancy zone protein complex was injected intravenously in rats and after 6 min uptake into cells of the liver and spleen was determined by electron microscopic autoradiography. The liver took up 68% of the injected radioactivity; 61% was in the hepatocytes and 7% was in the liver macrophages (Kupffer cells). The spleen took up 3-4% and nearly all the radioactivity was in the macrophages of the red pulp. The uptake per cell volume was several times higher in the macrophage than in the hepatocyte. The radioactivity associated with macrophages was largely in endocytotic vacuoles and lysosomes. Binding of labelled pregnancy zone protein complex to peritoneal macrophages at 4 degrees C was 2-3 times higher than binding of the homologous alpha 2-macroglobulin complex. The two ligands competed for binding to the same receptors and the difference was due to a higher affinity of the pregnancy zone protein complex (Kd approx. 60 pM). After binding to the receptor, this ligand was internalised within 2-3 min at 37 degrees C and radioactivity inside the cells largely represented intact pregnancy zone protein complex. Radioactivity was released from the cell as iodotyrosine after a lag time of about 10 min. It is concluded that pregnancy zone protein complex is bound with a high affinity to the alpha 2-macroglobulin receptors in rat macrophages followed by receptor-mediated endocytosis and degradation of the ligand in the lysosomes.

The complete amino acid sequence of human placental oxytocinase.

The complete amino acid sequence of human placental oxytocinase (placental leucine aminopeptidase) has been determined by cDNA cloning and sequencing. Oxytocinase is a type II integral membrane protein of 1025 amino acid residues, consisting of an acidic intracellular region of 110 amino acids followed by a hydrophobic transmembrane segment of 22 residues and 893 extracellular residues containing the characteristic Zn2+ coordination sequence element His-Glu-Xaa-Xaa-His-(18 residues)-Glu found in gluzincins. Two sets of cDNA clones with different 5'-ends were isolated and suggested to represent different spliced products of 3.6 kb (mature mRNA) and 12 kb, respectively. Oxytocinase mRNA is present in large amounts in placenta, heart and skeletal muscle and in small amounts in brain, kidney, liver and pancreas. A conserved sequence element, the GAMEN motif, which distinguishes the aminopeptidase family among gluzincins from other gluzincins, has been identified.

Membrane receptors for endocytosis in the renal proximal tubule.

The renal proximal tubule exhibits a very extensive apical endocytic apparatus consisting of an elaborate network of coated pits and small coated and noncoated endosomes. In addition, the cells contain a large number of late endosomes/prelysosomes, lysosomes, and so-called dense apical tubules involved in receptor recycling from the endosomes to the apical plasma membrane. This endocytic apparatus is involved in the reabsorption of molecules filtered in the glomeruli. The process is very effective as demonstrated by the fact that although several grams of protein are filtered daily in the human glomeruli, human urine is virtually devoid of proteins under physiological conditions. Several key receptors appear to be involved in this function, which serves not only to conserve protein as such for the organism but also to reabsorb vital substances such as different vitamins in complex with their binding proteins. Recent research has established megalin, a 600-kDa protein belonging to the LDL receptor family, as probably the most important receptor in this process in the proximal tubule mediating endocytosis of a large variety of ligands and therefore classifying it as a scavenger receptor. More specific receptors like the folate receptor, IGF-II/Man-6-P receptor, and gp280/IFR, identical to the intrinsic factor receptor, are also functioning in the apical endocytic pathway of renal proximal tubules. A better understanding of these receptors will give us new insight into these very important processes for the organism.

Predictive value of soluble haemoglobin scavenger receptor CD163 serum levels for survival in verified tuberculosis patients.

Pre-treatment serum levels of sCD163 were measured in a cohort of 236 suspected tuberculosis (TB) cases from Guinea-Bissau, with a median follow-up period of 3.3 years (range 0-6.4 years). In 113 cases, the diagnosis of TB was verified by positive sputum microscopy and/or culture. Among the verified TB cases, a decreased survival rate was found in 27 patients with sCD163 levels above the upper reference limit (3.95 microg/mL). The difference in survival was significant during TB treatment (log rank, p<0.02) and after long-term follow-up (log rank, p<0.001). The decrease in survival rate during TB treatment remained significant in a multivariate Cox model controlling for human immunodeficiency virus (HIV) status, age and gender, with a mortality increase of 1.19 (95% CI, 1.04-1.36) per microg of sCD163, and a hazard ratio (HR) for sCD163 levels above the upper reference limit of 4.18 (95% CI, 1.06-16.4). The difference was not significant after excluding patients with concomitant HIV-1 and HIV-2 infection in Kaplan-Meier analyses (log rank, p 0.11). In contrast, the difference in survival remained significant in Kaplan-Meier analyses after long-term follow-up, even after excluding patients with concomitant HIV-1 and HIV-2 infection (log rank, p 0.002). In the Cox model, the mortality increase per microg of sCD163 was 1.27 (95% CI, 1.14-1.40), with an HR for elevated sCD163 levels of 2.85 (95% CI, 1.44-5.63). The HRs for concomitant HIV-1 and HIV-2 infection were 6.92 (95% CI, 3.28-14.58) and 2.48 (95% CI, 1.09-5.67), respectively. Thus, sCD163 levels appeared to be an independent predictor of survival in verified TB patients.

Segmental distribution of the endocytosis receptor gp330 in renal proximal tubules.

The subcellular distribution and segmental variations in location of gp330, a scavenger receptor for filtered proteins in renal proximal tubules, was analyzed. Kidney tissue from rats (4 different strains), rabbits and humans were analyzed by light- and electron microscope immunocytochemistry, using cryosections or Lowicryl sections from cryosubstituted tissue. Gp330 was located mainly in apical coated pits, small and large endocytic vacuoles and in dense apical tubules in the proximal tubule cells. The labeling density was markedly higher in segments 1 and 2 as compared to segment 3 of the proximal tubule. In addition to the location in the early part of the endocytic pathway, gp330 was also present in lysosomes, especially in segments 1 and 2. The lysosomal labeling was not restricted to the membrane, but was also seen in the matrix. Localization of gp330 in lysosomes was confirmed on sections from purified lysosomal fractions from rat renal cortex. The brush border localization of gp330 in proximal tubules exhibited a characteristic segmental variation. In the initial part of segment 1, there was virtually no brush border labeling. In the remaining part of segment 1 and in segment 2, there was a distinct but sometimes patchy labeling of the brush border. In segment 3, groups of microvilli of approximately 10 as seen in sections were intensively labeled from bottom to tip and there were often more than one of these groups on a single cell, the remaining microvilli were unlabeled. No differences in the cellular and subcellular localization of gp330 were observed between species or rat strains. In conclusion, the present study demonstrates that in addition to its location in the early endocytic and recycling pathway, gp330 is also present in microvilli and the protein and degradation products thereof is present in lysosomes, consistent with its role as a protein scavenger receptor.

Purification of the human placental alpha 2-macroglobulin receptor.

The alpha 2-macroglobulin receptor was solubilized from human placental membranes, purified and characterized. Affinity cross-linking of labelled ligand to intact membranes showed a receptor size compatible with 400-500 kDa. The membranes were solubilized in 3-[(3-cholamidopropyl)dimethylammonio]propane sulfonate (CHAPS) and affinity chromatography was performed using Sepharose-immobilized alpha 2-macroglobulin-methylamine with elution in buffer containing 2 mM EDTA, pH 6.0. SDS-PAGE of the resulting receptor preparation showed a predominant approx. 440 kDa band (reducing conditions) and some minor accompanying proteins of 70-90 kDa and 40 kDa. The yield was 400-800 micrograms receptor preparation per placenta. The receptor preparation immobilized on nitrocellulose bound the alpha 2-macroglobulin-trypsin complex with a dissociation constant of about 400 pM. 125I-iodinated receptor preparation bound almost quantitatively to Sepharose-immobilized alpha 2-macroglobulin-methylamine in the presence of CHAPS alone, and bound 70-80% in the presence of 0.2% SDS. The labelled proteins were separated in the presence of 0.2% SDS by gel filtration or SDS-PAGE (unboiled samples). The 440 kDa protein accounted for the major part of the binding, although some approx. 80 kDa proteins, perhaps proteolytic degradation products, also showed binding activity.

Evidence that the newly cloned low-density-lipoprotein receptor related protein (LRP) is the alpha 2-macroglobulin receptor.

The human placental receptor (alpha 2MR) for alpha 2-macroglobulin-proteinase complexes contains 3 polypeptides of approx. 500 kDa, 85 kDa, and 40 kDa. N-terminal sequence analysis of the 500 kDa and 85 kDa polypeptides, analysis of a random selection of peptides convering 536 residues from these polypeptides, and analysis of a 1772 bp cDNA encoding part of the 500 kDa polypeptide provide evidence that the 500 kDa and 85 kDa chains are the alpha- and beta-subunits, respectively, of a recently cloned hepatic membrane protein, termed the low density lipoprotein receptor related protein (LRP) (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H. and Stanley, K.K. (1988) EMBO J. 7, 4119-4127; Herz, J., Kowal, R.C., Goldstein, J.L. and Brown, M.S. (1990) EMBO J. 9, 1769-1776). N-terminal sequence analysis of the 40 kDa polypeptide shows that it is of distinct genetic origin. It is suggested that LRP is the functional receptor for alpha 2-macroglobulin-proteinase complexes (alpha 2MR) and in addition may have as yet unsettled functions in lipoprotein metabolism.

Receptor-binding domain of human alpha 2-macroglobulin. Expression, folding and biochemical characterization of a high-affinity recombinant derivative.

A recombinant version of the receptor binding domain (RBDv) of human alpha 2-macroglobulin (alpha 2M) has been expressed in E. coli and refolded using a novel iterative procedure. RBDv (Val1299-Ala1451) is extended by 15 residues at the N-terminal side of the Lys1313-Glu papain cleavage site in human alpha 2M. RBDv contains the intra-chain bridge Cys1329-Cys1444 and is soluble and monomeric. Competition experiments with 125I-labelled methylamine-treated alpha 2M reveal that RBDv binds to the placental receptor for transformed alpha 2M with a Kd of 8 nM, i.e. the binding affinity of RBDv is of the same order of magnitude as the intrinsic affinity for binding of one domain in transformed alpha 2M to one receptor molecule.