Glycoprotein 330, a member of the low density lipoprotein receptor family, binds lipoprotein lipase in vitro

Glycoprotein 330 (gp330), a cell-surface protein that is localized in clathrin-coated pits, is structurally related to both the low density lipoprotein receptor (LDLR) and the LDLR-related protein/alpha 2-macroglobulin receptor (LRP). We recently demonstrated that gp330 and LRP may be functionally related as well; both bind the 39-kDa polypeptide referred to as receptor-associated protein (Kounnas, M. Z., Argraves, W. S., and Strickland, D. K. (1992) J. Biol. Chem. 267, 21162-21166). In this report, we tested several other LRP ligands for their ability to interact with human and rat gp330 in vitro. Gp330 did not exhibit detectable binding to the LRP ligands, alpha 2-macroglobulin protease complex or Pseudomonas aeruginosa exotoxin A. However, we found that gp330 (purified from human or rat) bound the lipolytic enzyme lipoprotein lipase (LPL) with high affinity (Kd = 6.1 and 2.7 nM, respectively). The binding was saturable, divalent cation dependent, and inhibited by heparin or receptor-associated protein. Because LRP has also been shown to bind LPL, the present findings further extend the functional similarities between gp330 and LRP. By analogy to the postulated role of the LRP-LPL interaction in facilitating hepatic clearance of LPL-associated lipoproteins from the blood (Beisiegel, U., Weber, W., and Bengtsson-Olivercrona, G. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8342-8346; Chappell, D. A., Fry, G. L., Waknitz, M. A., Iverius, P. H., Williams, S. E., and Strickland, D. K. (1992) J. Biol. Chem. 267, 25764-25767), we speculate that the gp330-LPL interaction described herein may contribute to the uptake of LPL-associated lipoproteins in tissues expressing gp330. Consistent with this possibility, we found that LPL promoted in vitro binding of 125I-lipoproteins to gp330.     

Cerebrovascular transport of Alzheimer's amyloid beta and apolipoproteins J and E: possible anti-amyloidogenic role of the blood-brain barrier

It is uncertain whether soluble circulating amyloid beta (sA beta) is the precursor of amyloid beta (A beta) found in cerebrovascular and parenchymal amyloid lesions in Alzheimer's Disease, and if so, how the transition to the filamentous form is brought about. Several lines of evidence suggest that apolipoprotein E (apoE) and apolipoprotein J (apoJ) may be involved in the regulation of amyloidogenesis. They both bind sA beta/A beta in vivo and in vitro. It has been suggested that apoE may modulate beta-pleated conformation of A beta and therefore act as a proamyloidogenic factor. On the other hand, apoJ as a major carrier protein of sA beta in body fluids may keep the peptide in a soluble form, thus having an anti-amyloidogenic effect. Using a well established guinea-pig brain perfusion model we have studied the blood-brain barrier (BBB) processes involved in the regulation of cerebral capillary sequestration, transport and metabolism of i) sA beta 1-40 and sA beta 1-42, synthetic peptides identical to the 40 and 42 residue forms of A beta, found primarily in vascular deposits and senile plaques, respectively; and ii) apoJ, apoE3 and apoE4 alone, and in a complex with sA beta. Specific saturable BBB luminal binding of both peptides was followed by transport into brain parenchyma and metabolism at the abluminal side of the BBB and/or in brain. The capillary sequestration of sA beta 1-40 was significant, while retention by the microvasculature of sA beta 1-42 was negligible. Binding to microvessels and blood-to-brain transport of both intact apoJ and sA beta 1-40 apoJ complexes were among the highest ever recorded for peptides and proteins at the BBB in vivo. These processes appear to be mediated by glycoprotein 330 (gp330/megalin), a receptor for multiple ligands, including apoJ. In contrast, capillary retention and transport of apoE3, apoE4 and sA beta 1-40-apoE3 complex were low to negligible, while blood-brain transport of sA beta 1-40-apoE4 was moderate. It is suggested that normal BBB may have predominantly anti-amyloidogenic functions by i) degrading sA beta during blood-to-brain transport; ii) favoring sequestration and transport of apoJ alone and in complex with sA beta via gp330 receptor-mediated mechanism and iii) excluding apoE3 and apoE4 isoforms from brain.     

Molecular analysis of ligand binding to the second cluster of complement-type repeats of the low density lipoprotein receptor-related protein. Evidence for an allosteric component in receptor-associated protein-mediated inhibition of ligand binding

The low density lipoprotein receptor-related protein (LRP), a member of the low density lipoprotein receptor gene family, mediates the cellular uptake of a diversity of ligands. A folding chaperone, the 39-kDa receptor-associated protein (RAP) that resides in the early compartments of the secretory pathway inhibits the binding of all ligands to the receptor and may serve to prevent premature binding of ligands to the receptor during the trafficking to the cell surface. To elucidate the molecular interactions that underlie the interplay between the receptor, RAP, and the ligands, we have analyzed and delineated the binding sites of plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (t-PA).PAI-1 complexes, RAP, and the anti-LRP Fab fragment Fab A8. To that end, we have generated a series of soluble recombinant fragments spanning the second cluster of complement-type repeats (C3-C10) and the amino-terminal flanking epidermal growth factor repeat (E4) of LRP (E4-C10; amino acids 787-1165). All fragments were expressed by stably transfected baby hamster kidney cells and purified by affinity chromatography. A detailed study of ligand binding to the fragments using surface plasmon resonance revealed the presence of three distinct, Ca2+-dependent ligand binding sites in the cluster II domain (Cl-II) of LRP. t-PA.PAI-1 complexes as well as PAI-1 bind to a domain located in the amino-terminal portion of Cl-II, spanning repeats E4-C3-C7. Adjacent to this site and partially overlapping is a high affinity RAP-binding site located on repeats C5-C7. Fab A8, a pseudo-ligand of the receptor, binds to a third Ca2+-dependent binding site on repeats C8-C10 at the carboxyl-terminal end of Cl-II. Next, we studied the RAP-mediated inhibition of ligand binding to LRP and to Cl-II. As expected, we observed a strong inhibition of t-PA.PAI-1 complex and Fab A8 binding to LRP by RAP (IC50 congruent with 0.3 nM), whereas in the reverse experiment, competition of t-PA. PAI-1 complexes and Fab A8 for RAP binding to LRP could only be shown at high concentrations of competitors (>/=1 microM). Interestingly, even though the equilibrium dissociation constants for the binding of RAP to LRP and to Cl-II are similar, the binding of the ligands to Cl-II is only prevented by RAP at concentrations that are at least 2 orders of magnitude higher than those required for inhibition of ligand binding to LRP. Our results favor models that propose RAP-induced allosteric inhibition of ligand binding to LRP that may require LRP moieties that are located outside Cl-II of the receptor.     

Megalin (gp330): a putative endocytic receptor for thyroglobulin (Tg)

Megalin (gp330) is a large glycoprotein receptor found mainly on a group of absorptive epithelial cells, including renal proximal tubule, epididymal and thyroid cells. Megalin has been shown to bind multiple, unrelated ligands, mainly in vitro, and to mediate endocytosis of ligandsin cultured cells. However, physiologic ligands of megalin are largely unknown. In the present study we have demonstrated that purified rat megalin binds rat thyroglobulin (Tg) in solid phase assays, with anestimated Kd of 9.2+/-0.6 nM. Binding was calcium dependent and was almost completely inhibited by excess Tg, by three megalin ligands - lactoferrin, lipoprotein lipase and apolipoprotein J- and by the receptor associated protein (RAP), which inhibits binding of all megalin ligands. Three anti-megalin antibodies partially inhibited Tg binding to megalin. 125I labeled Tg bound to megalin was released by EDTA and heparin; the released product was shown by SDS-PAGE and autoradiography to be 660 kD (dimeric) Tg. However, an immunoblotting experiment showed binding of megalin both to monomeric (330 kD) and dimeric Tg. We propose that megalin, which is known to mediate ligand endocytosis and is found on the apical surface of thyrocytes, may participate in the endocytosis of Tg from the colloid, a process that is required for hormone release from Tg.     

Lipoprotein lipase induces catabolism of normal triglyceride-rich lipoproteins via the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor in vitro. A process facilitated by cell-surface proteoglycans

Bovine milk lipoprotein lipase (LPL) induced binding, uptake, and degradation of 125I-labeled normal human triglyceride-rich lipoproteins by cultured mutant fibroblasts lacking LDL receptors. The induction was dose-dependent and occurred whether LPL and 125I-lipoproteins were added to incubation media simultaneously or LPL was allowed to bind to cell surfaces, and unbound LPL was removed by washing prior to the assay. Lipolytic modification of lipoproteins did not appear to be necessary for increased catabolism because the effect of LPL was not prevented by inhibitors of LPL's enzymatic activity, p-nitrophenyl N-dodecylcarbamate or phenylmethylsulfonyl fluoride. However, the effect was abolished by boiling LPL prior to the assay suggesting that major structural features of LPL were required. Also, LPL-induced binding to cells was blocked by an anti-LPL monoclonal antibody but not by antibodies that are known to block apolipoprotein E- or B-100-mediated binding to low density lipoprotein (LDL) receptors. This indicates that LPL itself mediated 125I-lipoprotein binding to cells. Cellular degradation of 125I-lipoproteins was partially or completely blocked by two previously described ligands for the LDL receptor-related protein/alpha 2-macroglobulin receptor (LRP): activated alpha 2-macroglobulin (alpha 2M*), and the 39-kDa receptor-associated protein. These data implicated LRP as mediating LPL-induced lipoprotein degradation and were confirmed by showing that LPL's effects were prevented by an immunoaffinity-isolated polyclonal antibody against LRP. Furthermore, LPL promoted binding of 125I-lipoproteins to highly purified LRP in a solid-phase assay. Heparin or heparinase treatment of cells markedly decreased LPL-induced binding, uptake, and degradation of lipoproteins, but had no effect on catabolism of alpha 2M*. Thus, cell-surface proteoglycans were obligatory participants in the effects of LPL but were not required for LRP-mediated catabolism of alpha 2M*. Taken together, these in vitro findings establish that through interaction with cell-surface proteoglycans, LPL induces catabolism of normal human triglyceride-rich lipoproteins via LRP.     

Financing long-term care for elderly persons: what are the options?

We report studies of the interaction of Alzheimer's amyloid beta protein (A beta) with normal human plasma high density lipoprotein (HDL), aiming to clarify to which lipoprotein (LP) structural constituent (apolipoprotein or lipid) soluble A beta is primarily bound. Purified HDLs were incubated with biotinylated A beta 1-40 followed by LP repurification by size exclusion (SE) HPLC. SDS-PAGE, immunoblot and N-terminal sequence analysis of the biotin-A beta positive protein bands revealed that A beta is bound to many apolipoproteins of the HDL, mainly apoA-I, apoA-II, apoE and apoJ. On the other hand, reconstituted, protein-free HDL lipid particles also bind A beta peptide and inhibit its aggregation, as intact HDL does. This was assessed by SE-HPLC, SDS-PAGE, immunoblot analysis, ultrastructural electron microscopy and Congo Red staining for beta amyloid fibrils. Our data imply that A beta binding to lipids may play an important role in maintaining the peptide in solution and thus be particularly relevant to A beta normal and pathologic biochemistry and physiology.     

Specificity of serine proteinase/serpin complex binding to very-low-density lipoprotein receptor and alpha2-macroglobulin receptor/low-density-lipoprotein-receptor-related protein

Very-low-density lipoprotein receptor (VLDLR) and alpha2-macroglobulin receptor/low-density-lipoprotein-receptor-related protein (alpha2MR/LRP) are multifunctional endocytosis receptors of the low-density lipoprotein receptor family. Both have been shown to mediate endocytosis and degradation of complex between plasminogen activators and type-1 plasminogen-activator inhibitor (PAI-1) by cultured cells. We have now studied the specificity of binding and endocytosis by VLDLR and alpha2MR/LRP among a variety of serine proteinase/serpin complexes, including various combinations of the serine proteinases urokinase-type and tissue-type plasminogen activators, plasmin, thrombin, human leukocyte elastase, cathepsin G, and plasma kallikrein with the serpins PAI-1, horse leukocyte elastase inhibitor, protein C inhibitor, C1-inhibitor, alpha2-antiplasmin, alpha1-proteinase inhibitor, alpha1-antichymotrypsin, protease nexin-1, heparin cofactor II, and antithrombin III. Binding was estimated with radiolabelled ligands in ligand blotting analysis and microtiter well assays. Endocytosis was estimated by measuring receptor-associated protein (RAP)-sensitive degradation of radiolabelled complexes by Chinese hamster ovary cells transfected with VLDLR cDNA and by COS-1 cells, which have a high endogenous expression of alpha2MR/LRP. We found that the receptors bind with high affinity to some, but not all, combinations of plasminogen activators and thrombin with PAI-1, protease nexin-1, protein C inhibitor, and antithrombin III, while complexes of many serine proteinases with their primary inhibitor, i.e. plasmin/alpha2-antiplasmin complex, do not bind, or bind with a very low affinity. Both the serine proteinase and the serpin moieties contribute to the binding specificity. The binding specificities of VLDLR and alpha2MR/LRP are overlapping, but not identical. The results suggest that VLDLR and alpha2MR/LRP have different biological functions by having different binding specificities as well as by being expressed by different cell types.     

The binding of receptor-recognized alpha2-macroglobulin to the low density lipoprotein receptor-related protein and the alpha2M signaling receptor is decoupled by oxidation

Receptor-recognized forms of alpha2-macroglobulin (alpha2M*) bind to two classes of cellular receptors, a high affinity site comprising approximately 1500 sites/cell and a lower affinity site comprising about 60,000 sites/cell. The latter class has been identified as the so-called low density lipoprotein receptor-related protein (LRP). Ligation of receptors distinct from LRP activates cell signaling pathways. Strong circumstantial evidence suggests that the high affinity binding sites are responsible for cell signaling induced by alpha2M*. Using sodium hypochlorite, a powerful oxidant produced by the H2O2-myeloperoxidase-Cl- system, we now demonstrate that binding to the high affinity sites correlates directly with activation of the signaling cascade. Oxidation of alpha2M* using 200 microM hypochlorite completely abolishes its binding to LRP without affecting its ability to activate the macrophage signaling cascade. Scatchard analysis shows binding to a single class of high affinity sites (Kd - 71 +/- 12 pM). Surprisingly, oxidation of native alpha2-macroglobulin (alpha2M) with 125 microM hypochlorite results in the exposure of its receptor-binding site to LRP, but the ligand is unable to induce cell signaling. Scatchard analysis shows binding to a single class of lower affinity sites (Kd - 0.7 +/- 0.15 nM). Oxidation of a cloned and expressed carboxyl-terminal 20-kDa fragment of alpha2M (RBF), which is capable of binding to both LRP and the signaling receptor, results in no significant change in its binding Kd, supporting our earlier finding that the oxidation-sensitive site is predominantly outside of RBF. Attempts to understand the mechanism responsible for the selective exposure of LRP-binding sites in oxidized native alpha2M suggest that partial protein unfolding may be the most likely mechanism. These studies provide strong evidence that the high affinity sites (Kd - 71 pM) are the alpha2M* signaling receptor.     

Ca2+ and receptor-associated protein are independently required for proper folding and disulfide bond formation of the low density lipoprotein receptor-related protein

The low density lipoprotein receptor-related protein (LRP) is a cysteine-rich, multifunctional receptor that binds and endocytoses a diverse array of ligands. Recent studies have shown that a 39-kDa receptor-associated protein (RAP) facilitates the proper folding and subsequent trafficking of LRP within the early secretory pathway. In the current study, we have examined the potential role of Ca2+ and its relationship to RAP during LRP folding. We found that depletion of Ca2+ following either ionomycin or thapsigargin treatment significantly disrupts the folding process of LRP. The misfolded LRP molecules migrate as high molecular weight aggregates under nonreducing SDS-polyacrylamide gel electrophoresis, suggesting the formation of intermolecular disulfide bonds. This misfolding is reversible because misfolded LRP can be re-folded into functional receptor molecules upon Ca2+ restoration. Using an LRP minireceptor representing the fourth ligand binding domain of LRP, we also observed significant variation in the conformation of monomeric receptor upon Ca2+ depletion. The role of Ca2+ in LRP folding is independent from that of RAP because RAP remains bound to LRP and its minireceptor following Ca2+ depletion. Furthermore, Ca2+ depletion-induced LRP misfolding occurs in RAP-deficient cells. Taken together, these results clearly demonstrate that Ca2+ and RAP independently participate in LRP folding.     

Domain organization of the 39-kDa receptor-associated protein

The 39-kDa receptor-associated protein (RAP) is an endoplasmic reticulum resident protein that binds to the low density lipoprotein receptor-related protein (LRP) as well as certain members of the low density lipoprotein receptor superfamily and antagonizes ligand binding. In order to identify important functional regions of RAP, studies were performed to define the domain organization and domain boundaries of this molecule. Differential scanning calorimetry (DSC) experiments revealed that the process of thermal denaturation of RAP is highly reversible and occurs in a broad temperature range with two well resolved heat absorption peaks. A good fit of the endotherm was obtained with four two-state transitions suggesting these many cooperative domains in the molecule. A number of recombinant fragments of RAP were expressed in bacteria, and their domain composition and stability were characterized by DSC, circular dichroism, and fluorescence spectroscopy. The results confirmed that RAP is composed of four independently folded domains, D1, D2, D3, and D4, that encompass residues 1-92, 93-163, 164-216, and 217-323, respectively. The first and the fourth domains preserved their structure and stability when isolated, whereas the compact structure of the fragment corresponding to D2 seems to be altered when isolated from the parent molecule. Isolated D3 was partially degraded during isolation from bacterial lysates. The isolated D4 was capable of binding with high affinity to LRP whereas neither D1 nor D2 bound. At the same time a fragment containing both D1 and D2 exhibited high affinity binding to LRP. These facts combined with the thermodynamic analysis of the melting process of the fragments containing D1 and D2 indicate that these two domains interact with each other and that the proper folding of the second domain into a native-like active conformation requires presence of the first domain.     

A new low density lipoprotein receptor related protein, LRP5, is expressed in hepatocytes and adrenal cortex, and recognizes apolipoprotein E

The isolation and characterization of rabbit and human cDNAs revealed a new low density lipoprotein receptor (LDLR)-related protein (LRP) designated as LRP5. Human LRP5 cDNA encodes a 1, 616-amino acid type I membrane-like protein with three ligand binding repeats in its extracellular region. LDLR-deficient cells transduced by recombinant adenovirus containing human LRP5 exhibited increased binding of apolipoprotein E (apoE)-enriched beta-migrating very low density lipoprotein. Northern blotting and in situ hybridization revealed a high level of LRP5 expression in hepatocytes and the adrenal gland cortex. In LDLR-deficient Watanabe heritable hyperlipidemic rabbits, LRP5 mRNA was increased in the liver and accumulated in cholesterol-laden foam cells of atherosclerotic lesions.     

Interaction of apolipoprotein J-amyloid beta-peptide complex with low density lipoprotein receptor-related protein-2/megalin. A mechanism to prevent pathological accumulation of amyloid beta-peptide

Apolipoprotein J (apoJ) has been shown to be the predominant amyloid beta-peptide (Abeta)-binding protein in cerebrospinal fluid. We have previously demonstrated that the endocytic receptor low density lipoprotein receptor-related protein-2/megalin (LRP-2), which is expressed by choroid plexus epithelium and ependymal cells lining the brain ventricles and neural tube, binds and mediates cellular uptake of apoJ (Kounnas, M. Z., Loukinova, E. B., Stefansson, S., Harmony, J. A., Brewer, B., Strickland, D. K., and Argraves, W. S. (1995) J. Biol. Chem. 270, 13070-13075). In the present study, we evaluated the ability of apoJ to mediate binding of Abeta1-40-apoJ complex to LRP-2 in vitro. Immunoblot analysis showed that incubation of apoJ with Abeta1-40 resulted in the formation of Abeta1-40-apoJ complex and the inhibition of the formation of Abeta1-40 aggregates. Using an enzyme-linked immunosorbent assay, an estimated dissociation constant (Kd) of 4.8 nM was derived for the interaction between Abeta1-40 and apoJ. Enzyme-linked immunosorbent assay was also used to study the interaction of the Abeta1-40-apoJ complex with LRP-2. The results showed that Abeta alone did not bind directly to LRP-2; however, when Abeta1-40 was combined with apoJ to form a complex, binding to LRP-2 took place. The binding interaction could be blocked by inclusion of the receptor-associated protein, an antagonist of apoJ binding to LRP-2. When LRP-2-expressing cells were given 125I-Abeta1-40, cellular uptake of the radiolabeled peptide was promoted by co-incubation with apoJ. When the cells were provided purified 125I-Abeta1-40-apoJ complex, the complex was internalized and degraded, and both processes were inhibited with polyclonal LRP-2 antibodies. Furthermore, chloroquine treatment inhibited the cellular degradation of the complex. The data indicate that apoJ facilitates Abeta1-40 binding to LRP-2 and that the receptor mediates cellular clearance of Abeta1-40-apoJ complex leading to lysosomal degradation of Abeta1-40. The findings support the possibility that LRP-2 can act in vivo to mediate clearance of the complex from biological fluids such as cerebrospinal fluid and thereby play a role in the regulation of Abeta accumulation.     

Receptors of the low density lipoprotein (LDL) receptor family in man. Multiple functions of the large family members via interaction with complex ligands

The LDL receptor family members are endocytic receptors composed of repeated protein modules, including clusters of ligand binding LDL receptor class A (LA) repeats. The large (approximately 600 kDa) members LRP and megalin bind numerous structurally unrelated and often complex ligands at different combinations of sites. LRP is expressed in a wide but restricted set of cell types including hepatocytes, macrophages, smooth muscle cells, and neurons of the CNS. Megalin is expressed in various epithelia including proximal kidney tubules, intestine, and ependymal cells. The two receptors share a multitude of ligands, and their function in vivo is therefore to a large extent determined by their expression pattern. For example, both receptors can endocytose lipoproteins, but this function appears mainly relevant for LRP. In addition, LRP helps regulating urokinase receptor expression on the cell surface via ligand-mediated internalization followed by return of the naked urokinase receptor to the cell surface. Both receptors also have specialist functions. LRP is specific for binding of alpha2-macroglobulin-proteinase complexes and provides clearance of the complexes and of peptides, e.g. cytokines, associated with the complex. Megalin has important functions in vitamin B12 homeostasis since it specifically mediates uptake of the vitamin B12-transcobalamin complex and helps building a storage pool for the vitamin in the kidneys. Moreover, megalin binds cubilin, the recently identified receptor for B12-intrinsic factor complex, thus providing a mechanism for uptake of dietary vitamin B12. Finally, megalin specifically mediates uptake of apolipoprotein J/clusterin, a binding protein for the Abeta peptide implicated in Alzheimer's disease. The binding of multiple complex ligands that belong to distinct physiological systems provides a challenge in future studies aiming at elucidating the role of LRP and megalin in disease mechanisms.     

Identification of the second cluster of ligand-binding repeats in megalin as a site for receptor-ligand interactions

Megalin is a large cell surface receptor that mediates the binding and internalization of a number of structurally and functionally distinct ligands from the lipoprotein and protease:protease inhibitor families. To begin to address how megalin is able to bind ligands with unique structurally properties, we have mapped a binding site for apolipoprotein E (apoE)-beta very low density lipoprotein (beta VLDL), lipoprotein lipase, aprotinin, lactoferrin, and the receptor-associated protein (RAP) within the primary sequence of the receptor. RAP is known to inhibit the binding of all ligands to megalin. We identified a ligand-binding site on megalin by raising mAb against purified megalin, selected for a mAb whose binding to megalin is inhibited by RAP, and mapped the epitope for this mAb. mAb AC10 inhibited the binding of apoE-beta VLDL, lipoprotein lipase, aprotinin, and lactoferrin to megalin in a concentration-dependent manner. When cDNA fragments encoding the four cysteine-rich ligand-binding repeats in megalin were expressed in a baculovirus system and immunoblotted with AC10, it recognized only the second cluster of ligand-binding repeats. The location of the epitope recognized by mAb AC10 within this domain was pinpointed to amino acids 1111-1210. From these studies we conclude that the binding of apoE-beta VLDL, lactoferrin, aprotinin, lipoprotein lipase, and RAP to megalin is either competitively or sterically inhibited by mAb AC10 suggesting that these ligands bind to the same or closely overlapping sites within the second cluster of ligand-binding repeats.     

Lipoprotein receptors in acute myelogenous leukemia: failure to detect increased low-density lipoprotein (LDL) receptor numbers in cell membranes despite increased cellular LDL degradation

The high-affinity degradation of low-density lipoprotein (LDL) is enhanced 3- to 100-fold in leukemic blood cells from patients with acute myelogenous leukemia (AML), suggesting an increased cellular LDL receptor expression. There are, however, inconsistencies regarding the published properties of LDL receptor regulation in AML cells, and previous data on this are indirect. In the present study the aim was to determine whether the LDL receptor number is increased in AML cells. The LDL receptor number was assayed by ligand blot with rabbit 125I-labeled beta-very-low-density lipoprotein (beta-VLDL) of transferred, SDS-polyacrylamide-gel-electrophoresis-separated AML cell membranes. Samples from 10 patients, six with AML, one with chronic myelogenous leukemia in blast crisis, and three with acute lymphoblastic leukemia, were investigated. The LDL receptor expression was strongly suppressed in all samples to levels lower than that of normal mononuclear cells. This was despite the fact that cells from one patient with AML of M4 subtype had a 50- to 100-fold higher 125I labeled LDL degradation compared with normal cells. Immunoblots with antibodies against gp330/megalin and the LDL-receptor-related protein (LRP) and ligand blot using 125I-labeled 39-kd receptor-associated protein (RAP) could not detect gp330/megalin or VLDL receptors. The LRP was abundant in AML samples of M4 and M5b subtype, as determined from both RAP ligand blot and immunoblot using an LRP-specific antibody. It is concluded that LDL receptors are suppressed in AML cells. It is possible that the high degradation of 125I-labeled LDL present in type M4 and M5 AML cells may involve another lipoprotein receptor.     

Hepatitis B virus surface antigen binds to apolipoprotein H

We have previously demonstrated that a plasma membrane-enriched fraction isolated from human liver is capable of binding recombinant hepatitis B surface antigen (rHBsAg) (P. Pontisso, M. A. Petit, M. Bankowski, and M. E. Peeples, J. Virol. 63:1981-1988, 1989). In this study we have separated the plasma membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used a ligand-blotting technique to identify a 46-kDa rHBsAg-binding protein. This protein could be removed from the membranes with a weakly acidic buffer, implying that it is peripherally bound. Examination of human serum revealed that the 46-kDa binding protein is a serum protein. Isolation of plasma lipoproteins revealed that the binding protein is in part associated with chylomicrons and high-density lipoproteins, both of which are targeted to the hepatocyte during the normal course of lipid metabolism. The binding protein was identified as apolipoprotein H (apo H), also known as beta 2-glycoprotein I, on the basis of copurification of the rHBsAg-binding activity with the apo H protein and the ability of cDNA-expressed apo H to bind rHBsAg. Serum-derived HBsAg also binds to apo H, indicating that binding is not unique to rHBsAg. Binding is saturable, requires only the small S protein of rHBsAg, and is inhibited by excess rHBsAg, antibodies to HBsAg, and antibodies to apo H. The binding activity of apo H is destroyed upon reduction, indicating that 1 or more of its 22 disulfide bonds are required for interaction with rHBsAg. The possibility that an interaction between hepatitis B virus particles and lipoprotein particles may facilitate entry of the virus into hepatocytes is discussed.     

Prenatal onset of axonopathy in Dystonia musculorum mice

CCR5 is a chemokine receptor expressed by T cells and macrophages, which also functions as the principal coreceptor for macrophage (M)-tropic HIV-1 strains to enter the host cells. In this study, we aim to better understand the ligand-binding profiles of CCR5 and the chemokine-receptor usage on leukocyte cells. We found that MCP-2 could bind to CCR5 transfectants with high affinity and cross-compete effectively with RANTES, MIP-1alpha, and MIP-1beta. MCP-2 is a true agonist for CCR5, eliciting a robust chemotactic response in CCR5 transfectants similar to that of the three known CCR5 ligands and exhibiting cross-desensitization with RANTES in the Ca2+ flux response. MCP-4 also bound to CCR5 with high affinity and was efficiently displaced by other CCR5 ligands. However, MCP-4 only partially displaced the binding of radiolabeled MIP-1alpha and caused a chemotactic response only at high concentrations. Furthermore, MCP-2 inhibited the binding of the M-tropic HIV-1 gp120 envelope glycoprotein to CCR5 and HIV-1 infection of peripheral blood mononuclear cells. More importantly, we found that MCP-2 could bind and elicit chemotaxis in CD3-activated and IL-2-maintained T cells, and most of these functions could be specifically inhibited by the anti-CCR5 mAb 2D7, whereas the responses mediated by MIP-1alpha or MCP-4 were only partially inhibited by 2D7. Thus, although MCP-2 can bind to and signal through CCR1, CCR2b, and CCR5, among which both CCR2 and CCR5 are expressed at high levels on activated T cells, it appears to preferably utilize CCR5 on these cells. In contrast, MIP-1alpha and MCP-4 seem to activate multiple receptors on the same cells.     

The effects of fibroblast growth factors in long-term primary culture of dystrophic (mdx) mouse muscle myoblasts

Neutralizing monoclonal antibodies specific for human interleukin-6 (IL-6) bind two distinct sites on the IL-6 protein (sites I and II). Their interference with IL-6 receptor binding suggested that site I is a receptor-binding site of IL-6, whereas site II is important for signal transduction. Mutagenesis of site II could therefore result in the isolation of IL-6 receptor antagonists. To test this hypothesis, a panel of IL-6 mutant proteins was constructed that did not bind to a site II-specific monoclonal antibody. One such site II mutant protein (with double substitution of Gln-160 with Glu and Thr-163 with Pro) was found to be an antagonist of human IL-6. It was inactive on human CESS cells, weakly active on human HepG2 cells, but active on mouse B9 cells. It could specifically antagonize the activity of wild-type IL-6 on CESS and HepG2 cells. The binding affinity of this variant for the 80-kDa IL-6 receptor was similar to that of wild-type IL-6. High affinity binding to CESS cells, however, was abolished, suggesting that the mutant protein is inactive because the complex of the 80-kDa IL-6 receptor and the mutant protein cannot associate with the signal transducer gp130. The human IL-6 antagonist protein may be potentially useful as a therapeutic agent.     

Nerve growth factor induces rapid increases in functional cell surface low density lipoprotein receptor-related protein

The low density lipoprotein receptor-related protein (LRP) is a large endocytic receptor that binds multiple ligands and is highly expressed in neurons. Several LRP ligands, including apolipoprotein E/lipoproteins and amyloid precursor protein, have been shown to participate either in Alzheimer's disease pathogenesis or pathology. However, factors that regulate LRP expression in neurons are unknown. In the current study, we analyzed the effects of nerve growth factor (NGF) treatment on LRP expression, distribution, and function within neurons in two neuronal cell lines. Our results show that NGF induces a rapid increase of cell surface LRP expression in a central nervous system-derived neuronal cell line, GT1-1 Trk, which was seen within 10 min and reached a maximum at about 1 h of NGF treatment. This increase of cell surface LRP expression is concomitant with an increase in the endocytic activity of LRP as measured via ligand uptake and degradation assays. We also found that the cytoplasmic tail of LRP is phosphorylated and that NGF rapidly increases the amount of phosphorylation. Furthermore, we detected a significant increase of LRP expression at the messenger RNA level following 24 h of NGF treatment. Both rapid and long term induction of LRP expression were also detected in peripheral nervous system-derived PC12 cells following NGF treatment. Taken together, our results demonstrate that NGF regulates LRP expression in neuronal cells.