The laying hen expresses two different low density lipoprotein receptor-related proteins

We have identified, by a combination of ligand, 45Ca2+, and immunoblotting, two large membrane proteins akin to the mammalian so-called low density lipoprotein (LDL) receptor-related protein (LRP) in chicken tissues. LRP has thus far been demonstrated only in mammalian species where it is thought to act as a receptor for proteinase-alpha 2-macroglobulin complexes and/or chylomicron remnants, lipoproteins not produced in birds. One of the chicken LRPs was demonstrated in liver, and has the same apparent Mr and hallmark biochemical properties as rat liver LRP. The other chicken LRP is smaller (approximately 380 kDa) and is expressed in ovarian follicles, but is undetectable in liver. Immunological analysis demonstrated a lack of cross-reactivity between the two LRPs, as well as between them and the previously identified chicken oocyte-specific 95-kDa receptor for the yolk precursors, very low density lipoprotein, and vitellogenin (Stifani, S., Barber, D. L., Nimpf, J., and Schneider, W. J. (1989) Proc. Natl. Acad. Sci. U.S.A. 87, 1955-1959). As shown by ligand blotting, both chicken LRPs have the ability to interact with vitellogenin, a property they share not only with rat LRP, but also with mammalian LDL receptors. To obtain independent confirmation of the ligand blotting results, the smaller (follicular) LRP was purified and high-affinity binding of vitellogenin to it was demonstrated by a solid-phase filtration binding assay. Amino acid sequences of tryptic fragments of the smaller LRP were obtained, and its homology with human LRP demonstrated through unambiguous alignment of three fragments. Both chicken LRPs, the chicken oocyte 95-kDa receptor, as well as rat LRP, could be shown by ligand blotting to interact specifically with chicken serum alpha 2-macroglobulin. In addition, human apolipoprotein E, a ligand implicated in receptor-mediated metabolism of chylomicron remnants, also binds to the smaller chicken LRP, further emphasizing the similarities between LDL receptors and related proteins from a variety of species. In analogy to the known dichotomy of chicken LDL receptors, which is characterized by the production of the 95-kDa oocyte-specific receptor on one hand and a 130-kDa LDL receptor that is exclusively expressed in somatic cells (Hayashi, K., Nimpf, J., and Schneider, W. J. (1989) J. Biol. Chem. 264, 3131-3139), it appears that the smaller and larger chicken LRPs also may be restricted to the oocyte and somatic cells, respectively.     

Unesterified fatty acids inhibit the binding of low density lipoproteins to the human fibroblast low density lipoprotein receptor

Micromolar concentrations of oleate were found to inhibit reversibly the binding of low density lipoprotein (LDL) to the human fibroblast LDL receptor. The decrease in LDL binding caused a parallel reduction of both 125I-LDL uptake and degradation at 37 degrees C. At 4 degrees C, oleate was also found to displace 125I-LDL already bound to the LDL receptor. The effect of oleate was rapid, reaching 70-80% of maximum displacement with 5-10 min of incubation, and was closely correlated to oleate-albumin molar ratios. Partition analysis of unesterified fatty acids between cells and LDL showed that the inhibitory effect of oleate resulted mainly from an interaction of unesterified fatty acids with the cell surface rather than with the LDL particles. Using different unesterified fatty acids and fatty acid analogs, we found that the inhibitory effect was modulated by both the length and the conformation of the monomeric carbon chain and was directly dependent on the presence of a negative charge on the carboxylic group. At 4 degrees C, the inhibitory effect of oleate never exceeded half of maximum binding capacity. This limitation was associated with the ability of oleate to interact only with part of the population of LDL receptors which spontaneously recycles in the absence of ligand, as demonstrated by the fact that oleate did not induce any reduction of LDL binding after cell treatment with monensin in the absence of LDL. Our results indicate that unesterified fatty acids could participate in the control of LDL catabolism in vivo by direct modulation of the ability of LDL receptor to bind LDL.     

Deletion of exon encoding cysteine-rich repeat of low density lipoprotein receptor alters its binding specificity in a subject with familial hypercholesterolemia

The proposed ligand binding domain of the low density lipoprotein (LDL) receptor consists of a 40-amino acid cysteine-rich unit that is repeated with some variation seven times. We describe here a mutant allele at the LDL receptor locus in which one of the seven repeats has been deleted. This mutation was found in a patient with the clinical syndrome of homozygous familial hypercholesterolemia. By molecular cloning, we show that the deletion arose by homologous recombination between repetitive Alu sequences in intron 4 and intron 5 of the gene. The deletion removes exon 5, which normally encodes the sixth repeat of the ligand binding domain. In the resultant mRNA, exon 4 is spliced to exon 6, preserving the reading frame. This mRNA produces a shortened protein that reaches the cell surface and reacts with anti-receptor antibodies but does not bind LDL, which contains apoprotein B-100 as its major protein component. Surprisingly, the deleted protein retains the ability to bind and internalize beta-migrating very low density lipoprotein, a lipoprotein that contains apoprotein E as well as apoprotein B-100. These data support the hypothesis that the seven repeated sequences in the receptor constitute the LDL binding domain. The data further indicate that the sixth repeat is required for binding of LDL, but not beta-migrating very low density lipoprotein, and that deletion of a single cysteine-rich repeat can alter the binding specificity of the LDL receptor.     

The low density lipoprotein receptor

The study of familial hypercholesterolemia at the molecular level has led to its advancement from a clinical syndrome to a fascinating experimental system. FH was first described 50 years ago by Carl Müller who concluded that the disease produces high plasma cholesterol levels and myocardial infarctions in young people, and is transmitted as an autosomal dominant trait determined by a single gene. The existence of two forms of FH, namely heterozygous and homozygous, was recognized by Khachadurian and Fredrickson and Levy much later. The value of FH as an experimental model system lies in the availability of homozygotes, because mutant genes can be studied without interference from the normal gene. The first and most important breakthrough was the realization that the defect underlying FH could be studied in cultured skin fibroblasts. Rapidly, the LDL receptor pathway was conceptualized and its dysfunction in cells from FH homozygotes was demonstrates. Isolation of the normal LDL receptor protein and studies on the biosynthesis and structure of abnormal receptors in mutant cell lines provided essential groundwork for elucidation of defects at the DNA level. The power of the experimental system, FH, became nowhere more obvious than in work that correlated structural information at the protein level with the elucidation of defined defects in the LDL receptor gene. In addition to revealing important structure-function relationships in the LDL receptor polypeptide and delineating mutational events, studies of FH have established several more general concepts. First, the tight coupling of LDL binding to its internalization suggested that endocytosis was not a non-specific process as suggested from early observations. The key finding was that LDL receptors clustered in coated pits, structures that had been described by Roth and Porter 10 years earlier. These investigators had demonstrated, in electron microscopic studies on the uptake of yolk proteins by mosquito oocytes, that coated pits pinch off from the cell surface and form coated vesicles that transport extracellular fluid into the cell. Studies on the LDL receptor system showed directly that receptor clustering in coated pits is the essential event in this kind of endocytosis, and thus established receptor-mediated endocytosis as a distinct mechanism for the transport of macromolecules across the plasma membrane. Subsequently, many additional systems of receptor-mediated endocytosis have been defined, and variations of the overall pathway have been described.(ABSTRACT TRUNCATED AT 400 WORDS)     

Very low density lipoprotein binding to cultured aortic endothelium

Because of very low density lipoprotein's (VLDL) potential atherogenicity and the demonstration that VLDL can bind to other cells, we examined the interaction of human VLDL with cultured porcine aortic endothelium. The lipoprotein-cell interaction had many properties similar to those seen with the binding of a ligand to a cell surface receptor. It was time and temperature dependent, saturable, and reversible. Scatchard analysis of competition data suggested that there may be more than one class of binding site. The affinity of the low affinity site was similar to that for low density lipoprotein (LDL). Also, the capacity of endothelial cells to bind VLDL was similar to that for LDL, when related to apo B (i.e., particle) concentration. Not only was unlabelled VLDL able to compete for VLDL binding sites, but so was LDL and high density lipoprotein (HDL). The maximal competition either by LDL or by HDL was less than that by VLDL. The maximal competition by HDL was more than by LDL. The VLDL binding was dependent on Ca2+. It was not changed by the content of lipoprotein in the medium in which cells were grown prior to the binding studies. These observations suggest that VLDL binding to endothelial cells is similar in some respects, but not in all, to the binding of LDL. Comparison of the data with endothelial cells to previous data with adipocytes also indicated differences between the interaction of these two cell types with VLDL. It is possible that this binding process may be involved in the formation of atherogenic remnants of triglyceride-rich lipoproteins on the endothelial surface of large blood vessels.     

Heparin binding to lipoprotein lipase and low density lipoproteins

Heparin was fractionated on an affinity column of bovine milk lipoprotein lipase (LpL) immobilized to Affi-Gel-15. The bound heparin, designated high-reactive heparin (HRH), enhanced LpL activity, presumably by stabilizing the enzyme against denaturation. The unbound heparin fraction had no observable effect on the initial rate of enzyme activity. However, at longer times of incubation there was inhibition of LpL activity. LpL-specific HRH also showed a high, Ca²⁺-dependent precipitating activity towards human plasma low density lipoproteins (LDL). Since LpL and LDL both bind to heparin-like molecules at the surface of the arterial wall, we suggest that their similar heparin-binding specificity may have physiological consequences as it relates to the development of atherosclerosis.

Heparin binding Lipoprotein lipase LDL Apolipoprotein Lipolysis     

The second and fourth cluster of class A cysteine-rich repeats of the low density lipoprotein receptor-related protein share ligand-binding properties.

The low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic cell-surface receptor that binds and internalizes a diverse array of ligands. The receptor contains four putative ligand-binding domains, generally referred to as clusters I, II, III, and IV. In this study, soluble recombinant receptor fragments, representing each of the four individual clusters, were used to map the binding sites of a set of structurally and functionally distinct ligands. Using surface plasmon resonance, we studied the binding of these fragments to methylamine-activated alpha(2)-macroglobulin, pro-urokinase-type plasminogen activator, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1, t-PA.plasminogen activator inhibitor-1 complexes, lipoprotein lipase, apolipoprotein E, tissue factor pathway inhibitor, lactoferrin, the light chain of blood coagulation factor VIII, and the intracellular chaperone receptor-associated protein (RAP). No binding of the cluster I fragment to any of the tested ligands was observed. The cluster III fragment only bound to the anti-LRP monoclonal antibody alpha(2)MRalpha3 and weakly to RAP. Except for t-PA, we found that each of the ligands tested binds both to cluster II and to cluster IV. The affinity rate constants of ligand binding to clusters II and IV and to LRP were measured, showing that clusters II and IV display only minor differences in ligand-binding kinetics. Furthermore, we demonstrate that the subdomains C3-C7 of cluster II are essential for binding of ligands and that this segment partially overlaps with a RAP-binding site on cluster II. Finally, we show that one RAP molecule can bind to different clusters simultaneously, supporting a model in which RAP binding to LRP induces a conformational change in the receptor that is incompatible with ligand binding.     

Mutational analysis of the ligand binding domain of the low density lipoprotein receptor

The ligand binding domain of the low density lipoprotein (LDL) receptor contains seven imperfect repeats of a 40-amino acid cysteine-rich sequence. Each repeat contains clustered negative charges that have been postulated as ligand-binding sites. The adjacent region of the protein, the growth factor homology region, contains three cysteine-rich repeats (A-C) whose sequence differs from those in the ligand binding domain. To dissect the contribution of these different cysteine-rich repeats to ligand binding, we used oligonucleotide-directed mutagenesis to alter expressible cDNAs for the human LDL receptor which were then introduced into monkey COS cells by transfection. We measured the ability of the mutant receptors to bind LDL, which contains a single protein ligand for the receptor (apoB-100), and beta-migrating very low density lipoprotein (beta-VLDL), which contains apoB-100 plus multiple copies of another ligand (apoE). The results show that repeat 1 is not required for binding of either ligand. Repeats 2 plus 3 and repeats 6 plus 7 are required for maximal binding of LDL, but not beta-VLDL. Repeat 5 is required for binding of both ligands. Repeat A in the growth factor homology region is required for binding of LDL, but not beta-VLDL. Repeat B is not required for ligand binding. These results support a model for the LDL receptor in which various repeats play additive roles in ligand binding, each repeat making a separate contribution to the binding event.     

Reconstitution of the low density lipoprotein receptor

The receptor for low density lipoprotein was purified from bovine adrenal cortex in the presence of the nonionic detergent octylglucoside. Receptors were incorporated into the bilayer of egg phosphatidylcholine vesicles by a detergent-dialysis method. Reconstituted receptors were functional in that they bound low density lipoprotein as well as a monoclonal antibody directed against the receptor in a specific, saturable fashion. Binding activity of reconstituted receptors was measured by a gel chromatography assay. The orientation of the receptor molecule within the phospholipid bilayer was investigated by binding assays following proteolytic digestion. Reconstituted receptors showed an orientation that was functionally indistinguishable from that of low density lipoprotein receptors in the plasma membrane of intact human fibroblasts.     

Effect of arginine 172 on the binding of apolipoprotein E to the low density lipoprotein receptor

The region of apolipoprotein E (apoE) that interacts directly with the low density lipoprotein (LDL) receptor lies in the vicinity of residues 136-150, where lysine and arginine residues are crucial for full binding activity. However, defective binding of carboxyl-terminal truncations of apoE3 has suggested that residues in the vicinity of 170-183 are also important. To characterize and define the role of this region in LDL receptor binding, we created either mutants of apoE in which this region was deleted or in which arginine residues within this region were sequentially changed to alanine. Deletion of residues 167-185 reduced binding activity (15% of apoE3), and elimination of arginines at positions 167, 172, 178, and 180 revealed that only position 172 affected binding activity (2% of apoE3). Substitution of lysine for Arg(172) reduced binding activity to 6%, indicating a specific requirement for arginine at this position. The higher binding activity of the Delta167-185 mutant relative to the Arg(172) mutant (15% versus 2%) is explained by the fact that arginine residues at positions 189 and 191 are shifted in the deletion mutant into positions equivalent to 170 and 172 in the intact protein. Mutation of these residues and modeling the region around these residues suggested that the influence of Arg(172) on receptor binding activity may be determined by its orientation at a lipid surface. Thus, the association of apoE with phospholipids allows Arg(172) to interact directly with the LDL receptor or with other residues in apoE to promote its receptor-active conformation.     

A macrophage receptor that recognizes oxidized low density lipoprotein but not acetylated low density lipoprotein

The formation of cholesterol-loaded macrophage foam cells in arterial tissue may occur by the uptake of modified lipoproteins via the scavenger receptor pathway. The macrophage scavenger receptor, also called the acetylated low density lipoprotein (Ac-LDL) receptor, has been reported to recognize Ac-LDL as well as oxidized LDL species such as endothelial cell-modified LDL (EC-LDL). We now report that there is another class of macrophage receptors that recognizes EC-LDL but not Ac-LDL. We performed assays of 0 degrees C binding and 37 degrees C degradation of 125I-Ac-LDL and 125I-EC-LDL by mouse peritoneal macrophages. Competition studies showed that unlabeled Ac-LDL could compete for only 25% of the binding and only 50% of the degradation of 125I-EC-LDL. Unlabeled EC-LDL, however, competed for greater than 90% of 125I-EC-LDL binding and degradation. Unlabeled Ac-LDL was greater than 90% effective against 125I-Ac-LDL; EC-LDL competed for about 80% of 125I-Ac-LDL binding and degradation. Copper-oxidized LDL behaved the same as EC-LDL in all the competition studies. Copper-mediated oxidation of Ac-LDL produced a superior competitor which could now displace 90% of 125I-EC-LDL binding. After 5 h at 37 degrees C in the presence of ligand, macrophages accumulated six times more cell-associated radioactivity from 125I-EC-LDL than from 125I-Ac-LDL, despite approximately equal amounts of degradation to trichloroacetic acid-soluble products, which may imply different intracellular processing of the two lipoproteins. Our results suggest that 1) there is more than one macrophage "scavenger receptor" for modified lipoproteins; and 2) oxidized LDL and Ac-LDL are not identical ligands with respect to macrophage recognition and uptake.     

The binding of chondroitin 6-sulfate to plasma low density lipoprotein

The interaction in vitro of several sulfated glycosaminoglycans with low density lipoproteins (LDL) has been studied. Chondroitin 6-sulfate and heparin were the only ones to produce turbidity when added to LDL in presence of Ca2+. However, when these two glycosaminoglycans were applied to LDL-affinity columns in presence of Ca2+, only chondroitin 6-sulfate was retained. Partially desulfated chondroitin 6-sulfate was not retained on LDL-affinity column, indicating the relevance of sulfate groups in the binding of LDL. Since chondroitin 4-sulfate and heparin, with a sulfate content respectively equal to and greater than that of chondroitin 6-sulfate, are not retained on LDL-affinity columns, the factors relevant to the binding of LDL are probably the conformation of the glycan in solution and the orientation of its sulfate groups.     

Mutation in apolipoprotein B associated with hypobetalipoproteinemia despite decreased binding to the low density lipoprotein receptor

Mutations in apolipoprotein B (APOB) may reduce binding of low density lipoprotein (LDL) to the LDL receptor and cause hypercholesterolemia. We showed that heterozygotes for a new mutation in APOB have hypobetalipoproteinemia, despite a reduced binding of LDL to the LDL receptor. APOB R3480P heterozygotes were identified among 9,255 individuals from the general population and had reduced levels of apoB-containing lipoproteins. Most surprisingly, R3480P LDL bound with lower affinity to the LDL receptor than non-carrier LDL in vitro, and these results were confirmed by turnover studies of LDL in vivo. In very low density lipoprotein (VLDL) turnover studies, the amount of VLDL converted to LDL in R3480P heterozygotes was substantially reduced, suggesting that this was the explanation for the hypobetalipoproteinemia observed in these individuals. Our findings emphasized the importance of combining in vitro studies with both human in vivo and population-based studies, as in vitro studies often have focused on very limited aspects of complex mechanisms taken out of their natural context.     

Defective receptor binding of low density lipoprotein from pigs possessing mutant apolipoprotein B alleles

We previously identified a defect in the in vivo catabolism of low density lipoprotein (LDL) from hypercholesterolemic pigs carrying a mutant apolipoprotein B allele. In the present studies, we examined the in vitro metabolism of mutant LDL in cultured pig fibroblasts. A 3-fold higher concentration of mutant LDL (compared to control) was needed to displace 50% of control 125I-LDL binding. Mutant LDL had a 6-fold higher dissociation constant than control LDL. Scatchard plots of the binding data were concave upward, suggesting multiple classes of binding sites or negative cooperativity. The mutant LDL degradation rate was reduced by 40%; this decrease could be attributed to a dense LDL subspecies. Mutant and control buoyant LDL subspecies were degraded more slowly than the corresponding dense LDL subspecies. Together, these studies show that diminished LDL receptor binding can result from mutations in apolipoprotein B and from changes in the lipid composition of LDL particles.     

A two-step binding model of PCSK9 interaction with the low density lipoprotein receptor

PCSK9 (proprotein convertase subtilisin-like/kexin type 9) is an emerging target for pharmaceutical intervention. This multidomain protein interacts with the LDL receptor (LDLR), promoting receptor degradation. Insofar as PCSK9 inhibition induces a decrease in plasma cholesterol levels, understanding the nature of the binding interaction between PCSK9 and the LDLR is of critical importance. In this study, the ability of PCSK9 to compete with apoE3 N-terminal domain-containing reconstituted HDL for receptor binding was examined. Whereas full-length PCSK9 was an effective competitor, the N-terminal domain (composed of the prodomain and catalytic domain) was not. Surprisingly, the C-terminal domain (CT domain) of PCSK9 was able to compete. Using a direct binding interaction assay, we show that the PCSK9 CT domain bound to the LDLR in a calcium-dependent manner and that co-incubation with the prodomain and catalytic domain had no effect on this binding. To further characterize this interaction, two LDLR fragments, the classical ligand-binding domain (LBD) and the EGF precursor homology domain, were expressed in stably transfected HEK 293 cells and isolated. Binding assays showed that the PCSK9 CT domain bound to the LBD at pH 5.4. Thus, CT domain interaction with the LBD of the LDLR at endosomal pH constitutes a second step in the PCSK9-mediated LDLR binding that leads to receptor degradation.     

The lipid-associated conformation of the low density lipoprotein receptor binding domain of human apolipoprotein E

Apolipoprotein E (apoE) is a 34-kDa exchangeable apolipoprotein that regulates metabolism of plasma lipoproteins by functioning as a ligand for members of the LDL receptor family. The receptor-binding region localizes to the vicinity of residues 130-150 within its independently folded 22-kDa N-terminal domain. In the absence of lipid, this domain exists as a receptor-inactive, globular four-helix bundle. Receptor recognition properties of this domain are manifest upon lipid association, which is accompanied by a conformational change in the protein. Fluorescence resonance energy transfer has been used to monitor helix repositioning, which accompanies lipid association of the apoE N-terminal domain. Site-directed mutagenesis was used to replace naturally occurring Trp residues with phenylalanine, creating a Trp-null apoE3 N-terminal domain (residues 1-183). Subsequently, tyrosine residues in helix 2, helix 3, or helix 4 were converted to Trp, generating single Trp mutant proteins. The lone cysteine at position 112 was covalently modified with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine, which serves as an energy acceptor from excited tryptophan residues. Fluorescence resonance energy transfer analysis of apoE N-terminal domain variants in phospholipid disc complexes suggests that the helix bundle opens to adopt a partially extended conformation. A model is presented that depicts a tandem arrangement of the receptor-binding region of the protein in the disc complex, corresponding to its low density lipoprotein receptor-active conformation.