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.     

Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins

Seven imperfect repeats of a 40-amino acid cysteine-rich sequence constitute the ligand binding domain of the low density lipoprotein (LDL) receptor. To assess the contribution of each repeat, three site-directed mutations were made individually in each repeat: 1) deletion of the repeat, 2) substitution of a conserved isoleucine with aspartic acid, and 3) substitution of a conserved aspartic acid with tyrosine. cDNAs containing these mutations were transfected into simian COS cells and assayed for their ability to bind LDL, which contains a 500-kDa protein ligand (apoB-100), and beta-migrating very low density lipoprotein (beta-VLDL), which contains multiple copies of a 33-kDa ligand (apoE). The results showed that binding of the two ligands required different combinations of repeats. LDL binding required repeats 3-7; deletion of any one of these repeats markedly reduced LDL binding. In contrast, beta-migrating very low density lipoprotein binding was insensitive to the loss of any single repeat with the important exception of repeat 5, whose loss reduced binding by 60%. The same effects were obtained when each of the repeats was altered by either of the two substitution mutations. The current findings suggest that a multiplicity of cysteine-rich repeats may allow a single protein to bind several different protein ligands by employing different combinations of repeats.     

A two-module region of the low-density lipoprotein receptor sufficient for formation of complexes with apolipoprotein E ligands

The low-density lipoprotein (LDL) receptor transports two different classes of cholesterol-carrying lipoprotein particles into cells: LDL particles, which contain a single copy of apolipoprotein B-100 (apoB-100), and beta-migrating very low-density lipoprotein (beta-VLDL) particles, which contain multiple copies of apolipoprotein E (apoE). The ligand-binding domain of the receptor lies at its amino-terminal end within seven adjacent LDL-A repeats (LA1-LA7). Although prior work clearly establishes that LA5 is required for high-affinity binding of particles containing apolipoprotein E (apoE), the number of ligand-binding repeats sufficient to bind apoE ligands has not yet been determined. Similarly, uncertainty exists as to whether a single lipid-activated apoE receptor-binding site within a particle is capable of binding to the LDLR with high affinity or whether more than one is required. Here, we establish that the LA4-5 two-repeat pair is sufficient to bind apoE-containing ligands, on the basis of binding studies performed with a series of LDLR-derived "minireceptors" containing up to four repeats. Using single chain multimers of the apoE receptor-binding domain (N-apoE), we also show that more than one receptor-binding site in its lipid-activated conformation is required to bind to the LDLR with high affinity. Thus, in addition to inducing a conformational change in the structure of N-apoE, lipid association enhances the affinity of apoE for the LDLR in part by creating a multivalent ligand.     

Characterization of hepatitis C virus (HCV) and HCV E2 interactions with CD81 and the low-density lipoprotein receptor

Hepatitis C virus (HCV) or HCV-low-density lipoprotein (LDL) complexes interact with the LDL receptor (LDLr) and the HCV envelope glycoprotein E2 interacts with CD81 in vitro. However, E2 interactions with LDLr and HCV interactions with CD81 have not been clearly described. Using sucrose gradient-purified low-density particles (1.03 to 1.07 g/cm(3)), intermediate-density particles (1. 12 to 1.18 g/cm(3)), recombinant E2 protein, or control proteins, we assessed binding to MOLT-4 cells, foreskin fibroblasts, or LDLr-deficient foreskin fibroblasts at 4 degrees C by flow cytometry and confocal microscopy. Viral entry was determined by measuring the coentry of alpha-sarcin, a protein synthesis inhibitor. We found that low-density HCV particles, but not intermediate-density HCV or controls bound to MOLT-4 cells and fibroblasts expressing the LDLr. Binding correlated with the extent of cellular LDLr expression and was inhibited by LDL but not by soluble CD81. In contrast, E2 binding was independent of LDLr expression and was inhibited by human soluble CD81 but not mouse soluble CD81 or LDL. Based on confocal microscopy, we found that low-density HCV particles and LDL colocalized on the cell surface. The addition of low-density HCV but not intermediate-density HCV particles to MOLT-4 cells allowed coentry of alpha-sarcin, indicating viral entry. The amount of viral entry also correlated with LDLr expression and was independent of the CD81 expression. Using a solid-phase immunoassay, recombinant E2 protein did not interact with LDL. Our data indicate that E2 binds CD81; however, virus particles utilize LDLr for binding and entry. The specific mechanism by which HCV particles interact with LDL or the LDLr remains unclear.     

Domain Swapping Reveals That Low Density Lipoprotein (LDL) Type A Repeat Order Affects Ligand Binding to the LDL Receptor

The low density lipoprotein receptor (LDLR) plays a key role in plasma cholesterol homeostasis by binding and internalizing lipoprotein ligands. Studies have revealed that one or more of the seven LDL type A repeats (LA1–LA7) in the receptor are responsible for apolipoprotein binding. In the present study, protein engineering was performed to swap or replace key LA repeats in a recombinant soluble LDLR (sLDLR). Although wild type sLDLR showed strong ligand binding activity, an sLDLR variant in which LA repeat 5 was replaced by a second copy of LA repeat 2 showed low binding activity. Likewise, a variant wherein LA repeats 2 and 5 were swapped displayed low binding activity. At the same time, substitution of LA repeat 2 with a second a copy of repeat 5 resulted in a receptor with ligand binding activity similar to wild type LDLR. When binding assays were conducted with human low density lipoprotein as ligand, LA repeat order was a less important determinant of binding activity. Taken together, the data indicate that the sequential order of LA repeats plays a key role in ligand binding properties of LDLR.The low density lipoprotein receptor (LDLR)³ plays an important role in plasma cholesterol homeostasis (1). A fundamental function of LDLR is transport of cholesterol-rich lipoproteins into cells via receptor-mediated endocytosis (2). Human LDLR is 839 amino acids in length and is comprised of five distinct modules that arose from gene duplication. At the N terminus of LDLR, there exists a series of seven imperfect, disulfide bond-rich, LDL type A (LA) repeats, each ∼40 amino acids in length. Calcium binding induces LA repeats to fold into a ligand binding-competent conformation (3). Adjacent to the ligand binding module is a ∼400-residue module that bears homology to epidermal growth factor (EGF) precursor. This module consists of two disulfide bond-rich EGF-like repeats (A and B) and a YWTD β-propeller motif followed by a third EGF-like repeat C (4). The third module of LDLR is distinguished by an abundance of O-linked sugars, whereas the fourth module is comprised of a single membrane-spanning sequence. Finally, a short intracellular C-terminal cytoplasmic domain, required for receptor internalization, is present (5).LDLR binds two apolipoprotein ligands, apolipoprotein (apo) E and apoB (6). Although these proteins do not share structural similarity, sequence elements rich in positively charged amino acid side chains are present in each that are required for binding. Deletion studies have demonstrated that specific LA repeats are required for apolipoprotein binding to LDLR (7, 8).Recently, another LDLR ligand, termed proprotein convertase subtilisin-like kexin type 9 (PCSK9), has emerged (9): PCSK9 serves to regulate cholesterol homeostasis by modulating LDLR processing. Unlike lipoprotein ligands, PCSK9 binds EGF repeat A and, apparently, is not released from the receptor at endosomal pH.LA1–LA7 are ∼40–50% identical in primary sequence. Each repeat contains a Ca²⁺ binding site and three disulfide bonds. The importance of these structural features for ligand binding is widely recognized. For example, it is known that LA5 is essential for optimal binding of apoB- and apoE-containing ligands (7, 8). On the other hand, deletion of LA2 had no effect on binding of apoE-containing lipoproteins. X-ray crystal structure information is available for isolated LA5 at pH 5.0 (10) as well as the entire ectodomain of LDLR (residues 1–699) at endosomal pH (11). Based on this structural information and complementary data on apolipoprotein ligands, it has been postulated that electrostatic interactions modulate LDLR conformation and ligand binding. Given this, it remains unclear whether the precise order of LA repeats within the ligand binding module may impact ligand binding.In the present study, protein engineering of a soluble LDLR (sLDLR) was performed to swap or replace specific LA repeats within the ligand binding module of sLDLR. Ligand binding to wild type (WT) and engineered sLDLR was then determined. The results show that LA repeat 5 must not only be present, it must exist in the correct context with respect to other LA repeats within the ligand binding module.     

Cooperation between fixed and low pH-inducible interfaces controls lipoprotein release by the LDL receptor

Low-density lipoprotein (LDL) receptors bind lipoprotein particles at the cell surface and release them in the low pH environment of the endosome. The published structure of the receptor determined at endosomal pH reveals an interdomain interface between its beta propeller and its fourth and fifth ligand binding (LA) repeats, suggesting that the receptor adopts a closed conformation at low pH to release LDL. Here, we combine lipoprotein binding and release assays with NMR spectroscopy to examine structural features of the receptor promoting release of LDL at low pH. These studies lead to a model in which the receptor uses a pH-invariant scaffold as an anchor to restrict conformational search space, combining it with flexible linkers between ligand binding repeats to interconvert between open and closed conformations. This finely tuned balance between interdomain rigidity and flexibility is likely to represent a shared structural feature in proteins of the LDL receptor family.     

Heterogeneity of apolipoprotein E epitope expression on human lipoproteins: importance for apolipoprotein E function

The conformations of apolipoproteins on the surfaces of lipoprotein particles affect their physiologic functions. The conformations of apoE on plasma lipoproteins were examined using a panel of eight anti-apoE monoclonal antibodies (MAbs). The antibodies, which reacted with the major isoforms of apoE (E2, E3, and E4), defined at least five epitopes on apoE. Proteolytic fragments and synthetic peptides of apoE were used in binding assays to assign antibody epitopes; the epitopes were all localized to the middle third of the apoE molecule. The expression of apoE epitopes on isolated apoE and on lipoproteins was probed in competitive microtiter plate immunoassays using the anti-apoE MAbs, 125I-labeled apoE as tracer, and isolated apoE, intermediate density (IDL), very low density (VLDL1-3), and high density (HDL2 and HDL3) lipoproteins as competitors. The antibodies determined the patterns of competition exhibited by the lipoprotein preparations. Antibodies of the IgM class (WU E-1, WU E-2, WU E-3) defined two sets of conformation-dependent epitopes that were assigned towards the middle and the carboxyl terminal of the middle third of apoE. Competition curves using these antibodies, apoE, and lipoproteins showed a large variability in ED50 values. MAbs WU E-4, WU E-7, and WU E-10 defined epitopes near the receptor recognition site on apoE. Competition curves demonstrated small ranges of ED50 values. MAbs WU E-11 and WU E-12, which defined epitopes toward the amino-terminal region of apoE, exhibited competition curves for apoE and lipoproteins that had consistent, but wider ranges of ED50 values. There was no strict relationship between lipoprotein flotation rates and epitope expression for any of the MAbs. Immunoaffinity chromatography of VLDL subfractions on four different MAb columns indicated that the differences in the competitive abilities of VLDL subfractions were partly due to heterogeneity of apoE epitope expression within any population of particles. VLDL particles specifically retained on two different anti-apoE MAb columns were better competitors than unretained fractions for 125I-labeled LDL binding to the apoB, E-receptor of cultured human fibroblasts, suggesting that increased accessibility of apoE on the surface of VLDL is associated with increased receptor recognition. These data suggest that individual epitopes of apoE can be modulated; epitope expressions are not determined solely by the sizes and/or densities of lipoprotein particles; and differences in apoE conformation have significant metabolic consequences.     

NMR solution structure of complement-like repeat CR3 from the low density lipoprotein receptor-related protein. Evidence for specific binding to the receptor binding domain of human alpha(2)-macroglobulin

We have used NMR methods to determine the structure of the calcium complex of complement-like repeat 3 (CR3) from the low density lipoprotein receptor-related protein (LRP) and to examine its specific interaction with the receptor binding domain of human alpha(2)-macroglobulin. CR3 is one of eight related repeats that constitute a major ligand binding region of LRP. The structure is very similar in overall fold to homologous complement-like repeat CR8 from LRP and complement-like repeats LB1, LB2, and LB5 from the low density lipoprotein receptor and contains a short two-strand antiparallel beta-sheet, a one turn alpha-helix, and a high affinity calcium site with coordination from four carboxyls and two backbone carbonyls. The surface electrostatics and topography are, however, quite distinct from each of these other repeats. Two-dimensional (1)H,(15)N-heteronuclear single quantum coherence spectra provide evidence for a specific, though relatively weak (K(d) approximately 140 microM), interaction between CR3 and human alpha2-macroglobulin receptor binding domain that involves a contiguous patch of surface residues in the central region of CR3. This specific interaction is consistent with a mode of LRP binding to ligands that uses contributions from more than one domain to generate a wide array of different binding sites, each with overall high affinity.     

Localization of the N-terminal domain of the low density lipoprotein receptor

The low density lipoprotein (LDL) receptor is a transmembrane glycoprotein performing "receptor-mediated endocytosis" of cholesterol-rich lipoproteins. At the N terminus, the LDL receptor has modular cysteine-rich repeats in both the ligand binding domain and the epidermal growth factor (EGF) precursor homology domain. Each repeat contains six disulfide-bonded cysteine residues, and this structural motif has also been found in many other proteins. The bovine LDL receptor has been purified and reconstituted into egg yolk phosphatidylcholine vesicle bilayers. Using gel electrophoresis and cryoelectron microscopy (cryoEM), the ability of the reconstituted LDL receptor to bind its ligand LDL has been demonstrated. After reduction of the disulfide-bonds in the N-terminal domain of the receptor, the reduced LDL receptor was visualized using cryoEM; reduced LDL receptors showed images with a diffuse density region at the distal end of the extracellular domain. Gold labeling of the reduced cysteine residues was achieved with monomaleimido-Nanogold, and the bound Nanogold was visualized in cryoEM images of the reduced, gold-labeled receptor. Multiple gold particles were observed in the diffuse density region at the distal end of the receptor. Thus, the location of the ligand binding domain of the LDL receptor has been determined, and a model is suggested for the arrangement of the seven cysteine-rich repeats of the ligand binding domain and two EGF-like cysteine-rich repeats of the EGF precursor homology domain.     

Stoichiometric binding of low density lipoprotein (LDL) and monoclonal antibodies to LDL receptors in a solid phase assay

The current paper describes a solid phase ligand binding assay for the low density lipoprotein (LDL) receptor that takes advantage of the domain structure of the protein. An antibody directed against one domain, e.g. the cytoplasmic tail, is adsorbed to a microtiter well. A detergent solution containing the LDL receptor is added, and the receptor is allowed to bind to the antibody. The wells are then washed, and one of the following radioiodinated ligands is added: 125I-LDL or an 125I-labeled monoclonal antibody directed against a different domain than the antibody adsorbed to the well. Under these conditions, the human LDL receptor shows high affinity for 125I-LDL and for 125I-IgG-HL1, a monoclonal antipeptide antibody directed against a 10-amino-acid "linker" between repeats 4 and 5 in the ligand binding domain. The binding affinity is the same at 4 degrees C and 37 degrees C. The binding of 125I-LDL and 125I-IgG-HL1 occurs with 1:1 molar stoichiometry, suggesting that the human LDL receptor binds 1 mol of LDL per mol of receptor. The acid-dependent dissociation of 125I-LDL and 125I-labeled monoclonal antibody from LDL receptors that is observed in intact cells was also shown to occur in the solid phase binding assay. We used the solid phase assay to demonstrate the secretion of LDL receptors from monkey cells that have been transfected with a cDNA encoding a truncated form of the human receptor that lacks the membrane-spanning domain. This assay may be useful in measuring the relative amounts of the intact LDL receptor in tissue extracts and the secreted receptor in transfected cells.     

The use of monoclonal antibodies to localize the low density lipoprotein receptor-binding domain of apolipoprotein B

Human apolipoprotein (apo) B-100 is composed of 4536 amino acids. It is thought that the binding of apoB to the low density lipoprotein (LDL) receptor involves an interaction between basic amino acids of the ligand and acidic residues of the receptor. Three alternative models have been proposed to describe this interaction: 1) a single region of apoB is involved in receptor binding; 2) groups of basic amino acids from throughout the apoB primary structure act in concert in apoB receptor binding; and 3) apoB contains multiple independent binding regions. We have found that monoclonal antibodies (Mabs) specific for a region that spans a thrombin cleavage site at apoB residue 3249 (T2/T3 junction) totally blocked LDL binding to the LDL receptor. Mabs specific for epitopes outside this region had either no or partial ability to block LDL binding. In order to define the region of apoB directly involved in the interaction with the LDL receptor we have tested 22 different Mabs for their ability to bind to LDL already fixed to the receptor. A Mab specific for an epitope situated between residues 2835 and 2922 could bind to its epitope on LDL fixed to its receptor whereas a second epitope between residues 2980 and 3084 is inaccessible on receptor-bound LDL. A series of epitopes near residue 3500 of apoB is totally inaccessible, and another situated between residues 4027 and 4081 is poorly accessible on receptor-bound LDL. In contrast, an epitope that is situated between residues 4154 and 4189 is fully exposed. Mabs specific for epitopes upstream and downstream of the region 3000-4000 can bind to receptor-bound LDL with a stoichiometry close to unity. Our results strongly suggest that the unique region of apoB directly involved in the LDL-receptor interaction is that of the T2/T3 junction.     

Monoclonal antibodies to human low density lipoprotein identify distinct areas on apolipoprotein B-100 relevant to the low density lipoprotein-receptor interaction.

We have characterized the epitopes for ten murine monoclonal antibodies (Mabs) to human low density lipoprotein (LDL) and studied their ability to interfere with the LDL-receptor interaction. The epitopes for the antibodies were defined by using the following approaches: 1) interaction with apoB-48; 2) interaction with apoB-100 thrombolytic fragments; and 3) interaction with beta-galactosidase-apoB fusion proteins spanning different areas of the apoB-100 sequence. The results obtained are consistent with the following map of epitopes: Mab 6E, amino acids (aa) 1-1297, Mabs 5A and 6B, aa 1480-1693, Mabs 2A, 7A, 3B, and 4B, aa 2152-2377, Mabs 8A and 9A, aa 2657-3248 and 3H, aa 4082-4306. Four Mabs (2A, 5A, 7A, and 9A) whose epitopes are located in three different areas of apoB, dramatically reduced (up to 95%) the LDL-receptor interaction on cultured human fibroblasts; Fab fragments were as effective as the whole antibodies. Mab 3H, on the other hand, increased LDL binding up to threefold. These findings are consistent with the hypothesis that several areas of apoB-100 are involved independently or in concert in modulating the apoprotein B conformation required for interaction with the LDL receptor.     

Native oligomeric human immunodeficiency virus type 1 envelope glycoprotein elicits diverse monoclonal antibody reactivities.

We synthesized and purified a recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein, lacking the gp120/gp41 cleavage site as well as the transmembrane domain, that is secreted principally as a stable oligomer. Mice were immunized with separated monomeric and oligomeric HIV-1 Env glycoproteins to analyze the repertoire of antibody responses to the tertiary and quaternary structure of the protein. Hybridomas were generated and assayed for reactivity by immunoprecipitation of nondenatured Env protein. A total of 138 monoclonal antibodies (MAbs) were generated and cloned, 123 of which were derived from seven animals immunized with oligomeric Env. Within this group, a significant response was obtained against the gp41 ectodomain; 49 MAbs recognized epitopes in gp41, 82% of which were conformational. The influence of conformation on gp120 antigenicity was less pronounced, with 40% of the anti-gp120 MAbs binding to conformational epitopes, many of which blocked CD4 binding. Surprisingly, less than 7% of the MAbs derived from mice immunized with oligomeric Env recognized the V3 loop. In addition, MAbs to linear epitopes in the C-terminal domain of gp120 were not obtained, suggesting that this region of the protein may be partially masked in the oligomeric molecule. A total of 15 MAbs were obtained from two mice immunized with monomeric Env. Nearly half of these recognized the V3 loop, suggesting that this region may be a less predominant epitope in the context of oligomeric Env than in monomeric protein. Thus, immunization with oligomeric Env generates a large proportion of antibodies to conformational epitopes in both gp120 and gp41, many of which may be absent from monomeric Env.     

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.     

Characterization of an immunodominant antigenic site on GB virus C glycoprotein E2 that is involved in cell binding

GB virus type C (GBV-C) is a human flavivirus that may cause persistent infection, although most infected individuals clear viremia and develop antibodies to the envelope glycoprotein E2. To study GBV-C E2 antigenicity and cell binding, murine anti-E2 monoclonal antibodies (MAbs) were evaluated to topologically map immunogenic sites on GBV-C E2 and for the ability to detect or block recombinant E2 binding to various cell lines. Five competition groups of MAbs were identified. Groups I and II did not compete with each other. Group III competed with both groups I and II. Group IV did not compete with group I, II, or III. One MAb competed with all of the other MAbs, suggesting that the epitopes bound by these MAbs are intimately related. Individually, none of the MAbs competed extensively with polyclonal human convalescent antibody (PcAb); however, combinations of all five MAb groups completely blocked PcAb binding to E2, suggesting that the epitopes bound by these MAbs form a single, immunodominant antigenic site. Only group I and III MAbs detected purified recombinant E2 bound to cells in binding assays. In contrast, group II MAbs neutralized the binding of E2 to cells. Both PcAb and MAbs were conformation dependent, with the exception of one group II MAb (M6). M6 bound to a five-amino-acid sequence on E2 if the peptide included four C-terminal or eight N-terminal residues, suggesting that the GBV-C E2 protein contains a single immunodominant antigenic site which includes a complex epitope that is involved in specific cellular binding.     

A PCSK9-binding antibody that structurally mimics the EGF(A) domain of LDL-receptor reduces LDL cholesterol in vivo

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.     

Recombinant hepatitis C virus-envelope protein 2 interactions with low-density lipoprotein/CD81 receptors

Hepatitis C virus (HCV) envelope protein 2 (E2) is involved in viral binding to host cells. The aim of this work was to produce recombinant E2B and E2Y HCV proteins in Escherichia coli and Pichia pastoris, respectively, and to study their interactions with low-density lipoprotein receptor (LDLr) and CD81 in human umbilical vein endothelial cells (HUVEC) and the ECV304 bladder carcinoma cell line. To investigate the effects of human LDL and differences in protein structure (glycosylated or not) on binding efficiency, the recombinant proteins were either associated or not associated with lipoproteins before being assayed. The immunoreactivity of the recombinant proteins was analysed using pooled serum samples that were either positive or negative for hepatitis C. The cells were immunophenotyped by LDLr and CD81 using flow cytometry. Binding and binding inhibition assays were performed in the presence of LDL, foetal bovine serum (FCS) and specific antibodies. The results revealed that binding was reduced in the absence of FCS, but that the addition of human LDL rescued and increased binding capacity. In HUVEC cells, the use of antibodies to block LDLr led to a significant reduction in the binding of E2B and E2Y. CD81 antibodies did not affect E2B and E2Y binding. In ECV304 cells, blocking LDLr and CD81 produced similar effects, but they were not as marked as those that were observed in HUVEC cells. In conclusion, recombinant HCV E2 is dependent on LDL for its ability to bind to LDLr in HUVEC and ECV304 cells. These findings are relevant because E2 acts to anchor HCV to host cells; therefore, high blood levels of LDL could enhance viral infectivity in chronic hepatitis C patients.     

Chiloscyllium plagiosum low-density lipoprotein receptor: Evolutionary conservation of five different functional domains

All five functional domains of the low-density lipoprotein (LDL) receptor were assembled in their modern form more than 450 million years ago, as revealed from the cloning and sequencing of an LDL receptor cDNA fromChiloscyllium plagiosum (banded cat shark). The shark LDL receptor has the same overall architecture as the mammalian and amphibian counterparts. Each of the seven cysteine-rich repeats in the ligand binding domain resembles its counterpart in the human LDL receptor more than it does the other repeats in the shark receptor as suggested by the presence of unique signature sequences, indicating that these repeats had already acquired their independent structures by the time of shark development. Furthermore, amino acid sequences of the entire ligand binding domain of shark LDL receptor show 35% identity over a stretch of 294 residues with aLymnaea stagnalis G-protein-linked receptor (LSGLR). The region of homology between these unrelated proteins includes conservation of most of the unique characteristics of the cysteine-rich repeats of LDL receptor at the expected positions in LSGLR. The results presented are consistent with the hypothesis that all seven repeats in the ligand binding domain of LDL receptor may have been lifted directly from an ancestral gene instead of being evolutionary duplications of a single repeat recruited by the primitive LDL receptor from another gene.The nucleotide sequence reported will appear in GenBank under accession number L36118     

Antigenic and immunogenic characterization of recombinant baculovirus-expressed severe acute respiratory syndrome coronavirus spike protein: implication for vaccine design

The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates the receptor interaction and immune recognition and is considered a major target for vaccine design. However, its antigenic and immunogenic properties remain to be elucidated. In this study, we immunized mice with full-length S protein (FL-S) or its extracellular domain (EC-S) expressed by recombinant baculoviruses in insect cells. We found that the immunized mice developed high titers of anti-S antibodies with potent neutralizing activities against SARS pseudoviruses constructed with the S proteins of Tor2, GD03T13, and SZ3, the representative strains of 2002 to 2003 and 2003 to 2004 human SARS-CoV and palm civet SARS-CoV, respectively. These data suggest that the recombinant baculovirus-expressed S protein vaccines possess excellent immunogenicity, thereby inducing highly potent neutralizing responses against human and animal SARS-CoV variants. The antigenic structure of the S protein was characterized by a panel of 38 monoclonal antibodies (MAbs) isolated from the immunized mice. The epitopes of most anti-S MAbs (32 of 38) were localized within the S1 domain, and those of the remaining 6 MAbs were mapped to the S2 domain. Among the anti-S1 MAbs, 17 MAbs targeted the N-terminal region (amino acids [aa] 12 to 327), 9 MAbs recognized the receptor-binding domain (RBD; aa 318 to 510), and 6 MAbs reacted with the C-terminal region of S1 domain that contains the major immunodominant site (aa 528 to 635). Strikingly, all of the RBD-specific MAbs had potent neutralizing activity, 6 of which efficiently blocked the receptor binding, confirming that the RBD contains the main neutralizing epitopes and that blockage of the receptor association is the major mechanism of SARS-CoV neutralization. Five MAbs specific for the S1 N-terminal region exhibited moderate neutralizing activity, but none of the MAbs reacting with the S2 domain and the major immunodominant site in S1 showed neutralizing activity. All of the neutralizing MAbs recognize conformational epitopes. These data provide important information for understanding the antigenicity and immunogenicity of S protein and for designing SARS vaccines. This panel of anti-S MAbs can be used as tools for studying the structure and function of the SARS-CoV S protein.