LDL Receptor Binding Domain Research Articles

Mimicking Nature‘S Nanocarrier: Synthetic Low-Density Lipoprotein-Like Nanoparticles for Cancer-Drug Delivery

Evaluation of: Nikanjam M, Blakely EA, Bjornstad KA, Shu X, Budinger TF, Forte TK: Synthetic nano-low density lipoprotein as targeted drug delivery vehicle for glioblastoma multiforme. Int. J. Pharm. 3287, 86–94 (2007) [1] . Low-density lipoproteins have long been recognized as a viable nanocarrier for targeted delivery of drug and imaging agents. Many groups have published promising initial findings; however, progress in this field has been impeded by the need to isolate low-density lipoproteins from fresh donor plasma. In a recent paper by Nikanjam and colleagues, synthetic low-density lipoprotein-like nanoparticles were prepared from commercial lipids and a bifunctional synthetic peptide containing the low-density lipoprotein receptor-binding domain and the lipid-binding motif. These particles were shown to behave similarly to native low-density lipoproteins and also to bind to the low-density lipoprotein receptor on cancer cells. Herein, we evaluate the utility of this novel delivery vehicle and discuss what role this technology may have in nanomedicine.

Targeted delivery of proteins across the blood–brain barrier

Treatment of many neuronal degenerative disorders will require delivery of a therapeutic protein to neurons or glial cells across the whole CNS. The presence of the blood-brain barrier hampers the delivery of these proteins from the blood, thus necessitating a new method for delivery. Receptors on the blood-brain barrier bind ligands to facilitate their transport to the CNS; therefore, we hypothesized that by targeting these receptors, we may be able to deliver proteins to the CNS for therapy. Here, we report the use of the lentivirus vector system to deliver the lysosomal enzyme glucocerebrosidase and a secreted form of GFP to the neurons and astrocytes in the CNS. We fused the low-density lipoprotein receptor-binding domain of the apolipoprotein B to the targeted protein. This approach proved to be feasible for delivery of the protein and could possibly be used as a general method for delivery of therapeutic proteins to the CNS.

Synthetic nano-low density lipoprotein as targeted drug delivery vehicle for glioblastoma multiforme

The low density lipoprotein (LDL) receptor has been shown to be upregulated in GBM tumor cells and is therefore a potential molecular target for the delivery of therapeutic agents. A synthetic nano-LDL (nLDL) particle was developed and tested to determine its utility as a drug delivery vehicle targeted to GBM tumors. nLDL particles were constructed by combining a synthetic peptide containing a lipid binding motif and the LDL receptor (LDLR) binding domain of apolipoprotein B-100 with a lipid emulsion consisting of phosphatidyl choline, triolein, and cholesteryl oleate. Composition analysis, fast protein liquid chromatography, and electron microscopy revealed that nLDL was highly reproducible and intermediate in size between high density lipoprotein and LDL particles (10.5 ± 2.8 nm diameter). The binding and uptake of fluorescently labeled nLDL particles was assessed using fluorescence microscopy. Uptake of nLDL was time dependent, exhibiting saturation at approximately 3 h, and concentration dependent, exhibiting saturation at concentrations greater than 5 μM peptide. Using Lysotracker as a cellular marker, nLDL co-localized with lysosomes. nLDL binding was eliminated by blocking LDLRs with suramin and nLDL inhibited binding of plasma LDL to LDLRs. Collectively these data strongly suggest that the synthetic nano-LDLs described here are taken up by LDLR and can serve as a drug delivery vehicle for targeting GBM tumors via the LDLR.

Characterization of low density lipoprotein receptor ligand interactions by fluorescence resonance energy transfer

The low density lipoprotein receptor (LDLR) is the prototype of a family of cell surface receptors involved in a wide range of biological processes. A soluble low density lipoprotein receptor (sLDLR) and a tryptophan (Trp)-deficient variant human apolipoprotein E3 (apoE3) N-terminal domain (NT) were used in binding studies. The sole cysteine in apoE3-NT was covalently modified with an extrinsic fluorescence probe, N-(iodoacetyl)-N'-(5-sulfo-1-napthyl)ethylenediamine (AEDANS), and the protein was complexed with lipid. Incubation of sLDLR with AEDANS-Trp-null apoE3-NT dimyristoylphosphatidylcholine (DMPC) disks, but not lipid-free AEDANS-apoE, induced an enhancement in AEDANS fluorescence emission intensity (excitation, 280 nm) consistent with intermolecular energy transfer from excited Trp in sLDLR to receptor-bound apoE. Ligand binding to sLDLR required calcium and was saturable. In competition binding assays, unlabeled apoE3-NT DMPC inhibited AEDANS-apoE DMPC binding to sLDLR more effectively than low density lipoprotein. Fluorescence changes in this system reflected pH-dependent ligand binding and release from sLDLR consistent with models derived from the X-ray crystal structure of the receptor at endosomal pH. Intermolecular energy transfer from excited Trp in LDLR family members to fluorescently tagged ligands represents a sensitive and convenient assay for the characterization of the myriad molecular interactions ascribed to this family of receptor.

The expression of apolipoprotein B epitopes is normal in LDL of diabetic and end-stage renal disease patients

When LDLs are exposed to glucose in vitro, glycation of apolipoprotein B100 (apoB) leads to a loss in its affinity for the LDL receptor and reproducible alterations in the immunoreactivity of specific apoB epitopes, including several epitopes close to the LDL receptor binding site. The aim of this work was to determine if similar immunological changes are observed in vivo in LDLs of diabetic and end-stage renal disease (ESRD) patients. The immunoreactivity of LDLs isolated from 14 diabetic patients with normal renal function and 13 patients with ESRD was studied with a panel of 25 well-characterised anti-apoB monoclonal antibodies. Although diabetic and ESRD LDLs showed evidence of glycation modification, none of the changes in the apoB immunoreactivity induced by glucose in vitro was observed in vivo, including those for epitopes close to the LDL receptor binding domain. These results suggest that in vivo glycation of LDLs is a complex process that is not mimicked by in vitro exposure of LDLs to high concentrations of glucose. This questions the clinical significance of the in vitro glycation studies used to understand the pathophysiological consequences of LDL glycation in diabetes and ESRD.

413. Targeting the Low-Density Lipoprotein Receptor on the Blood-Brain Barrier for Transport of β-Glucoronidase Can Treat the Neurological Degeneration of MPSVII

Inherited lysosomal storage disorders occur in approximately 1 in 8000 births worldwide resulting from deficient activity of a key enzyme involved in catalysis of glucosaminoglycans causing symptoms ranging from skeletal deformities to progressive neuronal degeneration. Enzyme replacement therapy has been used to treat the peripheral symptoms; however, no treatment effectively treats the neurological disorders associated with the diseases. We previously showed that fusion of the apolipoprotein B (ApoB) low-density lipoprotein receptor-binding domain to GFP resulted in transport of the protein across the blood brain barrier and uptake by neurons and astrocytes in the CNS.

A complete backbone assignment of the apolipoprotein E LDL receptor binding domain

Human apolipoprotein E (apoE) is a 299-residue exchangeable apolipoprotein that was initially recognized as a major determinant in lipoprotein metabolism and cardiovascular diseases. Recent evidence has indicated that apoE also plays critical roles in several other important biological processes not directly related to its lipid transport function, including Alzheimer's disease, cognitive function, immunoregulation, cell signaling, and possibly even infectious diseases. ApoE contains two structural/functional domains: A N-terminal domain spanning residues 1-191 that is responsible for apoE's LDL receptor binding activity and a C-terminal domain (residues 216-199) that is responsible for lipoprotein-binding (1). The x-ray crystal structure of the lipid-free apoE N-terminal domain was solved by Wilson et al in 1991 which represented the only high-resolution structure of this protein. This structure showed an unusually elongated four-helix bundle (2) that was organized in such 2 a way that its hydrophobic faces were directed towards the protein interior, whereas the hydrophilic faces were oriented towards the solvent. The major receptor-binding region, residues 130-150, was located on the fourth helix. The amphipathic a-helices were connected by short loops, giving rise to a compact, globular structure. However, this structure only contained residues 23-165. Recent studies have shown that residues beyond residues 23-165 are alsomore » very important to the apoE LDL receptor binding activity. For example, a mutation at position R172 reduces the receptor binding activity of apoE to only {approx}2% (3). In addition, an E3K mutant significantly increased the apoE receptor binding activity as well (4). While the x-ray crystal structure of the apoE N-terminal domain provided detailed structural information for most region of this domain, this structure does not provide an explanation of the above experimental results regarding the structural contribution to apoE's LDL receptor binding activity by these residues that are located in the region which is not seen in the x-ray crystal structure.« less

Screening of APOB Gene Mutations in Subjects with Clinical Diagnosis of Familial Hypercholesterolemia

Monogenic hypercholesterolemia is a group of lipid disorders, most of which have autosomal dominant transmission. Familial defective apoB (FDB) resulting from mutations in the APOB gene is a well-recognized cause of autosomal dominant monogenic hypercholesterolemia (ADMH). However, the frequency of FDB among patients with ADMH is not well established. The aim of our research was to screen for mutations responsible for FDB in subjects with a clinical diagnosis of familial hypercholesterolemia. We studied 408 patients from the Spanish Register of Familial Hypercholesterolemia, proportionally distributed among all Spanish regions. Abnormal SSCP patterns of the APOB gene were checked by DNA sequencing and restriction analysis. Three out of the 408 patients were carriers of the R3500Q mutation, and 2 subjects were carriers of the silent T3552T mutation; in both of these patients functional mutations in the LDL receptor gene were found. We conclude that FDB is not a common cause of ADMH in Spain; the R3500Q mutation is the only mutation in APOB causing FDB, and the LDL receptor binding domain of APOB is highly conserved in the studied sample.

P0171 PP CLINICAL AND BIOLOGICAL PREDICTORS FOR DOMINANTLY INHERITED HYPERCHOLESTEROLEMIA IN CHILDREN. VALIDATION BY MOLECULAR GENETICS.

Introduction: Hypercholesterolemia (HC) with autosomal dominant inheritance [Familial HC (FH) and familial deficiency in apolipoprotein B100 (FDB)] are frequent in childhood. Although possibly confused in clinical practice with more common forms of hyperlipidemia, they represent a group at much higher risk for cardiovascular disease. Case finding during childhood may allow timely treatment in order to prevent premature coronary disease or sudden death in adults. The aim of the study was to use genetic testing to define the most predictive clinical and biological characteristics for severe dominantly inherited forms of disease in children with HC. Methods: Inclusion criteria were: total cholesterol (TC) > 200mg/dl with LDL-C > 130mg/dl and age <12 years at diagnosis. In all children, direct sequencing of the LDL receptor (LDLR) gene and of the LDLR binding domain of APOB gene was performed on genomic DNA. Lipid measurements [12h fasting plasma TC, HDL-C and triglycerides] were performed before and after 6 months under adapted diet. In addition, family history for cardiovascular disease was documented together with fasting plasma lipids measurements in first degree relatives. Results: 82 children (40 girls) were studied [median age: 5 years (1–12 y), mean 5,5 „b 2,8 years]. Forty nine children heterozygous for a LDLR (n=43) or APOB (n=6) mutation, were compared with 33 non mutation carrier HC children. Significantly higher plasma TC and LDL-C were found in mutation carriers (328 „b 61 vs 254 „b 37 mg/dl and 255 „b 51 vs 181 „b 37 mg/dl respectively; p<0,0001) with a lower LDL-C decrease under diet (−12 „b 16 vs −22 „b 14%; p=0,027). A higher frequency of lipid lowering drugs regularly taken by parents (79% vs 33%; p<0,0001) and of premature cardiovascular disease in grandparents (44% vs 17%; p=0,014) was found in mutation carrier children. Biological criteria for dominantly inherited HC were 1) TC > 300 mg/dl (specificity = 93%, PPV = 92%, p< 0,0001) and LDL-C > 220 mg/dl (specificity = 91%, PPV = 92%, p< 0,0001) at diagnosis; 2) persistent LDL-C > 190 mg/dl after 6 months under diet (specificity=96%, PPV=97%, p<0,0001). Conclusion: The precise analysis of family history and biological characteristics in children with HC before and after a test diet, may readily identify those children with inherited HC whom will mostly benefit from early and adapted prevention of cardiovascular disease.

700. Targeted Delivery of a Recombinant Protein to Neurons and Astrocytes by Transport and Uptake Via the Low Density Lipoprotein (LDL) Receptor for Treatment of Neurodegenerative Disorders

Inherited lysosomal storage disorders occur in approximately 1 in 8000 births worldwide resulting from deficient activity of a key enzyme involved in catalysis of glucosaminoglycans resulting in symptoms ranging from skeletal deformities to progressive neuronal degeneration. Enzyme replacement therapy has been used to treat the peripheral symptoms; however, no treatment effectively treats the neurological disorders associated with the diseases. Gaucher's disease is caused by a deficiency of the lysosomal enzyme glucocerebrosidase (GC). To test the potential of gene therapy for this disorder, the GC gene was fused at the C-terminus with the LDL receptor-binding domain of Apolipoprotein B or Apolipoprotein E, thus targeting the protein for uptake via the LDL receptor and transport to the lysosome. These constructs were tested in vitro for their ability to express and secrete enzymatically active GC enzyme. They were then inserted into the lentivirus vector and delivered intra-peritonealy to 4–6 week old Gaucher heterozygote knockout mice. Plasma samples taken from these mice showed increasing GC activity throughout the 14 days of the experiment. At 14 days post-delivery, liver, lung, spleen and whole brain was removed and homogenized to form lysates, which were then tested for GC enzyme activity. GC assays of the liver, lung and spleen showed increased activity in all mice that were injected with the lentivirus expressing the GC protein; however, only those mice that had received the virus expressing the GC – ApoE or ApoB fusion proteins showed elevated enzyme activity in the brain. Immunofluorescence staining of the liver of injected animals showed the recombinant enzyme was expressed from sinusoidal cells. Coronal sections of the CNS of those mice that had received the GC fusion construct showed co-staining of the recombinant enzyme with Calretinin, GFAP, and lectin indicating the uptake of the protein via neurons, astrocytes and endothelial cells. In addition, co-staining with LAMP1 indicated delivery of the enzyme to the lysosome. These results suggest not only a possible treatment for the neurological aspects of Gaucher's disease but also a general method for protein delivery to neurons and astrocytes of the CNS via hepatic gene delivery.

Inhibition of nicotinic acetylcholine receptors by apolipoprotein E-derived peptides in rat hippocampal slices

Apolipoprotein E (ApoE) is a well-known genetic risk factor for Alzheimer's disease (AD). Dysfunctions in cholinergic signaling, and in particular in the function of neuronal nicotinic acetylcholine receptors (nAChRs), have also been linked with AD and cognition. To address whether there is a link between ApoE and nAChR function, we used electrophysiological techniques to test the effects of synthetic ApoE-mimetic peptides derived from the low-density lipoprotein receptor (LDLR) binding domain for the ability to modulate nAChR activity in hippocampal interneurons. ApoE 133–149 completely inhibited ACh-evoked responses in a dose-dependent manner, yielding an IC 50 value of 720±70 nM. A shorter peptide spanning residues 141–148 mimicked this effect while a second peptide spanning residues 133–140 was without effect, indicating that the arginine-rich domain is responsible for nAChR interaction. Inhibition of ACh-evoked responses was voltage-independent, and displayed partial receptor specificity as no effect on glycine- or GABA-evoked responses occurred. These results demonstrate that peptides derived from the LDLR binding domain of ApoE block the function of nAChRs in hippocampal slices, an interaction that may have implications for AD.

Apolipoprotein E gene expression correlates with endogenous lipid nutrition and yolk syncytial layer lipoprotein synthesis during fish development.

During embryogenesis of teleost fish, the formation of a yolk syncytial layer (YSL) enables the resorption of the yolk reserves and development up to the larval stage. We have examined the changes of the yolk cell structure in relation to yolk and oil-globule lipid utilization during development of the turbot (Scophthalmus maximus). After encapsulation by the YSL, resorption of the single, large oil globule occurred predominantly after yolk resorption and was slower in fasting larvae. The YSL was in contact with an enlarged perisyncytial space, but no vascular network or red blood cells were present within the walls of the yolk sac. Intrasyncytial channels infiltrated by pigmented lining cells were observed in the YSL surrounding the oil globule. Apolipoprotein E (apoE) has a prominent role in lipid metabolism because of its ability to interact with lipoprotein receptors. We performed molecular cloning of the putative low-density lipoprotein-receptor binding domain of turbot apoE. In situ hybridization analysis revealed a very high level of apoE transcripts in the YSL, while no expression could be detected in the intestine. YSL apoE expression was correlated with the synthesis of very low density lipoprotein (VLDL) particles. An extraordinarily high number of VLDL particles were poured into the perisyncytial space, and intrasyncytial channels enabled the transfer of yolk- and oil globule-derived lipids to the developing embryo or larva. The pattern of apoE mRNA distribution in relation to YSL lipoprotein synthesis indicates that apoE expression is a suitable molecular marker for monitoring endogenous lipid nutrition during the endoexotrophic period of teleost fish development.

Lipid binding-induced conformational changes in the N-terminal domain of human apolipoprotein E

The N-terminal domain of human apolipoprotein E3 (apoE3) adopts an elongated, globular four helix bundle conformation in the lipid-free state. Upon lipid binding, the protein is thought to undergo a significant conformational change that is essential for manifestation of its low density lipoprotein receptor recognition properties. We have used fluorescence resonance energy transfer (FRET) to characterize helix repositioning which accompanies lipid interaction of this protein. ApoE3(1-183) possesses a single cysteine at position 112 and four tryptophan residues (positions 20, 26, 34, and 39). Modification of Cys112 with the chromophore, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)etheylenediamine (AEDANS) was specific and did not alter the secondary structure content of the protein. The efficiency of energy transfer from donor Trp residues to the AEDANS moiety was 49% in buffer, consistent with close proximity of the chromophores. Guanidine HCl titration experiments induced characteristic changes in the efficiency of energy transfer, indicating that FRET data faithfully reports on the conformational status of the protein. Interaction of AEDANS-apoE3(1-183) with dimyristoylphosphatidylcholine to form disk particles, or with detergent micelles, resulted in large decreases in the efficiency of energy transfer. Distance calculations based on the FRET measurements revealed that lipid binding increases the average distance between the four Trp donors and the AEDANS acceptor from 23 A to 44 A. The results obtained demonstrate the utility of FRET to investigate conformational adaptations of exchangeable apolipoproteins and are consistent with the hypothesis that, upon lipid binding, apoE3(1-183) undergoes conformational opening, repositioning helix 1 and 3 to adopt a receptor-active conformation.

Anatomy by computer experiment of the exchange of a water molecule buried in human apolipoprotein E

NMR experiments show that even water molecules that are well ordered in a crystal structure exchange with the external solvent. Despite crucial progress on the understanding of the exchange of crystal-buried water molecules, the detailed pathways followed by a water molecule to escape from or penetrate into the protein interior are unknown. The exchange of a crystal water molecule buried in the low-density lipoprotein receptor-binding domain of human apolipoprotein E with a water molecule from the external solvent was detected and monitored in a molecular dynamics simulation. This simulation shows that the escape of the crystal water molecule from the protein interior and the penetration of the water molecule from the bulk occur by a single-pathway mechanism involving conformational fluctuations of arginine and tryptophan sidechains. Along the pathway the exchanging water molecule interacts specifically with protein atoms by way of a varying pattern of hydrogen bonds. The exchange pathway revealed by the molecular dynamics trajectory suggests a mechanism by which hydrogen bonds work in relay to permit either the penetration or the expulsion of a water molecule. This result may have important implications not only on the process of water exchange but also to probe ligand binding to proteins.

A new function for the LDL receptor: transcytosis of LDL across the blood-brain barrier.

Lipoprotein transport across the blood-brain barrier (BBB) is of critical importance for the delivery of essential lipids to the brain cells. The occurrence of a low density lipoprotein (LDL) receptor on the BBB has recently been demonstrated. To examine further the function of this receptor, we have shown using an in vitro model of the BBB, that in contrast to acetylated LDL, which does not cross the BBB, LDL is specifically transcytosed across the monolayer. The C7 monoclonal antibody, known to interact with the LDL receptor-binding domain, totally blocked the transcytosis of LDL, suggesting that the transcytosis is mediated by the receptor. Furthermore, we have shown that cholesterol-depleted astrocytes upregulate the expression of the LDL receptor at the BBB. Under these conditions, we observed that the LDL transcytosis parallels the increase in the LDL receptor, indicating once more that the LDL is transcytosed by a receptor-mediated mechanism. The nondegradation of the LDL during the transcytosis indicates that the transcytotic pathway in brain capillary endothelial cells is different from the LDL receptor classical pathway. The switch between a recycling receptor to a transcytotic receptor cannot be explained by a modification of the internalization signals of the cytoplasmic domain of the receptor, since we have shown that LDL receptor messengers in growing brain capillary ECs (recycling LDL receptor) or differentiated cells (transcytotic receptor) are 100% identical, but we cannot exclude posttranslational modifications of the cytoplasmic domain, as demonstrated for the polymeric immunoglobulin receptor. Preliminary studies suggest that caveolae are likely to be involved in the potential transport of LDL from the blood to the brain.

The apolipoprotein B Q3405E polymorphism has no effect on its low-density-lipoprotein receptor binding affinity.

A better understanding of the apolipoprotein B100 (apoB100) sequences involved in binding to the low-density lipoprotein (LDL) receptor will be achieved by studying the effects of polymorphisms and rare mutations of apoB100. Upon re-examination of apoB100 DNA sequencing discrepancies, a charge-change polymorphism, Q3405E, was found in the putative LDL receptor binding domain of the protein. Positively charged lysine and arginine side chains of the protein have been demonstrated to participate in the ligand. This led us to propose that the presence of an additional negative charge in close proximity could have an impact on the binding affinity. The polymorphism is the result of a C-to-G transition at nucleotide 10422. Population screening revealed 20 of the less common glutamate alleles at an allele frequency of 0.9%. The effect of the presence of one glutamate allele on the binding affinity of LDL for the LDL receptor was investigated in seven heterozygous individuals by a competitive dual-label fibroblast binding assay. One individual who was homozygous for the glutamate allele was discovered and her LDL examined in a competitive displacement binding assay. The additional negative charge at residue 3405 had no detectable affect on the binding affinity.

Three-dimensional structure of the LDL receptor-binding domain of the human apolipoprotein E2 (Arg 136 → Cys) variant

The familial lipoprotein disorder type III hyperlipoproteinemia (HLP) is usually inherited as a recessive trait. Indeed, more than 90% of affected individuals are homozygous for a receptor binding-defective isoform of apolipoprotein (apo) E, apo E2. However, some rare apo E variants have been described that dominantly (thus in a single dose) predispose to the disease. Amino acid substitutions, which are accompanied with the loss of positive charges within the proposed apo E binding-region to lipoprotein receptors, seem to be responsible in most of these cases for the dominance with respect to the expression of type III HLP. So far available data in the literature on the naturally occurring human apo E2 (Arg 136 → Cys) variant are not conclusive about its recessive or dominant character. We recently identified a subject heterozygous for this mutation, presenting the typical clinical and biochemical characteristics of type III HLP. In the present study we performed further analysis of the mutation on apo E structure and function based on computer modeling. Our combined data point to a dominant influence of the apo E2 (Arg 136 → Cys) variant with respect to the transmission of the disease.

Screening and identification of familial defective apolipoprotein B-100 in clinical samples by capillary gel electrophoresis

Familial defective apolipoprotein B-100 (FDB) is a dominantly inherited disorder. It is characterized by a decreased affinity of low density lipoprotein (LDL) for the LDL receptor, as a consequence of a substitution of adenine by guanine in exon 26 of the apolipoprotein B-100 gene, coding for the putative LDL receptor-binding domain of the mature protein. This disorder is associated with a strikingly high incidence of arteriosclerosis and tends to cause disease and premature death. In this communication we describe a rapid capillary gel electrophoretic method in combination with molecular biology techniques to facilitate the diagnosis of FDB. Mutation screening for FDB is performed by an allele-specific amplification followed by capillary gel electrophoresis (CGE). For the combined polymerase chain reaction (PCR)-CGE method, a total analysis time of only 3 h is needed, a period that is normally necessary for the run and for staining of the gel only, not including the time for PCR, gel casting, etc. In our pilot study 4 of 43 hypercholesterolemic patients were found to have the predominant apoB 3500 codon mutation. The verification is demonstrated by DNA-sequencing. This pilot study will be followed by a large cohort analysis of the south-west German population to determine the frequency of FDB in this area. The PCR-CGE method on the Dionex capillary electrophoresis system (CES I) allows rapid, fully automated detection of the mutation resulting in the unequivocal diagnosis of FDB.

Structure-Function Relationship of Lipoprotein Lipase-mediated Enhancement of Very Low Density Lipoprotein Binding and Catabolism by the Low Density Lipoprotein Receptor: FUNCTIONAL IMPORTANCE OF A PROPERLY FOLDED SURFACE LOOP COVERING THE CATALYTIC CENTER

We examined the structure-function relationship of human lipoprotein lipase (hLPL) in its ability to enhance the binding and catabolism of very low density lipoproteins (VLDL) in COS cells. Untransfected COS cells did not bind to or catabolize normal VLDL. Expression of wild-type hLPL by transient transfection enhanced binding, uptake, and degradation of the VLDL (a property of LPL that we call bridge function). Heparin pretreatment and a monoclonal antibody ID7 that blocks LDL receptor-binding domain of apoE both inhibited binding, and apoE2/E2 VLDL from a Type III hyperlipidemic subject did not bind. However, LDL did not reduce 125I-VLDL binding to the hLPL-expressing cells, whereas rabbit beta-VLDL was an effective competitor. By contrast, LDL reduced uptake and degradation of 125I-VLDL to the same extent as excess unlabeled VLDL or beta-VLDL. These data suggest that binding occurs by direct interaction of VLDL with LPL but the subsequent catabolism of the VLDL is mediated by the LDL receptor. Mutant hLPLs that were catalytically inactive, S132A, S132D, as well as the partially active mutant, S251T, and S172G, gave normal enhancement of VLDL binding and catabolism, whereas the partially active mutant S172D had markedly impaired capacity for the process; thus, there is no correlation between bridge function and lipolytic activity. A naturally occurring genetic variant hLPL, S447-->Ter, has normal bridge function. The catalytic center of LPL is covered by a 21-amino acid loop that must be repositioned before a lipid substrate can gain access to the active site for catalysis. We studied three hLPL loop mutants (LPL-cH, an enzymatically active mutant with the loop replaced by a hepatic lipase loop; LPL-cP, an enzymatically inactive mutant with the loop replaced by a pancreatic lipase loop; and C216S/C239S, an enzymatically inactive mutant with the pair of Cys residues delimiting the loop substituted by Ser residues) and a control double Cys mutant, C418S/C438S. Two of the loop mutants (LPL-cH and LPL-cP) and the control double Cys mutant C418S/C438S gave normal enhancement of VLDL binding and catabolism, whereas the third loop mutant, C216S/C239S, was completely inactive. We conclude that although catalytic activity and the actual primary sequence of the loop of LPL are relatively unimportant (wild-type LPL loop and pancreatic lipase loops have little sequence similarity), the intact folding of the loop, flanked by disulfide bonds, must be maintained for LPL to express its bridge function.

Site-specific modification of apolipoprotein B by advanced glycosylation end-products: implications for lipoprotein clearance and atherogenesis.

An AGE-modified form of LDL (AGE-LDL) circulates in patients with diabetes mellitus or renal insufficiency and shows impaired plasma clearance kinetics when injected into transgenic mice that express the human LDL receptor. The advanced glycosylation inhibitor aminoguanidine decreases plasma LDL in diabetic patients, further suggesting that the AGE modification of LDL contributes significantly to increased LDL in vivo. We utilized AGE-specific antibodies to identify the major site(s) of AGE modification within apolipoprotein B (apoB), which impairs the binding of AGE-LDL by human fibroblast LDL receptors. Despite the large size of apoB (4536 amino acid residues) and its high content of potentially reactive lysines, the predominant site of AGE-immunoreactivity was found to lie within a single 67 amino acid domain located 1791 residues N-terminal to the LDL receptor binding site. These data point to the high specificity and reactivity of this site toward AGE formation and to a significant structural interaction between this region of apoB and the LDL receptor binding domain. A low but detectable degree of AGE modification was found to affect this site in LDL which was isolated from normal, non-diabetic individuals, suggesting that advanced glycosylation may contribute to elevated LDL in the general population as well.