RESUMEN
Defective binding of apolipoprotein E (apoE) to heparan sulfate proteoglycans (HSPGs) is associated with increased risk of atherosclerosis due to inefficient clearance of lipoprotein remnants by the liver. The interaction of apoE with HSPGs has also been implicated in the pathogenesis of Alzheimer's disease and may play a role in neuronal repair. To identify which residues in the heparin-binding site of apoE and which structural elements of heparan sulfate interact, we used a variety of approaches, including glycosaminoglycan specificity assays, (13)C nuclear magnetic resonance, and heparin affinity chromatography. The formation of the high affinity complex required Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147 from apoE and N- and 6-O-sulfo groups of the glucosamine units from the heparin fragment. As shown by molecular modeling, using a high affinity binding octasaccharide fragment of heparin, these findings are consistent with a binding mode in which five saccharide residues of fully sulfated heparan sulfate lie in a shallow groove of the alpha-helix that contains the HSPG-binding site (helix 4 of the four-helix bundle of the 22-kDa fragment). This groove is lined with residues Arg-136, Ser-139, His-140, Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147. In the model, all of these residues make direct contact with either the 2-O-sulfo groups of the iduronic acid monosaccharides or the N- and 6-O-sulfo groups of the glucosamine sulfate monosaccharides. This model indicates that apoE has an HSPG-binding site highly complementary to heparan sulfate rich in N- and O-sulfo groups such as that found in the liver and the brain.
Asunto(s)
Apolipoproteínas E/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Animales , Apolipoproteínas E/química , Arginina/química , Sitios de Unión , Biotinilación , Encéfalo/metabolismo , Bovinos , Cromatografía de Afinidad , Relación Dosis-Respuesta a Droga , Glucosamina/química , Proteoglicanos de Heparán Sulfato/química , Heparina/química , Heparina/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Hígado/metabolismo , Lisina/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutación , Polisacáridos/metabolismo , Unión Proteica , Serina/química , Estreptavidina/química , Resonancia por Plasmón de Superficie , Factores de TiempoRESUMEN
Apolipoprotein E7 (apoE7) (apoE3 E244K/E245K) is a naturally occurring mutant in humans that is associated with increased plasma lipid levels and accelerated atherosclerosis. It is reported to display defective binding to low density lipoprotein (LDL) receptors, high affinity binding for heparin, and like apoE4, preferential association with very low density lipoproteins (VLDL). There are two potential explanations for the preference of apoE7 for VLDL: lysine mutations, which occur in the major lipid-binding region (residues 244-272) of the carboxy-terminal domain of apoE7, could either directly determine the lipoprotein-binding preference or could interact with negatively charged residues in the amino-terminal domain, resulting in a domain interaction similar to that in apoE4 (interaction of Arg-61 with Glu-255), which is responsible for the apoE4 VLDL preference. To distinguish between these possibilities, we determined the binding preferences of recombinant apoE7 and two amino-terminal domain mutants, apoE7 (E49Q/E50Q) and apoE7 (D65N/E66Q), to VLDL-like emulsion particles. ApoE7 and both mutants displayed a higher preference for the emulsion particles than did apoE3, indicating that the carboxy-terminal lysine mutations in apoE7 are directly responsible for its preference for VLDL. Supporting this conclusion, the carboxy-terminal domain 12-kDa fragment of apoE7 (residues 192;-299) displayed a higher preference for VLDL emulsions than did the wild-type fragment. In addition, lipid-free apoE7 had a higher affinity for heparin than did apoE. However, when apoE7 was complexed with dimyristoylphosphatidylcholine or VLDL emulsions, the affinity difference was eliminated. In contrast to previous studies, we found that apoE7 does not bind defectively to the LDL receptor, as determined in both cell culture and solid-phase assays. We conclude that the two additional lysine residues in the carboxy-terminal domain of apoE7 directly alter its lipid- and heparin-binding affinities. These characteristics of apoE7 could contribute to its association with increased plasma lipid levels and atherosclerosis.
Asunto(s)
Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Lipoproteínas VLDL/metabolismo , Lisina/genética , Mutación , Apolipoproteína E3 , Dimiristoilfosfatidilcolina/metabolismo , Emulsiones , Heparina/metabolismo , Humanos , Microscopía Electrónica , Mutagénesis Sitio-Dirigida , Tamaño de la Partícula , Receptores de LDL/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Conserved lysines and arginines within amino acids 140-150 of apolipoprotein (apo) E are crucial for the interaction between apoE and the low density lipoprotein receptor (LDLR). To explore the roles of amphipathic alpha-helix and basic residue organization in the binding process, we performed site-directed mutagenesis on the 22-kDa fragment of apoE (amino acids 1-191). Exchange of lysine and arginine at positions 143, 146, and 147 demonstrated that a positive charge rather than a specific basic residue is required at these positions. Consistent with this finding, substitution of neutral amino acids for the lysines at positions 143 and 146 reduced the binding affinity to about 30% of the wild-type value. This reduction corresponds to a decrease in free energy of binding of approximately 600 cal/mol, consistent with the elimination of a hydrogen-bonded ion pair (salt bridge) between a lysine on apoE and an acidic residue on the LDLR. Binding activity was similarly reduced when K143 and K146 were both mutated to arginine (K143R + K146R), indicating that more than the side-chain positive charge can be important.Exchanging lysines and leucines indicated that the amphipathic alpha-helical structure of amino acids 140-150 is critical for normal binding to the low density lipoprotein receptor.
Asunto(s)
Apolipoproteínas E/metabolismo , Receptores de LDL/metabolismo , Secuencia de Aminoácidos , Aminoácidos Diaminos/química , Apolipoproteínas E/química , Apolipoproteínas E/genética , Dicroismo Circular , Secuencia Conservada , Escherichia coli/genética , Humanos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
An amino-terminal fragment of human apolipoprotein E3 (residues 1-165) has been expressed and crystallized in three different crystal forms under similar crystallization conditions. One crystal form has nearly identical cell dimensions to the previously reported orthorhombic (P2(1)2(1)2(1)) crystal form of the amino-terminal 22 kDa fragment of apolipoprotein E (residues 1-191). A second orthorhombic crystal form (P2(1)2(1)2(1) with cell dimensions differing from the first form) and a trigonal (P3(1)21) crystal form were also characterized. The structures of the first orthorhombic and the trigonal form were determined by seleno-methionine multiwavelength anomalous dispersion, and the structure of the second orthorhombic form was determined by molecular replacement using the structure from the trigonal form as a search model. A combination of modern experimental and computational techniques provided high-quality electron-density maps, which revealed new features of the apolipoprotein E structure, including an unambiguously traced loop connecting helices 2 and 3 in the four-helix bundle and a number of multiconformation side chains. The three crystal forms contain a common intermolecular, antiparallel packing arrangement. The electrostatic complimentarity observed in this antiparallel packing resembles the interaction of apolipoprotein E with the monoclonal antibody 2E8 and the low density lipoprotein receptor. Superposition of the model structures from all three crystal forms reveals flexibility and pronounced kinks in helices near one end of the four-helix bundle. This mobility at one end of the molecule provides new insights into the structural changes in apolipoprotein E that occur with lipid association.
Asunto(s)
Apolipoproteínas E/química , Metabolismo de los Lípidos , Animales , Apolipoproteínas/química , Cristalografía por Rayos X , Electrones , Saltamontes , Humanos , Modelos Moleculares , Mariposas Nocturnas , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de LDL/química , Receptores de LDL/metabolismoRESUMEN
The crystal structure of the Fab fragment of 2E8, the monoclonal IgG1,kappa antibody specific for the low-density lipoprotein (LDL) receptor-binding region of apolipoprotein E (apoE), has been solved by molecular replacement and refined at 1.9 A resolution (PDB entry 12E8). Two 2E8 Fab molecules in the asymmetric unit are related by noncrystallographic symmetry and are hydrogen bonded through a beta-sheet-like intermolecular contact between the heavy-chain complementarity-determining regions 3 (CDRH3) of each molecule. The structure has been refined to an R value of 0.22 (Rfree = 0.27). The initially ill-defined heavy-chain constant domain (CH1) of 2E8 has been retraced with the aid of automatic refinement, confirming the beta-sheet tracing independently of any starting models. As a resolution better than 2 A is not common for Fab fragments, this model represents a well defined Fab structure and should prove useful in MR solution of other Fab fragments. Furthermore, in the absence of an LDL-receptor structure, the homology of the 2E8 CDRH2 to the ligand-binding domain of the LDL receptor has been exploited to model the apoE-LDL-receptor interaction.
Asunto(s)
Anticuerpos Monoclonales/química , Apolipoproteínas E/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Animales , Apolipoproteínas E/química , Apolipoproteínas E/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Electroquímica , Enlace de Hidrógeno , Ratones , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de Proteína , Receptores de LDL/química , Receptores de LDL/metabolismoRESUMEN
Both apolipoprotein (apo) E2 and apoE-Leiden (tandem repeat of amino acids 121-127) are associated with type III hyperlipoproteinemia and bind defectively to low density lipoprotein receptors. Removing the carboxyl terminus of both variants (residues 192-299) increases receptor-binding activity, suggesting that the carboxyl terminus modulates activity. To identify the region(s) that modulated binding activity, we produced carboxyl-terminal truncations in apoE2 and apoE-Leiden (terminating at positions 191, 223, 244, and 272) and in apoE3 (terminating at positions 191, 223, and 244) and compared their receptor-binding activities as dimyristoylphosphatidylcholine (DMPC) discs. The results suggest that the entire carboxyl terminus up to residue 272, not a discrete smaller segment, is responsible for the modulation in apoE2. Intact apoE-Leiden and the 223 and 244 variants displayed similar activities (approximately 25% of apoE3's), but the 191 variant's activity was identical to that of intact apoE3. ApoE-Leiden and its truncated variants formed larger DMPC discs than did intact or truncated apoE3 or apoE2. These discs contained more apoE molecules than apoE3 discs, suggesting that the apparently normal binding activity of the apoE-Leiden 191 variant results from an increased number of apoE molecules and that the binding activity is actually defective. Direct comparison in a solid-phase assay revealed that the binding activity of the apoE-Leiden fragment was defective (51.4+/-9.4%). Thus, the defective binding of apoE-Leiden results from a direct effect of the seven amino acid repeat on receptor-binding activity rather than from an indirect effect operating through the carboxyl terminus as previously believed.
Asunto(s)
Apolipoproteínas E/química , Apolipoproteínas E/metabolismo , Estructura Secundaria de Proteína , Receptores de LDL/sangre , Apolipoproteína E2 , Apolipoproteína E3 , Sitios de Unión , Unión Competitiva , Dimiristoilfosfatidilcolina , Variación Genética , Humanos , Cinética , Modelos Moleculares , Receptores de LDL/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The low density lipoprotein (LDL) receptor plays a key role in cholesterol homeostasis, mediating cellular uptake of lipoprotein particles by high affinity binding to its ligands, apolipoprotein (apo) B-100 and apoE. The ligand-binding domain of the LDL receptor contains 7 cysteine-rich repeats of approximately 40 amino acids; each repeat contains 6 cysteines, which form 3 intra-repeat disulfide bonds. As a first step toward determining the structure of the LDL receptor, both free and bound to its ligands, we produced in Escherichia coli a soluble fragment containing the ligand-binding domain (residues 1-292) as a thrombin-cleavable, heat-stable thioredoxin fusion. Modest amounts (5 mg/liter) of partially purified but inactive fragment were obtained after cell lysis, heat treatment, thrombin cleavage, and gel filtration under denaturing conditions. We were able to refold the receptor fragment to an active conformation with approximately 10% efficiency. The active fragment was isolated and purified with an LDL affinity column. The refolded receptor fragment was homogeneous, as determined by sodium dodecyl sulfate or non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing. The purified fragment did not react with fluorescein-5-maleimide, indicating that all 42 cysteines were disulfide linked. In addition, the refolded fragment exhibited properties identical to those of the intact native receptor: Ca2+-dependent binding and isoform-dependent apoE binding (apoE2 binding <5% of apoE3). Furthermore, antibodies to the fragment recognized native receptors and inhibited the binding of 125I-LDL to fibroblast LDL receptors. We conclude that we have produced a properly folded and fully active receptor fragment that can be used for further structural studies.
Asunto(s)
Pliegue de Proteína , Receptores de LDL/química , Apolipoproteína E2 , Apolipoproteína E3 , Apolipoproteína E4 , Apolipoproteínas E/metabolismo , Sitios de Unión , Escherichia coli , Humanos , Ligandos , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes/química , SolubilidadRESUMEN
The defective binding of apolipoprotein (apo) E2 to lipoprotein receptors, an underlying cause of type III hyperlipoproteinemia, results from replacement of Arg 158 with Cys, disrupting the naturally occurring salt bridge between Asp 154 and Arg 158. A new bond between Asp 154 and Arg 150 is formed, shifting Arg 150 out of the receptor binding region. Elimination of the 154-150 salt bridge by site-directed mutagenesis of Asp 154 to Ala restored the receptor binding activity to near normal levels. The X-ray crystal structure of apoE2 Ala 154 demonstrated that Arg 150 was relocated within the receptor binding region. Our results demonstrate that defective binding of apoE2 occurs by a novel mechanism of the replacement of one salt bridge with another.
Asunto(s)
Apolipoproteínas E/metabolismo , Hiperlipoproteinemia Tipo III/metabolismo , Receptores de LDL/metabolismo , Apolipoproteína E2 , Apolipoproteínas E/química , Apolipoproteínas E/genética , Cristalografía por Rayos X , Humanos , Hiperlipoproteinemia Tipo III/genética , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Receptores de LDL/químicaRESUMEN
Human apolipoprotein C-I, the smallest of the plasma apolipoproteins, is a component of several classes of plasma lipoproteins. Two crystalline forms of this protein, both suitable for high resolution X-ray diffraction analysis, have been obtained from mixtures of 2-methyl-2,4-pentanediol, sodium acetate, and octyl-beta-D-1-thioglucopyranoside at room temperature. The first form belongs to space group P2(1), with beta = 106 degrees and cell dimensions of a = 38.1 A, b = 50.8 A and c = 34.9 A. The asymmetric unit appears to contain two molecules of the protein. The second form belongs to space group P2(1)2(1)2(1), with cell dimensions of a = 34.5 A, b = 53.1 A, c = 71.1 A and it also appears to contain two molecules of the protein in the asymmetric unit.
Asunto(s)
Apolipoproteínas C/química , Apolipoproteína C-I , Apolipoproteínas C/aislamiento & purificación , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Cristalización , Cristalografía por Rayos X/métodos , Humanos , Conformación ProteicaRESUMEN
A family has been described in which type III hyperlipoproteinemia is associated with apo E phenotype E3/3 (Havel, R. J., L. Kotite, J. P. Kane, P. Tun, and T. Bersot. 1983. J. Clin. Invest. 72:379-387). In the current study, the structure of apo E from the propositus of this family was determined using both protein and DNA analyses. The propositus is heterozygous for two different apo E alleles, one coding for normal apo E3 and one for a previously undescribed variant apo E3 in which arginine replaces cysteine at residue 112 and cysteine replaces arginine at residue 142. Apo E gene analysis of nine other family members spanning four generations indicated that only those five members having type III hyperlipoproteinemia possess the variant apo E3. Like the propositus, all five are heterozygous for this variant, suggesting that the disorder in this family is transmitted in a dominant fashion. The variant apo E3 was defective in its ability to bind to lipoprotein receptors, and this functional defect probably contributes to the expression of type III hyperlipoproteinemia in this family.
Asunto(s)
Apolipoproteínas E/genética , Variación Genética , Hiperlipoproteinemia Tipo IV/genética , Adulto , Anciano , Secuencia de Aminoácidos , Apolipoproteínas E/aislamiento & purificación , Apolipoproteínas E/metabolismo , Secuencia de Bases , Niño , Femenino , Pruebas Genéticas , Humanos , Hiperlipoproteinemia Tipo IV/diagnóstico , Hiperlipoproteinemia Tipo IV/metabolismo , Focalización Isoeléctrica , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Fenotipo , Receptores de Superficie Celular/análisisRESUMEN
An underlying cause of type III hyperlipoproteinemia is the presence of variant forms of apolipoprotein (apo) E that are defective in binding to apo B,E low density lipoprotein receptors. This disorder is associated almost exclusively with the apo E2/2 phenotype. However, structural and functional heterogeneity have been demonstrated within this phenotype. The apo E2(Arg158----Cys) variant, displaying 1% of normal apo E3 binding activity, is the most defective known form. In this study, we describe a method in which a pair of 19-mer synthetic oligonucleotide probes were used to distinguish between DNA coding for arginine or cysteine at position 158 in apo E. The specificity of the probes was demonstrated by using DNA from subjects whose apo E protein sequence or phenotype was known. The probes were used to screen a French-Canadian population of 34 apo E2/2 subjects to determine the frequency of the apo E2(Arg158----Cys) variant. All 34 subjects, most of whom displayed clinical or biochemical features of type III hyperlipoproteinemia, were found to be homozygous for apo E2(Arg158----Cys), strongly suggesting that this variant is the most common form of apo E2 within this ethnic and clinical population. In addition, the utility of this approach in detecting new apo E mutants was demonstrated when DNA from one of the apo E3/3 control subjects, whose family has a history of hyperlipidemia and coronary artery disease, reacted with both probes. This result suggests that this subject is heterozygous for normal apo E3 and a new apo E3 variant that is likely to be functionally equivalent to apo E2(Arg158----Cys).
Asunto(s)
Apolipoproteínas E/genética , Arginina/genética , Cisteína/genética , Frecuencia de los Genes , Variación Genética , Hiperlipoproteinemia Tipo III/genética , Sondas de Oligonucleótidos , Adulto , Anciano , Secuencia de Aminoácidos , Apolipoproteína E2 , Apolipoproteínas E/sangre , Secuencia de Bases , ADN/genética , Femenino , Humanos , Hiperlipoproteinemia Tipo III/sangre , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , QuebecRESUMEN
Familial defective apolipoprotein (apo) B-100 is a recently described genetic disorder that appears to result from a mutation in the apoB-100 gene. This disorder is characterized by hypercholesterolemia resulting from elevated plasma concentrations of low density lipoprotein LDL. The disorder was first detected in three members of one family. The LDL from affected subjects binds defectively (approximately 30% of normal) to LDL receptors, retarding the clearance of LDL from plasma. In the present study, two other members of the affected family were found to possess abnormal LDL. In addition, abnormal LDL with a similar binding defect were found in a second, unrelated family. In both families, the defect is transmitted over three generations as an autosomal codominant trait and all affected members are heterozygotes. Since there is only one apoB-100 molecule per LDL particle, the abnormal LDL in heterozygous subjects is made up of two populations of particles: one that has normal binding activity to receptors and one that binds defectively. To localize the mutation in apoB-100, the binding of five apoB-100-specific monoclonal antibodies to abnormal LDL was assessed in a solid-phase RIA. Only antibody MB47, whose epitope is between residues 3350 and 3506, distinguished abnormal LDL from normal LDL isolated from control subjects with normal lipid levels; MB47 bound with a higher affinity (by approximately 60%) to abnormal LDL. In every individual with abnormal LDL, the MB47 antibody bound with a higher affinity. The convenience of this assay will facilitate screening of large populations to determine the frequency of this disorder.
Asunto(s)
Anticuerpos Monoclonales , Apolipoproteínas B/genética , Hiperlipoproteinemia Tipo II/genética , Lipoproteínas LDL/inmunología , Adulto , Anciano , Apolipoproteína B-100 , Arteriosclerosis/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Linaje , Polimorfismo GenéticoRESUMEN
A rapid procedure for determining apolipoprotein E genotype from genomic DNA has been developed. In this procedure, DNA is amplified by the polymerase chain reaction, and allele-specific oligonucleotide probes are used to detect the cysteine-arginine interchanges at residues 112 and 158 that distinguish the three common isoforms of apolipoprotein E. The method was tested with 68 subjects, representing the six common phenotypes, and yielded results consistent with the known phenotype.
Asunto(s)
Apolipoproteínas E/genética , ADN Polimerasa Dirigida por ADN , ADN/genética , Amplificación de Genes , Sondas de Oligonucleótidos , Alelos , Secuencia de Bases , Genotipo , Humanos , Hibridación de Ácido Nucleico , FenotipoRESUMEN
An effective preparative isoelectric focusing method has been developed using the LKB Immobiline system in a vertical slab gel apparatus. Advantages of this procedure are ease of sample application, excellent resolution, and the direct visualization of focused bands. Narrow pH gradients have been used to separate apolipoprotein E3 isoforms (pH gradient 4.9-5.9) and to resolve the apolipoprotein C mixture (pH gradient 4.0-5.0). Recoveries ranged from 40 to 70%. The method should be valuable for protein and isoform purification.