AIMilano apoprotein identification of the complete kindred and evidence of a dominant genetic transmission.

The AIMilano apoprotein variant is associated with a marked reduction of high density lipoprotein (HDL) cholesterol levels and with increased triglyceridemia. In spite of the low HDL-cholesterol (HDL-Ch), carriers do not generally show clinical signs of atherosclerosis. The biochemical disorder is linked to a molecular change in apoprotein AI, that is, an arg----cys substitution in the 173 position, thus allowing the formation of AIMilano-AIMilano dimers and AIMilano-AII complexes. The origin of the variant gene has been located in Limone sul Garda, a small community in Northern Italy (about 1,000 individuals). This community has a genetic, biochemical, and clinical individuality, consequent to its isolation up to a few years ago; the citizens show highly uniform alimentary habits and elevated consanguinity. The complete population of the small village was sampled, and, by the use of an analytical isoelectric focusing technique for the detection of the mutant, a total of 33 living carriers, ranging in age from 2 to 81 yrs, were identified. Analysis of the genealogic tree of the complete family groups showed that the apoprotein (apo) AIMilano is transmitted as an autosomal dominant trait, all carriers coming from a single mating couple, living in the eighteenth century. The carriers are heterozygous for the apoprotein variant.     

The functional characteristics of a human apolipoprotein E variant (cysteine at residue 142) may explain its association with dominant expression of type III hyperlipoproteinemia.

Type III hyperlipoproteinemia typically is associated with homozygosity for apolipoprotein (apo) E2(Arg158----Cys). Dominant expression of type III hyperlipoproteinemia associated with apoE phenotype E3/3 is caused by heterozygosity for a human apoE variant, apoE3(Cys112----Arg, Arg142----Cys). However, this apoE3 variant was not separable from the normal apoE3 in these patients' plasma because the two proteins have identical amino acid composition, charge, and molecular weight. Therefore, to determine the functional characteristics of this protein, we used recombinant DNA techniques to produce this apoE variant in bacteria. We also produced a non-naturally occurring variant, apoE(Arg142----Cys), that had only the cysteine substituted at residue 142. These two apoE variants were purified from cell lysates of the transfected Escherichia coli by ultracentrifugal flotation in the presence of phospholipid, by gel filtration chromatography, and by heparin-Sepharose chromatography. Both Cys142 apoE variants bound to lipoprotein receptors on human fibroblasts with only about 20% of normal binding activity. Therefore, cysteine at residue 142, not arginine at residue 112, is responsible for the decreased receptor binding activity of the variants. Cysteamine treatment and removal of the carboxyl-terminal domain had little effect on the binding activity, whereas both modulate the receptor binding activity of apoE2(Arg158----Cys). The mutation at residue 142 decreased the binding activity of apoE to both heparin and the monoclonal antibody 1D7 (this antibody inhibits receptor binding of apoE), whereas apoE2(Arg158----Cys), which is associated with recessive expression of type III hyperlipoproteinemia, binds normally to both. The Arg112, Cys142 variant predominantes 3:1 over normal apoE3 in the very low density lipoproteins of plasma from an affected subject, as assessed by differential reactivity with the antibody 1D7. The unique combination of functional properties of the Arg112, Cys142 variant provides a possible explanation for its association with dominant expression of type III hyperlipoproteinemia.     

Formation of micellar complexes of apolipoprotein A-I with dimyristoylphosphatidylcholine

Structural changes of apolipoprotein AI from human plasma high density lipoproteins in 2-chloroethanol solutions were studied using spin label and fluorescence techniques. Reversible changes in spectral parameters occur in 2-chloroethanol concentration range 0-40% and are affected by dimyristoylphosphatidylcholine, of 2-chloroethanol concentration does not exceed 30%. Dialysis experiments demonstrated the absence of binding of monomer phosphatidylcholine with apolipoprotein AI. It thus follows that formation of complexes of apolipoprotein AI with dimyristoylphosphatidylcholine is caused by lipid micella aggregation.     

Immunological discrimination between the human apolipoprotein E2(Arg158----Cys) and E3 isoforms.

A specific anti-apoE2(Arg158----Cys) monoclonal antibody was raised by means of immunization of mice with a variant specific synthetic peptide. The peptide sequences used were homologous to apolipoprotein E of human and mouse. Consequently, the mouse immune system was tolerant to most of the selected sequences. Immunization with only one of selected peptides (amino acids 154-172) evoked an anti-peptide and anti-native protein response. Surprisingly, this peptide was predicted to have a low antigenicity index, in contrast to the other used peptides. The variant specific anti-peptide MAb that was generated with this sequence, recognizes apoE2(Arg158----Cys) and not apoE3. We here describe a sensitive, time saving, and simple immunoblot assay to detect apoE2(Arg158----Cys) in human sera without prior isoelectric focusing of serum proteins.     

Apolipoprotein C-III/sphingomyelin recombinants: formation, isolation, and characterization

The association of apolipoprotein C-III (apoC-III) from human very low density lipoprotein with sphingomyelin from egg yolk (EYSM) has been studied at the transition temperature (Tc) of the phospholipid. Upon incubation of aliquots of the apoprotein with increasing amounts of sphingomyelin, the alpha-helical content of the apoprotein increased from 20% in the absence of EYSM to a limiting value of 67% at a protein:lipid molar ratio of 1:200. The tryptophan fluorescence spectrum of the apoprotein exhibited a gradual blue shift from 356 nm in the absence of EYSM to 348 nm when the protein:lipid ratio in the complex had reached 1:50. Gel filtration chromatography of complexes formed by incubating the apoprotein and phospholipid at differing apoC-III:EYSM ratios demonstrated a disintegration of sphingomyelin vesicles into particles of decreasing size with increasing proportion of protein. This effect was confirmed by sedimentation velocity experiments in which the observed sedimentation coefficient of EYSM decreased from 14.0 S (for vesicles) to a limiting value of 7.0 S when the apoprotein:phospholipid ratio reached 1:50 in the complex. Electron micrographs of negatively stained EYSM vesicles showed spherical particles of 380-A diameter. Addition of apoC-III led to the formation of disk-shaped structures whose diameter decreased to a limiting value of 204 +/- 34 A at a protein:lipid ratio of 1:50. In contrast, the disk thickness was relatively constant at 51 +/- 2 A for all isolated complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of alpha-tocopherol with model human high-density lipoproteins.

The effects of alpha-tocopherol on the properties of model high-density lipoproteins (HDLs), composed of human apolipoprotein A-I and dimyristoylphosphatidylcholine, were investigated by physicochemical methods. The intrinsic fluorescence of alpha-tocopherol and its effects on the polarization of fluorescence of 1,6-diphenyl-1,3,5-hexatriene, which probes the hydrocarbon region of the lipids, and 4-heptadecyl-7-hydroxycoumarin, which is a probe of lipid surfaces, suggest that alpha-tocopherol is located at the lipid-water interface. Relative to cholesterol, alpha-tocopherol in lipid surfaces is virtually inert physicochemically. Incorporation of alpha-tocopherol into HDLs induces only a modest increase in particle size, no change in the transition temperature, and little change in lipid polarity and lipid-lipid interactions. Moreover, alpha-tocopherol has only a negligible effect on the kinetic parameters of the lipophilic enzyme lecithin:cholesterol acyltransferase, which binds to phosphatidylcholine surfaces and forms cholesteryl esters. However, alpha-tocopherol has a dramatic inhibitory effect on the rate of association of apolipoprotein A-I with dimyristoylphosphatidylcholine, a process that occurs through the insertion of the protein into preformed defects in the lipid surface. It is proposed that alpha-tocopherol inhibits the rate of association of apolipoprotein A-I with dimyristoylphosphatidylcholine by inserting into defects within the lipid surface, thereby reducing the size and/or number of sites for insertion of apolipoprotein A-I.     

A novel apolipoprotein A-1 variant, Arg173Pro, associated with cardiac and cutaneous amyloidosis

An American kindred was found to have hereditary amyloidosis with cutaneous and cardiac involvement. Characterization of fibrils isolated from skin identified the amyloid protein as the N-terminal 90 to 100 residues of apolipoprotein A-1. Sequence of the apolipoprotein A-1 gene was normal except for a G/C transversion at position 1638 which predicts an Arg to Pro substitution at residue 173. This mutation, unlike previously described amyloidogenic mutations is not in the N-terminal fragment which is incorporated into the fibril. The mutation is at the same residue as in apolipoprotein A-1 Milano (Arg173Cys) which does not result in amyloid formation. Decreased plasma HDL cholesterol levels in carriers of the Arg173Pro mutation suggest an increased rate of catabolism as has been shown for the amyloidogenic Gly26Arg mutation. This suggests that altered metabolism caused by the mutation may be a significant factor in apolipoprotein A-1 fibrillogenesis.     

Immunochemical characterization of six monoclonal antibodies to human apolipoprotein A-I: epitope mapping and expression.

We have produced and characterized six murine monoclonal antibodies to human apolipoprotein A-I named A-I-9, A-I-12, A-I-15, A-I-16, A-I-19, and A-I-57. All monoclonal antibodies were specific for apolipoprotein A-I and bound between 55% and 100% of 125I-labeled high density lipoproteins (HDL) in a fluid phase radioimmunoassay. All antibodies possessed a higher affinity to apoA-I in HDL than to free, delipidated apoA-I. Two of them, particularly A-I-12 and A-I-15, which were directed to the same or very close epitopes on the molecule, recognized very poorly the delipidated protein. Binding of apoA-I to phospholipid restored the immunoreactivity of the monoclonal antibodies to the protein suggesting that lipids play an important role in determining the immunochemical structure of apoA-I. Using CNBr fragments and synthetic peptides, the epitopes for the antibodies were mapped as follows: A-I-19, CNBr fragment 1; A-I-12 and 15, CNBr fragment 2; A-I-9 and A-I-16, CNBr fragment 3; A-I-57, CNBr fragment 4. Antibody A-I-57 failed to recognized a mutant form of apoA-I, A-IMilano (Arg173----Cys) by immunoblotting and by competitive radioimmunoassay demonstrating that substitution of a single amino acid in human apoA-I may cause the loss of an antigenic determinant.     

Stoichiometry and specificity of lipid-protein interaction with myelin proteolipid protein studied by spin-label electron spin resonance

The interaction of spin-labeled lipids with the myelin proteolipid apoprotein in complexes with dimyristoylphosphatidylcholine of varying lipid/protein ratios has been studied with electron spin resonance spectroscopy. A first shell of approximately 10 lipids per 25 000-dalton protein is found to be motionally restricted by the protein interface. This stoichiometry is consistent with a hexameric arrangement of the protein in the membrane. A selectivity of the various spin-labeled lipids for the motionally restricted component at the protein interface is found in the order stearic acid greater than phosphatidic acid greater than cardiolipin approximately greater than phosphatidylserine greater than phosphatidylglycerol approximately equal to phosphatidylcholine greater than phosphatidylethanolamine greater than androstanol approximately greater than cholestane.     

Properties of discoidal complexes of human apolipoprotein A-I with phosphatidylcholines containing various fatty acid chains

In this study we demonstrate that apolipoprotein A-I determined the common size classes of discoidal particles formed with numerous phosphatidylcholines, and with ether analogs of phosphatidylcholines. We show furthermore, that the nature of the lipids dictates the distribution of particles among the different size classes. These experiments were performed with discoidal complexes containing various phospholipids (phosphatidylcholines with saturated and unsaturated fatty acid chains of different lengths and the ether analog of 1-palmitoyl-2-oleoylphosphatidylcholine), cholesterol, and human apolipoprotein A-I, prepared by the sodium cholate dialysis method, and fractionated by Bio-Gel A-5m gel-filtration chromatography. The complex preparations were analyzed in terms of their average composition, spectral properties of the apolipoprotein, and the dynamic behavior of the lipid domains. Nondenaturing gradient gel electrophoresis was used to analyze the size classes of particles present in the complex preparations. Starting with reaction mixtures containing around 100:1, phospholipid/apolipoprotein A-I molar ratios, complexes were isolated with molar ratios from 40:1 to 100:1. In most complexes apolipoprotein A-I had high levels of alpha-helical structure (65-77% alpha-helix), and tryptophan residues in a nonpolar environment. The lipid domains of complexes exhibited the dynamic behavior expected of the main phospholipid components. In the average size range from 90 to 100 A diameters, discrete particle classes with 80, 87, 102, 108, or 112 A Stokes diameters were observed for all the complexes containing different phospholipids. These discrete, recurring particle sizes are attributed to distinct apolipoprotein A-I conformations and variable lipid content.     

Human plasma high density apolipoprotein A-I: Effect of protein-protein interactions on the spontaneous formation of a lipid-protein recombinant

The effect of the self-association of apolipoprotein A-I on the dynamics of lipid-protein complex formation was studied. Treatment of self-associated apolipoprotein A-I with guanidine hydrochloride initially resulted in dissociation of the oligomers into monomers and subsequent denaturation of the monomers. The association of monomeric and oligomeric apolipoprotein A-I with dimyristoylphosphatidylcholine resulted in identical lipid-protein recombinants as determined by chemical analysis and gel-filtration column elution profiles. Denaturation of a recombinant with guanidine hydrochloride indicated that the protein is more stable in a lipid-protein recombinant than as an oligomer; however, self-association does decrease the rate of lipidprotein recombinant formation. Because apolipoprotein A-I is more stable when it is associated with lipid, we conclude that the association of this protein with a variety of lipids is subject to kinetic control.     

Examination of the peptide sequence requirements for lipid-binding. Alternative pathways for promoting the interaction of amphipathic alpha-helical peptides with phosphatidylcholine

To examine the relationship between peptide sequence and the interaction of amphipathic alpha-helical peptides with phosphatidylcholines, various methods of mixing the peptide and lipid were explored. A series of amphipathic alpha-helical peptides containing from 10 to 18 residues were synthesized by solid-phase techniques. An 18-residue peptide and two relatively hydrophobic 10-residue peptides did not disrupt dimyristoylphosphatidylcholine liposomes when added to the lipid in buffer. However, when the peptides were premixed with lipid in a suitable organic solvent and then reconstituted with aqueous buffer, clear micelles were formed, indicating association of the amphipathic alpha-helical peptide with lipid. In general, the best solvent for this purpose was trifluoroethanol. The circular dichroic and fluorescence spectra of peptides which readily formed clear mixtures when mixed in buffer with dimyristoylphosphatidylcholine liposomes were similar when prepared either by the alternative pathway technique using trifluoroethanol or by a cholate removal technique. For the peptides which did not clear liposomes in buffer, first mixing with dimyristoylphosphatidylcholine in trifluoroethanol resulted in an increase in the alpha-helicity of the peptides as judged by circular dichroic spectra and a blue-shift in the fluorescence emission maxima of the single tryptophan residue in each peptide. These data are consistent with formation of an amphipathic alpha-helix in lipid by peptides which based on mixing experiments with dimyristoylphosphatidylcholine liposomes in buffer at the phase transition temperature of the lipid would be considered ineffective in lipid binding. Thus, simple mixing of peptides with liposomes may give misleading results concerning the intrinsic affinity of a particular peptide sequence for lipid. In addition, the data demonstrate that relatively hydrophobic amphipathic alpha-helical peptides which do not form small micelles with dimyristoylphosphatidylcholine spontaneously in aqueous solution may interact with lipid as typical amphipathic alpha-helices when mixed by an alternative pathway.     

Reassembly of lipid-protein complexes of pulmonary surfactant. Proposed mechanism of interaction

We studied the interaction at 37 degrees C between a major apolipoprotein of pulmonary surfactant and 11 mixtures of lipids. The experiments were carried out in the presence of either 3 mM Ca2+ or 10 mM EDTA. The amount of apolipoprotein associated with lipid was independent of Ca2+. However the binding was sensitive to the percentage of gel-state lipid in the vesicles, and the amount of apolipoprotein in the recombinant lipoprotein complex decreased as the percentage of fully saturated phospholipid was reduced. Maximum association of the apolipoprotein occurred with lipid vesicles containing 85% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 15% 1,2-dipalmitoyl-sn-glycero-3-phospho-1-glycerol or 1,2-dipalmitoyl-sn-glycerol. Fluorescence measurements on the apolipoprotein indicated that the tryptophan side chains were in a relatively hydrophobic environment, and that the wavelength of maximum fluorescence emission was not changed upon the binding of lipid. The results suggest that the principal mode of interaction between the apolipoprotein and lipids of surfactant is hydrophobic bonding. The most extensive binding occurs with lamellar lipids in a gel that would be expected to have inhomogeneities in packing density due to the presence of acidic phospholipids or other glycerolipids. The role of Ca2+ in this interaction has not been fully determined. Although it is not needed to effect the binding of the lipids and the apolipoprotein, it does influence the physical state of the complex, and possibly the stoichiometry of lipid to protein. Some of the processes mediated by Ca2+ in this interaction may be analogous to those observed in membrane fusion. Thus, Ca2+ probably causes segregation of the lamellar phospholipids into domains, inducing vesicular disruption and fusion. This lipid aggregates about hydrophobic sites on the protein, thereby forming high molecular weight reassembly complexes.     

重组人载脂蛋白AI米兰突变体在大肠杆菌中的高表达

用RT-PCR法从人肝总RNA库中克隆出人载脂蛋白Al的cDNA序列,再通过重叠PCR将载脂蛋白AI的第179位精氨酸密码子突变成半胱氨酸密码子,即载胎蛋白AI米兰突变体基因。将此目的基因克隆至表达载体pQE30,重组质粒转化JMl09宿主菌,经表达试验筛选出高表达克隆;工程菌经诱导后表达出含6个氨基酸前肽的载脂蛋白AI米兰突变体。表达产物主要以可溶形式存在,但也有部分为包涵体。     

Expression of the human serum apolipoprotein AI and AII genes in Xenopus laevis oocytes. Lipid-associated secretion of gene products

The two major apolipoproteins of plasma high-density lipoproteins (HDL) are apolipoprotein AI (apo AI) and AII (apo AII). The apo AI and the correctly oriented apo CIII genes separated by 2.6 kb were obtained by fusion of two human lambda-genomic clones. The apo AII gene was isolated as a 3 kb clone. These apolipoprotein genes have been injected independently and together into Xenopus laevis oocytes and their expression studied. Both apolipoprotein genes were transcribed and translated into their preproforms and processed in Xenopus laevis oocytes to their proforms. They were secreted into the medium associated with newly synthesized phospholipids and neutral lipids as particles floating in the high-density lipoprotein range between 1.12 and 1.21 g/ml. Secreted apo AI is associated mainly with newly synthesized phosphatidylethanolamine and little triglyceride, apo AII with phosphatidylethanolamine, lysophosphatidylethanolamine and neutral lipids. Simultaneous injection of the apo AI and apo AII genes led to the secretion of both apoproteins which separated into two bands during CsCl-density gradient centrifugation. The heavier particles were associated with proapo AI and AII, phosphatidylethanolamine (greater than 90%) and traces of lysophosphatidylethanolamine as lipid components. Proapo AII was immunoprecipitated from the less dense fraction and found to be mainly associated with lysophosphatidylethanolamine. Radiolabelled newly synthesized apolipoproteins in secreted particles were characterized by immunoprecipitation after delipidation of the secreted lipoprotein particles. The oocyte-system proved very suitable for studies of the expression of serum apolipoprotein genes, the assembly of the apolipoproteins with specific lipids to lipoprotein particles and their secretion.     

Interaction between hepatic microsomal membrane lipids and apolipoprotein A-I

Incubation of apoprotein A-I (apo-A-I), the major protein component of human high density lipoprotein, with rat liver microsomal membranes under conditions of elevated pH and ionic strength leads to the production of a soluble protein:lipid complex (A-I/MM complex). The A-I/MM complex, as purified by density gradient centrifugation and agarose column chromatography, possesses a lipid composition similar to the hepatic microsomal membrane and a protein/lipid ratio similar to that of plasma high density lipoproteins, but markedly different from that of recombinant particles prepared with synthetic lipids. The A-I/MM complex constitutes a more physiological recombinant particle than can be formed using synthetic lipids and may be a suitable model for the newly assembled intracellular high density lipoproteins. Incubation of the erythrocyte plasma membranes with apo-A-I under the same conditions as used with microsomal membranes fails to generate any lipid:apoprotein complexes. This membrane specificity for forming soluble lipoprotein complexes suggests that the microsomal membranes possess a unique feature, possibly their lipid composition, which render them particularly suitable to serve as lipid donors to the apoproteins which are undergoing assembly within the endoplasmic reticulum/Golgi organelles.     

Ultrastructural immunogold labeling of lipid-laden enterocytes from patients with genetic malabsorption syndromes

Summary— Intestinal biopsies from patients having genetic disorders of lipoprotein assembly and secretion, such as abetalipoproteinemia (ABL) or Anderson's disease (AD), contain large amounts of lipids which are accumulated in the enterocytes. Determination of the intracellular sites in which the lipids accumulate and to which apolipoproteins the lipids are bound would help to identify the defects in these diseases and further elucidate the mechanisms by which lipoprotein assembly and secretion occur normally. Ultrastructural immunogold labeling, however, is hampered by the poor preservation of the lipids accumulated in the enterocytes of these patients. We have used routine electron microscopy (fixation and ultra-thin sectioning) along with three methods for immunogold labeling of lipid-laden enterocytes; ultrathin cryosectioning, low temperature freeze substitution with embedding in Lowicryl K4M, and ultra-low temperature freeze substitution with embedding in Lowicryl HM20, to establish a protocol for investigating the intestinal tissue from these patients. Ultracryosectioning, while preserving the overall morphology of the lipid laden enterocytes, did not preserve the lipid content and the immunogold labeling of apolipoprotein B (ApoB) appeared dislocated. Freeze substitution and low temperature embedding in Lowicryl K4M, in contrast, appeared to better preserve the lipid and lipoprotein structures; however, the antigenicity of both apoAI and apoB appeared to be lost and no specific labeling could be obtained. Freeze substitution and embedding in Lowicryl HM20 best preserved the lipid and lipoprotein structures while maintaining apoprotein antigenicity. In conclusion, immunogold labeling of apolipoproteins on lipid structures in the lipid-laden enterocytes of patients with ABL and AD is best obtained by freeze substitution and embedding in Lowicryl HM20.     

Mapping the structural transition in an amyloidogenic apolipoprotein A-I

The single amino acid mutation G26R in human apolipoprotein A-I (apoA-IIOWA) leads to the formation of beta-secondary structure rich amyloid fibrils in vivo. Here we show that full-length apoA-IIOWA has a decreased lipid-binding capability, an increased amino-terminal sensitivity to protease, and a propensity to form annular protofibrils visible by electron microscopy. The molecular basis for the conversion of apolipoprotein A-I to a proamyloidogenic form was examined by electron paramagnetic resonance spectroscopy. Our recent findings [Lagerstedt, J. O., Budamagunta, M. S., Oda, M. N., and Voss, J. C. (2007) J. Biol. Chem. 282, 9143-9149] indicate that Gly26 in the native apoprotein separates a preceding beta-strand structure (residues 20-25) from a downstream largely alpha-helical region. The current study demonstrates that the G26R variant promotes a structural transition of positions 27-56 to a mixture of coil and beta-strand secondary structure. Microscopy and staining by amyloidophilic dyes suggest that this alteration extends throughout the protein within 1 week of incubation in vitro, leading to insoluble aggregates of distinct morphology. The severe consequences of the Iowa mutation likely arise from the combination of losing the contribution of the native Gly residue in terminating beta-strand propagation and the promotion of beta-structure when an Arg is introduced adjacent to the succeeding residue of identical charge and size, Arg27.     

Structural features of apolipoprotein B synthetic peptides that inhibit lipoprotein(a) assembly

Lipoprotein(a) [Lp(a)] is assembled via an initial noncovalent interaction between apolipoprotein B100 (apoB) and apolipoprotein(a) [apo(a)] that facilitates the formation of a disulfide bond between the two proteins. We previously reported that a lysine-rich, alpha-helical peptide spanning human apoB amino acids 4372-4392 was an effective inhibitor of Lp(a) assembly in vitro. To identify the important structural features required for inhibitory action, new variants of the apoB4372-4392 peptide were investigated. Introduction of a central leucine to proline substitution abolished the alpha-helical structure of the peptide and disrupted apo(a) binding and inhibition of Lp(a) formation. Substitution of hydrophobic residues in the apoB4372-4392 peptide disrupted apo(a) binding and inhibition of Lp(a) assembly without disrupting the alpha-helical structure. Substitution of all four lysine residues in the peptide with arginine decreased the IC50 from 40 microM to 5 microM . Complexing of the arginine-substituted peptide to dimyristoylphosphatidylcholine improved its activity further, yielding an IC50 of 1 microM. We conclude that the alpha-helical structure of apoB4372-4392, in combination with hydrophobic residues at the lipid/water interface, is crucial for its interaction with apo(a). Furthermore, the interaction of apoB4372-4392 with apo(a) is not lysine specific, because substitutions with arginine result in a more effective inhibitor.     

The conformation of apolipoprotein A-I in high-density lipoproteins is influenced by core lipid composition and particle size: a surface plasmon resonance study

Plasma high-density lipoproteins (HDL) are a heterogeneous group of particles that vary in size as well as lipid and apoprotein composition. The effect of HDL core lipid composition and particle size on apolipoprotein (apo) A-I structure was studied using surface plasmon resonance (SPR) analysis of the binding of epitope-defined monoclonal antibodies. The association and dissociation rate constants of 12 unique apo A-I-specific monoclonal antibodies for isolated plasma HDL were calculated. In addition, the association rate constants of the antibodies were determined for homogeneous preparations of spherical reconstituted HDL (rHDL) that contained apo A-I as the sole apolipoprotein and differed either in their size or in their core lipid composition. This analysis showed that lipoprotein size affected the conformation of domains dispersed throughout the apo A-I molecule, but the conformation of the central domain between residues 121 and 165 was most consistently modified. In contrast, replacement of core cholesteryl esters with triglyceride in small HDL modified almost the entire molecule, with only two key N-terminal domains of apo A-I being unaffected. This finding suggested that the central and C-terminal domains of apo A-I are in direct contact with rHDL core lipids. This immunochemical analysis has provided valuable insight into how core lipid composition and particle size affect the structure of specific domains of apo A-I on HDL.