Dynamics of lipid-protein interactions. Interaction of apolipoprotein A-II from human plasma high density lipoproteins with dimyristoylphosphatidylcholine

ApoA-II and dimyristoylphosphatidylcholine (DMPC) spontaneously associate to give three different complexes whose structures are determined by the initial reactant concentration and by the reaction temperature with respect to Tc (23.9 degrees C), the gel to liquid crystalline transition temperature of DMPC. At an initial lipid to protein ratio of 45/1, a single complex (2.29 x 10(5) daltons) is quantitatively formed at all temperatures between Tc - 4 degrees C and Tc + 6 degrees C. When the 45/1 complex is mixed with DMPC liposomes there is lipid exchange but no net transfer of lipid, so that the structure of the complex remains unaltered. At an initial molar ratio of 100 to 300:1, the reaction scheme is more complex. At 24 degrees C a 240/1 complex (1.5 x 10(6) daltons) is formed from a precursor 75/1 complex (3.43 x 10(5) daltons) if excess (approximately 300 mol/mol) lipid is present. The 75/1 complex exhibits lipid exchange in the presence of added DMPC liposomes at 24 degrees C, and both the 75/1 and the 240/1 complex can be converted to smaller protein-rich complexes in the presence of added apoA-II. These results suggest that the initial lipid/protein ratio and the physical state of a lipid or lipid . protein complex determines the composition and structure of the resulting complex and support the view that lipid-protein interactions are stronger than protein-protein or lipid-lipid interactions.     

Abnormal interaction of the human apolipoprotein A-I variant [Lys107----0] with high density lipoproteins

Several isoforms of apoprotein A-I [apoA-I], the major apoprotein of high density lipoproteins [HDL], have been described. We compared the in vivo and in vitro properties of normal human apoA-I with those of apoA-I [Lys107----0]. Fluorescence and circular dichroic spectra showed that deletion of Lys107 decreases apoprotein self-association. In vivo metabolic studies in the rat indicated that the interaction of apoA-I [Lys107----0] with HDL was lower than normal. We conclude that deletion of Lys107 results in a reorganization of the apoprotein structure that decreases its potential to form hydrophobic associations.     

Very low density lipoprotein. Removal of Apolipoproteins C-II and C-III-1 during lipolysis in vitro.

In this study we have investigated the effects of very low density lipoprotein (VLDL) lipolysis on the removal of radiolabeled apolipoprotein C-II and apolipoprotein C-III-1 from in vitro lipolyzed lipoproteins. Lipolysis was carried out in vitro using lipoprotein lipase purified from bovine milk, and mixtures with or without plasma. Lipoproteins were isolated by ultracentrifugation and by gel filtration. Labeled apo-C-II and apo-C-III-1 distributed among plasma lipoproteins, predominantly VLDL and high density lipoprotein (HDL). Lipolysis induced transfer of apo-C-II and apo-C-III-1 from VLDL to HDL. The transfer was proportional to the extent of triglyceride hydrolysis, and similar for the two apoproteins. The apo-C-II/apo-C-III-1 radioactivity ratio did not change in either VLDL or the fraction of d greater than 1.006 g/ml during the progression of the lipolytic process. Similar observations were recorded while using plasma-devoid lipolytic systems. Gel filtration of incubation mixtures, on 6% agarose, revealed that the removal of labeled apo-C molecules from VLDL is not a consequence of either centrifugation or high salt concentration. These results suggest that there is no preferential removal of apo-C-II or apo-C-III-1 from lipolyzed VLDL particles. They further indicate that the ratio of apo-C-II to apo-C-III-1 does not regulate the extent of lipolysis of different VLDL particles, at least in VLDL isolated from normolipidemic humans.     

Primary sequence mapping of human apolipoprotein B-100 epitopes. Comparisons of trypsin accessibility and immunoreactivity and implication for apoB conformation

Differential trypsin-accessibility and monoclonal antibodies (Mabs) to human apolipoprotein (apo) B-100 are both important tools for probing apoB structure and conformation on low-density lipoproteins (LDL). In this study, we have mapped greater than 80% of the C-terminal region (720 residues) of LDL apoB-100 using trypsin digestion. Our results extend our previous data [Yang et al. (1986) Nature (Lond.) 323, 738-742] confirming that the C-terminal region of about 420 residues of apoB-100 is largely inaccessible to trypsin, whereas the part just preceding this region has interspersed trypsin-accessible and inaccessible peptides. We have determined the amino acid sequence of specific apoB-100 peptides containing epitopes recognized by four separate Mabs: two epitopes have been mapped to within 20 residues, one has been mapped to 36 residues, and the last to 80 residues. We used polyclonal antisera to identify 16 overlapping clones of varying lengths of apoB-100 cDNAs extending from the C-terminus of apoB-100 cloned in the expression vector, lambda gt11. These clones were then tested against individual Mabs. By nucleotide sequence analysis of overlapping clones that show differential reactivities to different Mabs, we have mapped the individual epitopes of each Mab to within about 50-150 amino acid residues predicted from the DNA sequences. Confirmation and further fine mapping were accomplished by competition for LDL binding using partially purified fusion proteins and chemically synthesized oligopeptides. Two epitopes (Mabs 7 and 22) were mapped to the C-terminal 20 amino acids of apoB-100, one (Mab 16) to residues 4154-4189, and another (Mab 20) to residues 3926-4005. Mab 16 precipitates more than 80% of LDL particles. Mab 20 precipitates only denatured apoB but not native LDL apoB [Milne et al. (1987) Mol. Immunol. 24, 435]. Mabs 7 and 22 are unique in that they precipitate LDL apoB modified by storage much better than freshly isolated LDL-apoB. Although epitope expression and trypsin-accessibility represent two useful probes for the study of protein conformation, there was no obvious correlation between these two parameters when applied to LDL apoB for the antibodies we have examined.     

Cholesterol metabolism in fibroblasts from rabbits resistant to diet-induced hypercholesterolemia

We have previously described a colony of New Zealand White rabbits that are resistant to hypercholesterolemia when fed a cholesterol-enriched diet. The present studies used skin fibroblasts obtained from normal and hypercholesterolemia-resistant rabbits to investigate cholesterol metabolism and lipid composition in vitro. The lipid compositions of the two cell lines after incubation in either fetal calf serum or lipoprotein-deficient serum were similar. The conversion of radiolabeled acetate into sterol and phospholipids was higher in resistant fibroblasts than in normal fibroblasts. In contrast, incorporation of radiolabeled oleic acid into cholesteryl ester was significantly lower in resistant fibroblasts than in normal cells. In parallel experiments, the 3-hydroxy-3-methylglutaryl coenzyme A reductase activity was higher and acyl-coenzyme A:cholesterol acyltransferase activity was lower in resistant cells compared to normal cells. Furthermore, binding, uptake, and degradation of normal rabbit 125I-labeled LDL (low density lipoproteins) were 30% higher in resistant than in normal fibroblasts. These observations are consistent with results from previous studies of cholesterol metabolism in the liver membranes of these rabbits. The results indicate that extrahepatic cells (such as fibroblasts) from the resistant rabbit exhibit the same altered cholesterol metabolism as that found in the hepatic tissues of these rabbits. These studies suggest that the resistant rabbit may provide an in vivo and in vitro system for studying the mechanisms by which some individuals of a species can minimize the effect of dietary cholesterol on the development of hypercholesterolemia and atherosclerosis.     

Relationship of the apolipoprotein E polymorphism with carotid artery atherosclerosis.

From the cohort taking part in the Atherosclerosis Risk in Communities (ARIC) study, a multicenter investigation of atherosclerosis and its sequelae in women and men ages 45-64 years, a sample of 145 subjects with significant carotid artery atherosclerosis but without clinically recognized coronary heart disease was identified along with 224 group-matched control subjects. The aim of this paper is to measure the association of the apolipoprotein (apo) E polymorphism with the prevalence of significant carotid artery atherosclerotic disease (CAAD) after considering the contribution of established risk factor variables. The first model used a stepwise selection procedure to define a group of significant physical and lifestyle characteristics and a group of significant plasma lipid, lipoprotein, and apolipoprotein variables that were predictive of CAAD status in this sample. Those variables selected included age (years), body mass index (BMI; kg/m2), consumption of cigarettes (CigYears; number of cigarettes/d x the number of smoking years), hypertension status, high-density lipoprotein (HDL)-cholesterol (mg/dl), total cholesterol (mg/dl), and Lp[a] (micrograms/ml). The second model was built by forcing into the equation an a priori set of demographic, anthropometric, and lipoprotein variables, which were age, BMI, CigYears, hypertensive status, LDL-cholesterol, and HDL-cholesterol. In both models, the apo E genotype epsilon 2/3 was related to CAAD status. For both models, the estimated odds ratio of being a CAAD case associated with the apo E genotype epsilon 2/3 was > 2:1. The mechanism of the observed association between the epsilon 2/3 genotype and carotid atherosclerosis is unknown, but it is likely due to the known effects of the E2 isoform in causing delayed clearance of triglyceride-rich lipoproteins.     

Diet and Health

The role of diet in personal health maintenance is important whether a person is trying to stay healthy or to treat diet-related diseases such as hypercholesterolemia, hypertension, diabetes mellitus or obesity. Dietary recommendations include limiting fat to 30% and protein to 20% of total calories, with the remaining 50% coming from carbohydrate. Maintaining dietary changes for long periods is very difficult for many persons. Specific self-management skills may ease the task.     

Lipid binding by fragments of apolipoprotein C-III-1 obtained by thrombin cleavage.

We have used thrombin to cleave apolipoprotein C-III-1 into two fragments constituting residues 1-40 (apoLP-C-III-A) and 41-79 (apoLP-C-III-B). The lipid binding properties of these fragments with dimyristoyl- and 1-palmitoyl-2-oleoylphosphatidylcholines have been determined using circular dichroic and intrinsic tryptophan fluorescence spectroscopy. The peptide-phospholipid mixtures were fractionated by density gradients of cesium chloride. ApoLP-C-III-A showed disordered structure in the absence and presence of DMPC and no significant amount of peptide-phospholipid complex was isolated. ApoLP-C-III-B showed conformational changes in the circular dichroic spectrum and a shift in the intrinsic tryptophan fluorescence spectrum. Ultracentrifugation in cesium chloride gradients yielded peptide-phospholipid complexes isolated between density 1.10 and 1.18. The molar ratio of lipid to protein was 12:1. The results of these studies and the examination of space filling models of apoLP-C-III provide evidence that an amphipathic alpha helix which contains a nonpolar face and a polar face is the basic structural unit for binding of phospholipid by the plasma apolipoproteins. These results also provide direct evidence that the hydrophobicity of the nonpolar face is important in lipid binding since the nonpolar face of residues 1-40 is considerably less hydrophobic than the nonpolar face of residues 41-79.