Predicting the structure of apolipoprotein A-I in reconstituted high-density lipoprotein disks

In reconstituted high-density lipoproteins, apolipoprotein A-I and phosphatidylcholines combine to form disks in which the amphipathic alpha-helices of apolipoprotein A-1 bind to the edge of a lipid bilayer core, shielding the hydrophic lipid tails from the aqueous environment. We have employed experimental data, sequence analysis, and molecular modeling to construct an atomic model of such a reconstituted high-density lipoprotein disk consisting of two apolipoprotein A-I proteins and 160 palmitoyloleoylphosphatidylcholine lipids. The initial globular domain (1-47) of apolipoprotein A-I was excluded from the model, which was hydrated with an 8-A shell of water molecules. Molecular dynamics and simulated annealing were used to test the stability of the model. Both head-to-tail and head-to-head forms of a reconstituted high-density lipoprotein were simulated. In our simulations the protein contained and adhered to the lipid bilayer while providing good coverage of the lipid tails.     

Structural analysis of apolipoprotein A-I: effects of amino- and carboxy-terminal deletions on the lipid-free structure

An amino-terminal deletion mutant (residues 1-43) and a carboxy-terminal deletion mutant (residues 187-243) of human apoliprotein A-I (apo hA-I) have been produced from a bacterial expression system to explore the importance of the missing residues for the conformation of apo hA-I. Our focus has been to study the lipid-free structure of apo hA-I to understand how discrete domains influence the conformational plasticity of the protein and, by inference, the mechanism of lipid binding. All spectral and physical measurements indicate that both apo delta(1-43)A-I and apo delta(187-243)A-I have folded, tertiary structures. These structures differ in the specific arrangement of helical domains based, in part, on their relative thermodynamic stability, near- and far-UV CD, limited proteolysis, and the accessibility of tryptophans to fluorescence quenchers. In addition, all data indicate that the folded domains of apo hA-I and apo delta(187-243)A-I are very similar. Results from analytical ultracentrifugation suggest that lipid-free apo hA-I and the deletion mutants each exist in a dynamic equilibrium between a loosely folded, helical bundle and an elongated monomeric helical hairpin. The conformational heterogeneity is consistent with significant ANS binding exhibited by all three proteins and could help to explain the facile lipid binding properties of apo hA-I.     

Isotope dilution--mass spectrometric quantification of specific proteins: model application with apolipoprotein A-I

An enzymatic hydrolysis isotope dilution-mass spectrometric method was developed for reference quantification of specific proteins. The analytical procedure involved measuring a reproducibly hydrolyzed peptide (serving as the primary standard) unique to a specific protein. This new mass spectrometric method was evaluated by assessing the concentration of apolipoprotein (apo) A-I in the European Community Bureau of Reference (BCR) lyophilized Certified Reference Material (CRM 393). We used the method to make 96 measurements (4 replicate analyses of 4 enzymatic digests of 6 vials of BCR-CRM 393), which gave an average total protein mass of 1.048 mg (+/- 1.0% at 99% confidence limits). The total overall analytical CV was 3.95%. The results of this evaluation of our model approach to determine the concentration of a specific protein in a purified preparation demonstrated that our new mass spectrometric method can be used to measure apolipoproteins and other specific proteins without the use of epitopic immunoassay methods.     

Interaction of N-methyl-anthraniloyl-labelled porcine apolipoprotein A-I with porcine lecithin: cholesterol acyltransferase: an energy transfer study

In an 8-month strictly controlled dietary study of 16 healthy young men, the long-term effect of a low-fat (26% of energy) high-fiber (4.5 g/MJ) diet on cardiovascular risk markers of the hemostatic system was assessed. Fasting blood sampling was performed during a 4-week baseline period and then monthly during the intervention. A matched control group of 16 men on habitual diets was also monitored. Median fibrinolytic activity of tissue-type plasminogen activator (t-PA) in plasma was significantly elevated (twofold to fourfold) by the experimental diet. A significant increase in the systemic fibrinolytic activity of the euglobulin fraction of plasma was also observed. Median plasma factor VII coagulant activity (F VIIc) was depressed by 5-10% during the first 2 months and the last month of the study period. The dietary change did not significantly affect plasma levels of fibrinogen, t-PA antigen, or plasminogen activator inhibitor type I antigen. In conclusion, young men who were switched from a typical Danish diet high in saturated fat to a low-fat/high-fiber diet showed a permanent increase in plasma fibrinolytic activity and a biphasic decrease in F VIIc. The dietary change thus had a favorable effect on cardiovascular risk markers of the hemostatic system.     

Relations between cardiac and vascular structure in patients with primary and secondary hypertension

BACKGROUND: Data on cardiac and vascular structure in secondary hypertension are generally scarce, and no data on the interrelations between cardiac mass and structural characteristics of the vessel wall, both in large and in small resistance arteries, are presently available. OBJECTIVES: The aim of this study was to investigate the relation between structural changes in subcutaneous small arteries, left ventricular mass and wall thickness of the common carotid artery in patients with primary and secondary hypertension. METHODS: Seventy-four subjects were included in the study: 11 patients with pheochromocytoma, 14 with primary aldosteronism (PA), 19 with renovascular hypertension (RVH), 18 with essential hypertension (EH) and 12 normotensive (NT) control subjects. All subjects were submitted to a biopsy of subcutaneous fat. Morphologic characteristics of subcutaneous small resistance arteries (relaxed diameter <300 microm) were directly evaluated using a micromyographic technique. All subjects were submitted to calculation of left ventricular mass index (LVMI) and common carotid artery intima-media thickness (CCIMT), using ultrasound technique. RESULTS: The correlation coefficients between the media to lumen ratio in subcutaneous small arteries (M/L) and LVMI or between M/L and CCIMT were closer in RVH than in pheochromocytoma, EH or NT; in PA the correlation coefficients were slightly less close than those in RVH. An excess prevalence of carotid plaques in RVH was observed. CONCLUSIONS: A close relation between small resistance artery morphology and cardiac or carotid artery structure may be observed in those hypertensive patients in whom the renin-angiotensin-aldosterone system is activated. In constrast, in NT, EH and pheochromocytoma no significant correlation between M/L and LVMI or CCIMT was observed.     

Secretion and processing of apolipoprotein A-I in the avian sciatic nerve during development

Apolipoprotein A-I (apo A-I), a major apolipoprotein synthesized by liver and intestine to facilitate transport of plasma lipids as lipoproteins, has been detected also in the avian sciatic nerve. The mRNA and protein levels of apo A-I have been shown to increase during the period of rapid myelination (LeBlanc et al.: J Cell Biol 109:1245-1256, 1989). In order to assess the synthesis of apo A-I protein and the processing of apo A-I isoforms during development, endoneurial slices of avian sciatic nerves from chicks during active myelination at 15 and 17 days embryonic and 1 day posthatch age were incubated with [35]S-methionine. The incubations were fractionated into secreted and intracellular fractions, and incorporation of the label was assessed for apo A-I protein. The pattern of labeling of Po protein, as a marker of myelination, was also determined in the intracellular and compact myelin fractions. Methionine incorporation into Po protein was highest in the intracellular compartment at the 15-day embryonic stage and decreased thereafter, with a corresponding increase in the myelin fraction. During these developmental periods, the levels of nascent apo A-I increased in both the secreted and intracellular fractions. The synthesis of apo A-I specifically increases in the secreted fraction compared with total protein synthesis. The processing of the pro-apo A-I is also developmentally regulated. In the intracellular compartment, there are approximately equal proportions of the acidic and basic isoforms. However, with increasing age, a higher proportion of the apo A-I is secreted as acidic isoforms. It is concluded that the secretion and processing of apo A-I is developmentally regulated in the chick sciatic nerve, in parallel with the process of active myelination.     

Peculiarities of primary and secondary structure of phage FI-1 DNA

The composition of nitrous bases of phage FI-1 DNA was studied. As is evidenced from the values of buoyant density in CsCl (p=1,7093 g/cm(3)), melting temperature (T degrees m=86,05 degrees), spectral parameters and direct chromatographic determination, the DNA analysed contains 41,5 mole% pairs of guanine-cystosine. 5-hydroxymethylcytosine and other anomalous bases were not found. Chemical identification and jaxtposition of data of buoyant density in CsCl and Cs2SO4 (p=1,4466 g/cm(3)) and T degrees m. showed the presence of the extra-sugar component in DNA, most likely in the form of hentibiose. Spectral character of thermal denaturation of DNA in different solvents is indicative of the double helixity of its structure. DNA is characterized by enthalpies of conformational transitions "helix coil" (deltaH=12,3 kcal/g) and (deltaH=10 kcal/g) for the solvents, 1 x SSC, and 0,1 x SSC, correspondingly. The presence of extra-sugar in DNA with standard set of nitrous bases is discussed.     

Apolipoprotein A-I complexed with phospholipid promotes hepatic lipoprotein and apolipoprotein secretion in the perfused hamster liver

BACKGROUND: Apolipoprotein A-I within high density lipoprotein (HDL) plays a significant role in the process of reverse cholesterol transport from peripheral tissues to the liver. However, additional roles are not well defined for it in hepatic cholesterol metabolism. We have previously shown in the hamster that dietary cholesterol supplementation resulted in enhancement of apolipoprotein A-I (Apo A-I) in secreted nascent hepatic very low density lipoprotein (VLDL), suggesting that apolipoprotein A-I itself may play a role in hepatic lipoprotein secretion. METHODS: Using the isolated hamster liver with Apolipoprotein A-I perfusion, we then examined the hypothesis that Apo A-I alone or in association with phosphotidylcholine (PC) i.e., Apo A-I/PC as a HDL-like particle, has effects upon hepatic lipoprotein and bile secretion. Ultracentrifugation was performed on perfusate samples at 3 hours on control vs treated livers (Apo A-I/PC, Apo A-I, or PC) to access lipid and protein concentration in VLDL, low density lipoprotein (LDL) and HDL. Four to thirty percent gradient SDS polyacrylamide electrophoresis (PAGE) and Western blot analysis were used on delipidated lipoprotein fractions and microsomes to assess apolipoproteins Apo B, A-I, II, and E. RESULTS: We found that perfusion of reconstituted HDL vesicles containing human apolipoprotein A-I and PC (Apo A-I/PC) 10 mg and 10 mg, respectively, in 22 mL for 3 hours into isolated hamster liver increased cholesterol (CH) and triglyceride (TG) components in secreted HDL; 45- and 6-fold, and in LDL; 15- and 2-fold, respectively. No significant changes occurred in VLDL or in biliary lipids. Concomitantly, Apo A-I/PC perfusion increased Apo E and Apo A-II and HDL and Apo B in LDL, while Apo E decreased in VLDL. Apo A-I/PC perfusion did not change the apolipoprotein content of hepatic microsomes of the perfused liver. Perfusion of apolipoprotein A-I (without PC) or PC (without apolipoprotein A-I) had none of these effects. CONCLUSION: These results indicate that the perfused discoidal apolipoprotein A-I/PC particle affects hepatic lipoprotein assembly and secretion, whereby both lipid and apolipoprotein components are enhanced in secreted HDL and LDL of hepatic origin.     

Protein secondary structure prediction by the analysis of variation and conservation in multiple alignments

A number of methods exist for the prediction of protein secondary structure from primary sequence. One method identifies variable charged and conserved hydrophobic residues within large multiple alignments as a means of indicating outside and inside sites respectively in the protein structure. These sites are then manually fitted to secondary structure templates to generate a secondary structure prediction. Using the existing theoretical bases of this method, we present an algorithm (STAMA) which automatically carries out the initial variation/conservation analysis of the alignment. We also test the accuracy of complete predictions carried out by manual fitting of the STAMA-derived assignments to structure templates, using five large multiple alignments each including a protein of known structure. The method was found on average to predict only 57% of residues in the correct secondary structure, and was only as accurate as predictions carried out using the established and automated method of Garnier, Osguthorpe and Robson (1978) applied to a single sequence. When used in conjunction with other secondary structure prediction methods, however, the resulting consensus predictions were found to be very accurate, with 78% of the elements (alpha helices or beta strands) for which a consensus could be obtained being predicted correctly. The algorithm presented here, plus the assessment of the accuracy of prediction generated by this method, should enable this predictive approach to receive informed general use.     

Cholestyramine and a fat-free diet lower apolipoprotein A-IV mRNA in jejunum and cholestyramine lowers apolipoprotein A-I mRNA in ileum of rats

To investigate the effect of bile acids or dietary lipid on the expression of intestinal apolipoproteins, the mRNA levels of apolipoprotein A-I and A-IV in the intestine from rats fed a diet containing cholestyramine or a fat-free diet were compared with those from rats fed a control diet containing 25% casein and 5% corn oil. Plasma total cholesterol concentration was lower after 16 h in rats fed a diet containing cholestyramine or a fat-free diet than in rats fed a control diet. In rats fed the fat-free diet, HDL cholesterol concentration also was lower than in those fed the control diet. The pool of bile acid in intestinal contents was significantly lower in rats fed cholestyramine than in both other groups. The relative abundance of jejunal apolipoprotein A-I mRNA did not differ between groups. Jejunal apolipoprotein A-IV mRNA abundance was significantly lower than in controls in rats fed the fat-free and cholestyramine-containing diets. Abundance of apolipoprotein A-I mRNA in ileal mucosa was comparable to controls in rats fed a fat-free diet but was significantly lower in rats fed cholestyramine. Ileal apolipoprotein A-IV mRNA tended to be lower in rats fed cholestyramine and a fat-free diet than in controls. We propose that decreased absorption of dietary lipid may modulate changes in jejunal apolipoprotein A-IV mRNA level and low levels of bile acids in the lumen may modulate changes in ileal apolipoprotein A-I mRNA level.     

Efflux of intracellular versus plasma membrane cholesterol in HepG2 cells: different availability and regulation by apolipoprotein A-I

We have compared the efflux of cholesterol from different cellular pools of human hepatoma cells HepG2 using intact cells or isolated membrane fractions. To label different pools, cells were incubated with either unesterified [14C]cholesterol that had been incorporated into high density lipoproteins ([14C]FC-HDL), low density lipoproteins ([14C]FC-LDL), or phosphatidylcholine liposomes ([14C]FC-PC), or with [14C]acetate. Cell fractionation revealed that labeling of cells with [14C]FC-PC resulted in the incorporation of [14C]cholesterol almost exclusively into the plasma membrane (PM), while incubation with [14C]FC-HDL resulted in the majority of [14C]cholesterol incorporation into the PM, but with a smaller component associated with lysosomes. Labeling with [14C]FC-LDL or [14C]acetate led to an accumulation of [14C]cholesterol predominantly in lysosomes or the endoplasmic reticulum (ER), respectively. When the kinetics of [14C]cholesterol efflux was analyzed after pulse-labeling of different cellular pools, half-times of cholesterol efflux from lysosomes and ER were significantly longer than that from PM. In another set of experiments, when both labeling and efflux times varied, efflux of [14C]cholesterol from the PM to human serum after 1.5 h pulse and chase incubations was double that from lysosomes and 8-fold that from ER. Extension of the incubation times from 1.5 to 3 h diminished the difference in cholesterol efflux from different membranes. Further incubation to 6 h almost abolished the different responses. Cell-free preparations of membranes, obtained from cells labeled with [14C]cholesterol, showed no differences in cholesterol efflux. No differences in the distribution of [14C]cholesterol released into serum among lipoprotein subfractions was observed. Pretreatment of the serum with Fab fragments of polyclonal rabbit anti-human apolipoprotein A-I antibodies reduced its ability to promote efflux of cholesterol from the ER by 77%, but had no effect on cholesterol efflux from the PM. Fab fragments of non-immune IgG had no effect on the efflux of both ER and PM cholesterol. We conclude that the availability of cellular cholesterol for efflux from HepG2 cells is strongly influenced by its subcellular location, and is regulated by apolipoprotein A-I.     

No common major gene for apolipoprotein A-I and HDL3-C levels: evidence from bivariate segregation analysis

Apolipoprotein A-I (apo A-I) is the most abundant protein in high-density lipoprotein (HDL) particles, and it plays an important role in HDL metabolism. Both apo A-I and HDL cholesterol (HDL-C) levels are inversely associated with risk of cardiovascular disease. Segregation analyses suggest apo A-I levels are under the control of one or more major loci. Since HDL particles are heterogeneous in their composition and size, genetic influence on its subfractions (i.e., HDL2 and HDL3) could vary. A previous report showed evidence of a major locus controlling HDL3-C levels in a subset of the current study population. Because quantitative trait loci involved in complex diseases are likely to have pleiotropic effects on several related traits, it is possible to have a common major gene involved in regulating apo A-I and HDL3-C levels. We performed a bivariate segregation analysis of apo A-I and HDL3-C levels in 1,006 individuals from 137 families ascertained through probands undergoing elective, diagnostic coronary angiography at the Johns Hopkins Hospital. The results showed significant genetic correlation between these two traits, but the hypothesis of a common major gene was rejected. Bivariate segregation analysis favored a model with two genes controlling apo A-I and a third gene independently controlling HDL3-C, and the genetic correlation between these two traits is due to residual additive polygenes. Overall, results from this study suggest that there are distinct genetic mechanisms for apo A-I and HDL3-C levels. Future studies, especially linkage analysis, should consider distinct genetic mechanisms and multiple major gene loci.     

Increased immunolocalization of paraoxonase, clusterin, and apolipoprotein A-I in the human artery wall with the progression of atherosclerosis

Using immunolocalization techniques, we have shown that paraoxonase (Pon), clusterin, and apolipoprotein (apo) A-I accumulate in the artery wall during the development of atherosclerosis. In normal aortas (n = 6) there were low levels of extracellular Pon, clusterin, and apoA-I, immunoreactivity. The cytoplasm of smooth muscle cells in the media showed granular positivity for both Pon and apoA-I, indicating that these proteins were undergoing lysosomal degradation. This activity was also indicated by the presence of both intact and degradation products of Pon in smooth muscle cells as shown by Western blotting. With the progression of disease from fatty streaks (n = 3) to advanced atherosclerosis (n = 8) there was an increase in Pon, apoA-I, and clusterin immunoreactivity, indicating the increasing presence of these proteins with disease progression. These proteins are the components of a specific HDL subspecies that has been implicated in the prevention of peroxidative damage to phospholipids in LDL and membranes. The increase in Pon, clusterin, and apoA-I during the development of atherosclerosis may therefore represent a protective response to the oxidative stress associated with the development of atherosclerosis.     

Impact of gender on the metabolism of apolipoprotein A-I in HDL subclasses LpAI and LpAI:AII in older subjects

The behavior of apolipoprotein (apo) A-I in lipoprotein (Lp) AI and LpAI:AII was studied in 11 postmenopausal females and 11 males matched for plasma triglyceride and total cholesterol levels. Subjects consumed a baseline diet [35% fat (14% saturated, 15% monounsaturated, and 7% polyunsaturated), 15% protein, 49% carbohydrate, and 147 mg cholesterol/1000 kcal] for 6 weeks before the start of the kinetic study. At the end of the diet period, using a primed-constant infusion of [5,5,5-2H3]leucine, residence times (RT) and secretion rates (SR) of apoA-I were determined in 2 subpopulations of high-density lipoprotein (HDL) particles, LpAI and LpAI:AII. Plasma total cholesterol, low-density lipoprotein cholesterol, and triglyceride concentrations were similar in males and females. The mean plasma HDL cholesterol concentration in males (1.14 +/- 0.23 mmol/L; mean +/- SD) was lower than in females (1.42 +/- 0.18 mmol/L; P =. 0034). Similarly, the mean plasma concentration of apoA-I in males (130 +/- 21 mg/dL) was lower than that in females (150 +/- 19 mg/dL; P = .0421). The RT of apoA-I in either LpAI or LpAI:AII was similar between men and women. Despite the higher plasma apo A-I levels in female compared with male subjects, total apoA-I and apoA-I in LpAI and LpAI:AII pool sizes were similar between the two groups, attributable to the lower body weight of the female subjects. The mean SR of total apoA-I in males (8.5 +/- 2.7 mg.kg-1.d-1) was 22% lower than in females (10.9 +/- 2.3 mg.kg-1.d-1; P = .0389). The SR of both apoA-I in LpAI and LpAI:AII was lower in males than females, although the differences did not reach statistical significance. These data suggest that the difference observed in HDL cholesterol concentration between males and females is attributable to SR of apoA-I and not the catabolic rate.     

The hydrophobic face orientation of apolipoprotein A-I amphipathic helix domain 143-164 regulates lecithin:cholesterol acyltransferase activation

Apolipoprotein A-I (apoA-I) activates the plasma enzyme lecithin:cholesterol acyltransferase (LCAT), catalyzing the rapid conversion of lipoprotein cholesterol to cholesterol ester. Structural mutants of apoA-I have been used to study the details of apoA-I-LCAT-catalyzed cholesterol ester formation. Several studies have shown that the alpha-helical segments corresponding to amino acids 143-164 and 165-186 (repeats 6 and 7) are essential for LCAT activation. In the present studies, we examined how the orientation of the hydrophobic face, independent of an increase in overall hydrophobicity, affects LCAT activation. We designed, expressed, and characterized a mutant, reverse of 6 apoA-I (RO6 apoA-I), in which the primary amino acid sequence of repeat 6 (amino acids 143-164) was reversed from its normal orientation. This mutation rotates the hydrophobic face of repeat 6 approximately 80 degrees. Lipid-free RO6 apoA-I showed a marked stabilization when denatured by guanidine hydrochloride, but showed significant destabilization to guanidine hydrochloride denaturation in the lipid-bound state compared with wild-type apoA-I. Recombinant high density lipoprotein discs (rHDL) formed from RO6 apoA-I, sn-1-palmitoyl-sn-2-oleoyl phosphati-dylcholine, and cholesterol were approximately 12 A smaller than wild-type apoA-I rHDL. The reduced size suggests that one of the repeats did not effectively participate in phospholipid binding and organization. The sn-1-palmitoyl-sn-2-oleoyl phosphatidylcholine RO6 rHDL were a less effective substrate for LCAT. Mapping the entire lipid-free and lipid-bound RO6 apoA-I with a series of monoclonal antibodies revealed that both the lipid-free and lipid-bound RO6 apoA-I displayed altered or absent epitopes in domains within and adjacent to repeat 6. Together, these results suggest that the proper alignment and orientation of the hydrophobic face of repeat 6 is an important determinant for maintaining and stabilizing helix-bilayer and helix-helix interactions.