Based on the assumption that severe alterations in the expression of genes known to be involved in high-density lipoprotein (HDL) metabolism may affect the expression of other genes, we screened an ...array of >5000 mouse expressed sequence tags for altered gene expression in the livers of two lines of mice with dramatic decreases in HDL plasma concentrations. Labeled cDNA from livers of apolipoprotein AI (apoAI)-knockout mice, scavenger receptor BI (SR-BI) transgenic mice, and control mice were cohybridized to microarrays. Two-sample
t
statistics were used to identify genes with altered expression levels in the knockout or transgenic mice compared with control mice. In the SR-BI group we found nine array elements representing at least five genes that were significantly altered on the basis of an adjusted
P
value < 0.05. In the apoAI-knockout group, eight array elements representing four genes were altered compared with the control group (adjusted
P
< 0.05). Several of the genes identified in the SR-BI transgenic suggest altered sterol metabolism and oxidative processes. These studies illustrate the use of multiple-testing methods for the identification of genes with altered expression in replicated microarray experiments.
The role of apolipoprotein A-II (apoA-II) in high density lipoprotein (HDL) structure and metabolism has been studied previously in transgenic mice overexpressing either human or murine apoA-II. ...These studies have shown differences between these two groups of transgenic animals in the levels of very low density, low density, and high density lipoproteins, in the HDL particle size distribution, and in the relationship between apoA-II levels and lipoprotein levels. To determine whether these differences are due to the fact that human apoA-II is dimeric and murine apoA-II monomeric, we have examined the effects of monomeric human apoA-II (hA-IImon) in transgenic mice. Site-directed mutagenesis (Cys6 -> Ser) was used to generate 15 transgenic founder lines of hA-IImon mice, that contained plasma hA-IImon concentrations over a 10-fold range (11 mg/dl to 185 mg/dl). The hA-IImon floated in the d < or = 1.21 g/ml fraction and migrated as an apoA-II monomer by nonreducing SDS-polyacrylamide gel electrophoresis. HDL levels were not correlated with hA-IImon levels (r = -0.26); HDL particle size and size distribution, as well as very low density and low density lipoprotein levels and sizes, were unchanged compared to nontransgenic control mice. These results suggest that differences between mice overexpressing human dimeric apoA-II and those overexpressing murine apoA-II are the result of sequence differences between these two apoA-II molecules and are not solely due to the fact that human apoA-II exists as a dimer.
Complexes of egg yolk phosphatidylcholine and apolipoprotein A-I were prepared by a detergent (sodium cholate)-dialysis method and characterized by gradient gel electrophoresis, gel filtration, ...electron microscopy and chemical analysis. Multicomponent electrophoretic patterns were obtained indicating formation of at least eight classes of discoidal complexes. The relative contribution of the different classes to the electrophoretic pattern was a function of the molar ratio of phosphatidylcholine:apolipoprotein A-I in the interaction mixture. Molar ratios of phosphatidylcholine:apolipoprotein A-I in isolated complexes were strongly and positively correlated with disc diameter obtained by electron microscopy. Incorporation of unesterified cholesterol into phosphatidylcholine/apolipoprotein A-I interaction mixtures also resulted in formation of unique complexes but with considerably different particle size distributions relative to those observed in the absence of cholesterol. One common consequence of cholesterol incorporation into interaction mixtures of 87.5:1 and 150:1 molar ratio of phosphatidylcholine:apolipoprotein A-I was the disappearance of a major complex class with diameter of 10.8 nm and the appearance of a major component with diameter of approximately 8.8 nm. Electrophoretic patterns of cholesterol-containing complexes showed a strong similarity to patterns recently published for high density lipoproteins from plasma of lecithin:cholesterol acyltransferase-deficient subjects, suggesting that the complexes formed in vitro by the detergent-dialysis method may serve as appropriate models for investigation of the origins of the HDL particle size distribution.
We investigated the effect of Cu2+ catalyzed peroxidation on the status of tryptophan (Trp) in protein moieties in HDL and LDL together with its effect on apolipoprotein-lipid association. Incubation ...of HDL with Cu2+ resulted in a rapid decrease of Trp fluorescence intensity with time with a concomitant increase in Trp maximum emission wavelength (lambda max). LDL incubated with Cu2+ also showed a rapid decrease in Trp fluorescence intensity with time, with no associated increase in lambda max. The status of apo HDL and apo LDL was investigated after 4 h oxidation (4h-oxHDL and 4h-oxLDL respectively). With 4h-oxHDL, the shift in lambda max was not associated with protein dissociation but rather with protein crosslinking and formation of larger HDL species. Progressive increase in lambda max was observed in 4h-oxHDL with increase in guanidine hydrochloride (GuHCl) concentration; this was not due to protein dissociation. Although oxidation of LDL did not produce an increase in lambda max, a significant increase in wavelength was observed when 4h-oxLDL was exposed to increasing concentration of GuHCl. SDS-polyacrylamide gel electrophoresis and nondenaturing gradient gel electrophoresis of the 4h-oxLDL indicated formation of smaller molecular weight protein fragments that were still associated with LDL. Ultracentrifugation of oxidized LDL in the presence and absence of GuHCl showed no dissociated protein. In summary, these data indicate the following: (a) lipid peroxidation has a direct effect on Trp residues in both HDL and LDL, (b) oxidation of HDL is associated with conformational change in apo HDL, crosslinking and formation of larger particles, (c) oxidized HDL have a more stable apolipoprotein-lipid association than native HDL, (d) oxidation of LDL is associated with changes in apo B, that by fluorescence are apparent only in presence of GuHCl and results in fragmentation of apo B without dissociation of protein or change in particle size, and (e) stability of apolipoprotein-lipid association is comparable in oxidized and native LDL.
Mouse and human plasma apolipoprotein A-I (apo A-Im and apo A-Ih, respectively) were investigated to compare their molecular properties in solution, their incorporation into ...palmitoyloleoylphosphatidylcholine-apo A-I (POPC-apo A-I) discoidal complexes; their structural stability in discoidal complexes and high-density lipoproteins (HDL), and their effect on structural rearrangement of discoidal complexes upon interaction with low-density lipoproteins (LDL). Unlike apo A-Ih, only minimal concentration-dependent self-association was observed for apo A-Im. While both apo A-Im and apo A-Ih formed discoidal complexes of distinct composition and size that reflected reassembly molar ratios of POPC/apo A-I, apo A-Im demonstrated specific deficiencies in formation of larger-sized complexes. Denaturation of both apo A-Im- or apo A-Ih-containing complexes and HDL with guanidine hydrochloride (GuHCl) indicated significantly reduced stabilization of apo A-Im by lipid in these particles. Interaction of apo A-Im- or apo A-Ih-containing discoidal complexes with human plasma LDL revealed a more extensive conversion of apo A-Im-complexes to smaller species. Mean hydrophobicities and mean hydrophobic moments of amphipathic helical segments in apo A-Im and apo A-Ih were compared; differences potentially contributing to differential lipid-binding properties between apo A-Im and apo A-Ih were identified. Our results demonstrate differences between apo A-Im and apo A-Ih that may contribute to the major changes in plasma HDL distribution and function observed in apo A-Ih transgenic mice.
The influence of sodium oleate (oleate) on complexing of apolipoprotein A-I (apo A-I) with egg yolk phosphatidylcholine (EYPC) was evaluated. Without the use of additional detergent such as sodium ...cholate, oleate facilitates formation of a single complex of unique stoichiometry, approx. 76:2:20, molar ratio EYPC/apo A-I/oleate, and mean size 7.4 nm with round to ellipsoidal morphology. Near complete reassembly of apo A-I into the complex occurs when the stoichiometry of the mixture approximates that of the complex itself. With increasing content of EYPC in the mixture, the same complex is formed but in decreasing yield; larger complexes are not formed. The rate of complex formation decreases with increase of EYPC in the mixture. Reduction of pH in the reassembly mixture from 8.0 to 5.4 results in a marked reduction in complex formation indicating that ionized oleate facilitates lipidation. Removal of oleate by interaction of the complex with fatty acid-free human serum albumin does not degrade the complex. Incorporation of increasing amounts of unesterified cholesterol into the EYPC-sonicate progressively inhibits oleate-facilitated complex formation. This study shows that oleate, a physiologically relevant lipolysis-derived product, facilitates reassembly of apo A-I with EYPC and promotes formation of a small lipid-poor particle similar to that observed in nascent HDL and during in vivo or in vitro lipolysis of triacylglycerol-rich lipoproteins in the presence of HDL.
Incubation studies were performed on plasma obtained from subjects selected for relatively low levels of high-density lipoprotein cholesterol (HDL-C) (no greater than 30 mg/dl) and particle size ...distributions enriched in the HDL3 subclass. Incubation (12 h, 37 degrees C) of plasma in the presence or absence of lecithin: cholesterol acyltransferase activity produces marked alteration in size profiles of both major apolipoprotein-specific HDL3 populations (HDL3(AI w AII), HDL3 species containing both apolipoprotein A-I and apolipoprotein A-II, and HDL3(AI w/o AII), HDL3 species containing apolipoprotein A-I) as isolated by immunoaffinity chromatography. In the presence or absence of lecithin: cholesterol acyltransferase activity, plasma incubation results in a shift of HDL3(AI w AII) species (initial mean sizes of major components, approx. 8.8 and 8.0 nm) predominantly to larger particles (mean size, 9.8 nm). A less prominent shift to smaller particles (mean size, 7.8 nm) accompanies the conversion to larger particles only when the enzyme is active. Combined shifts to larger (mean size, 9.8 nm) and smaller (mean size, 7.4 nm) particles are observed for HDL3(AI w/o AII) particles (mean size, 8.3 nm) also only in the presence of enzyme activity. However, in the absence of enzyme activity, HDL3(AI w/o AII) species, unlike the HDL3(AI w AII) species, are converted to smaller (mean size 7.4 nm) rather than to larger particles. Like native HDL2b(AI w/o AII) particles, the larger HDL3(AI w/o AII) conversion products exhibit a protein moiety with molecular weight equivalent to four apolipoprotein A-I molecules per particle; small HDL3(AI w/o AII) products are comprised predominantly of particles with two apolipoprotein A-I per particle. Incubation-induced conversion of HDL3 particles in the presence of lecithin: cholesterol acyltransferase activity is associated with increased binding of both apolipoprotein-specific HDL populations to low-density lipoproteins (LDL). The present studies indicate that, in the absence of lecithin: cholesterol acyltransferase activity, the two HDL3 populations follow different conversion pathways, possibly due to apolipoprotein-specific activities of lipid transfer protein or conversion protein in plasma. Our studies also suggest that lecithin: cholesterol acyltransferase activity may play a role in the origins of large HDL2b(AI w/o AII) species in human plasma by participating in the conversion of HDL3(AI w/o AII) particles, initially with three apolipoprotein A-I, to larger particles with four apolipoprotein A-I per particle.
The effects of repetitive ultracentrifugation on the physical and chemical properties of very low density lipoproteins (VLDL) were investigated. VLDL recentrifuged one to seven times were ...characterized by chemical analyses, analytical ultracentrifugation and electron microscopy. The VLDL content of triglyceride was increased and the proportion of phospholipid decreased by ultracentrifugation. Recentrifugation of VLDL decreased the number of S
f
o
20-100 particles and generated particles of S
f
o
> 400. The bulk of the material removed from VLDL by ultracentrifugation was lipoprotein having pre-g mobility on paper electrophoresis, flotation rates of S
f
o
10-100 and a particle size of 300-400 Å. Two ultracentrifugations separated an average of 14% of the starting VLDL protein. Characterization of the apoproteins in this material by polyacrylamide gel electrophoresis, gel chromatography, immuno precipitation and amino acid analysis demonstrated a relatively high proportion of B-apoprotein and relatively little C-apoproteins.
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