Trudy M. Forte

Characterization of human high-density lipoproteins by gradient gel electrophoresis

Gradient gel electrophoresis in conjunction with automated densitometry was applied to the identification and estimation of subpopulations of high-density lipoproteins (HDL) in the ultracentrifugal d less than or equal to 1.200 fraction from human plasma. The frequency distribution of relative migration distances (RF values) of subpopulation peaks in HDL patterns of a group (n = 194) of human subjects showed five apparent maxima: two in the RF range associated with the HDL2 subclass, and three in the RF range of the HDL3 subclass. HDL within RF intervals bounding these maxima were designated (HDL2b)gge, (HDL2a)gge, (HDL3a)gge, (HDL3b)gge and (HDL3c)gge and were shown to correspond approximately to material determined by analytic ultracentrifugation within the HDL2b, HDL2a and HDL3 components. Material represented by the HDL2a component, as resolved by three-component analysis of the ultracentrifugal Schlieren pattern, was found by gradient gel electrophoresis to be polydisperse in particle size. Mean hydrated densities and particle sizes of HDL corresponding to those with RF values of the frequency maxima were: 1.085 g/ml and 10.57 nm in the (HDL2b)gge; 1.115 g/ml and 9.16 nm in the (HDL2a)gge; 1.136 g/ml and 8.44 nm in the (HDL3a)gge; 1.154 g/ml and 7.97 nm in the (HDL3b)gge; and 1.171 g/ml and 7.62 nm in the (HDL3c)gge. The mean hydrated density values of the subpopulations within the (HDL3a)gge and (HDL3b)gge were comparable to those of the HDL3L and HDL3D components recently characterized by zonal ultracentrifugation. High order and statistically significant correlations between densitometric scans of the (HDL2b)gge, (HDL2a)gge and (HDL3)gge material, as obtained from gradient gels, and plasma concentrations of the HDL2b, HDL2a and HDL3 components, as obtained from analytic ultracentrifugation, were demonstrated.

Familial apolipoprotein E deficiency.

A unique kindred with premature cardiovascular disease, tubo-eruptive xanthomas, and type III hyperlipoproteinemia (HLP) associated with familial apolipoprotein (apo) E deficiency was examined. Homozygotes (n = 4) had marked increases in cholesterol-rich very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL), which could be effectively lowered with diet and medication (niacin, clofibrate). Homozygotes had only trace amounts of plasma apoE, and accumulations of apoB-48 and apoA-IV in VLDL, IDL, and low density lipoproteins. Radioiodinated VLDL apoB and apoE kinetic studies revealed that the homozygous proband had markedly retarded fractional catabolism of VLDL apoB-100, apoB-48 and plasma apoE, as well as an extremely low apoE synthesis rate as compared to normals. Obligate heterozygotes (n = 10) generally had normal plasma lipids and mean plasma apoE concentrations that were 42% of normal. The data indicate that homozygous familial apoE deficiency is a cause of type III HLP, is associated with markedly decreased apoE production, and that apoE is essential for the normal catabolism of triglyceride-rich lipoprotein constituents.

Particle distribution of human serum high density lipoproteins.

Density gradient ultracentrifugation of human serum high density lipoproteins (HDL) from both normolipemic males and females results in a distribution of HDL concentration versus subfraction hydrated density which has three maxima. Gradient gel electrophoresis of total HDL is characterized by three banding maxima, the positions of which suggest the presence of three particle size ranges: I. 10.8-12.0 nm, II. 9.7-10.7 nm, and III. 8.5-9.6 nm. Gradient gel electrophoresis of density gradient subfractions established an inverse relationship between particle size and particle hydrated density which was corroborated by electron microscopy and analytic ultracentrifugation. Comparison of male HDL from size ranges I, II, and III with female HDL from the same size ranges showed only small differences in the mean value of the peak F degrees 1.20 rate, size, molecular weight, protein weight percent, and weight protein/weight phospholipid. Major differences between males and females were seen in the relative amounts of HDL in density gradient subfractions 1-3 (size range I material) and 11-12 (size range III material); the percent total HDL in the group of subfractions 1-3 was greatly increased in female HDL while that of the group of subfractions 11-12 was increased in the male HDL. These studies indicate the presence of at least three major components in HDL instead of two (HDL2 and HDL3) and that peak F degrees 1.20 rate differences in HDL schlieren patterns between males and females are a function of the relative levels of these three components.

[26] Electron microscopy of negatively stained lipoproteins

Publisher Summary Negative staining is extremely useful in examining small macromolecular structures and, specifically, does not require interaction between the stain and the specimen. This chapter discusses the principle and application of electron microscopy of negatively stained lipoproteins. Negative staining is extremely useful for the examination of smaller, more dense lipoproteins, such as low-density lipoproteins (LDL) and high-density lipoproteins (HDL). The chapter also presents criteria for an ideal negative stain for electron microscopy, such as it must not react with the specimen to be examined, and it must have high electron density. Each of the lipoprotein classes constitutes a polydisperse spectrum of particle sizes and in certain pathological conditions, such as LCAT deficiency, Tangier disease, and fish eye disease. They are, as well, heterogeneous in geometry. Heterogeneity of particle size, and possibly morphology, must be kept in mind when comparing electron microscopic parameters with other techniques. The size of the LDL determined by the negative stain technique is in good agreement with gradient gel electrophoresis data.

Cholesteryl ester transfer protein and hepatic lipase activity promote shedding of apo A-I from HDL and subsequent formation of discoidal HDL.

The effects of lipid transfers and hepatic lipase (HL) on the concentration, composition, particle size distribution and morphology of high density lipoproteins (HDL) have been investigated. Human plasma supplemented with additional very low density lipoproteins (VLDL), cholesteryl ester transfer protein (CETP) and HL has been incubated at 37 degrees C for up to 8 h. The HDL became depleted of cholesteryl esters and reduced in particle size. Within 2 h of such incubation they had also lost about 30% of their apo A-I. However, with extension of the incubations beyond 2 h, the apo A-I returned progressively to the HDL fraction until, after 8 h, the concentration of apo A-I in HDL was identical to that in non-incubated samples. This return of apo A-I to the HDL density range was accompanied by a progressive appearance in electron micrographs of discoidal HDL particles. Thus, the depletion of the core lipid content and the reduction in particle size of HDL promoted by lipid transfers and HL activity in vitro is accompanied by a shedding of apo A-I which forms the nucleus of new discoidal HDL particles. The potential physiological importance of such a process is considerable.

Disruption of the Murine Lecithin:Cholesterol Acyltransferase Gene Causes Impairment of Adrenal Lipid Delivery and Up-regulation of Scavenger Receptor Class B Type I

Lecithin:cholesterol acyltransferase (LCAT) is the major determinant of the cholesteryl ester (CE) content of high density lipoprotein (HDL) in plasma. The selective uptake of HDL-CE is postulated to participate in delivery of tissue-derived cholesterol both to the liver and steroidogenic tissues. Recent studies comparing mice with similarly low levels of HDL, due to the absence of either of the two major HDL-associated apolipoproteins apoA-I and apoA-II, suggest that apoA-I is crucial in modulating this process, possibly through interaction with scavenger receptor class B type I (SR-BI). Because of the central role of LCAT in determining the size, lipid composition, and plasma concentration of HDL, we have created LCAT-deficient mice by gene targeting to examine the effect of LCAT deficiency on HDL structure and composition and adrenal cholesterol delivery. The HDL in the LCAT-deficient mice was reduced in its plasma concentration (92%) and CE content (96%). The HDL particles were heterogeneous in size and morphology and included numerous discoidal particles, mimicking those observed in LCAT-deficient humans. The adrenals of the male Lcat (−/−) mice were severely depleted of lipid stores, which was associated with a 2-fold up-regulation of the adrenal SR-BI mRNA. These studies demonstrate that LCAT deficiency, similar to apoA-I deficiency, is associated with a marked decrease in adrenal cholesterol delivery and supports the hypothesis that adrenal SR-BI expression is regulated by the adrenal cholesterol.

Optimized bacterial expression of human apolipoprotein A-I

Apolipoprotein A-I (apoA-I) serves critical functions in plasma lipoprotein metabolism as a structural component of high density lipoprotein, activator of lecithin:cholesterol acyltransferase, and acceptor of cellular cholesterol as part of the reverse cholesterol transport pathway. In an effort to facilitate structure:function studies of human apoA-I, we have optimized a plasmid vector for production of recombinant wild type (WT) and mutant apoA-I in bacteria. To facilitate mutagenesis studies, subcloning, and DNA manipulation, numerous silent mutations have been introduced into the apoA-I cDNA, generating 13 unique restriction endonuclease sites. The coding sequence for human apoA-I has been modified by the introduction of additional silent mutations that eliminate 18 separate codons that employ tRNAs that are of low or moderate abundance in Escherichia coli. Yields of recombinant apoA-I achieved using the optimized cDNA were 100+/-20 mg/L bacterial culture, more than fivefold greater than yields routinely obtained with the original cDNA. Site-directed mutagenesis of the apoA-I cDNA was performed to generate a Glu2Asp mutation in the N-terminal sequence of apoA-I. This modification, which creates an acid labile Asp-Pro peptide bond between amino acids 2 and 3, permits specific chemical cleavage of an N-terminal His-Tag fusion peptide used for rapid protein purification. The product proteins primary structure is identical to WT apoA-I in all other respects. Together, these changes in apoA-I cDNA and bacterial expression protocol significantly improve the yield of apoA-I protein without compromising the relative ease of purification.

The C-terminal domain of apolipoprotein A-I contains a lipid-sensitive conformational trigger

Exchangeable apolipoproteins can convert between lipid-free and lipid-associated states. The C-terminal domain of human apolipoprotein A-I (apoA-I) plays a role in both lipid binding and self-association. Site-directed spin-label electron paramagnetic resonance spectroscopy was used to examine the structure of the apoA-I C terminus in lipid-free and lipid-associated states. Nitroxide spin-labels positioned at defined locations throughout the C terminus were used to define discrete secondary structural elements. Magnetic interactions between probes localized at positions 163, 217 and 226 in singly and doubly labeled apoA-I gave inter- and intramolecular distance information, providing a basis for mapping apoA-I tertiary and quaternary structure. Spectra of apoA-I in reconstituted HDL revealed a lipid-induced transition of defined random coils and β-strands into α-helices. This conformational switch is analogous to triggered events in viral fusion proteins and may serve as a means to overcome the energy barriers of lipid sequestration, a critical step in cholesterol efflux and HDL assembly.

Electron microscopic study on reassembly of plasma high density apoprotein with various lipids.

Products resulting from the sonification of mixtures of plasma high density lipoprotein apoprotein and specific lipids were studied by electron microscopy using negative staining. Sonicates of apoprotein plus lecithin produced disc-shaped structures which stacked in aggregates with a 50–55-A repeat; the discs were 100–200 A in diameter. Incorporation of unesterified cholesterol into the mixture produced structures morphologically similar to those observed in sonicates of apoprotein plus lecithin. Disc-shaped particles from sonified mixtures of apoprotein, lecithin and unesterified cholesterol were ultracentrifugally isolated in the d 1.063–1.21 g/ml fraction and were incubated with a plasma d > 1.21 g/ml fraction containing lecithin: cholesterol acyltransferase activity. Electron microscopy following the incubation procedure showed a transformation of the disc-like structures into approximately spherical particles (50–100 A diameter). Similar spherical particles were also obtained after sonification of apoprotein-lecithin-unesterified cholesterol-cholesteryl ester mixtures. Results indicate a requirement for the presence of cholesteryl esters to maintain normal morphology of plasma high density lipoproteins.

Plasma Lipoproteins in Familial Lecithin:Cholesterol Acyltransferase Deficiency: Effects of Incubation with Lecithin: Cholesterol Acyltransferase in vitro

To study the effect of lecithin: cholesterol acyltransferase (LCAT) on the plasma lipoproteins of patients with familial LCAT deficiency, whole plasma or the lipoprotein fraction of d smaller than 1.006 g/ml (VLDL) was incubated in the presence of LCAT and subsequently examined by chemical, physical, and immunological techniques. The following occured upon incubating either hyperlipemic or nonlipemic plasma: The concentrations of polar lipids decreased, particulary in the large molecular weight lipoprotein subfraction of d 1.019-1.063 g/ml (LDL2) and in the lipoprotein fraction of 1.06301.25 g/ml (HDL). The concentration of cholesteryl ester (CE) increased, particularly in the VLDL and in the lipoprotein fractions of d 1.006-1.019 g/ml (LDL1) and LDL2. The concentration of arginine-rich apolipoprotein decreased in the HDL and increased in the VLDL and LDL1. The concentrations of the C-apoliproteins appeared to change in the opposite direction. The concentration of apolipoprotein B in the LDL increased concomitantly with an increase in the concentration and flotation rsate of the small LDL2. The concentration apolipoprotein A-I in the HDL increased; and a major component in the HDL fraction became identical in apperance to normal HDL. Upon incubating a patients isolated VLDL in the presence of LCAT, lipoproteins with properties similar to normal LDL2 were formed. These experiments show that the LCAT reaction can alter the apolipoprotein content and physical properties as well as the lipid content of the patients lipoproteins.