Effect of apoprotein B conformation on the activation of lysolecithin acyltransferase and lecithin: cholesterol acyltransferase. Studies with subfractions of low density lipoproteins.

In order to determine the role of apoprotein (apo) B conformation in the activation of the lysolecithin acyl-transferase reaction, we studied the activation of purified enzyme by various subfractions of low density lipoprotein (LDL), isolated by density gradient centrifugation. The activation of LAT correlated positively with the density of LDL and negatively with cholesterol/protein and triglyceride (TG)/protein ratios. The enzyme activation was also positively correlated with the number of trinitrobenzenesulfonic acid-reactive lysine amino groups, which increased with increasing density of LDL. The immunoaffinity of the LDL subfractions for B1B6, a monoclonal antibody directed to the receptor-binding region of apoB, increased with increasing density, while the affinity toward C1.4, a monoclonal antibody directed to the amino-terminal region of apoB, was not altered. Enrichment of normal whole LDL with TG resulted in a 45% reduction in enzyme activation, a 27% decrease in the number of trinitrobenzenesulfonic acid-reactive lysine groups, and a marked reduction in the immunoaffinity for B1B6. All these parameters reversed to normal when the TG-enriched LDL was treated with milk lipoprotein lipase, which specifically reduced the TG content of LDL. The LDL subfractions also supported cholesterol esterification by the purified enzyme, in parallel with lysolecithin esterification, indicating that apoB can also serve as an activator of the lecithin-cholesterol acyltransferase reaction. These results strongly suggest that the localized conformational change of apoB which occurs during the TG depletion of the precursor particle is critical for its activation of acyltransferase reactions, in a manner analogous to its interaction with the cellular receptors.     

Epinephrine potentiates activation of human platelets by low density lipoproteins.

Low density lipoproteins activate phosphoinositide turnover, increase free cytoplasmic calcium concentration and stimulate phosphorylation of 20- and 47-kDa proteins in blood platelets. All these effects are substantially potentiated by epinephrine.     

Cryopreservation with sucrose maintains normal physical and biological properties of human plasma low density lipoproteins.

The ability to preserve low density lipoprotein (LDL) preparations frozen for weeks and months without changes in structure or biological properties is of potential use in long-term comparative studies of LDL. We demonstrate that freeze-thawing of LDL causes marked alterations in its structure and biological behavior, and that such changes can be prevented by the addition of sucrose to the LDL solution prior to freezing. Freezing LDL at -70 degrees C in the absence of sucrose resulted in aggregation and fusion of particles as measured by electron microscopy, spectrophotometric absorption, and column gel filtration. This was associated with increased binding affinity of monoclonal antibodies at epitopes distant from the receptor binding region. Functional changes induced by freezing included 3- to 10-fold increases in binding at 4 degrees C and 37 degrees C, and uptake of LDL in fibroblasts, attributable mainly to increases in nonspecific binding processes. Cryopreservation of LDL in 10% sucrose (w/v) completely prevented the structural and functional changes incurred after short-term freezing, and LDL cryopreserved in sucrose for as long as 18 months displayed cell binding, uptake, and degradation very similar to that of freshly obtained LDL.     

Promotion of human T lymphocyte activation and proliferation by fatty acids in low density and high density lipoproteins

Mitogen-induced lymphocyte DNA synthesis measured by [3H]thymidine incorporation and lymphocyte proliferation assessed by counting the number of cells were reduced by greater than 95% when cells were cultured at low density in the absence of serum. Supplementation with either transferrin or lipoprotein alone only partially restored lymphocyte responses. Addition of both transferrin and lipoproteins of each major subclass permitted mitogen-induced lymphocyte DNA synthesis and proliferation equal to that observed in serum-containing medium. The degree of enhancement was dependent on the concentration of the lipoprotein added and could not be explained by the nonspecific addition of protein to the defined medium. The mechanisms of growth promotion by various lipoprotein fractions did not appear to be explained by provision of cholesterol to the cells. Neither cholesterol nor cholesteryl ester from endogenous sources or supplied exogenously was able to enhance mitogen-induced lymphocyte responses. In contrast, fatty acids, phospholipid, and triglyceride alone supported lymphocyte responses. Furthermore, lipoproteins retained the capacity to enhance lymphocyte responses following extraction of neutral lipid. Both low density lipoprotein and high density lipoprotein, subclass 3, increased the number of cells initially activated by mitogenic stimulation and supported the subsequent continued growth of the activated cells. Low density lipoprotein was more efficient than high density lipoprotein, subclass 3, in this latter regard. These results indicate that lipoproteins can promote maximal growth of mitogen-activated lymphocytes in transferrin-containing medium by providing growth factors other than cholesterol necessary for initial activation and required for continued lymphocyte proliferation.     

Interactions of high density lipoproteins with very low and low density lipoproteins during lipolysis

Interactions of high density lipoproteins (HDL) with very low (VLDL) and low (LDL) density lipoproteins were investigated during in vitro lipolysis in the presence of limited free fatty acid acceptor. Previous studies had shown that lipid products accumulating on lipoproteins under these conditions promote the formation of physical complexes between apolipoprotein B-containing particles (Biochim. Biophys. Acta, 1987. 919: 97-110). The presence of increasing concentrations of HDL or delipidated HDL progressively diminished VLDL-LDL complex formation. At the same time, association of HDL-derived apolipoprotein (apo) A-I with both VLDL and LDL could be demonstrated by autoradiography of gradient gel electrophoretic blots, immunoblotting, and apolipoprotein analyses of reisolated lipoproteins. The LDL increased in buoyancy and particle diameter, and became enriched in glycerides relative to cholesterol. Both HDL2 and HDL3 increased in particle diameter, buoyancy, and relative glyceride content, and small amounts of apoA-I appeared in newly formed particles of less than 75 A diameter. Association of apoA-I with VLDL or LDL could be reproduced by addition of lipid extracts of lipolyzed VLDL or purified free fatty acids in the absence of lipolysis, and was progressively inhibited by the presence of increasing amounts of albumin. We conclude that lipolysis products promote multiple interactions at the surface of triglyceride-rich lipoproteins undergoing lipolysis, including physical complex formation with other lipoprotein particles and transfers of lipids and apolipoproteins. These processes may facilitate remodeling of lipoproteins in the course of their intravascular metabolism.     

Structure and interactions of lipids in human plasma low density lipoproteins.

Temperature-dependent techniques (differential scanning calorimetry, polarizing microscopy, and x-ray scattering and diffraction techniques) were used to compare the properties of human plasma low density lipoproteins (LDL) with its extracted lipid classes. Three types of thermal transitions were characterized: (a) a reversible transition in intact LDL near body temperature associated with a liquid crystalline order-disorder phase change of cholesterol esters within the particles; (b) an irreversible high temperature transition (approximately 70-90 degrees) associated with LDL denaturation and release of cholesterol esters from the disrupted particles; and (c) low temperature transitions related to liquid crystalline and crystalline phase changes in these released esters. The temperature of the reversible transition in intact LDL varies among individual donors. Correlation analysis shows that the temperature of this transition negatively correlates with the amount of triglyceride relative to cholesterol ester in LDL. Studies on mixtures of cholesterol esters and triglycerides isolated from LDL show a similar effect, increasing amounts of triglycerides decreasing the temperature of the liquid leads to smectic liquid crystalline transition of the isolated esters. Thus, the amount of triglyceride in LDL influences the fluidity of the cholesterol esters in LDL. The enthalpy of the reversible transition in intact LDL is 0.69 cal/g of LDL cholesterol ester. This compares with 0.89 cal/g for the liquid leads to liquid crystalline transition of the cholesterol esters released from denatured LDL and 1.01 cal/g for the same transition in the extracted esters. Unlike the cholesterol esters released from denatured LDL, or isolated LDL esters, cholesterol ester in the intact LDL particle does not crystallize. These findings suggest that the behavior of cholesterol esters in intact LDL is constrained relative to their behavior when freed from the restrictions of the particle. These results together with experiments on partitioning of the individual lipid classes of LDL allow us to define the distribution and interaction of lipids in the intact LDL particle.     

Interaction of apoA-II from human high density lipoprotein with lysolecithin.

The effects of lysolecithin and hexadecyltrimethylammonium bromide on the structure and stability of apoA-II from human high density lipoprotein have been evalued by circular dichroism and fluorescence measurements. There is a profound enhancement in the stability of apoA-II to guanidinium hydrochloride denaturation when it forms a phospholipid complex with lysolecithin micelles. This complex is not only resistant to guanidinium hydrochloride denaturation, but it can be formed in a 6 M solution of this denaturant. The behavior of apoA-II in the native human high density protein is much closer to that of the lysolecithin apoA-II complex than to that of the free apoA-II.     

Activity of cholinephosphotransferase, lysolecithin: lysolecithin acyltransferase and lysolecithin acyltransferase in the developing mouse lung.

1. The present study presents the activity profiles of cholinephosphotransferase, lysolecithin:lysolecithin acyltransferase and lysolecithin acyltransferase at different stages of development of the mouse lung. 2. The specific activity of cholinephosphotransferase, a key enzyme in the de novo synthesis of phosphatidylcholine, increases during the later stages of fetal development until it reaches a maximal value at a gestational age of 17 days, i.e. 2 days before term. Thereafter, the activity of the enzyme declines again until around term. 2. The specific activity of lysolecithin:lysolecithin acyltransferase which catalyzes the transesterification between two molecules of 1-acyl-sn-glycero-3-phosphocholine, appears to be much lower than that of cholinephosphotransferase at gestational ages below 18 days. However, around day 18, the specific activity of lysolecithin:lysolecithin acyltransferase increases dramatically until it almost equals the maximal activity of cholinephosphotransferase measured on day 17. 4. The specific activity of lysolecithin acyltransferase, which catalyzes the direct acylation of 1-acyl-sn-glycero-3-phosphocholine, does not change significantly during the prenatal development and is lower than that of either lysolecithin:lysolecithin acyltransferase or cholinephosphotransferase at all stages of development. 5. These results are discussed in view of the possible role of these enzymes in the biosynthesis of pulmonary 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.     

Immunochemical study of the plasma low and high density lipoproteins in Tangier disease

Major disturbances of the lipoproteins in Tangier serum have been investigated using electrophoretic and immunochemical techniques. Previously described anomalies concerning the striking deficiency in HDL and the very low levels of apo A-I and apo A-II in Tangier patients are illustrated and explained. Anomalies concerning the fast LDL of Tangier serum are attributed to different forms of apo B not previously described. These data are strengthened by the features of a 2-dimensional electrophoresis method elaborated in the laboratory which allows apoproteins to separate in the second dimension. These apoproteins are obtained by the delipidation of the lipoproteins fractionated in a first polyacrylamide discontinuous gel. This method clearly shows the distribution of apoproteins in the first lipoprotein track and is in perfect accordance with the new concept of lipoprotein particles.     

Effects of guanidine hydrochloride on human plasma high density lipoproteins.

Denaturation of human plasma high density lipoproteins during ultracentrifugation in guanidine-HCl is characterized by: dissociation of apoA-I, in the range of 2-3 M guanidine-HCl, and dissociation of apoA-I and apoA-II in 5-6 M guanidine-HCl. Denaturation of high density lipoprotein species, during a sequence of timed exposure to guanidine-HCl followed first by removal of the denaturant by dialysis and then by ultracentrifugation, is characterized by:dissociation of lipid-poor apoA-I, which follows a time course similar to denaturation-related changes in reported spectroscopic parameters; and apparent formation of lipoprotein aggregation products depleted in apoA-I and relatively enriched in apoA-II. These studies indicate differential properties of the major apoproteins in stabilizing high density lipoprotein structure and characterize a mode of lipoprotein transformation and degradation which apparently results from apoprotein dissociation coupled with aggregation of denatured lipoprote species.     

Interaction of gangliosides with plasma low density lipoproteins

The role of gangliosides in the reception of low density lipoproteins (LDL) was studied using as targets mouse ascites hepatoma 22a (MAH) cells which bind LDL through a specific high affinity receptor. Low density lipoprotein binding and uptake by MAH cells decreased after brief treatment of the cells with neuraminidase to partially remove surface sialic acid residues. The LDL uptake capability of the neuraminidasetreated MAH cells was fully restored after incorporation of exogeneous G_M1- and G_D1a-gangliosides into the cell surface. In contrast, free (extracellular) gangliosides inhibited LDL uptake by native MAH cells. This inhibitory effect was seen at ganglioside concentrations corresponding to the ganglioside content of serum and was most pronounced with gangliosides whose sialic acids were linked to a terminal galactose residue (G_M3, G_D1a, G_T1b) but was smaller or absent with gangliosides whose sialic acids were attached to an internal galactose (G_M1, G_M2). The binding of gangliosides to LDL was structure and concentration dependent, saturable and trypsin sensitive. The LDL-ganglioside interaction was further investigated by steady state fluorescence spectroscopy. Changes in the LDL fluorescence polarization were observed with as little as 0.01 M concentrations of the gangliosides. The magnitude and nature of the effect depended on the type of ganglioside. We conclude that the LDL surface possesses sites recognizing specific carbohydrate sequences of glycoconjugates and that changes in the cell surface concentrations of sialic acids significantly modulate the LDL uptake. It is postulated that shedding of gangliosides into the blood stream may be a factor involved in regulation of cholesterol homeostasis.Abbreviations MAH mouse ascites hepatoma 22a - LDL low density lipoprotein - ASM anthrylvinyl-labeled sphingomyelin [N-12-(9-anthryl-trans-dodecanoyl-sphingosine-1-phosphocholine] - RITC rhodamine isothiocyanate. The designation of gangliosides follows the IUPAC-IUB recommendation [1]: G_M3, II³NeuAc-LacCer, II³-N-acetylneuraminosyllactosylceramide - G_M2 II³-NeuAc-GgOse₃Cer, II³-N-acetylneuraminosylgangliotriaosylceramide - G_M1 II³-NeuAc-GgOse₄Cer, II³-N-acetylneuraminosylgangliotetraosylceramide - G_D1a, II³ IV³(NeuAc)₂-GgOse₄Cer, II³, IV³-di(N-acetylneuraminosyl)gangliotetraosylceramide - G_T1b II³(NeuAc)₂, IV³ NeuAc-GgOse₄Cer, II³-di-N-acetylneuraminosyl, IV³-N-acetylneuraminosylgangliotetraosylceramide     

High density lipoproteins reduce the uptake of low density lipoproteins by human endothelial cells in culture.

Endothelial cells, explanted from human umbilical veins and cultured, maintained morphological characteristics of vascular endothelium. When exposed to human serum lipoproteins, the cells bound and took up low density lipoproteins in preference to high density lipoproteins. High density lipoproteins reduced markedly the uptake of low density lipoproteins and affected surface binding to a lesser extent. These data suggest that the different levels of high density lipoprotein encountered in normal plasma of males and females could modulate differently the transendothelial transport of low density lipoproteins and provide a possible explanation for the lesser severity of atheromatosis in the aortic intima of premenopausal females.