Role of phospholipids in the cytotoxic action of high density lipoprotein on trypanosomes.

Host range among the African trypanosomes, protozoa that cause fatal diseases both in humans and livestock, may be, in part, regulated by toxic properties associated with host high density lipoproteins (HDL). High density lipoproteins from hosts resistant (baboon, human) or susceptible (rabbit, rat) to Trypanosoma brucei infection were isolated and their trypanocidal activity was determined in in vitro cell lysis assays. Rabbit and rat HDL were not cytotoxic while baboon and human HDL rapidly lysed trypanosomes within 2 h at 37 degrees C. Analysis of the phospholipid composition of HDL preparations from these species suggested a correlation between trypanocidal activity and low phosphatidylinositol content. Phospholipase digestion of HDL resulted in a loss of trypanocidal activity, indicating the importance of native phospholipids in maintaining this biological activity of HDL. Cell lysis and loss of trypanosome infectivity induced by baboon HDL could be inhibited either by addition of rabbit or rat HDL to the incubation medium or by addition of purified phospholipids, phosphatidylinositol being the most effective inhibitor. Although the mechanism by which HDL lyses trypanosomes remains to be elucidated, these results suggest an important role for phospholipids in determining the specificity of this cytotoxic property of HDL.     

Endocytosis by African trypanosomes. II. Occurrence in different life-cycle stages and intracellular sorting

Horseradish peroxidase (HRP) and colloidal gold-labeled proteins enter many of the endocytic organelles of bloodstream forms of Trypanosoma brucei and T. congolense. However, the colloidal gold markers were excluded from substantial parts of the pathway that contained HRP. Morphometric studies revealed that HRP entered organelles that accounted for approximately 5% of the total cell volume while transferrin-gold entered organelles that comprised approximately 2% of the total cell volume. In addition, large colloidal gold particles were excluded from organelles that contained smaller gold particles. Antibodies, raised against the variable surface glycoprotein, when applied to thawed cryosections were found to label structures from which endocytosed colloidal gold coupled to bovine serum albumin (BSA) was excluded. Endocytosis was shown to occur in two in vitro propagated forms of trypanosomes, similar to those found in the insect vector (Glossina spp.). The mammal-infective metacyclic forms were similar to bloodstream forms in that they endocytosed HRP and colloidal gold markers but excluded colloidal gold from approximately 3% of the endocytic organelles. Estimation of the flagellar pocket volumes of bloodstream form T. brucei showed that this organelle occupied 0.5% to 1.4% of the total cell volume. The flagellar pocket volume of T. congolense varied between life-cycle stages, with a fractional volume of 4.4% for bloodstream forms, 2.3% for metacyclic forms and 1.4% for procyclic forms. Endocytosis of HRP, but not of protein-gold markers, occurred in procyclic (uncoated) forms. Endocytosis by procyclic forms has heretofore not been reported.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of high density lipoprotein by chicken liver: nature of nascent intracellular high density lipoprotein

Young chickens were administered L-[(3)H]leucine and after 10 or 30 min the livers were removed and fractioned into rough (RER) and smooth (SER) endoplasmic reticulum fractions and into light, intermediate, and heavy golgo cell fractions. The labeled high density lipoprotein (HDL), contained within these intracellular organelles was isolated either by immunoprecipitation using rabbit antiserum to rooster HDL, or by ultracentrifugal glotation between densities 1.063 and 1.21 g/ml. The radioactive apoproteins of nascent HDL were analyzed by SDS PAGE and detected by fluorography. Analyses of radioactive apoproteins obtained by immunoprecipitation from the contents of the RER, the SER, and the three golgi complex fractions revealed only one apoprotein, A1. The C peptide present in serum HDL was not detected intracellularly. The radioactive apoprotein A1 which is present within the cisternae of the RER and the SER fractions failed to float, whereas apoprotein A1, present within the golgi apparatus, readily floated between densities 1.063 and 1.21 g/ml. The HDL particles, isolated by flotation from the golgi apparatus content, were further characterized by lipid and protein analyses and by electron microscopy. Golgi HDL particles have the same density as serum HDL. On a percentage basis, golgi HDL contains less protein and more phospholipids than does serum HDL. Morphologically, golgi HDL is different in appearance from serum HDL. It is more heterogeneous in size, with most of the particles ranging 8.3-25 nm in diameter. The spherical particles contain small membrane tails. Occasionally, a few disk-shaped bilayer structures are also found within the golgi apparatus. These studies show that the newly synthesized apoprotein A1, present within the RER and the SER cell fractions, is not fully complexed with lipid and that apoprotein A1 does not acquire sufficient lipid to float at the proper HDL density until it enters the golgi apparatus. The difference in chemical composition and the heterogeneous size of golgi HDL may be attributed to the different stages of HDL maturation.     

Forms of human serum high density lipoprotein protein

Delipidation by ethanol-diethyl ether at -10 degrees C of human serum high-density lipoprotein (HDL, d 1.063-1.21) or of its subclasses HDL(2) (d 1.063-1.120) and HDL(3) (d 1.120-1.21), yielded proteins-alphaP, alphaP(2), and alphaP(3)-containing 3% phospholipid (largely lecithin) and 3.3% carbohydrate (glucosamine:L-fucose:D-galactose, D-mannose:sialic acid, 1.00:41 : 0.56:0.31). Solubility data and analytical ultracentrifugal analyses indicated that, upon lipid removal, HDL protein aggregates readily; the aggregation is dependent upon pH and ionic strength of the solvent medium. Subunits of 21,000 mol wt were obtained by acetylation or addition of sodium dodecyl sulfate (SDS). HDL and alphaP elicited in the rabbit a similar immunological response. By agar gel immunoelectrophoresis both anti-HDL and anti-alphaP sera detected a major and two minor antigenic determinants in HDL, HDL(3), alphaP, alphaP(2), and alphaP(3). HDL(2), antigenically homogeneous, gave an immunoelectrophoretic pattern of HDL(3) upon mixing with alphaP. alphaP, alphaP(2), and alphaP(3) exhibited a single antigenic determinant after treatment with SDS (0.5 M) or upon acetylation. Native or delipidated forms of HDL, HDL(2), and HDL(3) were separated by vertical starch gel electrophoresis into several components, which showed identical reactions against anti-HDL or anti-alphaP sera. The data suggest that (a) the proteins of HDL, HDL(2), and HDL(3) are made of subunits, probably identical, of an average molecular weight of 21,000; (b) the difference in antigenic behavior between HDL(2) and HDL(3) is due to the presence in the latter of a lipid-poor protein; (c) antigenic polymorphism of alphaP is probably related to the presence in solution of monomeric and polymeric forms having different reactivity against anti-HDL and anti-alphaP sera.     

Lipoxygenase-mediated transformation of human low density lipoprotein to an oxidized and cytotoxic complex

We have been studying the mechanisms involved in the oxidative modification of low density lipoprotein (LDL) that lead to its transformation to a cytotoxic complex. Here we examine the direct effect-of soybean lipoxygenase (SLO), a 15-lipoxygenase, on normal human LDL. SLO oxidized LDL and rendered it cytotoxic; agents known to interfere with lipoxygenase activity inhibited this reaction. Enhancement of both the SLO-mediated LDL oxidation and the conversion of LDL to a cytotoxin was observed when either superoxide dismutase or copper (II) (3,5,-diisopropylsalicylic acid)2, both of which dismute superoxide anion, were included during the incubation of SLO with LDL. In contrast, catalase inhibited this reaction in the presence or absence of agents that dismute superoxide anion. Thus, purified lipoxygenase can mediate LDL modification and superoxide anion inhibits this reaction, Furthermore, H2O2 is essential for SLO-mediated LDL oxidation and conversion of LDL to a cytotoxin.     

Dynamic properties of human high density lipoprotein apoproteins.

This study was designed to identify a method for the measurement of human high density lipoprotein subfraction (HDL2 and HDL3) metabolism. Apolipoproteins A-I, A-II, and C, the major HDL apoproteins, were radioiodinated and incorporated individually into HDL2 and HDL3 in vitro. Using a double label technique, the turnover of apoA-I in HDL2 and HDL3 was measured simultaneously in a normal male. The apoprotein exchanged rapidly between the two subfractions, evidenced by equilibration of their apoA-I specific activity. Radiolabeled apoA-II, incorporated into the subfractions, showed a similar exchange in vitro. Incubation of 131I-labeled very low density lipoproteins (VLDL) with HDL or its subfractions resulted in transfer of C proteins from VLDL to the HDL moiety. The extent of transfer was dependent on the HDL subfraction present; 50% of the VLDL apoC was transferred to HDL3, while the transfer to total HDL and HDL2 was 69% and 78%, respectively. ApoC also exchanged between HDL2 and HDL3, again showing a preference for the former and suggesting a primary metabolic relationship between VLDL and HDL2. Overall, the study indicates that apoA-I, apoA-II, and the C proteins exist in equilibrium between HDL2 and HDL3. This phenomenon precludes their use as probes for HDL subfraction metabolism in humans.     

Chromatofocusing of apolipoproteins from human serum high density lipoprotein

Human HDL was delipidated and the apolipoproteins were fractionated by chromatofocusing. Chromatofocusing, which separates proteins due to their differing isoelectric points, resulted in 8 peaks with corresponding pI values of 7.40, 6.92, 6.64, 5.48, 5.30, 5.18, 4.92 and 4.63. By one single chromatofocusing run four apolipoproteins were obtained in pure form. Two additional polypeptides could be purified during the desalting step using phenyl-Sepharose.     

Thermal behavior of human plasma high density lipoprotein.

Human plasma low density lipoprotein displays a reversible thermal transition between 20 and 40 degrees C, due to a phase transition of its core cholesterol ester from a smectic to a more liquid-like state. To determine if the cholesterol of high density lipoprotein (HDL) displays similar thermal behavior, the human lipoprotein and its extracted lipid have been examined by differential scanning calorimetry, low angle X-ray scattering and polarizing microscopy. Neither HDL2**(d 1.063--1.125--1.21 g/ml) nor HDL3(d1.125--1.21g/ml) show thermal transitions between O and 60 degrees C. By contrast cholesterol ester isolated from HDL and mixtures of cholesterol oleate and linoleate show reversible liquid crystalline transitions between 20 and 40 degreesC. X-ray scattering studies of HDL2 and HDL3 performed at 10 degreesC show no scattering fringes attributable to a smectic phase of cholesterol ester. When HDL is heated to temperatures above 60 degreesC a broad, double-peaked endotherm is observed. The first component (peak temperature=71 degreesC) corresponds to a selective release of apoprotein A-1 from the lipoprotein, and the second component (peak temperature=90 degreesC) to a more generalized disruption of lipoprotein structure with release of cholesterol ester and apoprotein A-2. Following the thermal disruption of HDL, reversible liquid crystalline transitions of cholesterol ester can be seen by differential scanning calorimetry and polarizing microscopy, showing the presence of large domains of cholesterol ester. The absence of cholesterol ester transitions in intact HDL may indicate an interaction of cholesterol ester molecules with the protein-phospholipid surface of HDL that prevents the formation of an organized lipid phase. The high temperature behavior of HDL indicates that apoprotein A-1 is less important than apoprotein A-2 in maintaining the HDL apolar lipids in the form of a stable miroemulsion.     

Interaction of bovine serum high density lipoprotein with mixed vesicles of phosphatidylcholine and cholesterol.

The interaction of sonicated, small vesicles of egg phosphatidylcholine and cholesterol (2:1, mol/mol) with bovine high density serum lipoproteins was examined in terms of lipid transfer between both types of particles and the resulting changes in lipoprotein structure. Saturation of high density lipoprotein preparations with vesicle lipids gave final lipoprotein particles with essentially unchanged protein content and composition, unchanged cholesterylester and nonpolar lipid content, but with markedly increased phospholipid content (59% increas by weight) and moderately increased cholesterol content (20% increase by weight). The lipoproteins enriched in lipid were relatively uniform, spherical particles, 110 +/- 3.6 A in diameter (6 A larger than the original lipoproteins); they had a markedly decreased intrinsic protein fluorescence, a red-shifted fluorescence wavelength maximum, and more fluid lipid domains. These results indicate that the direct addition of excess lipids from membranes or other lipoproteins is a possible mechanism for lipid transfer to high density lipoproteins. Also they suggest a structural flexibility of high density lipoproteins that allows the addition of significant amounts of surface components.     

Protein kinase C as a mediator of high density lipoprotein receptor-dependent efflux of intracellular cholesterol.

The interaction of high density lipoproteins (HDL) with the HDL receptor stimulates the translocation of cholesterol from intracellular pools to the plasma membrane where the cholesterol becomes available for removal by appropriate acceptors. The role of signal transduction through protein kinase C in HDL receptor-dependent cholesterol translocation and efflux was examined using cholesterol-loaded cultured human skin fibroblasts. Treatment of cells with HDL3 activated protein kinase C, demonstrated by a transient increase in membrane associated kinase activity. Kinase activation appeared to be dependent on binding of HDL3 to the HDL receptor, since tetranitromethane-modified HDL3, which does not bind to the receptor, was without effect. Translocation of intracellular sterol to the plasma membrane was stimulated by treatment of cells with the protein kinase C activators, dioctanoylglycerol and phorbol myristic acetate, and the calcium ionophore A23187. Conversely, treatment of cells with sphingosine, a protein kinase C inhibitor, reduced HDL3-mediated translocation and efflux of intracellular sterols. However, sphingosine had no effect on efflux of labeled cholesterol derived from the plasma membrane. Down-regulation of cellular protein kinase C activity by long term incubation with phorbol esters also inhibited HDL3-mediated efflux of intracellular sterols and abolished the ability of sphingosine to further inhibit HDL3-mediated efflux. These studies support the conclusion that HDL receptor-mediated translocation and efflux of intracellular cholesterol occurs through activation of protein kinase C.     

Induction of low density lipoprotein receptor synthesis by high density lipoprotein in cultures of human skin fibroblasts.

Further studies have been made of the effects of high density lipoprotein (HDL) on the surface binding, internalization and degradation of 125I-labeled low density lipoprotein (125I-labeled LDL) by cultured normal human fibroblasts. In agreement with earlier studies, during short incubations HDL inhibited the surface binding of 125I-labeled LDL. In contrast, following prolonged incubations 125I-labeled LDL binding was consistently greater in the presence of HDL. The increment in 125I-labeled LDL binding induced by HDL was: (a) associated with a decrease in cell cholesterol content; (b) inhibited by the addition of cholesterol or cycloheximide to the incubation medium; and (c) accompanied by similar increments in 125I-labeled LDL internalization and degradation. It is concluded that HDL induces the synthesis of high affinity LDL receptors in human fibroblasts by promoting the efflux of cholesterol from the cells.     

Distinction in the mode of receptor-mediated endocytosis between high density lipoprotein and acetylated high density lipoprotein: evidence for high density lipoprotein receptor-mediated cholesterol transfer

The interactions of high density lipoprotein (HDL) and acetylated high density lipoprotein (acetyl-HDL) with isolated rat sinusoidal liver cells have been investigated. Cellular binding of 125I-acetyl-HDL at 0 degrees C demonstrated the presence of a specific, saturable membrane-associated receptor. This receptor was affected neither by formaldehyde-treated albumin nor by low density lipoprotein modified either by acetylation or malondialdehyde, ligands known to undergo receptor-mediated endocytosis by the cells, indicating that the receptor for acetyl-HDL constitutes a distinct class among the scavenger receptors for chemically modified proteins. Parallel binding experiments using 125I-HDL also revealed the presence on these cells of a receptor for unmodified HDL. The ligand specificities of these two receptors were similar to each other except that the acetyl-HDL receptor was sensitive to polyanions such as dextran sulfate and fucoidin. Interaction of HDL with the cells at 37 degrees C was totally different from that of acetyl-HDL. Cellular binding of HDL was not accompanied by subsequent intracellular degradation of its apoprotein moiety, whereas its cholesterol moiety was significantly transferred to the cells. In contrast, acetyl-HDL was endocytosed and underwent lysosomal degradation as a holoparticle. This shift in receptor-recognition from the HDL receptor to the acetyl-HDL receptor was accomplished by acetylation of approximately 8% of the total lysine residues of HDL apoprotein. This unique difference in endocytic behavior between HDL and acetyl-HDL suggests a potential link of the HDL receptor to HDL-mediated cholesterol transfer in sinusoidal liver cells.     

Interaction of human serum high density lipoprotein apoprotein with phospholipids

The interaction of the apoprotein of human serum high density lipoprotein-3 (apo HDL₃)¹¹ with aqueous dispersion of natural and synthetic phospholipids (PL) was investigated at a temperature above the transitions of the PL hydrocarbon chains and also above their critical micellar concentration. This protein is known to contain two major polypeptides: apo A-I and apo A-II. The protein:PL mixtures (weight ratio, 2, 1 or 0.5) were subjected to sonic irradiation and then fractionated by either CsCl or D₂O-sucrose density gradient ultracentrifugation. Usually three bands were obtained, the relative mass distribution of which depended upon the nature of the PL and the ratio of the interacting components: one band contained the PL-poor protein (d 1.28 g/ml), another, the uncombined PL (d ? 1.08 g/ml), and the third band, both protein and PL. This band, which was considered to represent the actual complex, had a hydrated density intermediate between those of either apo HDL₃ or PL alone, a particle weight of 80,000 to 170,000 (i.e., less than that of intact HDL₃), a morphology by electron microscopy which depended on the materials and experimental conditions employed and circular dichroic spectra     

Lysis of Trypanosoma brucei by a toxic subspecies of human high density lipoprotein

Trypanosoma brucei brucei is an important pathogen of domestic cattle in sub-Saharan Africa and is closely related to the human sleeping sickness parasites, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. However, T. b. brucei is non-infectious to humans. The restriction of the host range of T. b. brucei results from the sensitivity of the parasite to lysis by toxic human high density lipoproteins (HDL) (Rifkin, M. R. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3450-3454). We show in this report that trypanosome lytic activity is not a universal feature of all human HDL particles but rather that it is associated with a minor subclass of HDL. We have purified the lytic activity about 8,000-fold and have identified and characterized the subspecies of HDL responsible for trypanosome lysis. This class of HDL has a relative molecular weight of 490,000, a buoyant density of 1.21-1.24 g/ml, and a particle diameter of 150-210 A. It contains apolipoproteins AI, AII, CI, CII, and CIII, and monoclonal antibodies against apo-AI and apo-AII inhibit trypanocidal activity. In addition to these common apolipoproteins, the particles also contain at least three unique proteins, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Treatment of the particles with dithiothreitol resulted in the disappearance of two of the proteins and abolished trypanocidal activity. Two-dimensional gel electrophoresis showed that these proteins were a disulfide-linked trimer of 45,000, 36,000, and 13,500-Da polypeptides and dimers of the 36,000- and 13,500-Da polypeptides or of 65,000- and 8,500-Da polypeptides. Studies on the lysis of T. b. brucei by the purified particle suggest that the lytic pathway may involve the uptake of the trypanocidal subspecies of HDL by endocytosis.     

Radioimmunoassay of human high density lipoprotein apo-protein A-1.

A double antibody radioimmunoassay technique was developed for the measurement of apolipoprotein A-I, the major apoprotein of human high density lipoproteins. Apolipoprotein A-I was prepared from human delipidated high density lipoprotein (d equal to 1.085-1.210) by gel filtration and ion-exchange chromatography. Purified apolipoprotein A-I antibodies were obtained by means of apolipoprotein A-I immunoadsorbent. Apolipoprotein A-I was radiolabeled with 125-I by the iodine monochloride technique. 65-80% of 125 I-labeled apolipoprotein A-I could be bound by the different apolipoprotein A-I antibodies, and more than 95% of the 125-I-labeled apolipoprotein A-I was displaced by unlabeled apolipoprotein A-I. The immunoassay was found to be sensitive for the detection of about 10 ng of apolipoprotein A-I in the incubation mixture, and accurate with a variability of only 3-5% (S.E.M.). This technique enables the quantitation of apolipoprotein A-I in whole plasma or high density lipoprotein without the need of delipidation. The quantitation of apolipoprotein A-I in high density lipoprotein was found similar to that obtained by gel filtration technique. The displacement capacity of the different lipoproteins and apoproteins in comparison to unlabeled apolipoprotein A-I was: very low density lipoprotein, 1.8%; low density lipoprotein, 2.6%; high density lipoprotein, 68%; apolipoprotein B, non-detectable; apolipoprotein C, 0.5%; and apolipoprotein A-II, 4%. The distribution of immunoassayable apolipoprotein A-I among the different plasma lipoproteins was as follows: smaller than 1% in very low density lipoprotein and low density lipoprotein; 50% in high density lipoprotein, and 50% in lipoprotein fraction of density greater than 1.21 g/ml. The amount of apolipoprotein A-I in the latter fraction was found to be related to the number of centrifugations.     

Effects of fat ingestion on high density lipoprotein profiles in human sera

Serum concentrations of triacylglycerol, apolipoprotein A-I, apolipoprotein A-II, HDL2, and HDL3 were determined in sera of nine normolipidemic adult males, just before and 3, 5, and 8 hr after ingestion of 250 ml of cream (100 g of triacylglycerol). In all individuals a rapid hypertriglyceridemic response was observed. Triacylglycerol concentrations increased from 624 +/- 124 mg/liter of serum to 1435 +/- 350 mg/liter of serum 3 hr after cream ingestion. In most individuals the hypertriglyceridemic response was followed by a decline in serum triacylglycerol concentration to below basic levels. As a result of cream ingestion, small but statistically highly significant increases in serum cholesterol and apolipoprotein A-I concentrations were observed that persisted till the end of the observation period. In most individuals a small rise in the apolipoprotein A-II concentration in serum was also present. Marked changes were observed in serum HDL as illustrated in the HDL absorption at 280 nm and cholesterol profiles obtained by single-spin rate-zonal density gradient ultracentrifugation of the sera. Due to a prominent increase in phospholipids (up to about 18%) and a smaller increase in protein (up to about 6%), flotation rates and concentrations of HDL2 as well as HDL3 increased. These changes in HDL subclass flotation characteristics and chemical composition are best explained by uptake of surface material from chylomicrons by existing HDL2 and HDL3 particles. The data do not support a previously proposed concept in which HDL3 is converted into HDL2 by uptake of surface remnants formed during catabolism of triglyceride-rich lipoproteins.