Lipid specificity and location of the sterol carrier protein-2 fatty acid-binding site: A fluorescence displacement and energy transfer study

Although it was recently recognized that sterol carrier protein-2 (SCP-2) interacts with fatty acids, little is known regarding the specificity of SCP-2 for long-chain fatty acids or branched-chain fatty-acid-like molecules. Likewise the location of the fatty-acid binding site within SCP-2 is unresolved. A fluorescent cis-parinaric acid displacement assay was used to show that SCP-2 optimally interacted with 14–22 carbon chain lipidic molecules: polyunsaturated fatty acids > monounsaturated, saturated > branched-chain isoprenoids > branched-chain phytol-derived fatty acids. In contrast, the other major fatty-acid binding protein in liver, fatty-acid binding protein (L-FABP), displayed a much narrower carbon chain preference in general: polyunsaturated fatty acids > branched-chain phytol-derived fatty acids > 14- and 16-carbon saturated > branched-chain isoprenoids. However, both SCP-2 and l-FABP displayed a very similar unsaturated fatty-acid specificity profile. The presence and location of the SCP-2 lipid binding site were investigated by fluorescence energy transfer. The distance between the SCP-2 Trp⁵⁰ and bound cis-parinaric acid was determined to be 40 Å. Thus, the SCP-2 fatty-acid binding site appeared to be located on the opposite side of the SCP-2 Trp⁵⁰. These findings not only contribute to our understanding of the SCP-2 ligand binding site but also provide evidence suggesting a potential role for SCP-2 and/or L-FABP in metabolism of branched-chain fatty acids and isoprenoids.     

Effect of Sterol Carrier Protein-2 Expression on Sphingolipid Distribution in Plasma Membrane Lipid Rafts/Caveolae

Although sphingolipids are highly important signaling molecules enriched in lipid rafts/caveolae, relatively little is known regarding factors such as sphingolipid binding proteins that may regulate the distribution of sphingolipids to lipid rafts/caveolae of living cells. Since early work demonstrated that sterol carrier protein-2 (SCP-2) enhanced glycosphingolipid transfer from membranes in vitro, the effect of SCP-2 expression on sphingolipid distribution to lipid rafts/caveolae in living cells was examined. Using a non-detergent affinity chromatography method to isolate lipid rafts/caveolae and non-rafts from purified L-cell plasma membranes, it was shown that lipid rafts/caveolae were highly enriched in multiple sphingolipid species including ceramides, acidic glycosphingolipids (ganglioside GM1); neutral glycosphingolipids (monohexosides, dihexosides, globosides), and sphingomyelin as compared to non-raft domains. SCP-2 overexpression further enriched the content of total sphingolipids and select sphingolipid species in the lipid rafts/caveolae domains. Analysis of fluorescence binding and displacement data revealed that purified human recombinant SCP-2 exhibited high binding affinity (nanomolar range) for all sphingolipid classes tested. The binding affinity decreased in the following order: ceramides > acidic glycosphingolipid (ganglioside GM1) > neutral glycosphingolipid (monohexosides, hexosides, globosides) > sphingomyelin. Enrichment of individual sphingolipid classes to lipid rafts/caveolae versus non-rafts in SCP-2 expressing plasma membranes followed closely with those classes most strongly bound to SCP-2 (ceramides, GM1 > the neutral glycosphingolipids (monohexosides, dihexosides, and globosides) > sphingomyelin). Taken together these data suggested that SCP-2 acts to selectively regulate sphingolipid distribution to lipid rafts/caveolae in living cells.     

Inhibition of rat hepatic sterol formation from squalene by plasma lipoproteins

The conversion of³H-squalene to sterols by rat liver microsomes and cytosol was inhibited by individual rat and human plasma lipoproteins at various concentrations. This inhibition was also observed with added human high density apolipoprotein, but triglycerides, cholesterol or cholesteryl esters had no inhibitory effects. Lipoproteins and apo high density lipoprotein (HDL) were demonstrated to bind³H-squalene in vitro. The binding of³H-squalene by apo HDL could be reversed by increasing concentration of liver cytosol containing sterol carrier protein.     

Sterol composition of ungerminated and germinated spores of the Vesicular-Arbuscular Mycorrhizal fungus,Glomus caledonius

The sterol composition of spores of the Vesicular-Arbuscular Mycorrhiza,Glomus caledonius, have been examined by combined gas liquid chromatography-mass spectrometry (GLC-MS). The sterols identified were cholesterol, 24-methylcholesterol, 24-methylenecholesterol and 24-ethylcholesterol. The sterols 24-methylcholesterol, 24-methylenecholesterol and 24-ethylcholesterol have not previously been reported as components of fungi in the Zygomycotina. The spores were germinated on agar and the major changes in sterol esters, free and bound sterols were studied over a period of 21 days. The total sterol content continually increased during the growth period; 24-methylcholesterol and cholesterol were shown to be the major sterols.     

The content and composition of sterols and sterol esters in low erucic acid rapeseed (Brassica napus)

The low temperature crystallization technique for the enrichment of “minor” components, such as sterols and sterol esters, from vegetable oils was applied to low erucic acid rapeseed oils. The recovery of free sterols and sterol esters was estimated by use of¹⁴C-cholesterol and¹⁴C-cholesterol oleate. 80% of the free sterols and 45% of the sterol esters were recovered in the liquid fraction, while in two studies total recoveries were 95% and 99%, respectively. This technique showed some selectivity toward the sterol bound fatty acids when compared to direct preparative thin layer chromatography (TLC) of the crude oil. Gas liquid chromatography (GLC) analysis of the free and esterified sterols as TMS-derivatives showed very little selectivity in the enrichment procedure. The fatty acid patterns of the sterol esters demonstrated, however, a preference in the liquid fraction for those sterol esters which have a high linoleic and linolenic acid content. The content of free sterols was 0.3–0.4% and that of sterol esters 0.7–1.2% of the rapeseed oils in both winter and summer types of low erucic acid rapeseed (Brassica napus) when the lipid classes were isolated by direct preparative TLC of the oils. The free sterols in the seven cultivars or breeding lines analyzed were composed of 44–55% sitosterol, 27–36% campesterol, 17–21% brassicasterol, and a trace of cholesterol. The esterified sterols were 47–57% sitosterol, 36–44% campesterol, 6–9% brassicasterol, and traces of cholesterol and Δ5-avenasterol. The fatty acid patterns of these esters were characterized by ca. 30% oleic acid and ca. 50% linoleic acid, whereas these acids constitute 60% and 20%, respectively, of the total fatty acids in the oil. Little or no variation in sterol and sterol ester patterns with locality within Sweden was observed for the one cultivar of summer rapeseed investigated by the low temperature crystallization technique.     

Increased amounts of cholesterol precursors in lipoproteins after ileal exclusion

Serum cholesterol precursor sterols reflect the activity of cholesterol synthesis. In this study, squalene, methyl sterol and lathosterol contents were studied in very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) of heterozygous familial hypercholesterolemia patients without and with ileal bypass. The contents of lathosterol and all methyl sterols (lanosterol, Δ^8,24-dimethylsterol, Δ⁸-dimetylsterol, Δ⁸-methostenol and methostenol), but not of squalene were increased in all lipoproteins by ileal bypass. The increase in the free methyl sterols was more marked than that in the esterified ones. The percentage esterification of the methyl sterols was highest in HDL and lowest in VLDL. Lipoprotein methyl sterol contents were positively correlated with each other and with cholesterol synthesis. The methyl sterols were slightly concentrated in LDL, and squalene strongly concentrated in VLDL. It is concluded that long-term stimulation of cholesterol synthesis increases the methyl sterols in all lipoproteins.     

Expression of fatty acyl-CoA binding proteins in colon cells: response to butyrate and transformation

Fatty acyl-CoA affect many cellular functions as well as serving as cellular building blocks. Several families of cytosolic fatty acyl-CoA binding proteins may modulate the activities of fatty acyl-CoA. Intestinal enterocytes contain at least three unique families of cytosolic proteins that bind fatty acyl-CoA: acyl-CoA binding protein (ACBP), fatty acid binding proteins (including the liver, L-FABP and intestinal, I-FABP), and sterol carrier protein-2 (SCP-2). Immortalized rat colon epithelial cell lines expressed only ACBP and SCP-2 at levels of 0.75±0.13 and 0.42±0.02 ng/μg protein. Ras and src transformation increased colon cell density and differentially altered ACBP and SCP-2 expression without affecting I-FABP or L-FABP levels. ACBP levels were 1.8-fold and 1.5-fold increased in ras- and src-transformed cells, respectively. In contrast, SCP-2 expression was significantly decreased 55 and 67% in ras- and src-transformed cells, respectively. Butyrate treatment of ras- and src-transformed cells decreased cell proliferation up to 60–85% as compared to 25–30% in control cells. Butyrate treatment decreased ACBP expression in all cell lines but had no effect on the levels of SCP-2, I-FABP, or L-FABP. These studies suggest that the differential expression of ACBP and SCP-2 in rat colonic cell lines, as well as their modulation by butyrate, may be altered by cell transformation.     

Thresholds for Sterol-Limited Growth of Daphnia magna: A Comparative Approach Using 10 Different Sterols

Arthropods are incapable of synthesizing sterols de novo and thus require a dietary source to cover their physiological demands. The most prominent sterol in animal tissues is cholesterol, which is an indispensable structural component of cell membranes and serves as precursor for steroid hormones. Instead of cholesterol, plants and algae contain a variety of different phytosterols. Consequently, herbivorous arthropods have to metabolize dietary phytosterols to cholesterol to meet their requirements for growth and reproduction. Here, we investigated sterol-limited growth responses of the freshwater herbivore Daphnia magna by supplementing a sterol-free diet with increasing amounts of 10 different phytosterols and comparing thresholds for sterol-limited growth. In addition, we analyzed the sterol composition of D. magna to explore sterol metabolic constraints and bioconversion capacities. We show that dietary phytosterols strongly differ in their potential to support somatic growth of D. magna. The dietary threshold concentrations obtained by supplementing the different sterols cover a wide range (3.5–34.4 μg mg C^?1) and encompass the one for cholesterol (8.9 μg mg C^?1), indicating that certain phytosterols are more efficient in supporting somatic growth than cholesterol (e.g., fucosterol, brassicasterol) while others are less efficient (e.g., dihydrocholesterol, lathosterol). The dietary sterol concentration gradients revealed that the poor quality of particular sterols can be alleviated partially by increasing dietary concentrations, and that qualitative differences among sterols are most pronounced at low to moderate dietary concentrations. We infer that the dietary sterol composition has to be considered in zooplankton nutritional ecology to accurately assess potential sterol limitations under field conditions.     

Sterols of the phylum Zygomycota: Phylogenetic implications

The sterol composition of 42 fungal species representing six of the eight orders of the Zygomycota was determined using gas-liquid chromatography-mass spectrometry to assess whether the distribution of major sterols in this phylum has taxonomic or phylogenetic relevance. Ergosterol, 22-dihydroergosterol, 24-methyl cholesterol, cholesterol, and desmosterol were detected as the major sterols among the species studied. Ergosterol was the major sterol of the Dimargaritales, Zoopagales, and 13 of the 14 Mucorales families included in this study. Desmosterol appeared to be the characteristic sterol of the Mortierellaceae (Mucorales). 24-Methyl cholesterol was the major sterol of the Entomophthorales genera Entomophthora, Conidiobolus and Basidiobolus, but cholesterol was the sole sterol detected in Delacroixia coronatus. The Kickxellales species analyzed in this study were characterized by 22-dihydroergosterol as the major sterol. These results suggest that certain orders of the Zygomycota may be distinguished on the basis of major sterol. Also, if sterol structure has phylogenetic implications, then orders might be arranged in the order Kickxellales (C₂₈Δ^5,7) → Dimargaritales, Zoopagales and Mucorales (C₂₈Δ^5,7,22) on the basis of evolution of the predominant and presumably most competent sterol, ergosterol. Although the Entomophthorales would be expected to be more primitive than the above orders based on the predominance of C₂₈Δ^5,, it is not apparent from these data that members of the Zygomycota with ergosterol or its precursors as major sterols evolved from this taxon or the Chytridiomycota.     

Acyl-CoA Binding Protein Gene Ablation Induces Pre-implantation Embryonic Lethality in Mice

Unique among the intracellular lipid binding proteins, acyl-CoA binding protein (ACBP) exclusively binds long-chain fatty acyl-CoAs (LCFA-CoAs). To test if ACBP is an essential protein in mammals, the ACBP gene was ablated by homologous recombination in mice. While ACBP heterozygotes appeared phenotypically normal, intercrossing of the heterozygotes did not produce any live homozygous deficient (null) ACBP^(−/−) pups. Heterozygous and wild type embryos were detected at all post-implantation stages, but no homozygous ACBP-null embryos were obtained—suggesting that an embryonic lethality occurred at a pre-implantation stage of development, or that embryos never formed. While ACBP-null embryos were not detected at any blastocyst stage, ACBP-null embryos were detected at the morula (8-cell), cleavage (2-cell), and zygote (1-cell) pre-implantation stages. Two other LCFA-CoA binding proteins, sterol carrier protein-2 (SCP-2) and sterol carrier protein-x (SCP-x) were significantly upregulated at these stages. These findings demonstrate for the first time that ACBP is an essential protein required for embryonic development and its loss of function may be initially compensated by concomitant upregulation of two other LCFA-CoA binding proteins, but only at the earliest pre-implantation stages. The fact that ACBP is the first known intracellular lipid binding protein whose deletion results in embryonic lethality suggests its vital importance in mammals.     

The biosynthesis of cholesterol and other sterols by brain tissue: II. A comparison of in vitro and in vivo methods

When microsomal + soluble preparations of adult or 15-day-old rat brains were incubated with 2-¹⁴C-mevalonic acid,¹⁴C-squalene accumulated. A metabolic block at the squalene to cholesterol stage was indicated. This prompted a comparison of all methods currently used to study cholesterol biosynthesis by brain tissue. Brain cell-free preparations from newborn, 15-day-old or adult rats accumulated¹⁴C-squalene in a similar manner, with either 2-¹⁴C-acetate or 2-¹⁴C-mevalonic acid as substrates. Homogenates and minced preparations from newborn or 15-day-old rats accumulated some 4,4-dimethyl sterols, but considerable conversion to free 4-desmethyl sterols (cholesterol) was evident. Sterol esters were also present in all the in vitro studies. In general, increased disruption of tissue resulted in decreased free 4-desmethyl sterol formation in vitro. Intraperitoneal injection of labeled acetate or mevalonate to newborn or 15-day-old rats produced labeled brain 4-desmethyl sterol with little accumulation of squalene or 4,4-dimethyl sterols, but the yields in brain were small compared to total amount of labeled material administered. At all ages intracerebral injection produced the best yield of labeled cholesterol for the amount of nonsaponifiable material formed. One of 12 papers being published from the Sterol Symposium, presented at the AOCS Meeting, New Orleans, April 1970.     

Sterol utilization and metabolism by Heliothis zea

Heliothis zea (corn earworm), an insect that fails to synthesize sterols de novo, was reared on an artificial diet treated with 18 different sterol supplements. Larvea did not develop on a sterol-less medium. Δ⁵-Sterols with a hydrogen atom, a methylene group, an E-or Z-ethylidene group, or an α- or β-ethyl group (cholesterol, ostreasterol, isofucosterol, fucosterol, sitosterol, and clionasterol, respectively) at position C-24, and Δ⁵-sterols doubly substituted in the side chain at C-24 with an α-ethyl group and at C-22 with a double bond (stigmasterol) supported normal larval growth to late-sixth instar (prepupal: maturity). The major sterol isolated from each of these sterol treatments was cholesterol, suggesting that H. zea operates a typical 24-dealkylation pathway. The sterol requirement of H. zea could not be met satisfactorily by derivatives of 3β-cholestanol with a 9β, 19-cyclopropyl group, gem dimethyl group at C-4, a Δ^5,7-bond or Δ⁸-bond, or by side-chain modified sterols that possessed a Δ²⁵⁽²⁷⁾-24β-ethyl group, Δ²³⁽²⁴⁾-24-methyl group, or 24-ethyl group, or Δ²⁴⁽²⁵⁾-24-methyl or 24-ethyl group. The major sterol recovered from the larvae (albeit developmentally arrested larvae) treated with a nonutilizable sterol was the test compound. Sterol absorption was related to the degree of sterol utilization. The most effective sterols absorbed by the insect ranged from 27 to 66 μg per insect, whereas the least effective sterols absorbed by the insect ranged from 0.6 to 6 μg per insect. Competition experiments using different proportions of cholesterol and 24-dihydrolanosterol (from 9:1 to 1:9 mixtures) indicated that abnormal development of H. zea may be induced on less than a 1 to 1 mixture of utilizable (cholesterol) to nonutilizable (24-dihydrolanosterol) sterols. The results demonstrate new structural requirements for sterol utilization and metabolism by insects, particularly with respect to the position of double bonds in the side chain and functionalization in the nucleus. The novel sterol specificities observed in this study appear to be associated with the dual role of sterols as membrane inserts (nonmetabolic) and as precursors to the ecdysteroids (metabolic).     

Utilization and metabolism of dietary sterols in the honey bee and the yellow fever mosquito

The honey bee,Apis mellifera, does not convert C₂₈ and C₂₉ phytosterols to cholesterol as found in most previous studies of phytophagous or omnivorous insects, but instead the workers and queens selectively transfer 24-methylenecholesterol, sitosterol and isofucosterol from their endogenous sterol pools to the brood larvae regardless of the sterol in the worker diet. Administering radiolabeled sterols by feeding and injection has made it possible to trace this selective transfer through a second generation of the honey bee. In further comparative sterol metabolism studies, the yellow fever mosquito,Aedes aegypti, was shown to be capable of dealkylating and converting a radiolabeled C₂₉ dietary sterol ([¹⁴C] sitosterol) to cholesterol. Metabolic studies with several radiolabeled dietary sterols and an inhibitor of steroid metabolism in the yellow fever mosquito further verified this capability.     

Sterols and their biosynthesis in some freshwater bivalves

A number of free sterols and sterol esters of three freshwater mussels was separated and identified. A slow rate of biosynthesisde novo of sterols was demonstrated inAnodonta cygnea. Injected cholesterol was found to undergo esterification, oxidation, Δ²²-dehydrogenation and C-24 alkylation. Methyl-[¹⁴C]methionine was proved to be incorporated in C-24 alkylsterols. Abnormally large amounts of cholesterol injected inA. cygnea were metabolized toward restoration of the normal composition of sterols. This was achieved by intensified metabolism of cholesterol, mainly by conjugation, oxidation and Δ²²-dehydrogenation.     

Sterol metabolism in the nematodePanagrellus redivivus

Panagrellus redivivus was propagated in media containing three structurally different sterols: 7-dehydrocholesterol, campesterol or stigmastanol. Nematodes propagated with 7-dehydrocholesterol contained mostly lathosterol and 7-dehydrocholesterol. Nematodes propagated with campesterol contained mostly cholesterol and cholestanol. Nematodes propagated with stigmastanol contained mostly cholestanol. The sterol ester fraction was enriched with 4α-methylsterols and contained the same sterols as the free sterol fraction except for nematodes propagated with 7-dehydrocholesterol, where no dietary sterol was found in the ester fraction.P. redivivus is capable of reducing the Δ⁵-bond, C−24 dealkylation and methylating the sterol nucleus at C−4.     

Inhibition of hepatic sterol and squalene biosynthesis in rats fed di-2-ethylhexyl phthalate

Di-2-ethylhexyl phthalate (DEHP), a commonly used plasticizer, was found to be an inhibitor of the biosynthesis of hepatic nonsaponifiable lipids in the rat. The addition of DEHP at levels of 0.5% or 1.0% to a stock diet of rats resulted in a decreased conversion of acetate-1-¹⁴C and mevalonate-5-³H into squalene, C₃₀ sterols, and C₂₇ sterols by liver minces or slices, in vitro. In studies conducted with 0.5% DEHP feeding from 2 to 11 days, the degree of inhibition was found to increase with the duration of DEHP feeding; the inhibition of³H-mevalonate conversion to squalene and sterols developed more slowly, being reduced to ca. 70% of control values in 11 days, whereas¹⁴C-acetate conversion was reduced to ca. 35% of control values during the same period.³H-mevalonate conversion to sterols and squalene was, however, found to be suppressable to the same extent as¹⁴C-acetate conversion when diets containing 1.0% DEHP were fed for 18 days. The inhibitory effect of dietary DEHP on sterol and squalene biosynthesis from¹⁴C-acetate and³H-mevalonate by rat liver preparations is unlikely to be accounted for by the negative feedback of cholesterol secondary to hepatic sterol accumulation since, in these studies, hepatic total lipid and hepatic total sterol levels were simialr in control and DEHP-fed rats.     

Growth modulation of low density lipoprotein receptor sterol sensitivity in cultured human fibroblasts

Sterols regulate low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene expressions by end product repression. Studies on cultured cells have shown that growing cells have a higher LDL uptake than quiescent cells and that incubation of cells with growth factors or mitogenic compounds leads to sterol-resistant upregulation of LDL receptor gene expression. The recent finding that elevated LDL receptor activity in acute myelogenous leukemia cells was characterized by a decreased sensitivity to downregulation by sterols raises the possibility that the mechanism behind this is related to the cellular growth rate. By using cultured human fibroblasts as a model system we therefore studied whether growth modulation of sterol sensitivity takes place in normal actively growing cells. Judging from the ability of sterols (25-hydroxycholesterol+cholesterol) to inhibit ¹²⁵I-LDL degradation, we found that the sensitivity to sterols varied markedly between cells of different densities. The lowest sensitivity to sterols and highest ¹²⁵I-LDL degradation rate were found in subconfluent cells, whereas sparse and confluent cells were the most sensitive ones. In contrast to the LDL receptor, HMG-CoA reductase sterol sensitivity did not appear to be growth regulated. We conclude that growth-dependent modulation of sterol sensitivity and LDL receptor activity takes place in normal human fibroblasts. Modulation of sterol sensitivity may be an important mechanism to ensure an adequate cholesterol supply in growing cells.     

Same Host-Plant,Different Sterols: Variation in Sterol Metabolism in an Insect Herbivore Community

Insects lack the ability to synthesize sterols de novo, which are required as cell membrane inserts and as precursors for steroid hormones. Herbivorous insects typically utilize cholesterol as their primary sterol. However, plants rarely contain cholesterol, and herbivorous insects must, therefore, produce cholesterol by metabolizing plant sterols. Previous studies have shown that insects generally display diversity in phytosterol metabolism. Despite the biological importance of sterols, there has been no investigation of their metabolism in a naturally occurring herbivorous insect community. Therefore, we determined the neutral sterol profile of Solidago altissima L., six taxonomically and ecologically diverse herbivorous insect associates, and the fungal symbiont of one herbivore. Our results demonstrated that S. altissima contained Δ⁷-sterols (spinasterol, 22-dihydrospinasterol, avenasterol, and 24-epifungisterol), and that 85% of the sterol pool existed in a conjugated form. Despite feeding on a shared host plant, we observed significant variation among herbivores in terms of their qualitative tissue sterol profiles and significant variation in the cholesterol content. Cholesterol was absent in two dipteran gall-formers and present at extremely low levels in a beetle. Cholesterol content was highly variable in three hemipteran phloem feeders; even species of the same genus showed substantial differences in their cholesterol contents. The fungal ectosymbiont of a dipteran gall former contained primarily ergosterol and two ergosterol precursors. The larvae and pupae of the symbiotic gall-former lacked phytosterols, phytosterol metabolites, or cholesterol, instead containing an ergosterol metabolite in addition to unmetabolized ergosterol and erogsterol precursors, thus demonstrating the crucial role that a fungal symbiont plays in their nutritional ecology. These data are discussed in the context of sterol physiology and metabolism in insects, and the potential ecological and evolutionary implications.     

Pollen sterols from three species of sonoran cacti

Cactus bees are important pollinators that contribute to the long-term stability of arid regions in the United States. Since all insects are dependent upon a dietary source of sterol for normal growth, development and reproduction, a study was undertaken to determine neutral sterols available to cactus bees. The total neutral sterol composition of hand-collected pollen was determined for three species of Sonoran cacti by gas-liquid chromatography and mass spectrometry. 24-Methylenecholesterol was the predominant pollen sterol in Engelmann's prickly pear,Opuntia phaeacantha, cholla,O. versicolor, and cardon,Pachycereus pringlei. Two pentacyclic triterpene alcohols, lupeol and moretenol, were also isolated. Since no cholesterol was detected in any of the pollen samples, cactus bees would have to utilize the 24-alkyl sterols unchanged or convert these sterols to cholesterolvia dealkylation.