Cholesterol Precursors Stabilize Ordinary and Ceramide-rich Ordered Lipid Domains (Lipid Rafts) to Different Degrees

Genetic disorders of cholesterol biosynthesis result in accumulation of cholesterol precursors and cause severe disease. We examined whether cholesterol precursors alter the stability and properties of ordered lipid domains (rafts). Tempo quenching of a raft-binding fluorophore was used to measure raft stability in vesicles containing sterol, dioleoylphosphatidylcholine, and one of the following ordered domain-forming lipids/lipid mixtures: dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM), a SM/cerebroside mixture or a SM/ceramide (cer) mixture. Relative to cholesterol, early cholesterol precursors containing an 8-9 double bond (lanosterol, dihydrolanosterol, zymosterol, and zymostenol) only weakly stabilized raft formation by SM or DPPC. Desmosterol, a late precursor containing the same 5-6 double bond as cholesterol, but with an additional 24-25 double bond, also stabilized domain formation weakly. In contrast, two late precursors containing 7-8 double bonds (lathosterol and 7-dehydrocholesterol) were better raft stabilizers than cholesterol. For vesicles containing SM/cerebroside and SM/cer mixtures the effect of precursor upon raft stability was small, although the relative effects of different precursors were the same. Using both detergent resistance and a novel assay involving fluorescence quenching induced by certain sterols we found cholesterol precursors were displaced from cer-rich rafts, and could displace cer from rafts. Precursor displacement by cer was inversely correlated to precursor raft-stabilizing abilities, whereas precursor displacement of cer was greatest for the most highly raft-stabilizing precursors. These observations support the hypothesis that sterols and cer compete for raft-association (Megha, and London, E. (2004) J. Biol. Chem. 279, 9997-10004). The results of this study have important implications for how precursors might alter raft structure and function in cells, and for the Bloch hypothesis, which postulates that sterol properties are gradually optimized for function along the biosynthetic pathway. Genetic disorders of cholesterol biosynthesis result in accumulation of cholesterol precursors and cause severe disease. We examined whether cholesterol precursors alter the stability and properties of ordered lipid domains (rafts). Tempo quenching of a raft-binding fluorophore was used to measure raft stability in vesicles containing sterol, dioleoylphosphatidylcholine, and one of the following ordered domain-forming lipids/lipid mixtures: dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM), a SM/cerebroside mixture or a SM/ceramide (cer) mixture. Relative to cholesterol, early cholesterol precursors containing an 8-9 double bond (lanosterol, dihydrolanosterol, zymosterol, and zymostenol) only weakly stabilized raft formation by SM or DPPC. Desmosterol, a late precursor containing the same 5-6 double bond as cholesterol, but with an additional 24-25 double bond, also stabilized domain formation weakly. In contrast, two late precursors containing 7-8 double bonds (lathosterol and 7-dehydrocholesterol) were better raft stabilizers than cholesterol. For vesicles containing SM/cerebroside and SM/cer mixtures the effect of precursor upon raft stability was small, although the relative effects of different precursors were the same. Using both detergent resistance and a novel assay involving fluorescence quenching induced by certain sterols we found cholesterol precursors were displaced from cer-rich rafts, and could displace cer from rafts. Precursor displacement by cer was inversely correlated to precursor raft-stabilizing abilities, whereas precursor displacement of cer was greatest for the most highly raft-stabilizing precursors. These observations support the hypothesis that sterols and cer compete for raft-association (Megha, and London, E. (2004) J. Biol. Chem. 279, 9997-10004). The results of this study have important implications for how precursors might alter raft structure and function in cells, and for the Bloch hypothesis, which postulates that sterol properties are gradually optimized for function along the biosynthetic pathway. De novo cholesterol biosynthesis proceeds through various intermediates. Conversion of lanosterol to cholesterol involves a series of demethylation, double bond isomerization, dehydrogenation, and reduction steps (Fig. 1). In genetic disorders of cholesterol biosynthesis mutations in genes coding for enzymes of the biosynthetic pathway lead to a loss or reduction in enzymatic function, and an accumulation of sterol precursors. Mutations in the 7-dehydrocholesterol (7-DHC) 2The abbreviations used are: 7-DHC, 7-dehydrocholesterol; Cer, ceramide (N-palmitoyl-d-erythro-sphingosine); DOPC, 1,2-dioleyl-sn-glycero-3-phosphocholine; DPH, 1,6-diphenyl-1,3,5-hexatriene; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DRM, detergent-resistant membrane; LcT-MADPH, 22-(diphenylhexatrienyl)docosyltrimethylammonium; LW peptide, acetyl-K2W2L8AL8W2K2-amide; SM, porcine brain sphingomyelin; tempo, 2,2,6,6-tetramethylpiperidine-1-oxyl; TX-100, Triton X-100; SUV, small unilamellar vesicles; MLV, multilamellar vesicles; SLOS, Smith-Lemli-Optiz syndrome. reductase gene, which catalyzes the conversion of 7-DHC to cholesterol, result in 7-DHC accumulation and Smith-Lemli-Optiz syndrome (SLOS) (1Irons M. Elias E.R. Salen G. Tint G.S. Batta A.K. Lancet. 1993; 341: 1414Abstract PubMed Scopus (324) Google Scholar). In desmosterolosis, a mutation in 24-dehydrocholesterol reductase gene results in accumulation of desmosterol (2Waterham H.R. Koster J. Romeijn G.J. Hennekam R.C. Vreken P. Andersson H.C. FitzPatrick D.R. Kelley R.I. Wanders R.J. Am. J. Hum. Genet. 2001; 69: 685-694Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Lathosterolosis is caused by a mutation in the lathosterol 5-desaturase gene, which results in the accumulation of lathosterol (3Brunetti-Pierri N. Corso G. Rossi M. Ferrari P. Balli F. Rivasi F. Annunziata I. Ballabio A. Russo A.D. Andria G. Parenti G. Am. J. Hum. Genet. 2002; 71: 952-958Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In addition, mutations in 3β-hydroxysteroid Δ8 sterol isomerase, terminating synthesis at zymosterol, have been implicated in CHILD syndrome and the disorder CDPX2 (4Braverman N. Lin P. Moebius F.F. Obie C. Moser A. Glossmann H. Wilcox W.R. Rimoin D.L. Smith M. Kratz L. Kelley R.I. Valle D. Nat. Genet. 1999; 22: 291-294Crossref PubMed Scopus (236) Google Scholar). All of these genetic disorders manifest themselves as broad malformation diseases with overlapping symptoms that include features such as microcephaly, skeletal abnormalities, and both overall growth and mental retardation (5Porter F.D. Curr. Opin. Pediatr. 2003; 15: 607-613Crossref PubMed Scopus (101) Google Scholar, 6Herman G.E. Hum. Mol. Genet. 2003; 12: R75-R88Crossref PubMed Google Scholar). Participation in the formation and function of ordered lipid domains (lipid rafts) is believed to be an important function of sterols in eukaryotic membranes. Lipid rafts are usually defined as sphingolipid and sterol-rich domains that exist in the liquid-ordered phase (Lo). In eukaryotic cell membranes they are thought to co-exist with liquid disordered (Ld) domains that are rich in lipids with unsaturated acyl chains. The Lo phase is an intermediate state with tightly packed lipids, like the solid-like gel phase, and a high lipid lateral diffusion rate, like that found in the Ld phase (7Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (638) Google Scholar, 8Brown D.A. London E. J. Membr. Biol. 1998; 164: 103-114Crossref PubMed Scopus (841) Google Scholar). Rafts have been proposed to be important for many cellular processes (9Zaas D.W. Duncan M. Rae Wright J. Abraham S.N. Biochim. Biophys. Acta. 2005; 1746: 305-313Crossref PubMed Scopus (94) Google Scholar, 10Edidin M. Annu. Rev. Biophys. Biomol. Struct. 2003; 32: 257-283Crossref PubMed Scopus (1138) Google Scholar, 11Holowka D. Gosse J.A. Hammond A.T. Han X. Sengupta P. Smith N.L. Wagenknecht-Wiesner A. Wu M. Young R.M. Baird B. Biochim. Biophys. Acta. 2005; 1746: 252-259Crossref PubMed Scopus (123) Google Scholar, 12Bollinger C.R. Teichgraber V. Gulbins E. Biochim. Biophys. Acta. 2005; 1746: 284-294Crossref PubMed Scopus (276) Google Scholar). Recent studies have revealed that ceramide(cer)-rich rafts can also form. We found that cer is a strong promoter of lipid raft formation (13Xu X. Bittman R. Duportail G. Heissler D. Vilcheze C. London E. J. Biol. Chem. 2001; 276: 33540-33546Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar), and cer has a high affinity for rafts (14Megha London E. J. Biol. Chem. 2004; 279: 9997-10004Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 15Wang T.Y. Silvius J.R. Biophys. J. 2003; 84: 367-378Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Cer-rich rafts appear to form large platforms when large amounts of cer are generated in plasma membranes in vivo by SMase action (16Grassme H. Jekle A. Riehle A. Schwarz H. Berger J. Sandhoff K. Kolesnick R. Gulbins E. J. Biol. Chem. 2001; 276: 20589-20596Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar). Cer-rich rafts have been reported to be involved in the initiation of certain types of apoptosis (17Grassme H. Schwarz H. Gulbins E. Biochem. Biophys. Res. Commun. 2001; 284: 1016-1030Crossref PubMed Scopus (161) Google Scholar, 18Miyaji M. Jin Z.X. Yamaoka S. Amakawa R. Fukuhara S. Sato S.B. Kobayashi T. Domae N. Mimori T. Bloom E.T. Okazaki T. Umehara H. J. Exp. Med. 2005; 202: 249-259Crossref PubMed Scopus (139) Google Scholar, 19Rotolo J.A. Zhang J. Donepudi M. Lee H. Fuks Z. Kolesnick R. J. Biol. Chem. 2005; 280: 26425-26434Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), in bacterial infection by Neisseria gonorrhoeae (20Grassme H. Gulbins E. Brenner B. Ferlinz K. Sandhoff K. Harzer K. Lang F. Meyer T.F. Cell. 1997; 91: 605-615Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar) and Pseudomonas aeruginosa (21Grassme H. Jendrossek V. Riehle A. von Kurthy G. Berger J. Schwarz H. Weller M. Kolesnick R. Gulbins E. Nat. Med. 2003; 9: 322-330Crossref PubMed Scopus (465) Google Scholar), and in rhinovirus infection (22Grassme H. Riehle A. Wilker B. Gulbins E. J. Biol. Chem. 2005; 280: 26256-26262Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Recently, we found that cholesterol is displaced from cer-rich rafts (14Megha London E. J. Biol. Chem. 2004; 279: 9997-10004Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). Displacement of cholesterol has now been confirmed by other groups, both in model membranes and cells (23Yu C. Alterman M. Dobrowsky R.T. J. Lipid Res. 2005; 46: 1678-1691Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 24Alanko S.M. Halling K.K. Maunula S. Slotte J.P. Ramstedt B. Biochim. Biophys. Acta. 2005; 1715: 111-121Crossref PubMed Scopus (90) Google Scholar). Because of the importance of cholesterol biosynthesis disorders and lipid rafts we examined the raft-stabilizing properties of combinations of a wide variety of cholesterol precursors and sphingolipids. A novel fluorescence assay (25Bakht O. London E. Biophys. J. 2004; 86: 202AGoogle Scholar) was employed to assess raft formation and stability in model membranes. This assay monitors the quenching of fluorescent probes bound to lipid bilayers by tempo, a small nitroxide-carrying quencher, which binds strongly to bilayers that are in the Ld state (26Kleemann W. McConnell H.M. Biochim. Biophys. Acta. 1976; 419: 206-222Crossref PubMed Scopus (160) Google Scholar). We find that there is a difference in the raft-stabilizing properties of different precursors, but stabilization does not improve gradually along the biosynthetic pathway. Instead, there is a distinct difference between raft stabilization by the early precursors and certain later precursors. We also show that substitution of precursors for cholesterol affects the sterol content of cer-rich rafts. These results have important implications for how sterol composition could affect raft structure and function in cells. Materials—Dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (porcine brain, SM), cholesterol, N-palmitoyl-d-erythro-sphingosine (C16:0 ceramide), total brain cerebrosides, and dioleoylphosphatidylcholine (DOPC) were purchased from Avanti Polar Lipids (Alabaster, AL). [3H]C16:0 ceramide was purchased from American Radiolabeled Chemicals (St. Louis, MO). Lipid (including radiolabeled lipid) purity was confirmed by TLC. 2,2,6,6-Tetramethylpiperidine-1-oxyl (tempo), 1,6-diphenyl-1,3,5-hexatriene (DPH), lanosterol ("97% pure"), and 7-dehydrocholesterol (7-DHC) (Fluka brand), were purchased from Sigma-Aldrich. Zymosterol, zymostenol (zymosterol with a saturated 24-25 double bond), dihydrolanosterol, desmosterol, and lathosterol were purchased from Steraloids Inc. (Newport, RI). Desmosterol was also obtained from Research Plus (Manasquan, NJ). 22-(Diphenylhexatrienyl)-docosyltrimethyl ammonium, (LcTMADPH) was a gift of G. Duportail and D. Heissler (Université Louis Pasteur, Strasbourg). Lipids and probes were stored dissolved in ethanol at −20 °C. Concentrations were determined by dry weight or (for LcTMADPH) absorbance using an ϵ of 88,000 cm−1 m−1 at 350 nm in ethanol. Acetyl-K2W2L8AL8W2K2-amide (LW peptide) purchased from Invitrogen (Carlsbad, CA) was used without further purification. Triton X-100 (scintillation grade) was from Yorktown Research (Hackensack, NJ). Sterol purity was confirmed by comparison of sterol melting temperatures to those reported by the manufacturers, and by TLC on HP-TLC plates (Merck & Co). Zymosterol and some desmosterol preparations contained impurities and were repurified by TLC. Approximately 1 mg of sterol was dissolved in ethanol, applied to an HP-TLC plate, and then chromatographed using a sequential solvent system. Solvent chambers were equilibrated with solvents for at least 2 h before chromatography. The first solvent (50:38:3:2 (v:v), chloroform/methanol/acetic acid/water) was allowed to migrate halfway up the plate. The plate was then dried, introduced into a second chamber containing the solvent system 1:1 hexane/ethyl acetate (v:v), and chromatographed until the solvent migrated to near the top of the plate. The plate was then dried and sprayed with 5% (w/v) cupric acetate dissolved in 8% (v/v) phosphoric acid in water. To detect sterol, plates were charred at 180 °C for 5 min. To purify sterol, the region of the plate containing the pure sterol (identified from a fragment of the plate in which sterol was charred) was scraped off, dissolved in 1:1 (v:v) chloroform/methanol, centrifuged to remove the silica particles, filtered on 0.22-μ nitrocellulose to remove residual silica, and then dried under nitrogen flow. Purity was confirmed as described above. Commercial desmosterol preparations with no impurities gave experimental results similar to those obtained with purified desmosterol. Vesicle Preparation—Ethanol dilution small unilamellar vesicles (SUV) and multilamellar vesicles (MLV) were prepared as described previously (14Megha London E. J. Biol. Chem. 2004; 279: 9997-10004Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). Vesicles containing the desired lipid mixtures were dispersed at 70 °C in 1 ml of phosphate-buffered saline (10 mm sodium phosphate, 150 mm NaCl, pH 7) at a final lipid concentration of 50 μm (for SUV) and 500 μm (for MLV). Fluorescence measurements were made after samples cooled to room temperature. For fluorescence measurements, vesicles also contained 0.1-0.2 mol % LcTMADPH or 0.5 mol % DPH. Background samples lacking fluorescent probe were also prepared. Fluorescence Measurements—A scaled-up stock sample of SUV, prepared as described above, was divided into four 1-ml aliquots. Two samples containing quencher (F samples) were prepared by adding tempo to a concentration of 2 mm (from a 353 mm stock solution of tempo dissolved in ethanol). Two samples lacking quencher (Fo samples) were prepared by adding a volume of pure ethanol equal to that added to the F samples. Both F and Fo samples were then incubated for 10 min at room temperature, after which fluorescence was measured. LcTMADPH or DPH fluorescence was measured at an excitation wavelength of 357 nm and emission wavelength 429 nm. Fluorescence was measured on a Fluorolog 3 spectrofluorimeter using quartz semimicro cuvettes (excitation path length: 10 mm, emission: 4 mm). Unless otherwise noted, slit bandwidths were 3.2-nm excitation and 6.3-nm emission. Fluorescence intensity was measured as a function of increasing temperature as described previously (14Megha London E. J. Biol. Chem. 2004; 279: 9997-10004Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). The ratio of fluorescence intensity in the presence of quencher to that in its absence (F/Fo) was calculated after subtraction of background values. Fluorescence anisotropy measurements were performed on SUV prepared as described previously, but using vesicles containing 0.5 mol % DPH or LcTMADPH (14Megha London E. J. Biol. Chem. 2004; 279: 9997-10004Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). Anisotropy measurements were made at room temperature using a SPEX automated Glan-Thompson polarizer accessory with slit bandwidths set at 4.2 nm (excitation) and 8.4 nm (emission). Peptide Quenching by Sterol—SUV containing 50 μm total lipid and 2 mol % LW peptide were prepared by ethanol dilution as described above. Peptide fluorescence was measured at 280-nm excitation and 340-nm emission wavelengths. Excitation and emission bandwidths were both 6.3 nm. Fluorescence from background samples lacking peptide was subtracted from that of peptide-containing samples to determine peptide fluorescence. Quenching of peptide fluorescence by sterol was assessed by calculation of F/Fo, where F samples contained 7-DHC, and Fo samples contained cholesterol in place of 7-DHC. Detergent Insolubility Assay—To MLV samples (500 nmol of total lipid dissolved in 950 ml of phosphate-buffered saline, pH 7) 50 μl of 10% (w/v) Triton X-100/phosphate-buffered saline, pH 7, was added. The samples were incubated for 2 h at 23°C, and then detergent-insoluble lipid was isolated by centrifugation as described previously (14Megha London E. J. Biol. Chem. 2004; 279: 9997-10004Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). The detergent-insoluble pellet, equivalent to the detergent-resistant membrane (DRM) fraction, was then resuspended in 100 μl of water, transferred to a glass vial, and dried under high vacuum for 45 min. The dried pellet was dissolved in 50 μl of 1:1 (v:v) chloroform/methanol. A 5-μl aliquot of this was then applied to a HP-TLC plate. Separate spots containing various amounts of the lipids used (0.25-4 μg) were also applied to the plate as standards. The sequential two-solvent system described above was used to separate lipids. Lipids were detected by charring as described above, and after the plate cooled, it was scanned as an image (CanonScan N124OU). Spot intensity was analyzed using the NIH/Scion Image program. Unknown lipid and sterol amounts were estimated by comparison to the standards, which generally exhibited a non-linear, but monotonic, increase in staining intensity as the amount of lipid applied was increased. The percent of each lipid species in the insoluble fraction was computed from the formula: (amount of lipid species in the pellet in nmol/total amount of lipid in pellet in nmol) × 100%. The total amount of lipid (including sterol) in the insoluble fraction was calculated in nmol, and total % insoluble lipid was computed from the formula: (total amount of insoluble lipid in nmol/total amount of lipid in initial sample (500 nmol)) × 100%. The amount of insoluble cer was determined by measurement of radioactivity in the pelleted lipid fraction as previously described (14Megha London E. J. Biol. Chem. 2004; 279: 9997-10004Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). Samples contained 0.25 μCi of [3H]C16:0 cer. How Raft Stability Is Assayed by Fluorescence Quenching—A fluorescence quenching assay was used to determine whether ordered domains/rafts were present in vesicles composed of lipid mixtures containing cholesterol precursors. We developed an assay in which the quencher tempo was used (25Bakht O. London E. Biophys. J. 2004; 86: 202AGoogle Scholar) instead of the nitroxide-labeled lipids we used previously (13Xu X. Bittman R. Duportail G. Heissler D. Vilcheze C. London E. J. Biol. Chem. 2001; 276: 33540-33546Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 25Bakht O. London E. Biophys. J. 2004; 86: 202AGoogle Scholar, 27Xu X. London E. Biochemistry. 2000; 39: 843-849Crossref PubMed Scopus (450) Google Scholar, 28Ahmed S.N. Brown D.A. London E. Biochemistry. 1997; 36: 10944-10953Crossref PubMed Scopus (615) Google Scholar). Tempo is water soluble, but binds strongly to disordered domains (26Kleemann W. McConnell H.M. Biochim. Biophys. Acta. 1976; 419: 206-222Crossref PubMed Scopus (160) Google Scholar). The fluorescent probe used was LcTMADPH, a derivative of DPH. LcTMADPH has a very high affinity for rafts, and is resistant to displacement from rafts by cer (14Megha London E. J. Biol. Chem. 2004; 279: 9997-10004Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). To examine the thermal stability of rafts, two sets of vesicles containing LcTMADPH were prepared: vesicles that contain tempo (F samples) and vesicles that lack tempo (Fo samples). In vesicles containing co-existing ordered and disordered domains LcTMADPH and tempo segregate, and F/Fo is high. As temperature increases the rafts melt, and the bilayer becomes progressively more homogenous. This ultimately abolishes segregation of LcTMADPH and tempo, and F/Fo decreases. For samples exhibiting a sigmoidal dependence of quenching upon temperature, melting temperature (Tm) can be defined: the more raft stabilizing the lipid mixture used, the higher the observed Tm (13Xu X. Bittman R. Duportail G. Heissler D. Vilcheze C. London E. J. Biol. Chem. 2001; 276: 33540-33546Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 27Xu X. London E. Biochemistry. 2000; 39: 843-849Crossref PubMed Scopus (450) Google Scholar, 29London E. Brown D.A. Xu X. Methods Enzymol. 2000; 312: 272-290Crossref PubMed Google Scholar). Tm values for rafts measured using tempo quenching were similar to those in analogous mixtures containing nitroxide-labeled lipids (13Xu X. Bittman R. Duportail G. Heissler D. Vilcheze C. London E. J. Biol. Chem. 2001; 276: 33540-33546Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 27Xu X. London E. Biochemistry. 2000; 39: 843-849Crossref PubMed Scopus (450) Google Scholar). Cholesterol Precursors Stabilize Raft Formation to Varying Degrees—The effect of cholesterol precursor structure upon the thermal stability of rafts was assayed using quenching. The sterols studied are shown in the schematic of the cholesterol biosynthetic pathway (Fig. 1). The two missing precursors were not available. Notice that there are two "vertical" branches to the pathway, which only differ in terms of whether the 24-25 double bond in the sterol "tail" is unsaturated (left branch) or reduced (right branch). In addition to sterols, the samples contained 1:1 mol:mol mixtures of a lipid that tend to form disordered domains at 23 °C (DOPC) with lipids that tend to form rafts at 23 °C (DPPC or sphingolipids). Fig. 2A shows the effect of temperature upon quenching of LcTMADPH fluorescence by tempo in vesicles composed of 3:3:2 DPPC/DOPC/sterol. Tm values for these mixtures are summarized in Table 1. Tm values in the presence of precursors and cholesterol are higher than in the absence of sterol. This shows that all precursors increase the thermal stability of rafts, i.e. they promote raft formation at temperatures where rafts are not stable in the absence of sterol. Cholesterol precursors formed during earlier steps in the cholesterol biosynthetic pathway, lanosterol, dihydrolanosterol, zymosterol, and zymostenol, stabilize ordered domain formation to a significantly lesser degree than cholesterol.TABLE 1Relationship between sterol precursor structure and raft melting temperature (Tm) in model membranes containing various sphingolipids or dipalmitoylphosphatidylcholine The data are derived from the samples used in the quenching experiments in Fig. 2. Samples contained mixtures of sphingolipid (or DPPC), DOPC and sterol in the proportions given in Figure 2. Tm was defined as the point of maximum slope in the sigmoidal melting curves (see Fig. 2). The average and range of Tm values from duplicate samples is shown.SterolTm (°C) of ordered domainsDPPCSMSM/CERBSM/CERNone25 ± 0.422.3 ± 0.932 ± 0.341.2 ± 0.3Lanosterol28 ± 0.327.7 ± 0.136.2 ± 0.338.3 ± 0.0Dihydrolanosterol26 ± 0.225.6 ± 1.035.3 ± 0.639.5 ± 1.0Zymosterol29.7 ± 0.231.4 ± 0.139.3 ± 0.638.7 ± 0.2Zymostenol31.4 ± 0.230.2 ± 0.336.6 ± 1.138.1 ± 0.5Desmosterol30.2 ± 0.327.6 ± 0.534.1 ± 0.336.7 ± 0.4Lathosterol42.9 ± 0.338.8 ± 1.841.5 ± 0.442.8 ± 0.77-DHC44.4 ± 0.138.5 ± 1.241.7 ± 0.244.8 ± 0.2Cholesterol34.1 ± 034 ± 0.437.6 ± 0.641.8 ± 0.8 Open table in a new tab Two precursors that occur late in the branch of the biosynthetic pathway lacking a 24-25 double bond, lathosterol and 7-DHC, stabilize ordered domain formation to a greater degree than cholesterol. In contrast, desmosterol, which is the last precursor in the branch in which the 24-25 double bond is present, and has a ring structure identical to that of cholesterol, stabilizes rafts to a much lesser degree than cholesterol. This is somewhat surprising, because Tm values show that the 24-25 double bond does not have a strongly destabilizing effect in the case of lanosterol and zymosterol. Cholesterol Precursors Stabilize Raft Formation to Varying Degrees: Effect of Sphingolipid Structure—Because rafts in cells should contain sphingolipid, whether sterol structure would affect the stability of rafts was also studied in vesicles containing various sphingolipids. First, the stability of rafts was measured in mixtures of mammalian brain SM/DOPC/sterol 3:3:2 (mol: mol). Fig. 2B and Table 1 show that the relative ability of different precursors to stabilize ordered domain formation in these mixtures is very similar to that observed in analogous mixtures containing DPPC. Experiments were then performed in mixtures containing mixed mammalian cerebrosides in place of almost one-third of the SM. This is of interest because glycosphingolipids can form a considerable fraction of total cellular sphingolipid under some conditions (30Ostermeyer A.G. Beckrich B.T. Ivarson K.A. Grove K.E. Brown D.A. J. Biol. Chem. 1999; 274: 34459-34466Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Our previous studies have shown that the presence of cerebrosides can lessen the effect of sterol on raft stability (13Xu X. Bittman R. Duportail G. Heissler D. Vilcheze C. London E. J. Biol. Chem. 2001; 276: 33540-33546Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). In agreement with this observation, the thermal stability of rafts in 2.04:0.96:3:2 SM/cerebrosides/DOPC/sterol (mol:mol) is only modestly affected by the presence of sterol or precursor structure (Fig. 2C). The former is illustrated most clearly by a comparison of the difference between Tm values in samples without sterol and those containing the most raft-stabilizing precursors. For the DPPC-containing samples this difference in Tm values is 19 °C, and for SM-containing samples the difference is 17 °C, but for the samples containing SM plus cerebroside the difference is only 10 °C. This behavior is consistent with the possibility that the cerebroside-rich rafts contain only low levels of sterol. If the rafts lack sterol, it could reduce the influence of sterol on their stability. Nevertheless, the overall pattern observed is similar to that in samples lacking cerebrosides, in that all sterols stabilize rafts to some degree, and the most stabilizing sterols are 7-DHC and lathosterol. One interesting difference in cerebroside-containing samples is the relatively strong stabilization of rafts by zymosterol. Finally, the effect of sterol precursor structure was examined in mixtures of 2.04:0.96:3:2 SM/C16:0 cer/DOPC/sterol (i.e. 12 mol % cer). These mixtures were of interest because of reports that cer-rich rafts can be physiologically important (12Bollinger C.R. Teichgraber V. Gulbins E. Biochim. Biophys. Acta. 2005; 1746: 284-294Crossref PubMed Scopus (276) Google Scholar). (Samples with 12 mol % cer were studied because at higher cer concentrations sterol displacement might be complete (14Megha London E. J. Biol. Chem. 2004; 279: 9997-10004Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar), and thus obscure differences between different cholesterol precursors.) As shown in Fig. 2D and Table 1, sterol presence and structure has only small effects on the stability of rafts in cercontaining samples (the difference between the Tm of samples without sterol and those containing the most raft-stabilizing sterol is 4 °C). This behavior is consistent with the possibility that the cer-rich rafts also contain only low levels of sterol. Fig. 2D shows early cholesterol precursors actually slightly destabilize ordered domain formation in cer-containing vesicles relative to those without sterol. In contrast, some stabilization of rafts in cer-containing samples is observed with lathosterol and 7-DHC, consistent with the great