Dec 5, 2017 - Fluorescent analogs of phosphatidylcholine (PC) and sphingomyelin (SM) labeled with diphenylhexatrie- nylpropionic acid (DPH) were ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY (0 1992 by The American Society for Biochemistry and Molecular Biology, Inc
Vol. 267, No. 34, Issue of December 5,pp. 24217-24222,1992 Printed in U.S.A .
Organization of Phosphatidylcholine and Sphingomyelin in the Surface Monolayer of LowDensity Lipoprotein and Lipoprotein(a)as Determined byTime-resolved Fluorometry* (Received for publication, December 19, 1991)
Andreas Sommer$, Elmar Prenner$, Roland Gorges$, Herbert Stutz$, Harald GrillhoferQ, Gerhard M. KostnerQ, Fritz Paltauf$, and Albin HerrnetterST From the tlnstitut furBiochemie und Lebensmittelchemie, Technische Uniuersitat Graz and the Slnstitut furMedizinische Biochemie: Univer.iitat Graz, A-8010 Graz, Austria
Fluorescent analogsof phosphatidylcholine (PC)and The hydrophobic core of lipoproteins consists of esterified sphingomyelin(SM)labeledwithdiphenylhexatriecholesterol and triglycerides. Low density lipoprotein (LDL) nylpropionic acid (DPH) were prepared and incorpo- possesses only one protein component, apolipoprotein B-100 rated into the surface layer of human lowdensity (apoB). Lipoprotein(a)(Lp(a))consists of a LDLparticle and lipoprotein (LDL) and lipoprotein(a) (Lp(a)). Fluores- an additional polypeptide, apolipoprotein(a) (apo(a)) (for a cence anisotropy measurementsof DPH-PC and DPH- review see Scanu, 1991). SM in both lipoprotein classes were carried out at Low density lipoprotein has been known for quite some differenttemperaturesrangingfrom 20 to 37 “C. time to be atherogenic. In the past few years Lp(a) has also DPH-PC as well as DPH-SM were shown to reside in been found to be an independent risk factor for coronary more rigid domains in Lp(a) than LDL in according to heart disease. Therefore, particular attention has been paid higher anisotropy values in Lp(a). In both LDL and to this lipoprotein class (Scanu and Fless, 1990). Lp(a), DPH-PC experienceda more rigid environment It was the aim of the present work to gain insight into the than DPH-SM, suggesting different environments of organization of the surface phospholipids of Lp(a) as comPC andSM in the surface shell of the lipoproteins. pared with LDL. In this respect, we were also interested in Fluorescencelifetimes of thelabeledlipoproteins the interactions of phosphatidylcholine and sphingomyelin were determined by phase and modulation fluoromwith apoproteins in LDL and Lp(a). In a fluorescence study etry. We found bimodal Lorentzian distributions for the decay times of DPH-PC and DPH-SM in LDL and with human high density lipoproteins, PC and SM have been Lp(a). Lifetime distribution centers for labeled lipids demonstrated to behave differently with respect to theiraffinwere very similar except for DPH-PC in Lp(a) whichity to apolipoprotein A-I (Molotkovsky et al., 1982). Similar was shifted to longer lifetimes, suggesting a less polar conclusions have also been reached on the basis of a study on environment of PC in Lp(a) than inLDL. The distri- the interaction of PC with apolipoprotein A-I from pig(Handa butional width of DPH-PC in Lp(a) was broader than et al., 1992). So far, the question has been left open as to i n LDL. Accordingly, phosphatidylcholine must belo- whether other apoproteins such as apoB would give rise to calized ina more homogeneous environment inLDL as preferential interactions with different phospholipid classes. compared with Lp(a).On the other hand, no difference Lipid domains of lipoproteins might be important in mainin distributional widths was observed for DPH-SM in taining proper apoprotein folding, thus modulating lipoprotein-lipoprotein and lipoprotein-cell receptor interactions. both lipoproteins, showing that SM organization in They might also play a role in protein-mediatedphospholipid Lp(a) is unaffected by apo(a). From the obtained fluorescence data we propose transfer that between lipoproteins, as well as between lipoproteins apoproteins discriminate between the choline phospho- and cell membranes, and in phospholipid degradation in the lipids and preferentially associate with phosphatidyl- lipoprotein surface. choline. This effect is enhanced in Lp(a) due to the We applied steady-stateand time-resolved (phase and presence of apolipoprotein(a). modulation) fluorometry in order to investigate the organization of the two major phospholipid classes in LDL and Lp(a). Phosphatidylcholine and sphingomyelin carrying the 1,6Phosphatidylcholine (PC)’ andsphingomyelin (SM) are the major phospholipids of human lipoproteins. Both choline diphenyl-1,3,5-hexatrienylpropionylresidue (DPH) as a fluorescence marker were incorporated into the outer layer of lipids are located in the surface monolayer of the particles LDL and Lp(a).We determined continuous fluorescence lifetogether with the major part of free cholesterol and proteins. time distributions for the respective systems, assuming that * This work was supported by grants,from the Fondszur Forderung the lifetime homogeneity of the labeled lipids would reflect der wissenschaftlichen Forschung in Osterreich, Projects S 4602 (to the homogeneity of their environment in the lipoprotein surface. In addition, fluorescence anisotropies were determined G. M. K.) and S 4615 (to A. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This as a measure of phospholipid side chain mobility in the lipid article must therefore be hereby marked “aduertisement” in accordmonolayer. We found that DPH-PC and DPH-SMwere not ance with 18 U.S.C. Section 1734 solely t o indicate this fact. equally distributed in LDL and Lp(a).We suppose that DPH(I To whom correspondence should be addressed. PC preferentially interacts with the apoproteins, whereas SM ’ The abbreviations used are: PC, phosphatidylcholine; PE, phos- probably resides in the “bulk” lipid phase. phatidylethanolamine; SM, sphingomyelin; POPC,l-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine; DPH, 1,6-diphenyl-1,3,5-hexaEXPERIMENTALPROCEDURES triene; Lp(a), lipoprotein(a); LDL, low density lipoprotein; apo(a), apolipoprotein(a); apoB, apolipoproteinB-100; TLC, thinlayer chroMaterials-The following fluorescent analogs of phospholipids matography. l-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexawere prepared
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i n LDL Lp(a)and
trienyl)phenyl]ethyl]carbonyl]-sn-glycero-3-phosphocholine(DPH- Shimadzu RF-540 spectrofluorometer. The time-dependent increase phosphatidylcholine) was prepared as described (Kalb et al., 1989). of fluorescence intensity (due to dequenching) a t 37 'C upon incuN-[[2-[4-(6-Phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]bation of lipoproteins withDPH lipids wasmeasured a t 430 nm using trans-4-sphingenine-1-phosphocholine (DPH-sphingomyelin) was an excitation wavelength of 360 nm. Excitation and emission slits synthesized by acylation of sphingosylphosphocholine with 1,6-di- were set 10 and 5 nm, respectively. Complete uptake of fluorescent phenyl-1,3,5-hexatrienylpropionicacid (Lambda Fluoreszenz Tech- phospholipids into the lipoproteinswas confirmed in control experinologie, Graz, Austria) asdescribed for the preparation of fluorescent ments. After addition of 1% Triton X-100(Sigma,Deisenhofen, phosphatidylcholine(Kalb et al., 1989). Sphingosylphosphocholine Germany) to the incubation mixture, no further increase in fluoreswas prepared from bovine brain sphingomyelin (Sigma, Deisenhofen, cence intensity was observed. The detergentwould solubilize remainGermany) by acidic hydrolysis, using a modified version (the reaction ing donor vesicles and dilute fluorogenic lipids into mixed micelles. time was three times as long as indicated in the literature) of the Fluorescence Anisotropies-For the measurement of the fluoresprocedure described by Kaller (1961) and characterized according to cence anisotropies of DPH-phosphatidylcholine and DPH-sphingoCohenet al. (1984). l-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocho-myelin in phospholipid vesicles, LDL and Lp(a), the excitation and line (POPC) was synthesized as described (Hermetter et al., 1989); emission wavelengths were set 360 nm (5-nm slits) and430 nm (20chicken egg yolk sphingomyelin containing primarily palmitic acid nm slits), respectively. Fluorescence anisotropies r were determined was purchased from Sigma, Deisenhofen, Germany. according to thefollowing equations. Isolation of Lipoproteins-Lipoprotein(a) and low density lipoproI,, - GIvh tein were isolated from plasma of fasting normolipemic volunteers r= (Eq. 1) selected according to their plasma Lp(a) concentrations. Lp(a) was I v v -k 2GIvh purified essentially as described earlier (Steyrer and Kostner, 1990). Immediately after blood drawing and centrifugation, the plasmawas G = -I h v (Eq. 2) stabilized with EDTA and sodiumazide (1 mg/ml) and subjected to Ihh density gradient ultracentrifugation in a S W 40 rotor (Beckman) for relative 24 h a t 40,000 rpm (Knipping et al., 1986). The fraction a t density I,, and I v h are thefluorescence intensities parallel and normal 1.070-1.125 g/ml was passed over an immunoadsorber specific for to thevertically polarized excitation light. Ihv and I h h are the fluoresemissionpolarizer oriented apo(a). Adsorbed Lp(a) was eluted with glycine-hydrochloride buffer, cence intensitiesdeterminedwiththe vertically and horizontally when the excitation polarizer was set in p H 2.5, yielding preparations of more than 95% purity. In control experiments Lp(a)was prepared by lysine-Sepharose chromatography the horizontal position. Measurements were carried out at tempera(Karadi et al., 1988). LDL was harvested from a fraction correspond- tures rangingfrom 20 to 37 "C, the sample temperature in the cuvette ing to densities 1.025-1.055 g/ml of the density gradient and recen- being maintained by an external thermostatic bath. Fluorescence Lifetimes-Lifetime measurements were performed trifuged under identical conditions. All buffers and solutionsused forlipoprotein preparation contained using a GREG 200 (ISS, Champaign, IL) variable frequency crossa frequency range EDTA andsodium azide (1mg/ml) and were deoxygenated in vacuum correlation phase and modulation fluorometer with after saturation withnitrogen. All purification steps were performed from 1 to 200 MHz (Gratton and Limkeman, 1984). The measurements were carried out at 37 "C. The temperature in the sample at 4 "C, and preparations were used within 1 week. The purity of the Lp(a) and LDL fractions was assayed by double- compartment was controlled by an external thermostatic bath. A He-Cd laser (Liconix 4207 NB) was used as an excitation light decker rocket immunoelectrophoresis and SDS-polyacrylamide gel electrophoresisas described (Laurell, 1966; Gaubatz et al., 1983; source a t 325 nm. The incident light was modulated by a Pockel's cell. p-bis(5-Phenyloxazol-2-yl)benzenein ethanol (Lakowicz et al., Armstrong et al., 1985). Phospholipid Analysis-Total lipids were isolated from LDL and 1981) served as a lifetime reference (7 = 1.35 f 0.2 ns). Fluorescence Lp(a) obtained from the same plasma by chloroform/methanol (2/1, wasobserved through acutoff filter (KV370; Schott, Mainz, Gerwere determined by the crossv/v) extraction (Folch et al., 1957) and separated by TLC on silica many). Phase shifts and demodulations 1969) a t modulation fregel plates, using chloroform/methanol/water(65/25/4, v/v/v) as a correlation method (Spencer and Weber, solvent. Spots containing ethanolamine glycerophospholipids, choline quencies of 10, 15,20,25, 50, 70, 100, 140, and 200 MHz. Fluorescence lifetimes were determined from the measured phase glycerophospholipids, or sphingomyelin were scraped off the plate and their phosphorus content was determined according to Broek- angles and demodulations, using a least squares program from I.S.S. huyse (1968). For the determination of alkenylacyl subclasses (plas- minimizing xzRas described (Jameson and Gratton, 1983). First, the malogens), ethanolamine and choline glycerophospholipid spots were experimental data were analyzed in terms of a double exponential decay. The obtained values for lifetime centers and fractions were separated by TLC as described above, and lipids were eluted from then used as starting valuesfor continuous distributional lifetime silica gel by chloroform/methanol (1/4, v/v). From the respective analyses. The experimental errors for phase angles and demodulalipid extracts, cholineplasmalogens (Hoerrmann et al., 1991) and tions were 0.4" and 0.008", respectively. Bimodal continuous Lorenethanolamine plasmalogens (Kates, 1986) were analyzed as described. tzian lifetime distributions were determined for the labeled lipoproVesicle Preparations-Unilamellar vesicles were prepared by the teins, characterized by the lifetime distribution centers ( T ) , the disethanol injection method(BatzriandKorn, 1973; Kremer et al., tribution widths a t half-height (w),and the fractional intensities( f ) 1977). DPH-PC or DPH-SM were used as fluorescent markers, and (Alcala et al., 1987). POPC or chicken egg yolk sphingomyelin served as matrix lipids. A chloroform/methanol (2/l,v/v) solution containing 3.3 nmol of the RESULTS fluorescent phospholipid and 1 pmol of matrix phospholipid were brought to dryness under a stream of nitrogen, and residual solvent Phospholipid Compositions of LDL and Lp(a)-The relative was removed under vacuum. Lipids were dissolved in 25 pl of ethanol, amounts of phosphatidylcholine, sphingomyelin, as well as and the resulting solutionwas injected with a Hamilton syringe into 3 ml of 10 mM Tris-HC1 buffer, pH 7.4, a t 37 "C under stirring. The phosphatidylethanolamine, are very similar in LDL and Lp(a) (Table I). Furthermore, plasmalogens were determined in molar ratio of matrix phospholipid to thelabel was 3001. Vesicles for the labeling of lipoproteins contained only fluorescent both lipoprotein classes. Almost nocholine plasmalogen could phospholipid (DPH-PC or DPH-SM) and were prepared using the be detected in the investigated lipoproteins (0.3% of PC in same injection method.Fluorescentphospholipid (1.4 nmol) was LDL, 0.2% of PC in Lp(a)), whereas ethanolamine phosphodried, dissolved in 10 p1 of ethanol, and the resultant solution was lipids contain a very high percentage of plasmalogen (68.8% injected into 0.8 ml of 10 mM Tris-HC1 buffer a t 37 "C under stirring. All vesicle preparations were deoxygenated aftersaturationwith TABLEI argon and stored in the dark notlonger than 3 days at 4 "C. Phospholipid composition (percent of total phospholipid) Lipoprotein Labeling-Immediately before use the lipoprotein fracof LDL and Lp(a) tions were disssolved in 2 ml of 10 mM Tris-HC1, p H 7.4, and desalted by gel chromatography using a Sephadex G-25 column (5 X 1.5 cm, Phospholipid LDL Ma) from Pharmacia LKB, Freiburg, Germany). Then, 0.8 ml of fluoro64.8 f 0.2 66.0 f 0.2 Phosphatidylcholine genic vesicles were added to 2 ml of a lipoprotein suspension with a
