Structure of low density lipoprotein (LDL) particles: Basis for understanding molecular changes in modified LDL

Biochimica et Biophysica Acta 1488 (2000) 189^210 www.elsevier.com/locate/bba

Review

Structure of low density lipoprotein (LDL) particles: Basis for understanding molecular changes in modi¢ed LDL Tiia Hevonoja, Markku O. Pentika«inen, Marja T. Hyvo«nen, Petri T. Kovanen *, Mika Ala-Korpela Wihuri Research Institute, Kalliolinnantie 4, FIN-00140 Helsinki, Finland Received 21 June 2000; received in revised form 23 August 2000; accepted 24 August 2000

Abstract Low density lipoprotein (LDL) particles are the major cholesterol carriers in circulation and their physiological function is to carry cholesterol to the cells. In the process of atherogenesis these particles are modified and they accumulate in the arterial wall. Although the composition and overall structure of the LDL particles is well known, the fundamental molecular interactions and their impact on the structure of LDL particles are not well understood. Here, the existing pieces of structural information on LDL particles are combined with computer models of the individual molecular components to give a detailed structural model and visualization of the particles. Strong evidence is presented in favor of interactions between LDL lipid constituents that lead to specific domain formation in the particles. A new three-layer model, which divides the LDL particle into outer surface, interfacial layer, and core, and which is capable of explaining some seemingly contradictory interpretations of molecular interactions in LDL particles, is also presented. A new molecular interaction model for the Lsheet structure and phosphatidylcholine headgroups is introduced and an overall view of the tertiary structure of apolipoprotein B-100 in the LDL particles is presented. This structural information is also utilized to understand and explain the molecular characteristics and interactions of modified, atherogenic LDL particles. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Apolipoprotein B-100; Computer models domain formation; Low density lipoprotein; Modi¢ed low density lipoprotein; Molecular interaction

1. Introduction Low density lipoprotein (LDL) particles are the main carriers of cholesterol in the human circulation

and are thus key players in cholesterol transfer and metabolism. Both physical and physiological characteristics have led to a common convention for de¢ning LDL particles as lipoprotein particles within the

Abbreviations: LDL, low density lipoprotein; DSC, di¡erential scanning calorimetry; NMR, nuclear magnetic resonance; CD, circular dichroism; apo, apolipoprotein; HDL, high density lipoprotein; TG, triglyceride ; CE, cholesteryl ester; UC, unesteri¢ed cholesterol; PC, phosphatidylcholine; SM, sphingomyelin; lyso-PC, lysophosphatidylcholine; DAG, diacylglycerol; CER, ceramide; PE, phosphatidylethanolamine; VLDL, very low density lipoprotein; SMase, sphingomyelinase; IDL, intermediate density lipoprotein; PLC, phospholipase C; PLA2 , phospholipase A2 ; GAG, glycosaminoglycan; CEase, cholesterol esterase; HG, phosphocholine headgroup; FFA, free fatty acid; MDA, malondialdehyde * Corresponding author. Fax: +358-9-637-476; E-mail: petri.kovanen@wri.¢ 1388-1981 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 8 - 1 9 8 1 ( 0 0 ) 0 0 1 2 3 - 2

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density limits of 1.019^1.063 g/ml. Therefore, LDL forms a heterogeneous group of particles varying greatly in size, composition and structure. During the recent decades, various biophysical methodologies such as electron microscopy, di¡erential scanning calorimetry (DSC), nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, circular dichroism (CD), £uorescence spectroscopy, X-ray small angle and neutron scattering techniques have been used to characterize the structure of LDL particles. Model systems, e.g., unilamellar vesicles and emulsion particles, have also been employed in order to overcome some of the di¤culties faced in studies of large and compositionally complex LDL particles. Interestingly, the ¢rst three-dimensional models and atomic-level molecular dynamics simulations of apolipoprotein A-I (apoA-I) and discoidal high density lipoprotein (HDL) particles have recently become available [1,2]. Unfortunately, however, the large size of LDL particles presently excludes the application of corresponding methodologies to resolve the structure of apoB-100 and LDL particles (apoB-100 of 4536 amino acid residues plus approximately 3000 lipid molecules in an LDL particle in contrast to two apoA-I molecules of 243 amino acid residues plus approximately 200 lipid molecules in discoidal HDL). Current knowledge and understanding of the LDL particle structure is therefore far from trivial. Steim and co-workers gave convincing evidence in 1968 [3] for the micellar model of lipoprotein particles. In the mid 1970s the LDL surface was suggested to be a trilayer or a bilayer, the protein moiety existing in the core of the lipoprotein particle [4]. Piece by piece, however, the data have led to the development of the current picture of the spherical lipoprotein particle as an amphipathic monolayer surrounding a hydrophobic lipid core. Moreover, a biophysical model has recently been developed for micellar lipoprotein particles [5]. More speci¢cally, LDL particles have an average diameter of 22 nm, the core consisting of about 170 triglyceride (TG) and 1600 cholesteryl ester (CE) molecules and the surface monolayer comprising about 700 phospholipid molecules and a single copy of apoB-100 [6]. In addition, the particles contain about 600 molecules of unesteri¢ed cholesterol (UC), of which about one-third is located in the core

and two-thirds in the surface [7]. It should also be noted that a few percent of the TG and CE molecules penetrate toward the surface. The main phospholipid components are phosphatidylcholine (PC) (about 450 molecules/LDL particle) and sphingomyelin (SM) (about 185 molecules/LDL particle). The LDL particles also contain lysophosphatidylcholine (lyso-PC) (about 80 molecules/LDL particle) [6], phosphatidylethanolamine (PE) (about 10 molecules/ LDL particle) [8], diacylglycerol (DAG) (about 7 molecules/LDL particle) [9], ceramide (CER) (about 2 molecules/LDL particle) [10] and some phosphatidylinositol [11]. In addition to lipids, LDL particles also carry lipophilic antioxidants, such as K-tocopherol (about 6 molecules/LDL particle) and minute amounts of Q-tocopherol, carotenoids, oxycarotenoids and ubiquinol-10 [6]. The particles are in a dynamic state, their structure and physical properties being dependent on their lipid composition as well as on the conformation of apoB-100. A characteristic phenomenon of early atherogenesis is extracellular accumulation of LDL-derived lipids in the form of small lipid droplets and vesicles, which can lead to the development of atherosclerotic lesions in the arterial intima [12^14]. Modi¢cations in the structure of native LDL that are capable of inducing aggregation and/or fusion of the particles are currently recognized to be a prerequisite for the initiation of lipid accumulation [15,16]. Detailed knowledge of the molecular structure of LDL particles is therefore essential for understanding the pathological processes of intimal lipid accumulation, in addition to the physiology of cholesterol metabolism. In this review we will explore the various molecular aspects that in£uence the structure of LDL particles. As a combination of the existing structural information on LDL particles and closely related model systems, we present new models of the structures of native and modi¢ed LDL particles. The rationale for gathering the molecular details of LDL particles into a coherent setting is twofold. Firstly, to provide a better understanding of the structural aspects of native particles, and secondly, to build a ¢rm basis for interpreting the structural changes in variously modi¢ed LDL particles. This will enable an understanding of the molecular characteristics that determine their fate in early atherogenesis, namely aggregation and fusion.

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Fig. 1. Schematic molecular model of an LDL particle at around body temperature (above the phase transition of core CEs). The depicted particle has a diameter of 20 nm, including a surface monolayer of 2 nm (yellowish background), and an average composition of 20% protein, 20% phospholipids, 40% CEs, 10% UC, and 5% TGs. The molecular components of the particle are drawn in both the correct percentages and size ratios. Note the di¡erent domains illustrated at the particle surface and the interpenetration of core and surface lipids. The individual molecules were built using Cerius2 software (MSI Molecular Simulations Inc.). The chain compositions are illustrated as follows: SM (16:0); PC (16:0/18:2v9;12 ); TG (16:0/18:2v9;12 /14:0); CE (18:2v9;12 ).

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A schematic molecular model of a native LDL particle is given in Fig. 1. The evidence for the model is discussed in detail in the following paragraphs. Moreover, the tertiary structure of apoB-100 in an LDL particle as well as schematic molecular models for various atherogenic modi¢cations of LDL particles will also be presented and discussed. 2. Outer surface and interfacial layers A widespread convention is to divide the LDL particle into two structurally distinct regions: core and surface. This simple division is in good accord with the micellar model and the known molecular constituents of the particles. However, experiments employing di¡erent types of probes to obtain information on the interactions between the presumed surface and core components have led to seemingly contradictory results. Thus, a coupling between the surface phospholipids and core CE and TG was suggested based on the behavior of lipid fatty acid chains [17^19] and a direct connection between core lipids and apoB-100 has also been shown [20,21]. On the other hand, many investigators have interpreted their experiments in favor of quite an independent core and surface regions of LDL particles. Thus, in recent £uorescence spectroscopy experiments, Saito and co-workers indicated that the ability of exchangeable apos (apoE and apoCII) to bind to lipid emulsion particles is closely correlated with the arrangement of UC in the particles as well as with the mobility of the core lipids [22]. Nevertheless, the authors concluded that the e¡ect of the core lipids on the surface is due to redistribution of UC rather than to a direct interaction between core and surface lipids. Moreover, Kroon has stated that, in reconstituted LDL particles, the motional states of core and surface lipids are relatively independent [23]. Supporting evidence for this claim comes from proton NMR spectroscopy experiments on LDL particles showing that, while the core CEs change from a liquid-like state to a more ordered liquid crystalline state, the phospholipids surrounding the core remain relatively £uid [24]. Finally, proton NMR spectroscopy experiments on LDL have indicated that the core phase transition (which is clearly observed from the aliphatic fatty acid protons) can-

Fig. 2. Division of an LDL particle into three radially dissimilar structural layers: the outer surface layer, the interfacial layer, and the core. ApoB-100 is depicted in gray, phospholipid headgroups in blue, and lipid fatty acids and cholesterol in reddish (interfacial layer) or orange/yellow (core). The conventional division into surface and core is shown below the diagram. Note that the molecules in each of the three radial layers show distinct orientational behavior: random in the core, radial in the interfacial, and tangential on the outer surface layer.

not be detected from the phosphocholine headgroup resonances of the surface phospholipids [25]. When the experimental details, and particularly the locations of the various molecular probes in the lipid particles studied, are carefully considered, the apparent contradiction turns out to result from an inconsistency in the terminology used for the LDL particle structure. Clearly, the terms core and surface do not re£ect the details of the LDL particle structure accurately enough. Therefore, a new three-layer model of an LDL particle is introduced. 2.1. A three-layer model The new model depicted in Fig. 2 involves division of the surface region into an outer surface layer consisting mainly of the phospholipid headgroups and an interfacial layer consisting of interpenetrating core and surface lipids [22,26]. The pure core layer consists of molecules that are not in direct contact with the surface monolayer. The apoB-100 is expected to have some parts in each of these three layers of the LDL particle. Interestingly, each of the three structural layers is characterized by a distinct average orientation: the phospholipid headgroups in the outer surface layer are oriented nearly parallel to the surface, and the fatty acid chains in

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the interfacial layer are radially and the core lipids randomly oriented (at 37³C). 2.2. Nanoenvironments The interfacial layer is likely to have a crucial role in regulating the molecular properties of the LDL particles that a¡ect their interactions with other particles and molecules. On the basis of the present knowledge, the lipid constituents (and apoB-100) of LDL are illustrated in Fig. 1. It is apparent that the lipid constituents of the LDL particles are not homogeneously distributed in the interfacial layer but form distinct local molecular environments. These environments involve variable numbers of individual lipid molecules and are expected to have characteristic molecular properties relating to the principal interactions leading to their formation. In the LDL particles, these local environments are nano-sized and will therefore be logically called nanoenvironments. In addition to the mainly apoB-100-containing environments, two di¡erent types of lipid environments are likely to exist in the LDL particles, namely, one rich in PC and poor in UC and the other rich in both SM and UC. Environments of these kinds, locally enriched with a particular combination of molecules, may have an important physiological role in controlling lipid transfer and lipolysis, i.e., in the recognition and activation of lipid transfer proteins and lipid-hydrolyzing enzymes. In fact, the PC rich and UCpoor environment, in contrast to the SM- and UC rich environment, is likely to favor penetration of core lipids toward the water environment, thus making it possible for the water-soluble enzymes to reach the hydrophobic core lipids in the LDL particles. It should be kept in mind, however, that the LDL particles are in a dynamic state, i.e., that in none of the environments the composition or structure is constant. A more detailed discussion of the various lines of evidence supporting the nanoenvironment picture of the LDL particles will be given in the following paragraphs: the mainly lipid-related aspects will be presented in this section and the apoB-100-related aspects in a separate section. 2.3. UC and phospholipids The in£uence of UC on the structure of the phos-

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pholipid systems is substantial and has been widely studied in bilayer membranes [27,28] but not in such detail in lipoprotein particles or lipoprotein model systems. Nevertheless, close similarities are to be expected in the main e¡ects of UC on phospholipid bilayers and on the LDL surface monolayer. Phosphorous NMR studies have given evidence that UC molecules are not associated with the polar headgroups of the phospholipids, but that the OH group of UC is likely to reside at the height of the ester carbonyl oxygen of the PC molecules in a membrane [29,30]. However, UC molecules do not form a tight complex with phospholipids, since the rotation rate of UC about its long axis is faster than that of phospholipids [31]. In addition, UC is known to increase the order of phospholipid hydrocarbon chains in the liquid crystalline state [32] and, at high concentrations, to completely eliminate the gel to liquid crystalline phase transition of phospholipids [33]. The phospholipid headgroup interactions are also diminished in the presence of UC, because of the increased intermolecular distances between the phospholipids [34^36]. UC prefers interactions with SM to PC, with PC to PE, with saturated to unsaturated fatty acids, and with 16-carbon to 14-carbon fatty acid chains [28,37]. Deuterium NMR spectroscopy experiments of very low density lipoprotein (VLDL), LDL, and microemulsions have demonstrated that the surface phospholipids exist in two distinct environments, with higher and lower order, at the surface monolayer of the particles [18]. The existence of these environments appear not to depend on the protein components and were therefore interpreted as an indication of a cholesterol de¢cient and a cholesterol rich environment. 2.4. UC and SM Clear evidence for the strong preference of UC to interact with SM rather than with PC comes from experiments on the homeostasis of UC in plasma membranes [38]: on depletion of the cell surface SM with sphingomyelinase (SMase), the e¥ux of UC to an extracellular UC acceptor was found to increase dramatically, while the corresponding hydrolysis of PC had almost no e¡ect on the e¥ux. The resynthesis of SM was found to reduce the UC e¥ux to control levels. Studies with model mono-

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layers have also shown that UC appears to interact favorably with SMs [39]. In addition, it has recently been veri¢ed that UC molecules can potentially interact with all the physiologically relevant SM species in membranes [40]. The more favorable interaction between UC and SM is likely to be due to the more favorable hydrophobic interactions between these two lipids, i.e., short-distance van der Waals interactions between the cholesterol ring and the saturated fatty acid chains of SM [39^42]. The fatty acid chains in SM are, in fact, more saturated than in PC, which might partly explain the preferential interaction between UC and SM. It is also possible, however, that the hydroxyl group of UC can form hydrogen bonds with the amide group in SM [40]. 2.5. UC and neutral lipids UC has been found to decrease the solubility of neutral lipid molecules to phospholipid bilayers, the e¡ect being more pronounced for TG than for CE [43,44]. The e¡ect of UC may result from competition between neutral lipids and UC for the same space or from the known condensing e¡ect of UC on phospholipid monolayers, which can hinder interpenetration [45]. Since the LDL particle surface is rich in UC molecules that prefer to interact with SM, it is possible that interpenetration of TG and CE molecules may occur predominantly in the PC rich areas, which are expected to have low local UC concentrations. This is also supported by Chana and co-workers, who pointed out the possible disordering e¡ect of the interpenetration of fatty acid chains of TGs and surface phospholipids and suggested that in VLDL and LDL particles the more ordered cholesterol rich domain should also be TG de¢cient [18]. In a recent £uorescent spectroscopy study of emulsion particles Saito and co-workers [22] found that the core lipid composition a¡ects the distribution of UC in the particles. In TG^PC emulsions, over 80% of the UC molecules were associated with the PCs, whereas in CE^PC emulsions the corresponding percentage was only about 50%. The partitioning of UC into CE rich LDL particles, two-thirds associated with the phospholipids versus one-third within the core [7], is in good accord with the results of these extreme model systems. The lipid composition of the

LDL particle core might therefore, by a¡ecting the distribution of UC, have considerable in£uence on the molecular properties of the interfacial layer. 2.6. PC headgroup orientation The orientational behavior of the PC headgroups a¡ects the general electrostatic properties of the LDL particle surface. Although we are not aware of any direct information on LDL, it appears most likely that the PC headgroups have an average orientation closely parallel to the particle surface in a manner similar to PC bilayers [34,46^50]. However, the headgroups are highly mobile parts of the molecules. In fact, molecular dynamics simulations have demonstrated that the PC headgroup orientation can vary, on a nanosecond time scale, between the water environment and the PC bilayer interior, indicating a dynamically rough outer surface [51^53]. In pure phospholipid systems, the nitrogen and phosphorus atoms of the headgroups have an optimum interaction distance (that gives rise to the observed average orientation). Therefore, any intervening molecules or changes in the charge characteristics of the surroundings potentially a¡ect the orientation [35,36,54]. The surface curvature is also expected to contribute: in the smaller lipoprotein particles, the surface is highly curved and therefore likely to disturb the PC headgroup interactions. 2.7. Lipid packing Saito and co-workers have indicated that interpenetration of core lipids (TG and CE) and phospholipid fatty acid chains increases the orientational order in the interfacial layer of PC-based emulsion particles [22]. TG and CE increase the order in a similar manner, but in the CE^PC particles the rotational motion of the PCs is more restricted than in the TG^PC particles. This may be due either to a more ordered core in the CE^PC emulsion particles as compared with TG^PC emulsions or to interpenetration of the cholesterol moiety and the phospholipid fatty acid chains. However, despite the interpenetration of neutral lipids and phospholipid fatty acid chains, £uorescence spectroscopy experiments do not indicate a tight connection between the motional states of the core lipids and the phospholipid mono-

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layer [22,23]. X-ray small-angle scattering experiments support this conclusion, since, below the core lipid phase transition temperature, the LDL particle sizes were found to be slightly larger [55]. This would imply decreased interpenetration of the core lipids and the phospholipid fatty acid chains and so would explain why the rigidity of the highly oriented core phase does not overrule the motion of the phospholipid molecules. It has been shown that UC molecules in LDL and HDL particles, in contrast to cell membranes, are readily susceptible to oxidation by cholesterol oxidase [56]. This indicates that the molecular packing of lipids in the monolayer of LDL and HDL is looser than the comparable packing in biological bilayer membranes and may be due to the spherical nature and curved membranous surface of the particles. The surface pressure has been estimated to be higher for native LDL than for native HDL particles [56]. Also, in LDL particles, in comparison to HDL, the phospholipid layer has been indicated to have tighter lateral molecular packing [7,57]. A probable explanation for these ¢ndings comes from the more saturated phospholipid composition as well as from higher UC and SM contents of the LDL particles. Increasing numbers of studies point out that the packing and interpenetration of lipid molecules in lipoprotein particles are profound structural characteristics that have a crucial role in many physiological processes, for example, in controlling apolipoprotein binding and enzyme activity [22,57,58]. 3. Core The core compartment of an LDL particle is composed mainly of CE molecules and small amounts of TG and UC. Calorimetric studies and proton NMR spectroscopy have shown that the core lipids exhibit a reversible broad phase transition at around 30³C (having a width of about 20³C and a variation of as much as 10³C in the peak temperature). Thus, above the phase transition, i.e., at a physiological temperature, the core lipids exist in a liquid-like state and, below the phase transition, in a highly ordered smectic-like liquid crystal state. The transition temperature has been indicated to be inversely proportional to the TG/CE ratio of the particles [26]. In addition,

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increased unsaturation of the core lipid fatty acid chains lowers the transition temperature [26,59,60]. Interestingly, in DSC experiments, g-3 fatty acid supplementation was shown to decrease the transition temperature by 2³C (29.5³C versus 27.6³C) [59]. The DSC experiments of Parks and Hauser [60] with LDL particles isolated from nonhuman primates fed diets containing lard or ¢sh oil showed a bigger di¡erence in transition temperatures, namely 33³C for the ¢sh oil group versus 40³C for the lard group. These authors also used proton NMR spectroscopy and concluded that the increase in the lipid £uidity of ¢sh oil LDL compared to lard LDL primarily re£ects a change in the CE fatty acid composition and its in£uence on the physical state of the CE core. However, the g-3 fatty acid enrichment seemed to have little e¡ect on the motional properties of the LDL phospholipid monolayer [60]. 3.1. Low temperature nanoenvironments X-ray small-angle scattering experiments, combined with chemical analysis and geometric calculations, have given a clear picture of the overall molecular arrangement in the LDL particle core [55]. Below the phase transition temperature, the data suggest a highly ordered arrangement in which the cholesterol moieties of CEs are localized in two radial shells. Such organization of CE molecules implies that, in each shell, approximately half of the fatty acid chains of CE point toward the center and the other half toward the surface of the particle. Above the phase transition temperature, the arrangement of the CEs in the core appears to be much less ordered [26,55]. Information on the structural organization of the LDL particle core has recently been obtained also with the aid of electron spin resonance measurements and the use of spin-labeled CE and TG molecules [61]. Pregetter and co-workers found that the calorimetric transition temperature was almost constant at CE/TG mass ratios higher than 7:1 but that below this molecular ratio, TG had a strong e¡ect on the calorimetric transition. On the basis of geometrical argumentation that a volume ratio of 7:1 can be obtained in a spherical particle when the radius of the inner core is 3.6 nm and the surrounding shell extends to 7.2 nm, and of all the experimental results,

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Fig. 3. Schematic molecular model of an LDL particle at low temperature (below the phase transition of core CEs). Note the existence of liquid-like TGs at the center of the particle surrounded by highly oriented layers of CEs (drawn according to the results in [55,61]). The key to the molecular models is given in Fig. 1.

they proposed a new model for the organization of the CE and TG molecules in the LDL particle core. To illustrate the present knowledge of the low temperature core organization, the results of both studies above have been visualized in Fig. 3. Assuming that the TG molecules occupy the inner core and the CE molecules the concentric outer compartment, a CE/ TG ratio greater than 7:1 would mean that the TG molecules do not perturb the radial smectic arrangement of CEs below the phase transition temperature. This would allow optimal CE^CE interactions in the liquid crystal phase and explain the high phase transition temperatures observed [61]. On the other hand, if the CE/TG ratio is smaller than 7:1, there are not enough CE molecules to form a pure CE phase around the TGs, which therefore leads to mixing of these core components. Interestingly, these ¢ndings also support the idea of the existence of TG and CE nanoenvironments in the core of LDL particles above the phase transition temperature, particularly if the CE/TG ratio is close to 7:1, since the TG compartment was found to remain in the same physical state, namely a liquid resembling olive oil, throughout the temperature range measured, from well below to well above the LDL core phase transition.

The presence of di¡erent environments in the core of chylomicra and VLDL particles of non-human primates above a phase transition temperature was shown by proton NMR [62]. Hamilton and co-workers indicated TG species of di¡erent melting points: the TG molecules that remained liquid at low temperatures were found to have mainly two or three monounsaturated chains or polyunsaturated chains. Their results also suggested that in the lipoprotein particles the e¡ect of `solid' TGs were transmitted to the `liquid' TGs, i.e., at low temperature the presence of `solid' TG can cause restrictions on the motions of the `liquid' TGs [62]. In addition, Bell and co-workers have found by proton and carbon NMR that g-3 fatty acid enrichment of VLDL and LDL particles leads to signi¢cant alterations in their molecular architecture [63]. On the basis of relaxation behavior of g-3 fatty acids in the lipoprotein particles, the authors suggested formation of environments rich in g-3 fatty acids in the lipoprotein particles. 4. ApoB-100 ApoB-100 is one of the largest monomeric proteins known, consisting of 4536 amino acid residues [64^ 66]. In the apolipoprotein family, apoB-100 and apoB-48 are the only nonexchangeable members. The single molecule of apoB-100 that accompanies each LDL particle therefore originates from a VLDL particle that is modi¢ed and transformed into an LDL particle in the metabolic processes in the circulation. The apoB-100 molecule must be able to adjust for the structural and compositional changes taking place in the carrier particle; for example, the diameter of 80^200 nm of a VLDL particle is reduced to approximately 22 nm for an LDL particle. ApoB-100 also has a particular role in maintaining the structural integrity and controlling the interactions of LDL particles. 4.1. Functional domains Several functional domains have been identi¢ed in apoB-100. The binding site for the LDL receptor is located in the residues 3359^3369 [67]. The R3500Q mutation of apoB-100 leads to inability of the LDL

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particles to interact with the LDL receptor, causing the disease `familial defective apoB-100'. This has been shown to result from blocking of the LDL receptor binding site by the C-terminus of apoB-100, which is kept away from the binding site by arginine 3500 in wild-type LDL [67]. The N-terminus of apoB-100 has been suggested to bind the enzyme lipoprotein lipase [68], and the C-terminus has been shown to mediate the association of the enzyme platelet-activating factor acetylhydrolase with LDL particles [69]. The positively charged lysine and arginine residues of apoB-100 are known to interact with glycosaminoglycans (GAGs) and thereby mediate matrix^LDL interactions. At least eight potential GAG binding sites have been identi¢ed in apoB100 [70^72]. Interestingly, the binding of LDL particles to GAGs was severely impaired when human LDL with the mutation of lysine 3363 of apoB-100 was expressed in transgenic mice [73]. This ¢nding suggests, perhaps slightly surprisingly, that, in the apoB-100 of native LDL particles, there is only a single crucial GAG binding site. 4.2. Secondary and tertiary structure The huge size and insoluble nature of apoB-100 hinder structural studies. However, the evidence that has recently accumulated allows the presentation of a consistent view of the tertiary structure of apoB100 on an LDL particle. The immunoelectron microscopic studies of Chatterton et al. [74] have shown that apoB-100 does not encircle an LDL particle in a straightforward manner, but is formed of kinks and can be described as having `a ribbon and bow conformation'. Cryo-electron microscopy experiments of LDL particles are also in general agreement with `the ribbon and bow model', even though a bow structure could not be resolved [75]. On the other hand, the extensive computational analyses of the secondary structures of apoB-100 by Segrest and co-workers [76,77] have revealed a pentapartite structure, composed of both amphipathic K-helical domains and amphipathic L-stranded domains. In order to outline the K-helices and L-sheets of apoB-100, we need information on the size of these structures. Therefore, we constructed molecular models based on the N-terminal residues 24^38 and 44^56 to form an antiparallel L-sheet and the C-ter-

Fig. 4. Molecular models for K-helical and L-sheet structures of apoB-100. On the basis of the work by Segrest et al. [77], residues 4437^4463 of apoB-100 were chosen to model an K-helical structure (upper panel) and residues 24^38 combined with residues 44^56 to construct an antiparallel L-sheet (lower panel). The `snorkel model' interaction between an K-helix and a PC molecule is illustrated in the upper panel [133]. In the lower panel, a possible mode of interaction between a L-sheet structure and a PC molecule is presented. Note that, unlike the Khelix-PC interaction, the L-sheet-PC interaction could lead to ¢rm attachment of PC molecules to apoB-100 and to immobilization of the phosphocholine headgroups. The modeling was done with QUANTA software (MSI Molecular Simulations Inc.). The coloring of the K-helix is according to the charge of each amino acid: red for positively charged, blue for negatively charged, and pink for nonpolar amino acids. The atoms in the PC and in the L-sheet are colored as follows: green for carbon, white for hydrogen, red for oxygen, yellow for phosphorus and blue for nitrogen.

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minal residues 4437^4463 to represent an K-helix. These models, together with a PC molecule (16:0/ 18:2), are shown in Fig. 4. The modeling led to the following approximate dimensions: an K-helix is a cylindrical structure with a diameter of 1.3 nm, while a L-sheet (two antiparallel L-strands) is an upside down V-shaped structure with a height of 0.8 nm and a width of 1.2 nm. These values are naturally a¡ected by the speci¢c amino acid sequences and serve here only as estimates providing a means to assess the tertiary structure of the apoB-100 on an LDL particle. In the case of an K-helix, 3.6 amino acids result in a turn that is 0.54 nm in length, i.e., an K-helix progresses 0.15 nm per amino acid. The corresponding value for a L-strand (and a L-sheet) is approximately twice as large, i.e., 0.34 nm per amino acid. An additional important fact for the demonstration of the tertiary structure of apoB-100 is the length of the paths of apoB-100 domains on the particle surface. According to the results of Chatterton et al. [74], an approximate path length of 110^120 nm is obtained. It is notable that this value is almost twice the circumference of a sphere with a diameter of 22 nm (i.e., 69 nm). On the basis of the above considerations and the structural information on apoB-100 provided by Segrest and co-workers [76,77], we now present an approximate model for the tertiary structure of apoB-100 on an LDL particle. The model is illustrated in Fig. 5 and as animation at BBA Multimedia Library (http://www.elsevier.nl/homepage/sah/multimedia/lsvl.htm). The principles of construction are brie£y explained below. The pentapartite structure of apoB-100 consists of three K-helical and two L-structured domains [76,77]: K1 (residues 58^795), L1 (827^2001), K2 (2045^2587), L2 (2571^4032), and K3 (4017^4515). In addition, the small LN -region at the N-terminus has been considered separately [77]. The K-helical domains K2 and K3 are structurally similar (a mean length of 19.5^ 22.0 þ 9.2 amino acid residues per K-helix) and distinct from the K1 domain (a mean length of 15.5 þ 4.4 amino acid residues per K-helix). The K2 domain consists of about 500 amino acids and takes a path of about 15 nm on the surface of an LDL particle [74]. Therefore, approximately a dozen closely situated K-helices (of a diameter of 1.3 nm and of a length of about 5 nm) would be enough to cover

this path. In practice, the number of helices is likely to be slightly higher in this region (according to the mean length of K-helices) and the length of an individual K-helix may vary from about 3 to 6 nm. Accordingly, in the region of K3 about 4 and 12 Khelices (with a diameter of 1.3 nm and a length of about 4 nm) would cover the paths of 12 nm (from 89 to 92% of apoB-100 sequence) and 17.5 nm (from 92 to 100% of apoB-100 sequence) on an LDL particle surface, respectively [74]. The characteristics of the K1 domain suggest a preference for protein^protein interactions and a close resemblance to the globular 4-helix bundle domain of apoE [78]: the K1 domain is therefore illustrated in Fig. 5 as a combination of several 4-helix bundles. The L1 - and L2 -structures cover approximately the paths of 31 nm (from 18 to 45% of the apoB-100 sequence) and 29 nm (from 56 to 89% of the apoB100 sequence) on an LDL particle surface, respectively [74]. The L1 -area consists of about 1250 and the L2 -area of about 1450 amino acid residues, and thus the numbers of L-sheets (with widths of 1.2 nm) needed to cover these paths on the LDL particle surface are about 26 and 24, respectively. If the antiparallel amphipathic L-sheet structure were homogeneous, as shown in Fig. 5, its width would be approximately 7 and 9 nm in the L1 and L2 regions, respectively. One L-sheet is also drawn in Fig. 5 to illustrate the small LN region near the N-terminal end of apoB-100. 4.3. Structure^function relationships The distinct structural aspects of the di¡erent Khelical and L-structured domains and their possible metabolic roles deserve a few remarks. The structure of the K1 domain suggests that it might act as an anchor of apoB-100 for the lipid environment in the early stages of synthesis [66]. This view is supported by the ¢nding that the highly linear relationship between the lipoprotein radius and apoB size seems to be valid only after the K1 domain in the apoB-100 sequence [66]. The K2 domain is likely to form a £exible lipid-associating domain that can adjust to the variation in particle size via reversible protein^lipid interactions. This is supported by recent ¢ndings on the conformation of an K-helical apoA-I in solution and their extrapolation to discoi-

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Fig. 5. A three-dimensional model (tertiary structure) of apoB-100 at the surface of an LDL particle. The semitransparent particle in the middle is surrounded by views from six orientations that demonstrate the sequence of domains in the protein structure: LN -K1 -L1 K2 -L2 -K3 (according to Segrest et al. [76,77] and Chatterton et al. [74]). The blue-toned bars represent antiparallel L-sheets, the redtoned bars amphipathic K-helices, and the red circles transmembrane K-helices. This model clearly illustrates that apoB-100 should not be considered to be a homogeneous belt surrounding an LDL particle, but a highly organized molecule consisting of structurally and functionally distinct areas. Note also that one hemisphere of LDL is mostly covered by apoB-100, whereas there are large amounts of lipids exposed on the other hemisphere. The principles of the model construction are explained in the text (in Section 4). See also animation at http://www.elsevier.nl/homepage/sah/multimedia/lsvl.htm.

dal HDL particles. In this model, two apoA-I molecules form a beltwise wrapped antiparallel dimer that circulates and covers the hydrophobic fatty acid tails of the phospholipids [1,2]. The structural arrangement of the apoA-I molecules in mature spherical HDL particles is suggested to be similar to that in the discoidal particles [2,79]. Therefore, since the K2 domain of apoB-100 and apoA-I might be expected to have matching structural characteristics, the individual K-helices in the K2 -domain could adjust for the size changes in the particles by adopting a more beltlike conformation in the larger VLDL and intermediate density lipoprotein (IDL) particles than in the smaller LDL particles. The K3 domain is structurally similar to the K2 domain, but, since it

crosses the receptor-binding domain of apoB-100, it may have a crucial role in controlling the cellular uptake of apoB-containing particles in the circulation [74]. In fact, there are data to suggest that, in normal VLDL particles, the K3 domain could inhibit binding to the LDL receptor, but in IDL and LDL particles the conformation of the K3 domain would be changed so that it would not mask the LDL receptor binding site [74]. 4.4. ApoB-100^lipid-interactions Segrest and co-workers [77] have suggested that whether the individual L-strands or L-sheets interact with lipids would be determined by local conditions.

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They have also proposed that large anti-parallel amphipathic L-sheets amount to an in¢nitely high lipid a¤nity. The L-sheet regions of apoB-100 would therefore be key structural areas for the integrity of LDL particles and represent irreversibly lipid-associating domains. This view is supported by the infrared spectroscopy work of Goormaghtigh et al. [80], which showed that the apoB-100 peptides remaining in the LDL particles after exposure to di¡erent proteases were rich in L-sheet structures and that some, at least, of the L-sheets were associated with the phospholipid components of the particles. Moreover, the results of proton, phosphorus, and carbon NMR spectroscopy studies of LDL and HDL particles have demonstrated that in LDL (but not in HDL, where all the apo components lack L-structures), a portion of the PC headgroups is immobilized [7,81,82]. Surprisingly, immobilization of PC in LDL was not observed by phosphorus spin-lattice relaxation in LDL [19]. Time-resolved £uorescence spectroscopy of £uorescent analogs of PC and SM incorporated in LDL particles have suggested that apoB-100 discriminates between the phospholipids and that PC is more closely associated with the protein than SM [8,83]. Correspondingly, recent 1 H NMR spectroscopy of LDL particles of indicated distinct behavior for the phosphocholine headgroups of PC and SM and preferential immobilization of this particular molecular group in PC [84]. On average, about 27% of the PC headgroups were immobile in native LDL (corresponding to a value of 19% for the total phospholipid pool) but modest proteolysis of apoB-100 was su¤cient to make a portion of these PC molecules NMR-visible. A possible mode of interaction between the L-sheet structures and the PC molecules, capable of resulting in immobilization of the HGs and explaining the e¡ect of proteolysis, is presented in Fig. 4. The causes of the di¡erence in the interactions of PC and SM with apoB-100 are not currently clear. As indicated in Fig. 2, it is likely that parts of apoB-100 are in contact with the lipids in all the three concentric layers of LDL particles. Therefore, it would not be surprising if the various lipid constituents and the lipid composition of LDL in£uenced the conformation of apoB-100. In fact, there is evidence that the amount of TG in LDL particles a¡ects the conformation of apoB-100 [20,85,86]. The

compositional changes are interconnected with changes in the size in LDL subpopulations, which, in turn, leads to an inevitable adjustment of apoB100 conformation [87]. However, McKeone and coworkers have shown that LDL particles enriched in TG, but unchanged in size, exhibited the same dosedependent changes in binding to the B/E-receptor on HepG2 cells as LDL isolated from hypertriglyceridemic subjects [86]. It is therefore likely that TG also has a size-independent e¡ect on the tertiary structure of LDL apoB-100. Since the CD spectra indicate that there are no changes in the secondary structure of apoB-100 due to TG enrichment, the conformational di¡erences are likely to be rather small and/or of a local character [20,86]. Further evidence for lipid-induced changes in apoB-100 conformation comes from the infrared spectroscopy study of Banuelos and co-workers, who showed that cooling the temperature of LDL particles below the phase transition temperature of the core lipids results in reversible changes in the apoB-100 conformation, namely, a decrease in the number of K-helices, L-strands, and unordered regions and an increase in the number of L-sheets and L-turns [88]. 5. Modi¢ed LDL Numerous hydrolytic enzymes and pro-oxidative agents are present in the arterial intima. Thus, the arterial intima has been shown to contain proteases like mast cell chymase and tryptase [89], plasmin [90], matrix metalloproteinases [91^94] and lysosomal proteases [95], lipases like secretory SMase [10,96] and phospholipase A2 (PLA2 ) [97,98], and cholesterol esterase (CEase) [99,100]. Furthermore, at least myeloperoxidase and 15-lipoxygenase but also other oxidative systems are likely to contribute to lipoprotein oxidation in the arterial intima [101]. These modifying substances have a key role in transforming the LDL particles into the extracellular lipid droplets and vesicles found in the intima in early atherogenesis [16,102]. Indeed, in vitro studies have demonstrated that treatment of LDL particles with either proteolytic or lipolytic enzymes and also with extensive oxidation will induce aggregation and fusion of the particles [14,103^106]. In the following paragraphs, illustrated by Fig. 6, the structural e¡ects

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Fig. 6. Schematic illustration of characteristic structural aspects in various modi¢ed LDL particles. The key to the molecules used in the modeling is given in the box at the bottom. (A) Native LDL particle. (B) An LDL particle proteolysed by K-chymotrypsin. Hydrolysis of apoB-100 and the release of its pieces from the particle are expected to loosen the surface and result in reorganization of the lipids in the particle. (C) An LDL particle lipolysed with SMase that hydrolyzes SM into CER and phosphocholine headgroups. Being hydrophilic, the latter leave the particle, whereas the CER molecules are likely to cluster and form a potentially hydrophobic spot at the surface of the particle. (D) An LDL particle lipolysed with PLC that hydrolyzes PC into DAG and HG. Similar to SMase, PLC may produce hydrophobic surface areas and lead to a more hydrophobic LDL particle as well as to some reorganization of the lipid molecules, including partitioning of the DAG in the core. (E) An LDL particle lipolysed with PLA2 in the presence of albumin. PLA2 hydrolyzes the PC molecules into lyso-PC and FFA molecules. The latter are mainly sequestered from the particles by albumin. This modi¢cation induces tighter packing of the particle surface and also results in a slight decrease in particle size. (F) A GAG-bound LDL particle lipolysed with PLA2 in the presence of albumin. On the basis of observations that treatment of GAGbound LDL particles with PLA2 can produce particle aggregation and fusion, the LDL particle here is visualized as not shrunken and as being less rigid than the particle in the absence of GAG. (G) An LDL particle modi¢ed with trypsin and CEase. Trypsin proteolysis induces loosening of the particle structure to such extent that CEase can e¡ectively lipolyse the (increased number of) CE molecules from the interfacial layer of the particle. Hydrolysis of CE molecules leads to the formation of large amounts of UC which, together with PC and SM, can form an UC rich bilayer membrane and induce vesicle formation. (H) Some structural features of an LDL particle oxidized to an intermediate degree. Lipid fatty acids throughout the particle have been oxidized to contain large amounts of lipid radicals and some lipid hydroperoxides. Some PC molecules have been transformed to lyso-PC (via intrinsic PLA2 activity) and some lipid dimers have also been formed. Decomposition of oxidized lipids have created lipid aldehydes, some of which interact with apoB-100. Oxidation also leads to fragmentation of apoB-100.

of various modi¢cations on LDL particles are discussed. An important focus will be on the structural di¡erences that induce aggregation of native-sized particles in contrast to irreversible fusion and generation of enlarged particles.

5.1. Proteolytic modi¢cations Proteolysis of LDL particles has been studied with several di¡erent proteases, including plasmin, kallikrein, and thrombin, which induce only fragmenta-

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tion of apoB-100, and with trypsin, K-chymotrypsin and pronase, which degrade apoB-100 extensively [107]. An important ¢nding was that fusion of LDL particles requires not only fragmentation of apoB-100 but also loss of the apoB-100 fragments. The structural e¡ects of extensive apoB-100 proteolysis on an LDL particle are illustrated in Fig. 6B. The loosening of the lipid packing on the surface is likely to result in increased penetration of the hydrophobic core molecules toward the particle surface, thereby increasing the hydrophobicity of the proteolysed particles. This, in turn, is expected to lead to enhanced hydrophobic interactions between the particles, allowing their aggregation and subsequent fusion. The fusion processes generate an excess of amphipathic lipid material that has a tendency to form multilayered structures which remain attached to the fused LDL particles [108,109]. In fact, carbon NMR spectroscopy experiments of LDL particles and human aortic tissue with ¢brous plaques have suggested at least partially disrupted LDL structures present in the intima [110]. 5.2. SMase Treatment of LDL particles with SMase from Bacillus cereus has been shown to induce both aggregation and fusion of the particles [103,104,111]. SMase cleaves a SM molecule into a CER and a phosphocholine headgroup (HG). The latter, being hydrophilic, are released and the former, being hydrophobic, accumulate in the LDL particles. The structural e¡ects of SMase treatment on an LDL particle are illustrated in Fig. 6C. The aggregation and fusion of SMase-treated LDL particles has been shown to depend on the accumulation of CER within the particles [10]. Most likely the key molecular mechanism behind the observed changes in a SMase-treated LDL population is formation of CER-enriched domains that are likely to be tighter than the various nanoenvironments present in the native LDL particles. SMase has recently been shown to induce formation of CER-enriched domains in a PC^SM membrane [112]. Therefore, the CER-enriched domains, once formed, could act as nonpolar spots at the surface of the particles and lead to particle aggregation through hydrophobic associations between the domains in di¡erent

particles [103,111]. It has been observed that substantial aggregation and fusion of LDL particles occur only after the majority of the SM molecules of the particles have been hydrolyzed [103]. This supports the view that the driving force for the particle aggregation is formation of CER-enriched domains. The SM molecules on an LDL particle cover only approximately 15% of the surface volume and, therefore, a substantial proportion of the SM molecules must be hydrolyzed before the CER-enriched domains can organize. In addition, it has been observed that treatment of LDL particles with SMase at low temperature (15³C) does not lead to aggregation of the particles, even though SM is hydrolyzed [111]. However, if these SMase-treated particles are subsequently incubated at 37³C, they aggregate and fuse. This is understood from the interactions of the lipid molecules in and between the core and interfacial layers of the LDL particles. At 15³C, the core lipids of LDL are in a radially ordered liquid crystalline phase and at 37³C in a liquid-like state. Therefore, at the lower temperature, the oriented rigid core lipids can order the interfacial layer and thereby inhibit lateral di¡usion of the surface phospholipids. Consequently, formation of the CER-enriched domains should also be at least partially inhibited. This argument is also supported by the ¢ndings that the reorganization process is much slower than the enzymatic formation of CER [112]. Thus, the observed behavior of SMasetreated LDL particles below and above the transition temperature of the core lipids gives further support to the idea that the initial SMase-induced aggregation of LDL particles takes place via the formation of hydrophobic CER-enriched domains in the modi¢ed LDL particles. The observed fusion of SMase-treated LDL particles is also likely to relate to the molecular properties of CER. A transition from a lamellar to an inverted hexagonal phase in phospholipid bilayers is facilitated by CER, most likely because the volume requirement of the fatty acid chains is large compared with that of the headgroup [113]. Moreover, formation of an inverted hexagonal phase has been suggested to induce membrane fusion at an early stage [114] and under certain conditions CER is known to induce fusion of liposomes [115].

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5.3. Phospholipase C (PLC) PLC hydrolyzes a PC molecule into a DAG and a phosphocholine headgroup (HG). Like SMasetreated LDL, the hydrophilic headgroups are released, while the hydrophobic DAG molecules accumulate in the particles. PLC treatment of LDL particles has been shown to result in instability of the particles [116], increased turbidity of the sample [117] and both aggregation and fusion of the particles [118]. The structural e¡ects of PLC treatment on an LDL particle are illustrated in Fig. 6D. The hydrophobicity of the DAG molecules is likely to lead to their distribution to both the interfacial and core layers of the LDL particles, in the same way as insect lipoproteins, in which DAG molecules are the main constituents of the particles [119]. This generation of DAG from the amphipathic PC molecules will strongly increase the hydrophobicity of the LDL particles. Even though the redistribution of part of the generated DAG pool into the core regions of the particles could decrease the surface hydrophobicity, it is likely that many of the places left open by the DAG molecules in the interfacial layer can only be occupied by hydrophobic core lipids. Therefore, e¡ective hydrophobic attachment of PLC-treated LDL particles seems inevitable. This view is supported by the ¢ndings that, by binding to the hydrophobic surface areas, exchangeable apos A-I, E [120] and Manduca sexta apolipophorin III [118] can inhibit PLC-induced aggregation and fusion of LDL particles. In addition, treatment of LDL with PLC at low temperature (15³C), in contrast to the SMase-treatment, is su¤cient to lead to particle aggregation suggesting a predominant role for the breakdown of the main surface lipid component and for the accompanying increase in the hydrophobicity of the particles (Pentika«inen et al., unpublished). 5.4. PLA2 PLA2 hydrolyzes mainly fatty acid esters in the sn2 position of PC and thereby generates lyso-PC and free fatty acid (FFA) molecules. In the presence of a physiological albumin concentration, the fatty acid and lyso-PC molecules formed have a tendency to move from the LDL particles to albumin [121]. The

203

structural e¡ects of PLA2 treatment on an LDL particle (in the presence of albumin) are illustrated in Fig. 6E. Lipolysis of LDL particles by PLA2 , particularly if albumin is present, has been shown to alter the structure of the particles: both conformational changes in the apoB-100 component and a reorganization of lipids have been reported [121,122]. PLA2 lipolysis of LDL has been shown to lead to aggregation of some of the lipolysed particles in the presence of albumin, but does not trigger particle fusion [106]. This ¢nding indicates that even though PLA2 lipolysis is able to a¡ect the outcome of LDL particle interactions, the resultant structural changes are not su¤cient to promote fusion of the particles. There is evidence that PLA2 hydrolysis and loss of the PC fatty acids results in tighter packing and decreased mobility of the surface lipids by increasing interpenetration of the lyso-PC fatty acid chains and the core lipids [122]. This is also supported by the ¢ndings that the hydrolysis of LDL phospholipids by PLA2 slightly reduces the size of the LDL particles [106,121,123]. Therefore, whereas the hydrophobicity of such modi¢ed LDL particles may have increased their tendency to aggregate, it seems that the enhanced structural rigidity of the particles stabilizes the aggregates and precludes particle fusion. In contrast to lipolysis of native LDL, lipolysis of heparin-bound and even heparin-treated LDL particles has been shown to induce fusion among the aggregated particles [106]. An LDL particle hydrolyzed by PLA2 in the presence of heparin (and albumin) is illustrated in Fig. 6F. The interactions of LDL particles with proteoglycans and GAG have also been reported to cause changes in both the apoB-100 component and the lipid pool of the particles [124^128]. The interactions of LDL particles with GAGs have been shown to induce conformational changes in apoB-100 that increase the exposure of the arginine- and lysine-containing segments [126] and, in contrast to the PLA2 e¡ect on the lipid pool of LDL, decrease the organization of the core and surface regions of the particles [124,126]. The structural changes induced by GAGs do not by themselves lead to aggregation or fusion of LDL particles, but they have been shown to accelerate both proteolytic and oxidative modi¢cations of the particles [108,129]. The relative importance of the

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structural changes of LDL apoB-100 and the reorganization of LDL lipids on particle aggregation is unclear. It seems likely, however, that the (irreversible) destabilization of the particles induced by heparin is an important event that outweighs the rigidifying e¡ect of PLA2 on LDL, and thereby triggers fusion of the lipolysed particles [106]. 5.5. CEase Hydrolysis of LDL CEs with CEases produces lipid vesicles and small lipid droplets from the LDL particles [130,131]. For hydrolysis of the CEs in LDL particles, this enzyme requires some disruption of the apoB-100 component of the particles, e.g., proteolysis of apoB-100 by trypsin. These ¢ndings suggest that, in a native LDL particle, the number of CE molecules penetrating toward the surface and thereby accessible to the enzyme is not su¤cient or that the surface pressure is too high and that a certain degree of loosening due to proteolysis is necessary for e¤cient CE hydrolysis. Depletion of the hydrophobic CE core of LDL and the concomitant generation of UC results in an increase in the hydrophilic character of the particles. In addition, the excessive loss of core hydrophobicity leads to an energetically unfavorable situation for the hydrophobic fatty acid chains of the surface phospholipids. This, together with the fact that the UC molecules have a tendency to solubilize in membrane-like structures, explains the generation of liposomes and complex multilamellar structures from CEase-treated LDL particles [130,131]. An LDL particle hydrolyzed by (trypsin and) cholesteryl esterase is illustrated in Fig. 6G. 5.6. Oxidation Oxidation leads to changes of several types in the LDL particles. Depending on the initiator of oxidation, the early changes in the LDL particles are somewhat di¡erent. However, irrespective of the type of initiator, the progression of oxidation leads to loss of endogenous antioxidant molecules and polyunsaturated lipid fatty acids, and also to formation of Shi¡'s bases between aldehydes and lysines of apoB-100 and hydrolysis of apoB-100 (reviewed by Esterbauer) [6]. In addition, a wide range of biolog-

ically active products, such as peroxides, aldehydes, lyso-PC, and oxysterols, are produced. Some molecular aspects that contribute to intermediately oxidized LDL particles are illustrated in Fig. 6H. On the basis of the numerous changes occurring during oxidation, the changes in the structure of LDL particles have also been di¤cult to analyze. A mild degree of oxidation has already been shown to induce aggregation of the particles, an e¡ect likely to be due to cross-linking of the particles by lipid aldehydes [132]. Extensive oxidation of LDL, in turn, has been shown to lead to loss of particle integrity. Thus, in electron microscopy, fused particles, lipid vesicles, and pieces of membranous material have been observed [14,105]. 6. Concluding remarks Current knowledge of the molecular characteristics and structural details of LDL enables understanding of important relationships between structure and function in these particles. It has become apparent that interaction preferences between the di¡erent molecular components lead to the formation of speci¢c nanoenvironments in the particles. These nanoenvironments, are not only of structural importance, but most likely they also in£uence many processes of LDL metabolism. For example, by a¡ecting the properties of the interfacial layer of the particles, the intrinsic organization of lipids can control, for instance, the functioning of lipid transfer proteins and the activity of lipolytic enzymes. Even though the current molecular model of native LDL particles provides a comprehensive view that accords with the experimental evidence, there are numerous unanswered questions which call for further experimentation. The tertiary structure of apoB-100 in LDL particles is still the most approximated aspect in the presented models. For apoA-I and discoidal HDL particles, in contrast to LDL, detailed models have been recently developed, which have underlined the impact that atomic level models can have for the understanding of lipoprotein structure and function. Unfortunately, the size and complexity of LDL particles, and the accompanying experimental and computational problems, currently preclude all-inclusive atomic models of LDL. It

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seems likely, however, that more detailed models of molecular assemblies characteristic of LDL particles will become available in the near future. Extensive knowledge of the structure of native LDL particles can also be utilized to understand and explain the molecular characteristics of variously modi¢ed, potentially atherogenic LDL particles. Many factors capable of modifying LDL particles have been found in the arterial intima. Therefore, in the arterial wall the LDL particles undergo not only a single modi¢cation but a combination of several. Thus, for understanding the complex processes that are likely to occur in vivo, it is necessary to understand the molecular interactions occurring in native and variously modi¢ed LDL particles.

[8]

[9]

[10]

[11]

Acknowledgements The Wihuri Research Institute is maintained by the Jenny and Antti Wihuri Foundation. This work was supported by grants from the Academy of Finland, the Juselius Foundation, the Federation of Finnish Insurance Companies, and the Ida Montin Foundation. References

[12]

[13]

[14]

[1] J.P. Segrest, M.K. Jones, A.E. Klon, C.J. Sheldahl, M. Hellinger, H. De Loof, S.C. Harvey, A detailed molecular belt model for apolipoprotein A-I in discoidal high density lipoprotein, J. Biol. Chem. 274 (1999) 31755^31758. [2] J.P. Segrest, L. Li, G.M. Anantharamaiah, S.C. Harvey, K.N. Liadaki, V. Zannis, Structure and function of apolipoprotein A-I and high-density lipoprotein, Curr. Opin. Lipidol. 11 (2000) 105^115. [3] J.M. Steim, O.J. Edner, F.G. Bargoot, Structure of human serum lipoproteins nuclear magnetic resonance supports a micellar model, Science 162 (1968) 909^911. [4] R.L. Jackson, J.D. Morrisett, A.M. Gotto Jr., Lipoprotein structure and metabolism, Physiol. Rev. 56 (1976) 259^ 316. [5] J. Lounila, M. Ala-Korpela, J. Jokisaari, M.J. Savolainen, Y.A. Kesa«niemi, E¡ects of orientational order and particle size on the NMR line positions of lipoproteins, Phys. Rev. Lett. 72 (1994) 4049^4052. [6] H. Esterbauer, J. Gebicki, H. Puhl, G. Ju«rgens, The role of lipid peroxidation and antioxidants in oxidative modi¢cation of LDL, Free Radic. Biol. Med. 13 (1992) 341^390. [7] S. Lund-Katz, M.C. Phillips, Packing of cholesterol mole-

[15]

[16]

[17]

[18]

[19]

[20]

205

cules in human low-density lipoprotein, Biochemistry 25 (1986) 1562^1568. A. Sommer, E. Prenner, R. Gorges, H. Stu«tz, H. Grillhofer, G.M. Kostner, F. Paltauf, A. Hermetter, Organization of phosphatidylcholine and sphingomyelin in the surface monolayer of low density lipoprotein (a) as determined by timeresolved £uorometry, J. Biol. Chem. 267 (1992) 24217^ 24222. F. Lalanne, V. Pruneta, S. Bernard, G. Ponsin, Distribution of diacylglycerols among plasma lipoproteins in control subjects and in patients with non-insulin-dependent diabetes, Eur. J. Clin. Invest. 29 (1999) 139^144. S.L. Schissel, J. Tweedie-Hardman, J.H. Rapp, G. Graham, K.J. Williams, I. Tabas, Rabbit aorta and human atherosclerotic lesions hydrolyze the sphingomyelin of retained low-density lipoprotein. Proposed role for arterial-wall sphingomyelinase in subendothelial retention and aggregation of atherogenic lipoproteins, J. Clin. Invest. 98 (1996) 1455^1464. A. Ravandi, A. Kuksis, N.A. Shaikh, Glycated phosphatidylethanolamine promotes macrophage uptake of low density lipoprotein and accumulation of cholesteryl esters and triacylglycerols, J. Biol. Chem. 274 (1999) 16494^16500. N. Simionescu, E. Vasile, F. Lupu, G. Popescu, M. Simionescu, Prelesional events in atherogenesis. Accumulation of extracellular cholesterol-rich liposomes in the arterial intima and cardiac valves of the hyperlipidemic rabbit, Am. J. Pathol. 123 (1986) 109^125. J.S. Frank, A.M. Fogelman, Ultrastructure of the intima in WHHL and cholesterol-fed rabbit aortas prepared by ultrarapid freezing and freeze-etching, J. Lipid Res. 30 (1989) 967^978. D. Tirziu, A. Dobrian, C. Tasca, M. Simionescu, N. Simionescu, Intimal thickenings of human aorta contain modi¢ed reassembled lipoproteins, Atherosclerosis 112 (1995) 101^ 114. K.J. Williams, I. Tabas, The response-to-retention hypothesis of atherogenesis reinforced, Curr. Opin. Lipidol. 9 (1998) 471^474. ë o«rni, M. Ala-Korpela, P.T. KovaM.O. Pentika«inen, K. O nen, Modi¢ed LDL-trigger of atherosclerosis and in£ammation in the arterial intima, J. Intern. Med. 247 (2000) 359^ 370. G.S. Ginsburg, D.M. Small, D. Atkinson, Microemulsions of phospholipids and cholesterol esters. Protein-free models of low density lipoprotein, J. Biol. Chem. 257 (1982) 8216^ 8227. R.S. Chana, W.D. Treleaven, Y.I. Parmar, R.J. Cushley, Dynamic structure of the lower density lipoproteins. II. Deuterium NMR studies of the monolayer of very low and low density lipoproteins, Biochem. Cell Biol. 68 (1990) 189^198. D.B. Fenske, R.S. Chana, Y.I. Parmar, W.D. Treleaven, R.J. Cushley, Structure and motion of phospholipids in human plasma lipoproteins. A 31 P NMR study, Biochemistry 29 (1990) 3973^3981. M. Aviram, S. Lund-Katz, M.C. Phillips, A. Chait, The

BBAMCB 55697 6-11-00 Cyaan Magenta Geel Zwart

206

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28] [29]

[30]

[31]

[32]

[33]

[34]

[35]

T. Hevonoja et al. / Biochimica et Biophysica Acta 1488 (2000) 189^210 in£uence of the triglyceride content of low density lipoprotein on the interaction of apolipoprotein B-100 with cells, J. Biol. Chem. 263 (1988) 16842^16848. G.C. Chen, M.J. Chapman, J.P. Kane, Secondary structure and thermal behavior of trypsin-treated low-density lipoproteins from human serum, studied by circular dichroism, Biochim. Biophys. Acta 754 (1983) 51^56. H. Saito, T. Minamida, I. Arimoto, T. Handa, K. Miyajima, Physical states of surface and core lipids in lipid emulsions and apolipoprotein binding to the emulsion surface, J. Biol. Chem. 271 (1996) 15515^15520. P.A. Kroon, Fluorescence study of the motional states of core and surface lipids in native and reconstituted low density lipoproteins, Biochemistry 33 (1994) 4879^4884. P.A. Kroon, The order^disorder transition of the core cholesteryl esters of human plasma low density lipoprotein. A proton nuclear magnetic resonance study, J. Biol. Chem. 256 (1981) 5332^5339. M. Ala-Korpela, J. Oja, J. Lounila, J. Jokisaari, M.J. Savolainen, Y.A. Kesa«niemi, Structural changes of lipoprotein lipids by 1 H NMR, Chem. Phys. Lett. 242 (1995) 95^100. R.J. Deckelbaum, G.G. Shipley, D.M. Small, Structure and interactions of lipids in human plasma low density lipoproteins, J. Biol. Chem. 252 (1977) 744^754. T.P.W. McMullen, R.N. McElhaney, Physical studies on cholesterol^phospholipid interactions, Curr. Opin. Coll. Interf. Sci. 1 (1996) 83^90. P.L. Yeagle, Cholesterol and the cell membrane, Biochim. Biophys. Acta 822 (1985) 267^287. P.L. Yeagle, W.C. Hutton, C.H. Huang, R.B. Martin, Headgroup conformation and lipid^cholesterol association in phosphatidylcholine vesicles: a 31 P(1 H) nuclear Overhauser e¡ect study, Proc. Natl. Acad. Sci. USA 72 (1975) 3477^ 3481. P.L. Yeagle, R.B. Martin, L. Pottenger, R.G. Langdon, Location and interactions of phospholipid and cholesterol in human low density lipoprotein from 31 P nuclear magnetic resonance, Biochemistry 17 (1978) 2707^2710. P.L. Yeagle, Cholesterol rotation in phospholipid vesicles as observed by 13 C-NMR, Biochim. Biophys. Acta 640 (1981) 263^273. M.W. Rooney, Y. Lange, J.W. Kau¡man, Acyl chain organization and protein secondary structure in cholesterolmodi¢ed erythrocyte membranes, J. Biol. Chem. 259 (1984) 8281^8285. S. Mabrey, P.L. Mateo, J.M. Sturtevant, High-sensitivity scanning calorimetric study of mixtures of cholesterol with dimyristoyl- and dipalmitoylphosphatidylcholines, Biochemistry 17 (1978) 2464^2468. P.L. Yeagle, W.C. Hutton, C. Huang, R.B. Martin, Phospholipid head-group conformations; intermolecular interactions and cholesterol e¡ects, Biochemistry 16 (1977) 4344^ 4349. M.F. Brown, J. Seelig, In£uence of cholesterol on the polar region of phosphatidylcholine and phosphatidylethanolamine bilayers, Biochemistry 17 (1978) 381^384.

[36] W.-G. Wu, S.-W. Leu, C.-H. Hsieh, L.-M. Chi, The order of phosphatidylcholine headgroup in sonicated cholesterol^ phospholipid vesicles as revealed by 1 H-NMR, Chem. Phys. Lipids 58 (1991) 241^248. [37] P.L. Yeagle, J.E. Young, Factors contributing to the distribution of cholesterol among phospholipid vesicles, J. Biol. Chem. 261 (1986) 8175^8181. [38] H. Ohvo, C. Olsio, J.P. Slotte, E¡ects of sphingomyelin and phosphatidylcholine degradation on cyclodextrin-mediated cholesterol e¥ux in cultures ¢broblasts, Biochim. Biophys. Acta 1349 (1997) 131^141. [39] P. Mattjus, J.P. Slotte, Does cholesterol discriminate between sphingomyelin and phosphatidylcholine in mixed monolayers containing both phospholipids, Chem. Phys. Lipids 81 (1996) 69^80. [40] B. Ramstedt, J.P. Slotte, Interaction of cholesterol with sphingomyelin and acyl-chain-matched phosphatidylcholines: a comparative study of the e¡ect of the chain length, Biophys. J. 76 (1999) 908^915. [41] C.C. Kan, Z.S. Ruan, R. Bittman, Interaction of cholesterol with sphingomyelin in bilayer membranes evidence that the hydroxy group of sphingomyelin does not modulate the rate of cholesterol exchange between vesicles, Biochemistry 30 (1991) 7759^7766. [42] S. Lund-Katz, H.M. Laboda, L.R. McLean, M.C. Phillips, In£uence of molecular packing and phospholipid type on rates of cholesterol exchange, Biochemistry 27 (1988) 3416^ 3423. [43] P.J. Spooner, J.A. Hamilton, D.L. Gantz, D.M. Small, The e¡ect of free cholesterol on the solubilization of cholesteryl oleate in phosphatidylcholine bilayers: A 13 C-NMR study, Biochim. Biophys. Acta 860 (1986) 345^353. [44] P.J. Spooner, D.M. Small, E¡ect of free cholesterol on incorporation of triolein in phospholipid bilayers, Biochemistry 26 (1987) 5820^5825. [45] Q.-T. Li, W.H. Sawyer, E¡ect of unesteri¢ed cholesterol on the compartmentation of a £uorescent cholesteryl ester in a lipoprotein-like lipid microemulsion, J. Lipid Res. 33 (1992) 503^512. [46] H. Akutsu, T. Nagamori, Conformational analysis of the polar headgroup in phosphatidylcholine bilayers a structural change induced by cations, Biochemistry 30 (1991) 4510^ 4516. [47] G. Bu«ldt, R. Wohlgemuth, The headgroup conformation of phospholipids in membranes, J. Membr. Biol. 58 (1981) 81^ 100. [48] H. Hauser, I. Pascher, R.H. Pearson, S. Sundell, Preferred conformation and molecular packing of phosphatidylethanolamine and phosphatidylcholine, Biochim. Biophys. Acta 650 (1981) 21^51. [49] J. Seelig, H.-U. Gally, R. Wohlgemuth, Orientation and £exibility of the choline head group in phophatidylcholine bilayers, Biochim. Biophys. Acta 467 (1977) 109^119. [50] J. Seelig, A. Seelig, Lipid conformation in model membranes and biological membranes, Q. Rev. Biophys. 13 (1980) 19^ 61.

BBAMCB 55697 6-11-00 Cyaan Magenta Geel Zwart

T. Hevonoja et al. / Biochimica et Biophysica Acta 1488 (2000) 189^210 [51] D.P. Tieleman, S.J. Marrink, H.J. Berendsen, A computer perspective of membranes: molecular dynamics studies of lipid bilayer systems, Biochim. Biophys. Acta 1331 (1997) 235^270. [52] D.J. Tobias, K. Tu, M.L. Klein, Atomic-scale molecular dynamics simulations of lipid membranes, Curr. Opin. Coll. Interf. Sci. 2 (1997) 15^26. [53] M.T. Hyvo«nen, T.T. Rantala, M. Ala-Korpela, Structure and dynamic properties of diunsaturated 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine lipid bilayer from molecular dynamics simulation, Biophys. J. 73 (1997) 2907^2923. [54] P.G. Scherer, J. Seelig, Electric charge e¡ects on phospholipid headgroups. Phosphatidylcholine in mixtures with cationic and anionic amphiphiles, Biochemistry 28 (1989) 7720^ 7728. [55] M.W. Baumstark, W. Kreutz, A. Berg, I. Frey, J. Keul, Structure of human low-density lipoprotein subfractions, determined by X-ray small-angle scattering, Biochim. Biophys. Acta 1037 (1990) 48^57. [56] J.P. Slotte, L. Gro«nberg, Oxidation of cholesterol in low density and high density lipoproteins by cholesterol oxidase, J. Lipid Res. 31 (1990) 2235^2242. [57] J.A. Ibdah, S. Lund-Katz, M.C. Phillips, Molecular packing of high-density and low-density lipoprotein surface lipids and apolipoprotein A-I binding, Biochemistry 28 (1989) 1126^1133. [58] I. Arimoto, H. Saito, Y. Kawashima, K. Miyajima, T. Handa, E¡ects of sphingomyelin and cholesterol on lipoprotein lipase-mediated lipolysis in lipid emulsions, J. Lipid Res. 39 (1998) 143^151. [59] M.S. Nenseter, A.C. Rustan, S. Lund-Katz, E. Soyland, G. Maelandsmo, M.C. Phillips, C.A. Drevon, E¡ect of dietary supplementation with n-3 polyunsaturated fatty acids on physical properties and metabolism of low density lipoprotein in humans, Arterioscler. Thromb. 12 (1992) 369^379. [60] J.S. Parks, H. Hauser, Low density lipoprotein particle size and core cholesteryl ester physical state a¡ect the proton NMR magnetic environment of fatty acid methylene and methyl nuclei, J. Lipid Res. 37 (1996) 1289^1297. [61] M. Pregetter, R. Prassl, B. Schuster, M. Kriechbaum, F. Nigon, J. Chapman, P. Laggner, Microphase separation in low density lipoproteins. Evidence for a £uid triglyceride core below the lipid melting transition, J. Biol. Chem. 274 (1999) 1334^1341. [62] J.A. Hamilton, D.M. Small, J.S. Parks, 1 H NMR studies of lymph chylomicra and very low density lipoproteins from nonhuman primates, J. Biol. Chem. 258 (1983) 1172^ 1179. [63] J.D. Bell, M.L. Barnard, H.G. Parkes, E.L. Thomas, C.H. Brennan, S.C. Cunnane, P.C. Dagnelie, E¡ects of n-3 fatty acids on the NMR pro¢le of plasma lipoproteins, J. Lipid Res. 37 (1996) 1664^1674. [64] T.J. Knott, R.J. Pease, L.M. Powell, S.C. Wallis, S.C. Rall Jr., T.L. Innerarity, B. Blackhart, W.H. Taylor, Y. Marcel, R. Milne, Complete protein sequence and identi¢cation of

[65]

[66]

[67]

[68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

207

structural domains of human apolipoprotein B, Nature 323 (1986) 734^738. C.Y. Yang, S.H. Chen, S.H. Gianturco, W.A. Bradley, J.T. Sparrow, M. Tanimura, W.H. Li, D.A. Sparrow, H. DeLoof, M. Rosseneu, Sequence, structure, receptor-binding domains and internal repeats of human apolipoprotein B100, Nature 323 (1986) 738^742. V.N. Schumaker, M.L. Phillips, J.E. Chatterton, Apolipoprotein B and low-density lipoprotein structure: implications for biosynthesis of triglyceride-rich lipoproteins, Adv. Protein Chem. 45 (1994) 205^248. J. Bore¨n, I. Lee, W. Zhu, K. Arnold, S. Taylor, T.L. Innerarity, Identi¢cation of the low density lipoprotein receptorbinding site in apolipoprotein B100 and the modulation of its binding activity by the carboxyl terminus in familial defective apo-B100, J. Clin. Invest. 101 (1998) 1084^1093. P. Sivaram, S.Y. Choi, L.K. Curtiss, I.J. Goldberg, An amino-terminal fragment of apolipoprotein B binds to lipoprotein lipase and may facilitate its binding to endothelial cells, J. Biol. Chem. 269 (1994) 9409^9412. D.M. Sta¡orini, L.W. Tjoelker, S.P. McCormick, D. Vaitkus, T.M. McIntyre, P.W. Gray, S.G. Young, S.M. Prescott, Molecular basis of the interaction between plasma plateletactivating factor acetylhydrolase and low density lipoprotein, J. Biol. Chem. 274 (1999) 7018^7024. K.H. Weisgraber, S.C. Rall Jr., Human apolipoprotein B100 heparin-binding sites, J. Biol. Chem. 262 (1987) 11097^ 11103. N. Hirose, D.T. Blankenship, M.A. Krivanek, R.L. Jackson, A.D. Cardin, Isolation and characterization of four heparinbinding cyanogen bromide peptides of human plasma apolipoprotein B, Biochemistry 26 (1987) 5505^5512. G. Camejo, S.O. Olofsson, F. Lopez, P. Carlsson, G. Bondjers, Identi¢cation of apoB-100 segments mediating the interaction of low density lipoproteins with arterial proteoglycans, Arteriosclerosis 8 (1988) 368^377. J. Bore¨n, K. Olin, I. Lee, A. Chait, T.N. Wight, T.L. Innerarity, Identi¢cation of the principal proteoglycan-binding site in LDL. A single-point mutation in apo-B100 severely a¡ects proteoglycan interaction without a¡ecting LDL receptor binding, J. Clin. Invest. 101 (1998) 2658^2664. J.E. Chatterton, M.L. Phillips, L.K. Curtiss, R. Milne, J.C. Fruchart, V.N. Schumaker, Immunoelectron microscopy of low density lipoproteins yields a ribbon and bow model for the conformation of apolipoprotein B on the lipoprotein surface, J. Lipid Res. 36 (1995) 2027^2037. R. van Antwerpen, G.C. Chen, C.R. Pullinger, J.P. Kane, M. LaBelle, R.M. Krauss, C. Luna-Chavez, T.M. Forte, J.C. Gilkey, Cryo-electron microscopy of low density lipoprotein and reconstituted discoidal high density lipoprotein: imaging of the apolipoprotein moiety, J. Lipid Res. 38 (1997) 659^ 669. J.P. Segrest, M.K. Jones, V.K. Mishra, G.M. Anantharamaiah, D.W. Garber, apoB-100 has a pentapartite structure composed of three amphipathic K-helical domains alternating with two amphipathic beta-strand domains. Detection by

BBAMCB 55697 6-11-00 Cyaan Magenta Geel Zwart

208

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

[87]

[88]

[89]

T. Hevonoja et al. / Biochimica et Biophysica Acta 1488 (2000) 189^210 the computer program LOCATE, Arterioscler. Thromb. 14 (1994) 1674^1685. J.P. Segrest, M.K. Jones, V.K. Mishra, V. Pierotti, S.H. Young, J. Boren, T.L. Innerarity, N. Dashti, Apolipoprotein B-100: conservation of lipid-associating amphipathic secondary structural motifs in nine species of vertebrates, J. Lipid Res. 39 (1998) 85^102. C. Wilson, M.R. Wardell, K.H. Weisgraber, R.W. Mahley, D.A. Agard, Three-dimensional structure of the LDL receptor-binding domain of human apolipoprotein E, Science 252 (1991) 1817^1822. D.W. Borhani, D.P. Rogers, J.A. Engler, C.G. Brouillette, Crystal structure of truncated human apolipoprotein A-I suggests a lipid-bound conformation, Proc. Natl. Acad. Sci. USA 94 (1997) 12291^12296. E. Goormaghtigh, V. Cabiaux, J.D. Meutter, M. Rosseneu, J.-M. Ruysschaert, Secondary structure of the particle associating domain of apolipoprotein B-100 in low-density lipoprotein by attenuated total re£ection infrared spectroscopy, Biochemistry 32 (1993) 6104^6110. E.G. Finer, R. Henry, R.B. Leslie, R.N. Robertson, NMR studies of pig low- and high-density serum lipoproteins. Molecular motions and morphology, Biochim. Biophys. Acta 380 (1975) 320^337. P.L. Yeagle, R.G. Langdon, R.B. Martin, Phospholipid^ protein interactions in human low density lipoprotein detected by 31 P nuclear magnetic resonance, Biochemistry 16 (1977) 3487^3491. A. Hermetter, A. Sommer, R. Gorges, E. Prenner, G.M. Kostner, F. Paltauf, Apoprotein^phospholipid interactions in Lp(a), Chem. Phys. Lipids 67/68 (1994) 115^119. H.C. Murphy, M. Ala-Korpela, J.W. White, A. Raoof, J.D. Bell, M.L. Barnard, S.P. Burns, R.A. Iles, Evidence for distinct behaviour of phosphatidylcholine and sphingomyelin at the low density lipoprotein surface, Biochem. Biophys. Res. Commun. 234 (1997) 733^737. M. Kinoshita, E.S. Krul, G. Schonfeld, Modi¢cation of the core lipids of low density lipoproteins produces selective alterations in the expression of apoB-100 epitopes, J. Lipid Res. 31 (1990) 701^708. B.J. McKeone, J.R. Patsch, H.J. Pownall, Plasma triglycerides determine low density lipoprotein composition, physical properties, and cell-speci¢c binding in cultured cells, J. Clin. Invest. 91 (1993) 1926^1933. J.R. McNamara, D.M. Small, Z. Li, E.J. Schaefer, Di¡erences in LDL subspecies involve alterations in lipid composition and conformational changes in apolipoprotein B, J. Lipid Res. 37 (1996) 1924^1935. S. Banuelos, J.L. Arrondo, F.M. Goni, G. Pifat, Surface^ core relationships in human low density lipoprotein as studied by infrared spectroscopy, J. Biol. Chem. 270 (1995) 9192^9196. M. Kaartinen, A. Penttila«, P.T. Kovanen, Mast cells of two types di¡ering in neutral protease composition in the human aortic intima. Demonstration of tryptase- and tryptase/chymase-containing mast cells in normal intimas, fatty streaks,

and the shoulder region of atheromas, Arterioscler. Thromb. 14 (1994) 966^972. [90] W.L. Hendriks, L.C. van Vark, K. Schoonderwoerd, H. Jansen, L.M. Havekes, Not the mature 56 kDa lipoprotein lipase protein but a 37 kDa protein copurifying with the lipase mediates the binding of low density lipoproteins to J774 macrophages, Biochem. J. 330 (1998) 765^769. [91] A.M. Henney, P.R. Wakeley, M.-J. Davies, K. Foster, R. Hembry, G. Murphy, S. Humphries, Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization, Proc. Natl. Acad. Sci. USA 88 (1991) 8154^ 8158. [92] Z.S. Galis, G.K. Sukhova, M.W. Lark, P. Libby, Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques, J. Clin. Invest. 94 (1994) 2493^2503. [93] Z.S. Galis, G.K. Sukhova, P. Libby, Microscopic localization of active proteases by in situ zymography detection of matrix metalloproteinase activity in vascular tissue, FASEB J. 9 (1995) 974^980. [94] G.K. Sukhova, G.P. Shi, D.I. Simon, H.A. Chapman, P. Libby, Expression of the elastolytic cathepsins S and K in human atheroma and regulation of their production in smooth muscle cells, J. Clin. Invest. 102 (1998) 576^583. [95] D.S. Leake, S.M. Rankin, J. Collard, Macrophage proteases can modify low density lipoproteins to increase their uptake by macrophages, FEBS Lett. 269 (1990) 209^212. [96] S. Marathe, G. Kuriakose, K.J. Williams, I. Tabas, Sphingomyelinase, an enzyme implicated in atherogenesis, is present in atherosclerotic lesions and binds to speci¢c components of the subendothelial extracellular matrix, Arterioscler. Thromb. Vasc. Biol. 19 (1999) 2648^2658. [97] M. Menschikowski, M. Kasper, P. Lattke, A. Schiering, S. Schiefer, H. Stockinger, W. Jaross, Secretory group II phospholipase A2 in human atherosclerotic plaques, Atherosclerosis 118 (1995) 173^181. [98] E. Hurt-Camejo, S. Andersen, R. Standahl, B. Rosengren, P. Sartipy, E. Stadberg, B. Johansen, Localization of nonpancreatic secretory phospholipase A2 in normal and atherosclerotic arteries. Activity of the isolated enzyme on lowdensity lipoproteins, Arterioscler. Thromb. Vasc. Biol. 17 (1997) 300^309. [99] T. Sakurada, H. Orimo, H. Okabe, A. Noma, M. Murakami, Puri¢cation and properties of cholesterol ester hydrolase from human aortic intima and media, Biochim. Biophys. Acta 424 (1976) 204^212. [100] R. Shamir, W.L. Johnson, K. Morlock-Fitzpatrick, R. Zolfaghari, L. Li, E. Mas, D. Lombardo, D.W. More, E.A. Fisher, Pancreatic carboxyl ester lipase: a circulating enzyme that modi¢es normal and oxidized lipoproteins in vitro, J. Clin. Invest. 97 (1996) 1696^1704. [101] D. Steinberg, Low density lipoprotein oxidation and its pathobiological signi¢cance, J. Biol. Chem. 272 (1997) 20963^20966. ë o«rni, M.O. Pentika«inen, M. Ala-Korpela, P.T. Kova[102] K. O nen, Aggregation, fusion, and vesicle formation of modi¢ed

BBAMCB 55697 6-11-00 Cyaan Magenta Geel Zwart

T. Hevonoja et al. / Biochimica et Biophysica Acta 1488 (2000) 189^210

[103]

[104]

[105]

[106]

[107]

[108]

[109]

[110]

[111]

[112]

[113]

[114]

[115]

LDL particles: molecular mechanisms and e¡ects on matrix interactions, J. Lipid Res. 41 (2000) 1703^1714. X.X. Xu, I. Tabas, Sphingomyelinase enhances low density lipoprotein uptake and ability to induce cholesteryl ester accumulation in macrophages, J. Biol. Chem. 266 (1991) 24849^24858. K. Paananen, P.T. Kovanen, Proteolysis and fusion of low density lipoprotein particles independently strengthen their binding to exocytosed mast cell granules, J. Biol. Chem. 269 (1994) 2030^2031. M.O. Pentika«inen, E.M.P. Lehtonen, P.T. Kovanen, Aggregation and fusion of modi¢ed low density lipoprotein, J. Lipid Res. 37 (1996) 2638^2649. ë o«rni, M. Ala-Korpela, P.T. Kovanen, J.K. Hakala, K. O Lipolytic modi¢cation of LDL by phospholipase A2 induces particle aggregation in the absence and fusion in the presence of heparin, Arterioscler. Thromb. Vasc. Biol. 19 (1999) 1276^1283. M. Piha, L.K. Lindstedt, P.T. Kovanen, Fusion of proteolyzed low-density lipoprotein in the £uid phase: a novel mechanism generating atherogenic lipoprotein particles, Biochemistry 34 (1995) 10120^10129. ë o«rni, S. Lusa, P. M.O. Pentika«inen, E.M.P. Lehtonen, K. O Somerharju, M. Jauhiainen, P.T. Kovanen, Human arterial proteoglycans increase the rate of proteolytic fusion of low density lipoprotein particles, J. Biol. Chem. 272 (1997) 25283^25288. M. Ala-Korpela, M.O. Pentika«inen, A. Korhonen, T. Hevonoja, J. Lounila, P.T. Kovanen, Detection of low density lipoprotein particle fusion by proton nuclear magnetic resonance spectroscopy, J. Lipid Res. 39 (1998) 1705^ 1712. J.A. Hamilton, E.H. Cordes, C.J. Glueck, Lipid dynamics in human low density lipoproteins and human aortic tissue with ¢brous plaques, J. Biol. Chem. 254 (1979) 5435^5441. ë o«rni, J.K. Hakala, A. Annila, M. Ala-Korpela, P.T. K. O Kovanen, Sphingomyelinase induces aggregation and fusion, but phospholipase A2 only aggregation, of low density lipoprotein (LDL) particles. Two distinct mechanisms leading to incresed binding strenght of LDL to human aortic proteoglycans, J. Biol. Chem. 273 (1998) 29127^29134. J.M. Holopainen, M. Subramanian, P.K.J. Kinnunen, Sphingomyelinase induces lipid microdomain formation in a £uid phosphatidylcholine/sphingomyelin membrane, Biochemistry 37 (1998) 17562^17570. M.P. Veiga, J.L. Arrondo, F.M. Gon¬i, A. Alonso, Ceramides in phospholipid membranes: e¡ects on bilayer stability and transition to nonlamellar phases, Biophys. J. 76 (1999) 342^350. D.P. Siegel, R.M. Epand, The mechanism of lamellar-toinverted hexagonal phase transitions in phosphatidylethanolamine: implications for membrane fusion mechanisms, Biophys. J. 73 (1997) 3089^3111. G. Basa¨n¬ez, M.B. Ruiz-Arguello, A. Alonso, F.M. Goni, G. Karlsson, K. Edwards, Morphological changes induced by phospholipase C and by sphingomyelinase on large uni-

[116]

[117]

[118]

[119]

[120]

[121]

[122]

[123]

[124]

[125]

[126]

[127]

[128]

[129]

209

lamellar vesicles: a cryo-transmission electron microscopy study of liposome fusion, Biophys. J. 72 (1997) 2630^2637. H. Pollard, A.M. Scanu, E.W. Taylor, On the geometrical arrangement of the protein subunits of human serum lowdensity lipoprotein: evidence for a dodecahedral model, Proc. Natl. Acad. Sci. USA 64 (1969) 304^310. A.G. Suits, A. Chait, M. Aviram, J.W. Heinecke, Phagosytosis of aggregated lipoprotein by macrophages: low density lipoprotein receptor-dependent foam-cell formation, Proc. Natl. Acad. Sci. USA 86 (1989) 2713^2717. H. Liu, D.G. Scraba, R.O. Ryan, Prevention of phospholipase-C-induced aggregation of low density lipoprotein by amphipathic apolipoproteins, FEBS Lett. 316 (1993) 27^33. J. Wang, H. Liu, B.D. Sykes, R.O. Ryan, Identi¢cation and localization of two distinct microenvironments for the diaglycerol component of lipophorin particles by 13 C NMR, Biochemistry 34 (1995) 6755^6761. J.C. Khoo, E. Miller, P. McLoughlin, D. Steinberg, Prevention of low density lipoprotein aggregation by high density lipoprotein or apolipoprotein A-I, J. Lipid Res. 31 (1990) 645^652. Y. Kleinman, E.S. Krul, M. Burnes, W. Aronson, B. P£eger, G. Schonfeld, Lipolysis of LDL with phospholipase A2 alters the expression of selected apoB-100 epitopes and the interaction of LDL with cells, J. Lipid Res. 29 (1988) 729^743. I.N. Gorshkova, M. Menschikowski, W. Jaross, Alterations in the physiochemical characteristics of low and high density lipoproteins after lipolysis with phospholipase A2 , Biochim. Biophys. Acta 1300 (1996) 103^113. L. Aggerbeck, F.J. Kedzy, A.M. Scanu, Enzymatic probes of lipoprotein structure, J. Biol. Chem. 251 (1976) 3823^ 3830. é vila, G. Camejo, V. Leo©n, N. Liscano, L. Mateu, E.M. A The structural stability of low-density lipoprotein. A kinetic X-ray scattering study of its interaction with arterial proteoglycans, Biochim. Biophys. Acta 795 (1984) 525^534. G. Camejo, E. Hurt, G. Bondjers, E¡ect of proteoglycans on lipoprotein^cell interactions: possible contribution to atherogenesis, Curr. Opin. Lipidol. 1 (1990) 431. G. Camejo, E. Hurt, O. Wiklund, B. Rosengren, F. Lopez, G. Bondjers, Modi¢cations of low-density lipoprotein induced by arterial proteoglycans and chondroitin-6-sulfate, Biochim. Biophys. Acta 1096 (1991) 253^261. G.M. Cherchi, M. Formato, P. Demuro, M. Masserini, I. Varani, G. DeLuca, Modi¢cations of low density lipoprotein induced by the interaction with human plasma glycosaminoglycan^protein complexes, Biochim. Biophys. Acta 1212 (1994) 345^352. E. Hurt-Camejo, U. Olsson, O. Wiklund, G. Bondjers, G. Camejo, Cellular consequences of the association of apoB lipoproteins with proteoglycans. Potential contribution to atherogenesis, Arterioscler. Thromb. Vasc. Biol. 17 (1997) 1011^1017. E. Hurt-Camejo, G. Camejo, B. Rosengren, F. Lopez, C.

BBAMCB 55697 6-11-00 Cyaan Magenta Geel Zwart

210

T. Hevonoja et al. / Biochimica et Biophysica Acta 1488 (2000) 189^210

Ahlstrom, G. Fager, E¡ect of arterial proteoglycans and glycosaminoglycans on low density lipoprotein oxidation and its uptake by human macrophages and arterial smooth muscle cells, Arterioscler. Thromb. 12 (1992) 569^583. [130] F.F. Chao, E.J. Blanchette-Mackie, V.V. Tertov, S.I. Skarlatos, Y.J. Chen, H.S. Kruth, Hydrolysis of cholesteryl ester in low density lipoprotein converts this lipoprotein to a liposome, J. Biol. Chem. 267 (1992) 4992^4998. [131] S. Bhakdi, B. Dorweiler, R. Kirchmann, J. Torzewski, E. Weise, J. Tranum-Jensen, I. Walev, E. Wieland, On the

pathogenesis of atherosclerosis: enzymatic transformation of human low density lipoprotein to an atherogenic moiety, J. Exp. Med. 182 (1995) 1959^1971. [132] H.F. Ho¡, J. O'Neil, Lesion-derived low density lipoprotein and oxidized low density lipoprotein share a lability for aggregation, leading to enhanced macrophage degradation, Arterioscler. Thromb. 11 (1991) 1209^1222. [133] C.G. Brouillette, G.M. Anantharamaiah, Structural models of human apolipoprotein A-I, Biochim. Biophys. Acta 1256 (1995) 103^129.

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