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Rev. Biochem. 1978. 47:751-77 Copyright © 1978 by Annual Reviews. Inc. All rights reserved
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Annu. Rev. Biochem. 1978.47:751-777. Downloaded from www.annualreviews.org by Duke University on 05/16/12. For personal use only.
THE PLASMA LIPOPROTEINS:
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STRUCTURE AND METABOLISM Louis C. Smith, Henry J. Pownall, and Antonio M. Gotto Jr. Department of Medicine, Baylor College of Medicine and The Methodist Hospital, Houston, Texas 71030
CONTENTS PERSPECTIVES AND SUMMARY ............................................................................
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INTRODUCTION ..........................................................................................................
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APOPROTEINS..............................................................................................................
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Natural Distribution ... .......................................................................................... Structure and Properties........................................................................................
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LIPOPROTEIN STRUCTURE ....................................................................................
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High Density Lipoprotein (HDL) .................... ... ................................................. Low Density Lipoprotein (LDL) .............. ....................................... .. ....... ......... Very Low Density Lipoprotein (VLDL) . ................ .......... ................... ............ Lipoprotein-X (LP-X) ..... . ....... ........ . ............................................. .... . ........ . .........
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LIPOPROTEIN BIOGENESIS......................................................................................
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Chylomicrons............................................................................................................ Very Low Density Lipoprotein (VLDL) ........ . .. ...... . ... ... .. . .. .. ......... .... ... ..... Low Density Lipoprotein (LDL) ............... .................. .. ....... ... ....... ......... ............. High Density Lipoprotein (HDL) ...... ................ ................... .................... ......... .
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758 760 760 761 761 762 762
LIPOPROTEIN CATABOLISM ..................................................................................
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Exchange and Trans/er .......................... ........ .. ..... . ... .. ................ ................... Enzymatic Modification .. ....... . .... .... ..... ......... ......... ........ ....... ..... .. .... .... ...
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Lecithin cholesterol acyltrans/erase (LeATj........................................................ Lipoprotein lipase (LPL) ... ... .... .. . . .. .... . . . ..... . . ..... . .... .. ........ .. ..... .. Hepatic lipase........................................................................................................ Catabolism 0/ Chylomicrons and VLDL ........................... ............................... Uptake and Degradation by Cultured Cells ........ ......................... ...... ........ .... . .
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PERSPECTIVES AND SUMMARY
In plasma, lipids are integral components of several macromolecular lipid protein complexes, termed lipoproteins, which have characteristic sizes, densities, and compositions. All lipoproteins contain protein components, called apoproteins, and polar lipids in a surface film surrounding a neutral lipid core. The apoproteins range in molecular weight from 5,700 to 75,000 and are distributed among lipoproteins of different density classes; the primary amino acid sequence of five of the eight major apoproteins is known. Plasma lipoproteins function to transport lipids in a water-soluble form. Specifically, chylomicrons carry dietary triglyceride from the intestine to nonhepatic tissues for utilization or storage; very low density lipoproteins (VLDL) contain triglyceride made primarily in the liver; the low density lipoproteins (LDL) derive from VLDL catabolism, and the high density lipoproteins (HDL), made in the liver, contain the bulk of the plasma cholesterol. The enzymes, lipoprotein lipase and lecithin cholesterol acyl transferase, modify the structure of lipoproteins by catalyzing the hydroly sis of triglyceride and by forming cholesteryl ester from cholesterol and phosphatidylcholine, respectively. Furthermore, LDL may regulate de novo cholesterol synthesis in nonhepatic tissues; HDL may promote choles terol transport from peripheral tissues to the liver. Physical studies of the apoproteins have shown that they have a low energy of stabilization. These findings suggest that hydrophobic residues are exposed to the aqueous phase, and that these exposed residues may be involved in hydrophobic self-association and in lipid binding. All of the apoproteins sequenced to date contain amphipathic helical regions that are thought to be essential for apoprotein-phospholipid interaction. In LDL, but not in HDL or VLDL, sufficient amounts of cholesteryl ester exist as a separate phase that undergoes cooperative melting. The microviscosity of the apolar regions of lipoprotein increases with increasing apoprotein con tent. Studies of lipoprotein biogenesis have shown that rat intestine synthesizes apoA-I as a component of chylomicrons. The hepatic origin of the "argi nine-rich" protein has been demonstrated. At the subcellular level, an mRNA for a major apoprotein of chicken VLDL has been translated in vitro. The catabolism of triglyceride-rich lipoproteins is substantially differ ent in humans than it is in rats, the experimental animal principally studied. The primary structure of apoC-1I has been determined. The minimal se quence of apoC-II necessary for activation of lipoprotein lipase is contained in the COOH-terminal half of the apoprotein and involves two different portions of the activator. Triglyceride hydrolysis is maximal at a molar ratio
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Annu. Rev. Biochem. 1978.47:751-777. Downloaded from www.annualreviews.org by Duke University on 05/16/12. For personal use only.
of enzyme to apoC-II of 1: 1. Lipid transfer between lipoproteins can occur by way of a monomolecular species in the aqueous solution. In future years, the dynamics of lipid: apoprotein interaction and trans port will be correlated with the structure of apoproteins and lipids. Delinea tion of the chemical structure of the lipoprotein receptors on cell membranes, and the events associated with intracellular cholesterol synthe sis,will lead to correlation of the in vitro studies of lipoprotein metabolism with normal and pathological phenomena. INTRODUCTION
The plasma lipoproteins are lipid-protein complexes that transport lipids in the circulation and regulate lipid synthesis and catabolism. Recent studies of lipoproteins, published since this topic was last reviewed by Morrisett et al (1), have substantially expanded the body of information about the composition and structural properties of lipoproteins and have provided in vitro evidence that correlates lipid structure and function. Herein, we con sider recent developments in (a) apoprotein structure and (b) lipoprotein metabolism. Our review focuses on the distribution, composition, structure, and metabolism of the plasma lipoproteins in various species, with the major emphasis on man. We shall use the A, B, C, etc nomenclature for identifying the apoproteins (2). We have compiled and related the different nomenclatures for lipoproteins and apoproteins in Table 1. Many other aspects of lipoprotein structure and metabolism are covered in recent re views (1, 3-7,49,154,155). Table 1
Composition of human plasma lipoproteins
Propertie s
Chylomicrons
Major apoproteins
ApoA-I
ApoB
ApoBa
ApoC-I
ApoC
ApoC-II
VLDL
LDL ApoB
HDL ApoA-I ApoA-II
ApoC-I1I ApoE
Minor apoproteins
ApoA-II ApoEb
PRpC
ApoA-I ApoA-II ApoDd
ApoC
ApoC-I ApoC-II ApoC-III ApoD ApoE
a Also termed apoLDL. bAlso termed arginine-rich protein (52).
c Proline-rich protein (10). dAlso termed "thin-line" protein (9) and apoA-III (9).
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SMITH,
POWNALL & GOTTO
APOPROTEINS
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Natural Distribution The distribution of the major human plasma apoproteins is well-known (1). Isolation and characterization of a number of apoproteins of lesser abun dance have been reported. McConathy & Alaupovic (8) have studied the properties of apoD and LP-D, a distinct class or subclass of lipoproteins isolated from HDL3. They obtained apoD by successive treatment of HDL3 with neuraminidase, chromatography of the product on concanavalin A-Sepharose 4B, elution of the retained LP-D with 0.2 M methyl-a.-D glucopyranoside, and final chromatography on hydroxyapatite-cellulose. An alternative procedure combines chromatography of HDL3 on an im munosorber containing anti-apoD antibodies followed by hydroxyapatite chromatography. With either procedure, they have judged that the purified LP-D is homogeneous, based on a single, symmetrical boundary in the analytical ultracentrifuge, a single band on gel electrophoresis, and unique antigenic properties. The composition is about 70% protein and 30% lipid, the former being a single glycoprotein called apoD with a molecular weight of 22,100. Kostner uses a similar procedure but finds traces of apoA-I in all of the purified fractions and therefore he assigns this protein to the A-family and designates the purified component as apoA-III rather than apoD (9). The same protein has been given the trivial name "thin-line" apoprotein because of its characteristic thin precipitin line near the antigen well when tested against antibodies (9). Sata et al (10) have described the isolation of a proline-rich protein (PRP) from human plasma. After removal of most of the lipoproteins from plasma by centrifugation at d 1.21 g/ml, the infranatant fraction is bound to Intralipid. After recentrifugation the resulting supernatant is chromato graphed on a 4% agarose gel and the eluted Intralipid-protein complex delipidated and chromatographed on 4% agarose and in 6 M urea on DEAE-cellulose. The purified protein aggregate, of>106 daltons, is rich in proline (8.9 mole %) and forms a single subunit of molecular weight 74,000 in polyacrylamide gel electrophoresis in sodium dodecyl sulfate. PRP is found in plasma only in a lipid-free state and in chylomicrons. Its plasma concentration varies between 12 and 41 mg/dl. Its importance in lipid metabolism is not known and we consider it an open question whether it should be classified with the plasma apoproteins. =
Structure and Properties The primary structure of apoA-I, apoA-II, apoC-I, and apoC-III have been reviewed previously (1). Little progress has been reported on the
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PLASMA LIPOPROTEINS
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structure of apoB and there are widely varying reports as to its molecular weight. Jackson et al (11) have completed the primary structure of apoC-II (Figure 1). The calculated molecular weight for the 78 amino acids is 8837. Three of the four prolines occur within the first 12 residues; these "helix breaking" amino acids would prevent helix formation in this part of the protein. Except for an additional proline at residue 42, the remainder of the apoprotein can readily form an a-helical structure. The presence of four adjacent and three 14 pairs of oppositely charged amino acid residues in apoC-II may be important in the stabilization of its amphipathic helical structure within VLDL (1). Using spectroscopic methods, Gwynne et al (12) have observed a revers ible thermal unfolding of apoA-I between 43 and 7SoC; this transition is absent in HDL where the apoprotein presumably is stabilized by lipid protein interactions. Tall et al (13, 14) have studied the structural stability of apoA-I when challenged by various chemical and thermal changes. They identify a reversible two-state thermal transition between 43 and 71°C that has an enthalpy of protein unfolding of 64 kcallmole. From the unusually low free energy of stabilization of apoA-I at 37°C (-2.4 kcallmole), they suggest that native apoA-I has a loosely folded tertiary structure in whiCh Thr -Glu -GI n -Pro -GI n - GI n -Asp -GI u-Met- Pro-Ser -Pro-Thr-Phe- Leu-
5
ill
B
Thr-Glu -Val-Lys -Glu -Trp-Leu - Ser-Ser-Tyr- Gln - Ser -Ala-Lys-Thr-
�
�
�
Ala - Ala-Gin -Asn -Leu-Tyr-Glu -Lys-Thr-Tyr-Ieu -Pro-Ala-Val-Asp-
�
�
e
GIu -Lys-Leu -Arg-Asp-Leu -Tyr-Ser-Lys- Ser-Thr-Ala-Ala - Met-Ser-
50
55
60
Thr-Tyr-Thr- Gly - lie - Phe -Thr-Asp- Gin -Val-leu - Ser-Val-leu -lys-
�
ro
Gly -Glu -.Glu 78 Figure I
Amino acid sequence of apoC-II.
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a large number of hydrophobic areas of the protein are exposed to water. Reynolds (15) has arrived at a similar conclusion based upon the fact that low concentrations of guanidine-HCI unfold apoA-I and apoA-II. The exposed hydrophobic areas of the protein may be important in the phos pholipid-binding and self-associative properties of apoproteins. Relevant to this concept are the monolayer experiments of P hillips et al (16) which show that apoA-I, in contrast to other hydrophobic proteins such as casein, forms a loose helical structure at the water-lipid interface. McLachlan (17) has hypothesized that the helical pattern in apoA-I is a basic repeating unit of 22 amino acids that appears eight times. He has further suggested that residues 47-240 have appeared by gene duplication from an ancestral unit of 22 amino acids. Stone & Reynolds (18) have stated that apoA-I dimerizes with an associa tion constant of 1.3 X 1()4 M-'. Vitello & Scanu (19) have found a monomer dimer-tetramer-octamer model for apoA-I and their results are similar to those of Stone & Reynolds. V itello & Scanu also find that the self-associa tion is relatively insensitive to changes in pH and ionic strength and there fore discount electrostatic effects. The self-association of apoA-1I (18, 20, 21), of reduced apoA-1I (22), and of reduced carboxymethylated (RCM) apoA-II (23) has also been studied. Stone & Reynolds (18) find only noncovalent dimers of apoA-1I between 0.4 and 1.0 mg/ml. Gwynne et al (20), on the other hand, observe an apoA-II dimer that maximally associates at 25°C and has an association constant "'-'3 X 1()4 M-l. In contrast, V itello & Scanu (21) report successive monomer-dimer-trimer equilibria for the self-association of apoA-II at con centrations between 0.8 and 1.5 mg/ml. They obtain similar results with reduced apoA-II (22) and with RCM apoA-II (23). These latter results raise the question as to whether the disulfide linkage is necessary for the func tional integrity of apoA-II, an impression enforced by the fact that apoA-II from other species has serine substituted for the cysteine at residue six (24). In the self-association studies of apoA-II, the fluorescence depolarization and mean residue ellipticity of the monomeric and self-associated apo protein are substantially different (20, 22, 23). With increasing protein concentration, the negative mean residue ellipticity at 222 nm and the polarization of apoA-II fluorescence both increase in magnitude. These findings suggest that oligomeric apoA-II has a different secondary structure than the monomer. The qualitative differences in the extent of self-associa tion of apoA-I and apoA-II as measured in various laboratories may be due to the buffer systems used. From the studies we have reviewed, we conclude that apoA-I and apoA-II do self-associate and that lipid-protein interac tions must compete effectively with protein-protein interactions to form a stable lipid-protein complex.
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LIPOPROTEIN STRUCTURE
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High Density Lipoprotein (HDL) Investigations of the composition, structure, and properties of intact lipo protein have refined the earlier models for HDL . The recent studies have focused on the microscopic structure of lipoproteins, and physicochemical methods have, understandably, been an important component of these in vestigations. The high field nuclear (proton) magnetic resonance (NMR) spectra of whole HDL have revealed several distinctive features. In porcine HDL3, Hauser (25) has observed that the chemical shifts of the phosphatidylcho line (PC) protons in the polar head group and first two -CH2-units of the acyl chain are different from those of pure egg PC; the remainder of the protons have chemical shifts similar to egg pc. He attributes this result to lipid-protein interaction between the phospholipid polar head group and apoprotein within HDL3• Analysis of the linewidth data suggests that the phospholipids are not completely immobilized within HDL (28) . Based on paramagnetic ion-induced shifts, practically all of the phospholipid polar head groups are on the surface of HDL (26, 27), a concept supported by the kinetics of phospholipase A2 HDL hydrolysis (29). Immunochemical and cross-linking experiments also show that a substantial percentage of the protein is located at the HDL surface. Schonfeld et al (30) detect only 10% of the ApoA-I in HDL by RIA. By contrast, Mao et al (31) obtain values for apoA-1I in HDL that exceed 100% of the apoA-1I measured by chemi cal analysis. The apoproteins of HDL are readily accessible to the bi functional cross-linking reagent, 1, 5-difluoro-2, 4-dinitrobenzene (32, 33). ApoA-I can be cross-linked with apoA-II, but no cross-linking products are found containing the same apoproteins. From these results, Grow & Fried (32, 33) suggest that within HDL , apoA-I and apoA-1I have specific topo graphical distributions in which all of the apoA-1I molecules are adjacent to an apoA-I molecule but with little or no self-association of either apoA-I or apoA-II. It is difficult to relate this result to the published molar ratio of apoA-I to apoA-1I of 2: 1 (1, 3, 34) and 1 : 1 (35). Grow & Fried's study raises the possibility that within HDL there may be a specific stoichiometry and topographic relationship of apoA-I and apoA-1I and that the associa tion of apoA-I with apoA-II could contribute to this relationship. The microscopic properties of intact HDL have been investigated by fluorescence depolarization of the probe, 1, 6-diphenyl-l, 3, 5-hexatriene (36), by pyrene excimer fluorescence (37) , and by partitioning of the ESR probe, 2,2,6, 6-tetramethylpiperidine- l -oxyl (TEMPO) (37) . The depolarization data indicate that the lipid region of HDL is more viscous than that of lipids extracted from HDL (36). TEMPO, which partitions preferentially into
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fluid regions of phospholipids, does not partition into HOL as well as it does into lipids extracted from HOL (37). Similarly, the rate of diffusion of pyrene in HOL is much lower than that observed in fluid phospholipids. These findings suggest that the apoproteins may restrict the mobility of small molecules in the lipid domain of HOL. Tall et al (38) have analyzed the thermal behavior of HOL by differential scanning calorimetry (O SC), by low angle X-ray diffraction, and by polar ized light microscopy. They find no thermal transitions in HOL between o and 60°C, whereas the extracted lipids of HOL exhibit well-defined thermal transitions that coincide with the melting of cholesteryl esters. The absence of a cholesteryl ester transition in HOL suggests that this lipid is confined to domains containing less than the requisite number of molecules required for cooperative melting. When HOL was heated above 71°C, a broad endothermic peak corresponding to the irreversible release of apoA-I was observed (38). In contrast, no release of apoA-II occurs until HOL is heated above 90°C. One interpretation of these results is that apoA-II is more tightly bound to HOL than is apoA-I. On the basis of these studies, several statements can be made about the structure of HOL. First, the phospholipid polar head groups are probably located at the surface of HOL. Second, the apoproteins are also at or near the surface, although photoaffinity labeling indicates that penetration of hydrophobic segments into the particle core may occur (39). Third, the interior of the HOL particle is highly viscous and contains its component lipids in domains smaller than those required for cooperative melting.
Low Density Lipoprotein (LDL) Small and co-workers have studied the thermal properties of LOL by a variety of techniques (40-43). Calorimetric and X-ray scattering data reveal a broad reversible transition between 20 and 45°C (40, 41), that represents a liquid crystalline � liquid phase transition involving cholesteryl esters. This assignment requires that the cholesteryl esters of LOL exist in a separate domain containing enough esters to exhibit cooperative melting. Studies of mixtures of triglycerides and cholesteryl esters and of LOL having different triglyceride content show that an increase in the triglycer ide to cholesteryl ester ratio decreases the thermal transition temperature. It is probable that a significant quantity of the triglyceride of intact LOL is confined to the same lipid domain as cholesteryl ester. \3C-NMR studies ofLOL over the temperature range between 20 and 45°C showed that there is increased mobility of the steroid moiety of the cholesteryl esters asLOL is raised above its phase transition (42). The thermally-induced changes in the molecular structure of LOL cholesteryl esters previously observed by
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small angle X-ray scattering have been reevaluated by Atkinson et a1(43), who infer that the cholesteryl esters are radially distributed in the core of LOL in an arrangement similar to that of the cholesteryl ester, cholesteryl myristate (CM). The similarity of the intense 1/36 A-I scattering from LOL and CM at lOoC is evidence that the LOL cholesteryl esters are in a smectic layered organization in which pairs of ester molecules are translated parallel to their molecular long axis; the pairs of opposed steroid groups with interlocked CIS and CI9 angular methyl groups observed in the crystal structure are retained in this model of the smectic phase. Mateu et al (44) report a reversible low temperature thermal transition « ooq in LOL in which the 1/36 A-I scattering is absent; the significance of this finding remains to be shown. Jonas (36) has found, on the basis of fluorescence depolarization ofOPH, that, of the human plasma lipoproteins, LOL has the most viscous (6 Poise) apolar region. The microviscosity of the extracted lipids of LOL is lower (2.4 Poise) than that of LOL , perhaps because the lipid-protein interactions restrict the movement of OPH. The thermal data show no discontinuities characteristic of a phase transition, a finding at variance with directly measured thermal transitions (40, 41). This discrepancy may be due to the localization of the OPH to regions of LOL that do not undergo a thermal transition. The topographical distributions of phospholipids in LOL have been stud ied by NMR. On the basis of the quenching of the 31p signal of human LOL by a Mn2+/EOT A complex, Henderson et al (26, 27) assign 50% of the phospholipid polar head groups to the surface of the particle. Finer et al (28), in studies of porcine LOL by IH-NMR at 220 MHz, find that two thirds of the choline groups have a sharp resonance with the remaining one third too immobilized to give a signal in the absence of detergent; all of the resonances of the choline groups in porcine LOL are accessible to the shift reagent, sodium ferricyanide. Finer et al conclude that the phospholipids of LOL are in two different chemical environments and propose a model in which a core of protein is surrounded by an inverted monolayer contain ing one third of the phospholipids having their head groups tightly packed and in contact with 15% of the protein. The outer surface of the particle is a phospholipid monolayer containing the rest of the phospholipids and apoB. The neutra1lipids are assigned to a central layer with some interdigi tation with opposing phospholipid acyl chains on the outside and in the protein core. The resulting model may be represented as a trilayer struc ture. In contrast, Shen et al (45) suggest, on the basis of the size and composi tion of lipoproteins, that all lipoprotein structures can be unified into a
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simple model in which the neutral lipids are in a central core and the proteins and phospholipids are in a monolayer on the particle surface. In their model cholesterol is located just beneath the protein layer. The loca tion of the protein on the surface is also consistent with the find ings of Harmony & Cordes (46) that the glycoprotein component of LDL is ex posed to the extent that it reacts with concanavalin A. Although adequate support for a model of LDL is not yet available, several conclusions can be made in light of recent studies. First, LDL contain cholesteryl esters in separate domains in which some LDL triglyc erides are probably solubilized . A part of the cholesteryl esters is in a d omain such that it participates in a reversible thermal transition. Second, much of the LDL protein is at or near the surface in association with a phospholipid monolayer. Some of the phospholipid is relatively immobil ized but the mechanism by which this immobilization occurs is unclear. Third, cholesterol is probably partitioned between the surface monolayer and the neutral core with a partition coefficient that is dependent on the composition of these two regions. These three generalizations apply only to where the
major fraction of these components reside. The location of
minor
amounts of lipids or a hydrophobic segment of the apoB in other regions of the particle cannot be exclud ed. It is important to keep in mind that the components are very likely in a state of dynamic equilibrium.
Very Low Density Lipoprotein (VLDL) Little is known concerning the structure of VLDL. Deckelbaum et al (47) have noted that cholesteryl esters in VLDL are probably interspersed throughout the rest of the neutral lipids since no transition due to melting of a separate cholesteryl ester domain can be detected by DSC; separate solubility studies show that cholesteryl esters are soluble in triglyceride.
Lipoprotein-X (LP-X) Patsch et al (48) have reported a detailed structural analysis of LP-X, found in the plasma of patients with advanced obstructive liver d isease. Three different fractions, LP-X1 (d = 1.038 glml), LP-X2 (d = 1.049 glml), and LP-X3 (d 1.058 glml) can be isolated by zonal ultracentrifugation. All three populations are rich in phospholipids (65%) and free cholesterol (25%) and are relatively poor in triglycerides (--5%). LP-X \,2,3, respec tively, exhibit flotation rates of 17.3,9.7, and 3.2 Svedbergs, and Stokes radii of 339, 343, and 249 A. In all three particles, the protein constituents contain a large fraction of a-helical structure (41-65%) and the fluidities of the lipid regions of the particles are very low. All three contain serum albumin and C-peptides but only LP-X\,2 contain apoA-I and apoE. =
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LIPOPROTEIN BIOGENESIS
The assembly and secretion of the plasma lipoproteins occurs only in the liver and the intestine. The liver secretes "nascent" VLOL, containing triglycerides of endogenous origin, and nascent HOL. The intestine synthe sizes and secretes dietary triglyceride, transported largely as chylomicrons and, to a lesser extent, as VLOL. Once secreted, the nascent lipoproteins undergo rapid modification in the plasma by physical transfer of lipid and apoprotein components and by enzymatic modification by lecithin choles terol acyltransferase (LCAT) and lipoprotein lipase (LPL). By the action of these enzymes, HOL and LOL, the most abundant and long-lived lipo proteins in the plasma, are formed. Molecular details of the synthesis and assembly of lipoprotein components, the secretion of intact lipoproteins, and the mechanisms affecting hormonal and d ietary regulation of these processes are largely unknown. Lipoprotein biogenesis has been compre hensively reviewed; we use the earlier reviews (3, 49) as a starting point.
Chy/omicrons ApoA, apoB, and apoC are the major protein constituents of chylomicrons in the lymph (50, 51). ApoE (52) and the proline-rich protein are also present (10). ApoB is synthesized by the intestine, whereas apoC and apoE are probably acquired by transfer from HOL (3, 53). Glickman & Green (54) have shown by pH]leucine incorporation that during lipid absorption, the rat intestine actively synthesizes apoA-I, most of which (85 %) is present in lipoproteins with d < 1.006, as determined by quantitative immunoelec trophoresis. Some apoA-I of lymph HOL is produced in the intestine, but filtration of plasma HOL has precluded quantification of the intestinal contribution. The coordinate synthesis of apoprotein and lipid components of chylomicrons has not been studied systematically. Such studies are neces sary to clarify the regulatory mechanisms that control the incorporation of dietary cholesterol into chylomicrons, which likely involves coenzyme A dependent esterification of cholesterol (55).
Very Low Density Lipoprotein (VLDL) An mRNA for a major apoprotein of chicken VLDL, apoVLDL-II (56), has been partially purified and translated in a heterologous cell-free system (57). Chan et al have shown that estrogen stimulation of VLDL synthesis in the cockerel produces an increased amount of translatable mRNA for apoVLDL-II. The number of specific nuclear binding sites for estrogen rapidly increases. This change is associated with enhanced activities of RNA polymerase I and II and with an increase in the number of RNA
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synthesis initation sites (58). In this system, the control of VLDL synthesis by estrogen resides, in part, at the level of gene transcription. Synthesis of immunoprecipitable VLDL and the stimulation by estrogen have also been demonstrated in nonproliferating chicken liver in cell cultures (59). Isolated rat hepatocytes in suspension also synthesize a material which, by immuno logical, flotation, and electrophoretic criteria, appears to be VLDL (60). The site within the hepatic cell at which VLDL is synthesized is the subject of dispute (61, 62). Alexander et al (62) have suggested that both triglycerides and phospholipids are made in the smooth endoplasmic reticu lum, independently of apoprotein synthesis in the rough endoplasmic reticulum. According to Nestruck & Rubinstein (63), apoB combines with the lipid components in the first stage of VLDL assembly. In their view, apoC proteins are acquired subsequently during or after secretion into the space of Disse. Glucosamine incorporation occurs during passage through the Golgi (64). VLDL of intestinal origin contains apoA, while hepatic VLDL reportedly does not (3). The basis for this difference, which exists in spite of apoprotein exchange, has not been studied.
Low Density Lipoprotein (LDL) P lasma LDL is produced primarily by the action of lipoprotein lipase on triglyceride-rich lipoproteins and is discussed later in this review as well as elsewhere (49, 5).
High Density Lipoprotein (HDL) Hamilton et al (65) have isolated from the perfused rat liver newly secreted, nascent HDL that are disc-shaped in negatively stained electron micro graphs, and differ from plasma HDL by a low content of esterified choles terol (4.3 versus 23.8%) and by apoprotein composition; the amount of apoE is much greater than that of apoA-I (66). However, the discs are still highly active as a substrate of LCAT (65). LIPOPROTEIN CATABOLISM
Catabolism of plasma lipoproteins proceeds by three distinct processes: (a) physical processes of transfer and exchange of lipid and apoprotein compo nents; (b) enzymatic changes in composition involving LeAT and LPL, and (c) receptor-mediated cellular uptake and passive endocytosis. The dynamics and the contributions of each process (Table 2) under various nutritional and physiological states are poorly defined, and compli cated further by the appreciable differences in lipoprotein composition and metabolism in various species studied. With new information about lipo protein structure and catabolic mechanisms, rapid progress in the under standing of lipoprotein metabolism should ensue.
PLASMA LIPOPROTEINS Table 2
763
Lipoprotein synthesis and metabolism
Lipoprotein Chylomicron
Process
Major components
assembly, secretion
apoA, apoB, phospholipid, choles-
Location Intestine
terol, cholesteryl ester, triglyc-
Annu. Rev. Biochem. 1978.47:751-777. Downloaded from www.annualreviews.org by Duke University on 05/16/12. For personal use only.
eride Lymph
transfer
apoC, apoEa
Plasma
transfer
apoC, cholesterol
Endothelial
b hydrolysis
triglyceride, phospholipid
transfer
fatty acid, cholesterol, phospho-
assembly, secretion
apoB, apoC, cholesterol, phospho-
cell lipid, apoC, apoA, apoEa VLDL
Liver
lipid, triglyceride, cholesteryl ester Plasma
transfer
apoCa, apoEa, cholesteryl ester
Endothelial
hydrolysisa
triglyceride, phsopholipid
transfer
fatty acid, cholesterol, phospho-
cell lipid, apoC, apoEa LOL
Plasma
Peripheral tissues,
, formation
from VLOL and chylomicrons
exchange
cholesterol, phospholipid
uptake, degradation,
cholesterol, cholesteryl ester
regulation
livera HOL
Liver
assembly, secretion
apoA, apoE, phospholipid,
Plasma
formationa
from chylomicronsa
acyltransferc
phosphatidyJcholine, cholesterol
transfer
cholesteryl ester, apoC, apoEa
exchange
apoC, cholesterol, phospholipid
uptake, degradation
cholesterol, cholesteryl ester
cholesterol
Liver, peripheral tissuesa
a Inferred but not determined, bLipoprotein lipase (triacylglycerol hydrolyase, apoC-II activated). cLecithin cholesterol acyltransferase.
Exchange and Transfer It has been known for many years that phospholipid and cholesterol ex change takes place among lipoproteins and various blood components. However, the mechanisms of these transfers are poorly understood. Pub lished values for the rates of transfer of many lipids are of questionable validity since the times required to isolate the lipoproteins for analysis were
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764
SMITH, POWNALL & GOTIO
much greater than the half-times of exchange. The use of high concentra tions of salt (67) for ultracentrifugal separation of reactants,and of heparin MnCh to precipitate lipoproteins (68) creates further ambiguity concerning the interpretation of the experiments, since the effects of these procedures on lipoprotein structure are uncertain. For further discussion the reader is referred to Bruckdorfer (69) and Bell (70). Pyrene transfer between HDL has provided a model of lipid exchange (71). When pyrene-Iabeled HDL are mixed with unlabeled lipoproteins, pyrene excimer fluorescence decreases with a half-time of approximately 3 msec. The fact that changes in the concentration of either pyrene-Iabeled or unlabeled HDL do not affect the half-time of pyrene exchange is compell ing evidence that the limiting step is the dissociation of the lipid into the aqueous solvent. Similar phenomena have been shown with pyrene-contain ing lipids, 1O-(3-pyrenyl)-decanoic acid (72), l -oleyl-2-[4(3-pyrenyl)buta noyl]glycerol, a fluorescent alkylacylglycerol (73), and 3'-pyrenemethyl23,24-dinor-5-cholen-22-oate-3,B-ol, a cholesterol analog (74). Exchange of these lipids between lipoproteins appears to be a physiochemical process that may not involve apoproteins. IH-NMR evidence excludes fusion of phosphatidylcholine vesicles as a mechanism of cholesterol transfer (75). The mechanism of phospholipid exchange between lipoproteins has not been studied (70). However, in an artificial, protein-free system, Martin & MacDonald (76) find that transfer between phosphatidylcholine vesicles occurs by way of a monomolecular species in the aqueous solution. A similar mechanism has been demonstrated for lysophosphatidylcholine, from true or micellar solution, which transfers into LDL or HDL across a dialysis membrane (77). Proteins that catalyze the exchange of phos pholipids between various organelle membranes in vitro have been de scribed (78). A similar function for apoproteins or other plasma proteins in promoting lipid transfer or exchange between lipoproteins remains an inter esting but untested possibility. The report that a high molecular weight plasma protein stimulates cholesteryl ester exchange between VLDL and LDL (79) also merits further attention. Both rapid exchange and net trans fer of apoC between chylomicrons or VLDL and HDL (49,80-83) occur in vivo and in vitro, but no studies of mechanism or kinetics have been conducted. It is not known whether lipids are involved in these transfers of apoproteins. The physiological significance of lipid exchange remains to be deter mined. The equilibrium of lipid components of lipoproteins with the plasma compartment may regulate physical properties such as fluidity of the lipo protein and membrane surfaces which in tum may influence enzymatic degradation. In addition,cholesterol and other lipids may be removed by transfer apart from endocytosis of the carrier lipoprotein. An important
PLASMA LIPOPROTEINS
765
task of future studies will be to define the contribution of the physical, enzymatic, and endocytotic processes of lipid removal from plasma in various tissues.
Enzymatic Modification LCAT is an extracellular enzyme of hepatic (84) and intestinal origin (85) that circulates in the plasma and catalyzes the transfer of the C-2' fatty acid from phos phatidylcholine to cholesterol (86). LCAT is activated by apoA-I (87) and by apoC-I (88). Activation by apoA-I is greatest with unsaturated PC, and exceeds by fourfold the stimulation by apoC-I, which is equally effective with both saturated and unsaturated PC (88). One report claims that apoD activates LeAT (89). The transfer of apoE from HDL to VLDL occurs concomitantly with LeAT action (90). The relationship of these apo proteins to LCAT catalysis awaits clarification by quantitative studies with LeAT preparations which have no contaminating apoproteins. The reac tion catalyzed by LeAT is poorly understood because there is no stable homogeneous enzyme preparation; various attempts yielded only a partially purified enzyme (89, 91, 92). In a complex set of procedures, Albers et al (93) prepared an enzyme of high purity, the stability of which was not reported. They removed contaminating apoD with an immobilized anti body to this protein. The apparent molecular weight of the purified LCAT is 68,000. One approach with great potential for studying apoprotein activation of LCAT is that of solid-phase peptide synthesis of activators. ApoC-I, synthe sized independently by Sparrow and associates (94) and by Harding et al (95), spontaneously forms a lipid-protein complex when mixed with syn thetic PC (94). Synthetic apoC-I activates LCAT indistinguishably from the naturally occurring apoprotein. A synthetic fragment composed of residues 17-57 is as active as the entire apoprotein in enhancing LeAT activity, while synthetic fragment 24-57 is only 60% as active (96). These findings suggest that the residues 17-57 contain the determinants for LeAT activa tion. Thus by peptide synthesis of native and model apoproteins, function ally important regions of apoproteins can be localized. The exact role of LeAT in lipoprotein metabolism remains to be defined. The presence of apoA-I in intestinal chylomicrons (50, 54) suggests that LCAT has an important role in the processing of the HDL components PC, cholesterol, and apoA-I-originating from chylomicrons during cata bolism (98). A lipoprotein deficient in cholesteryl ester appears in the HDL density range in the postprandial plasma of LCAT-deficient patients (97). LCAT may remove cholesterol and PC in excess of the amount assimilated
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LECITHIN CHOLESTEROL ACYLTRANSFERASE (LCAT)
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by cellular membranes.LCAT action may follow transfer of substrates and apoA to preexisting HDL. Alternatively, by analogy with the studies in perfused liver systems, there may be coordinate release of a nascent lipo protein from which HDL (66) is formed byLCAT action. Characterization and thorough kinetic studies of the various lipoprotein substrates will lead to an understanding of the physiological role of this enzyme. The transfer of cholesteryl ester from HDL to VLDL has been reported (99); the quanti tative importance is not known. The major pathway for removal of choleste ryl ester formed by LCAT action appears to be uptake of the intact HDL in the liver (1�102). The hydrolysis of di- and triglyceride in chylomi crons and VLDL in extrahepatic tissues is accomplished by an exoenzyme, lipoprotein lipase, at the luminal surface of the endothelial cell (103, 104). For maximal activity, this enzyme requires apoC-II, a component of the surface film of the lipoprotein substrate ( l05, 106). Interaction ofLPL with apoC-II produces an extremely stable complex. The calculated equilibrium dissociation constant for the enzyme:apoC-II complex is
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Rev. Biochem. 1978. 47:751-77 Copyright © 1978 by Annual Reviews. Inc. All rights reserved
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THE PLASMA LIPOPROTEINS:
+988
STRUCTURE AND METABOLISM Louis C. Smith, Henry J. Pownall, and Antonio M. Gotto Jr. Department of Medicine, Baylor College of Medicine and The Methodist Hospital, Houston, Texas 71030
CONTENTS PERSPECTIVES AND SUMMARY ............................................................................
752
INTRODUCTION ..........................................................................................................
753
APOPROTEINS..............................................................................................................
754
Natural Distribution ... .......................................................................................... Structure and Properties........................................................................................
754
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754
LIPOPROTEIN STRUCTURE ....................................................................................
754
High Density Lipoprotein (HDL) .................... ... ................................................. Low Density Lipoprotein (LDL) .............. ....................................... .. ....... ......... Very Low Density Lipoprotein (VLDL) . ................ .......... ................... ............ Lipoprotein-X (LP-X) ..... . ....... ........ . ............................................. .... . ........ . .........
757
LIPOPROTEIN BIOGENESIS......................................................................................
761
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Chylomicrons............................................................................................................ Very Low Density Lipoprotein (VLDL) ........ . .. ...... . ... ... .. . .. .. ......... .... ... ..... Low Density Lipoprotein (LDL) ............... .................. .. ....... ... ....... ......... ............. High Density Lipoprotein (HDL) ...... ................ ................... .................... ......... .
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758 760 760 761 761 762 762
LIPOPROTEIN CATABOLISM ..................................................................................
762
Exchange and Trans/er .......................... ........ .. ..... . ... .. ................ ................... Enzymatic Modification .. ....... . .... .... ..... ......... ......... ........ ....... ..... .. .... .... ...
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Lecithin cholesterol acyltrans/erase (LeATj........................................................ Lipoprotein lipase (LPL) ... ... .... .. . . .. .... . . . ..... . . ..... . .... .. ........ .. ..... .. Hepatic lipase........................................................................................................ Catabolism 0/ Chylomicrons and VLDL ........................... ............................... Uptake and Degradation by Cultured Cells ........ ......................... ...... ........ .... . .
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PERSPECTIVES AND SUMMARY
In plasma, lipids are integral components of several macromolecular lipid protein complexes, termed lipoproteins, which have characteristic sizes, densities, and compositions. All lipoproteins contain protein components, called apoproteins, and polar lipids in a surface film surrounding a neutral lipid core. The apoproteins range in molecular weight from 5,700 to 75,000 and are distributed among lipoproteins of different density classes; the primary amino acid sequence of five of the eight major apoproteins is known. Plasma lipoproteins function to transport lipids in a water-soluble form. Specifically, chylomicrons carry dietary triglyceride from the intestine to nonhepatic tissues for utilization or storage; very low density lipoproteins (VLDL) contain triglyceride made primarily in the liver; the low density lipoproteins (LDL) derive from VLDL catabolism, and the high density lipoproteins (HDL), made in the liver, contain the bulk of the plasma cholesterol. The enzymes, lipoprotein lipase and lecithin cholesterol acyl transferase, modify the structure of lipoproteins by catalyzing the hydroly sis of triglyceride and by forming cholesteryl ester from cholesterol and phosphatidylcholine, respectively. Furthermore, LDL may regulate de novo cholesterol synthesis in nonhepatic tissues; HDL may promote choles terol transport from peripheral tissues to the liver. Physical studies of the apoproteins have shown that they have a low energy of stabilization. These findings suggest that hydrophobic residues are exposed to the aqueous phase, and that these exposed residues may be involved in hydrophobic self-association and in lipid binding. All of the apoproteins sequenced to date contain amphipathic helical regions that are thought to be essential for apoprotein-phospholipid interaction. In LDL, but not in HDL or VLDL, sufficient amounts of cholesteryl ester exist as a separate phase that undergoes cooperative melting. The microviscosity of the apolar regions of lipoprotein increases with increasing apoprotein con tent. Studies of lipoprotein biogenesis have shown that rat intestine synthesizes apoA-I as a component of chylomicrons. The hepatic origin of the "argi nine-rich" protein has been demonstrated. At the subcellular level, an mRNA for a major apoprotein of chicken VLDL has been translated in vitro. The catabolism of triglyceride-rich lipoproteins is substantially differ ent in humans than it is in rats, the experimental animal principally studied. The primary structure of apoC-1I has been determined. The minimal se quence of apoC-II necessary for activation of lipoprotein lipase is contained in the COOH-terminal half of the apoprotein and involves two different portions of the activator. Triglyceride hydrolysis is maximal at a molar ratio
PLASMA LIPOPROTEINS
753
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of enzyme to apoC-II of 1: 1. Lipid transfer between lipoproteins can occur by way of a monomolecular species in the aqueous solution. In future years, the dynamics of lipid: apoprotein interaction and trans port will be correlated with the structure of apoproteins and lipids. Delinea tion of the chemical structure of the lipoprotein receptors on cell membranes, and the events associated with intracellular cholesterol synthe sis,will lead to correlation of the in vitro studies of lipoprotein metabolism with normal and pathological phenomena. INTRODUCTION
The plasma lipoproteins are lipid-protein complexes that transport lipids in the circulation and regulate lipid synthesis and catabolism. Recent studies of lipoproteins, published since this topic was last reviewed by Morrisett et al (1), have substantially expanded the body of information about the composition and structural properties of lipoproteins and have provided in vitro evidence that correlates lipid structure and function. Herein, we con sider recent developments in (a) apoprotein structure and (b) lipoprotein metabolism. Our review focuses on the distribution, composition, structure, and metabolism of the plasma lipoproteins in various species, with the major emphasis on man. We shall use the A, B, C, etc nomenclature for identifying the apoproteins (2). We have compiled and related the different nomenclatures for lipoproteins and apoproteins in Table 1. Many other aspects of lipoprotein structure and metabolism are covered in recent re views (1, 3-7,49,154,155). Table 1
Composition of human plasma lipoproteins
Propertie s
Chylomicrons
Major apoproteins
ApoA-I
ApoB
ApoBa
ApoC-I
ApoC
ApoC-II
VLDL
LDL ApoB
HDL ApoA-I ApoA-II
ApoC-I1I ApoE
Minor apoproteins
ApoA-II ApoEb
PRpC
ApoA-I ApoA-II ApoDd
ApoC
ApoC-I ApoC-II ApoC-III ApoD ApoE
a Also termed apoLDL. bAlso termed arginine-rich protein (52).
c Proline-rich protein (10). dAlso termed "thin-line" protein (9) and apoA-III (9).
754
SMITH,
POWNALL & GOTTO
APOPROTEINS
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Natural Distribution The distribution of the major human plasma apoproteins is well-known (1). Isolation and characterization of a number of apoproteins of lesser abun dance have been reported. McConathy & Alaupovic (8) have studied the properties of apoD and LP-D, a distinct class or subclass of lipoproteins isolated from HDL3. They obtained apoD by successive treatment of HDL3 with neuraminidase, chromatography of the product on concanavalin A-Sepharose 4B, elution of the retained LP-D with 0.2 M methyl-a.-D glucopyranoside, and final chromatography on hydroxyapatite-cellulose. An alternative procedure combines chromatography of HDL3 on an im munosorber containing anti-apoD antibodies followed by hydroxyapatite chromatography. With either procedure, they have judged that the purified LP-D is homogeneous, based on a single, symmetrical boundary in the analytical ultracentrifuge, a single band on gel electrophoresis, and unique antigenic properties. The composition is about 70% protein and 30% lipid, the former being a single glycoprotein called apoD with a molecular weight of 22,100. Kostner uses a similar procedure but finds traces of apoA-I in all of the purified fractions and therefore he assigns this protein to the A-family and designates the purified component as apoA-III rather than apoD (9). The same protein has been given the trivial name "thin-line" apoprotein because of its characteristic thin precipitin line near the antigen well when tested against antibodies (9). Sata et al (10) have described the isolation of a proline-rich protein (PRP) from human plasma. After removal of most of the lipoproteins from plasma by centrifugation at d 1.21 g/ml, the infranatant fraction is bound to Intralipid. After recentrifugation the resulting supernatant is chromato graphed on a 4% agarose gel and the eluted Intralipid-protein complex delipidated and chromatographed on 4% agarose and in 6 M urea on DEAE-cellulose. The purified protein aggregate, of>106 daltons, is rich in proline (8.9 mole %) and forms a single subunit of molecular weight 74,000 in polyacrylamide gel electrophoresis in sodium dodecyl sulfate. PRP is found in plasma only in a lipid-free state and in chylomicrons. Its plasma concentration varies between 12 and 41 mg/dl. Its importance in lipid metabolism is not known and we consider it an open question whether it should be classified with the plasma apoproteins. =
Structure and Properties The primary structure of apoA-I, apoA-II, apoC-I, and apoC-III have been reviewed previously (1). Little progress has been reported on the
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PLASMA LIPOPROTEINS
755
structure of apoB and there are widely varying reports as to its molecular weight. Jackson et al (11) have completed the primary structure of apoC-II (Figure 1). The calculated molecular weight for the 78 amino acids is 8837. Three of the four prolines occur within the first 12 residues; these "helix breaking" amino acids would prevent helix formation in this part of the protein. Except for an additional proline at residue 42, the remainder of the apoprotein can readily form an a-helical structure. The presence of four adjacent and three 14 pairs of oppositely charged amino acid residues in apoC-II may be important in the stabilization of its amphipathic helical structure within VLDL (1). Using spectroscopic methods, Gwynne et al (12) have observed a revers ible thermal unfolding of apoA-I between 43 and 7SoC; this transition is absent in HDL where the apoprotein presumably is stabilized by lipid protein interactions. Tall et al (13, 14) have studied the structural stability of apoA-I when challenged by various chemical and thermal changes. They identify a reversible two-state thermal transition between 43 and 71°C that has an enthalpy of protein unfolding of 64 kcallmole. From the unusually low free energy of stabilization of apoA-I at 37°C (-2.4 kcallmole), they suggest that native apoA-I has a loosely folded tertiary structure in whiCh Thr -Glu -GI n -Pro -GI n - GI n -Asp -GI u-Met- Pro-Ser -Pro-Thr-Phe- Leu-
5
ill
B
Thr-Glu -Val-Lys -Glu -Trp-Leu - Ser-Ser-Tyr- Gln - Ser -Ala-Lys-Thr-
�
�
�
Ala - Ala-Gin -Asn -Leu-Tyr-Glu -Lys-Thr-Tyr-Ieu -Pro-Ala-Val-Asp-
�
�
e
GIu -Lys-Leu -Arg-Asp-Leu -Tyr-Ser-Lys- Ser-Thr-Ala-Ala - Met-Ser-
50
55
60
Thr-Tyr-Thr- Gly - lie - Phe -Thr-Asp- Gin -Val-leu - Ser-Val-leu -lys-
�
ro
Gly -Glu -.Glu 78 Figure I
Amino acid sequence of apoC-II.
�
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SMITH, POWNALL & Gorro
a large number of hydrophobic areas of the protein are exposed to water. Reynolds (15) has arrived at a similar conclusion based upon the fact that low concentrations of guanidine-HCI unfold apoA-I and apoA-II. The exposed hydrophobic areas of the protein may be important in the phos pholipid-binding and self-associative properties of apoproteins. Relevant to this concept are the monolayer experiments of P hillips et al (16) which show that apoA-I, in contrast to other hydrophobic proteins such as casein, forms a loose helical structure at the water-lipid interface. McLachlan (17) has hypothesized that the helical pattern in apoA-I is a basic repeating unit of 22 amino acids that appears eight times. He has further suggested that residues 47-240 have appeared by gene duplication from an ancestral unit of 22 amino acids. Stone & Reynolds (18) have stated that apoA-I dimerizes with an associa tion constant of 1.3 X 1()4 M-'. Vitello & Scanu (19) have found a monomer dimer-tetramer-octamer model for apoA-I and their results are similar to those of Stone & Reynolds. V itello & Scanu also find that the self-associa tion is relatively insensitive to changes in pH and ionic strength and there fore discount electrostatic effects. The self-association of apoA-1I (18, 20, 21), of reduced apoA-1I (22), and of reduced carboxymethylated (RCM) apoA-II (23) has also been studied. Stone & Reynolds (18) find only noncovalent dimers of apoA-1I between 0.4 and 1.0 mg/ml. Gwynne et al (20), on the other hand, observe an apoA-II dimer that maximally associates at 25°C and has an association constant "'-'3 X 1()4 M-l. In contrast, V itello & Scanu (21) report successive monomer-dimer-trimer equilibria for the self-association of apoA-II at con centrations between 0.8 and 1.5 mg/ml. They obtain similar results with reduced apoA-II (22) and with RCM apoA-II (23). These latter results raise the question as to whether the disulfide linkage is necessary for the func tional integrity of apoA-II, an impression enforced by the fact that apoA-II from other species has serine substituted for the cysteine at residue six (24). In the self-association studies of apoA-II, the fluorescence depolarization and mean residue ellipticity of the monomeric and self-associated apo protein are substantially different (20, 22, 23). With increasing protein concentration, the negative mean residue ellipticity at 222 nm and the polarization of apoA-II fluorescence both increase in magnitude. These findings suggest that oligomeric apoA-II has a different secondary structure than the monomer. The qualitative differences in the extent of self-associa tion of apoA-I and apoA-II as measured in various laboratories may be due to the buffer systems used. From the studies we have reviewed, we conclude that apoA-I and apoA-II do self-associate and that lipid-protein interac tions must compete effectively with protein-protein interactions to form a stable lipid-protein complex.
PLASMA LIPOPROTEINS
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LIPOPROTEIN STRUCTURE
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High Density Lipoprotein (HDL) Investigations of the composition, structure, and properties of intact lipo protein have refined the earlier models for HDL . The recent studies have focused on the microscopic structure of lipoproteins, and physicochemical methods have, understandably, been an important component of these in vestigations. The high field nuclear (proton) magnetic resonance (NMR) spectra of whole HDL have revealed several distinctive features. In porcine HDL3, Hauser (25) has observed that the chemical shifts of the phosphatidylcho line (PC) protons in the polar head group and first two -CH2-units of the acyl chain are different from those of pure egg PC; the remainder of the protons have chemical shifts similar to egg pc. He attributes this result to lipid-protein interaction between the phospholipid polar head group and apoprotein within HDL3• Analysis of the linewidth data suggests that the phospholipids are not completely immobilized within HDL (28) . Based on paramagnetic ion-induced shifts, practically all of the phospholipid polar head groups are on the surface of HDL (26, 27), a concept supported by the kinetics of phospholipase A2 HDL hydrolysis (29). Immunochemical and cross-linking experiments also show that a substantial percentage of the protein is located at the HDL surface. Schonfeld et al (30) detect only 10% of the ApoA-I in HDL by RIA. By contrast, Mao et al (31) obtain values for apoA-1I in HDL that exceed 100% of the apoA-1I measured by chemi cal analysis. The apoproteins of HDL are readily accessible to the bi functional cross-linking reagent, 1, 5-difluoro-2, 4-dinitrobenzene (32, 33). ApoA-I can be cross-linked with apoA-II, but no cross-linking products are found containing the same apoproteins. From these results, Grow & Fried (32, 33) suggest that within HDL , apoA-I and apoA-1I have specific topo graphical distributions in which all of the apoA-1I molecules are adjacent to an apoA-I molecule but with little or no self-association of either apoA-I or apoA-II. It is difficult to relate this result to the published molar ratio of apoA-I to apoA-1I of 2: 1 (1, 3, 34) and 1 : 1 (35). Grow & Fried's study raises the possibility that within HDL there may be a specific stoichiometry and topographic relationship of apoA-I and apoA-1I and that the associa tion of apoA-I with apoA-II could contribute to this relationship. The microscopic properties of intact HDL have been investigated by fluorescence depolarization of the probe, 1, 6-diphenyl-l, 3, 5-hexatriene (36), by pyrene excimer fluorescence (37) , and by partitioning of the ESR probe, 2,2,6, 6-tetramethylpiperidine- l -oxyl (TEMPO) (37) . The depolarization data indicate that the lipid region of HDL is more viscous than that of lipids extracted from HDL (36). TEMPO, which partitions preferentially into
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SMITH, POWNALL & GOTTO
fluid regions of phospholipids, does not partition into HOL as well as it does into lipids extracted from HOL (37). Similarly, the rate of diffusion of pyrene in HOL is much lower than that observed in fluid phospholipids. These findings suggest that the apoproteins may restrict the mobility of small molecules in the lipid domain of HOL. Tall et al (38) have analyzed the thermal behavior of HOL by differential scanning calorimetry (O SC), by low angle X-ray diffraction, and by polar ized light microscopy. They find no thermal transitions in HOL between o and 60°C, whereas the extracted lipids of HOL exhibit well-defined thermal transitions that coincide with the melting of cholesteryl esters. The absence of a cholesteryl ester transition in HOL suggests that this lipid is confined to domains containing less than the requisite number of molecules required for cooperative melting. When HOL was heated above 71°C, a broad endothermic peak corresponding to the irreversible release of apoA-I was observed (38). In contrast, no release of apoA-II occurs until HOL is heated above 90°C. One interpretation of these results is that apoA-II is more tightly bound to HOL than is apoA-I. On the basis of these studies, several statements can be made about the structure of HOL. First, the phospholipid polar head groups are probably located at the surface of HOL. Second, the apoproteins are also at or near the surface, although photoaffinity labeling indicates that penetration of hydrophobic segments into the particle core may occur (39). Third, the interior of the HOL particle is highly viscous and contains its component lipids in domains smaller than those required for cooperative melting.
Low Density Lipoprotein (LDL) Small and co-workers have studied the thermal properties of LOL by a variety of techniques (40-43). Calorimetric and X-ray scattering data reveal a broad reversible transition between 20 and 45°C (40, 41), that represents a liquid crystalline � liquid phase transition involving cholesteryl esters. This assignment requires that the cholesteryl esters of LOL exist in a separate domain containing enough esters to exhibit cooperative melting. Studies of mixtures of triglycerides and cholesteryl esters and of LOL having different triglyceride content show that an increase in the triglycer ide to cholesteryl ester ratio decreases the thermal transition temperature. It is probable that a significant quantity of the triglyceride of intact LOL is confined to the same lipid domain as cholesteryl ester. \3C-NMR studies ofLOL over the temperature range between 20 and 45°C showed that there is increased mobility of the steroid moiety of the cholesteryl esters asLOL is raised above its phase transition (42). The thermally-induced changes in the molecular structure of LOL cholesteryl esters previously observed by
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small angle X-ray scattering have been reevaluated by Atkinson et a1(43), who infer that the cholesteryl esters are radially distributed in the core of LOL in an arrangement similar to that of the cholesteryl ester, cholesteryl myristate (CM). The similarity of the intense 1/36 A-I scattering from LOL and CM at lOoC is evidence that the LOL cholesteryl esters are in a smectic layered organization in which pairs of ester molecules are translated parallel to their molecular long axis; the pairs of opposed steroid groups with interlocked CIS and CI9 angular methyl groups observed in the crystal structure are retained in this model of the smectic phase. Mateu et al (44) report a reversible low temperature thermal transition « ooq in LOL in which the 1/36 A-I scattering is absent; the significance of this finding remains to be shown. Jonas (36) has found, on the basis of fluorescence depolarization ofOPH, that, of the human plasma lipoproteins, LOL has the most viscous (6 Poise) apolar region. The microviscosity of the extracted lipids of LOL is lower (2.4 Poise) than that of LOL , perhaps because the lipid-protein interactions restrict the movement of OPH. The thermal data show no discontinuities characteristic of a phase transition, a finding at variance with directly measured thermal transitions (40, 41). This discrepancy may be due to the localization of the OPH to regions of LOL that do not undergo a thermal transition. The topographical distributions of phospholipids in LOL have been stud ied by NMR. On the basis of the quenching of the 31p signal of human LOL by a Mn2+/EOT A complex, Henderson et al (26, 27) assign 50% of the phospholipid polar head groups to the surface of the particle. Finer et al (28), in studies of porcine LOL by IH-NMR at 220 MHz, find that two thirds of the choline groups have a sharp resonance with the remaining one third too immobilized to give a signal in the absence of detergent; all of the resonances of the choline groups in porcine LOL are accessible to the shift reagent, sodium ferricyanide. Finer et al conclude that the phospholipids of LOL are in two different chemical environments and propose a model in which a core of protein is surrounded by an inverted monolayer contain ing one third of the phospholipids having their head groups tightly packed and in contact with 15% of the protein. The outer surface of the particle is a phospholipid monolayer containing the rest of the phospholipids and apoB. The neutra1lipids are assigned to a central layer with some interdigi tation with opposing phospholipid acyl chains on the outside and in the protein core. The resulting model may be represented as a trilayer struc ture. In contrast, Shen et al (45) suggest, on the basis of the size and composi tion of lipoproteins, that all lipoprotein structures can be unified into a
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simple model in which the neutral lipids are in a central core and the proteins and phospholipids are in a monolayer on the particle surface. In their model cholesterol is located just beneath the protein layer. The loca tion of the protein on the surface is also consistent with the find ings of Harmony & Cordes (46) that the glycoprotein component of LDL is ex posed to the extent that it reacts with concanavalin A. Although adequate support for a model of LDL is not yet available, several conclusions can be made in light of recent studies. First, LDL contain cholesteryl esters in separate domains in which some LDL triglyc erides are probably solubilized . A part of the cholesteryl esters is in a d omain such that it participates in a reversible thermal transition. Second, much of the LDL protein is at or near the surface in association with a phospholipid monolayer. Some of the phospholipid is relatively immobil ized but the mechanism by which this immobilization occurs is unclear. Third, cholesterol is probably partitioned between the surface monolayer and the neutral core with a partition coefficient that is dependent on the composition of these two regions. These three generalizations apply only to where the
major fraction of these components reside. The location of
minor
amounts of lipids or a hydrophobic segment of the apoB in other regions of the particle cannot be exclud ed. It is important to keep in mind that the components are very likely in a state of dynamic equilibrium.
Very Low Density Lipoprotein (VLDL) Little is known concerning the structure of VLDL. Deckelbaum et al (47) have noted that cholesteryl esters in VLDL are probably interspersed throughout the rest of the neutral lipids since no transition due to melting of a separate cholesteryl ester domain can be detected by DSC; separate solubility studies show that cholesteryl esters are soluble in triglyceride.
Lipoprotein-X (LP-X) Patsch et al (48) have reported a detailed structural analysis of LP-X, found in the plasma of patients with advanced obstructive liver d isease. Three different fractions, LP-X1 (d = 1.038 glml), LP-X2 (d = 1.049 glml), and LP-X3 (d 1.058 glml) can be isolated by zonal ultracentrifugation. All three populations are rich in phospholipids (65%) and free cholesterol (25%) and are relatively poor in triglycerides (--5%). LP-X \,2,3, respec tively, exhibit flotation rates of 17.3,9.7, and 3.2 Svedbergs, and Stokes radii of 339, 343, and 249 A. In all three particles, the protein constituents contain a large fraction of a-helical structure (41-65%) and the fluidities of the lipid regions of the particles are very low. All three contain serum albumin and C-peptides but only LP-X\,2 contain apoA-I and apoE. =
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LIPOPROTEIN BIOGENESIS
The assembly and secretion of the plasma lipoproteins occurs only in the liver and the intestine. The liver secretes "nascent" VLOL, containing triglycerides of endogenous origin, and nascent HOL. The intestine synthe sizes and secretes dietary triglyceride, transported largely as chylomicrons and, to a lesser extent, as VLOL. Once secreted, the nascent lipoproteins undergo rapid modification in the plasma by physical transfer of lipid and apoprotein components and by enzymatic modification by lecithin choles terol acyltransferase (LCAT) and lipoprotein lipase (LPL). By the action of these enzymes, HOL and LOL, the most abundant and long-lived lipo proteins in the plasma, are formed. Molecular details of the synthesis and assembly of lipoprotein components, the secretion of intact lipoproteins, and the mechanisms affecting hormonal and d ietary regulation of these processes are largely unknown. Lipoprotein biogenesis has been compre hensively reviewed; we use the earlier reviews (3, 49) as a starting point.
Chy/omicrons ApoA, apoB, and apoC are the major protein constituents of chylomicrons in the lymph (50, 51). ApoE (52) and the proline-rich protein are also present (10). ApoB is synthesized by the intestine, whereas apoC and apoE are probably acquired by transfer from HOL (3, 53). Glickman & Green (54) have shown by pH]leucine incorporation that during lipid absorption, the rat intestine actively synthesizes apoA-I, most of which (85 %) is present in lipoproteins with d < 1.006, as determined by quantitative immunoelec trophoresis. Some apoA-I of lymph HOL is produced in the intestine, but filtration of plasma HOL has precluded quantification of the intestinal contribution. The coordinate synthesis of apoprotein and lipid components of chylomicrons has not been studied systematically. Such studies are neces sary to clarify the regulatory mechanisms that control the incorporation of dietary cholesterol into chylomicrons, which likely involves coenzyme A dependent esterification of cholesterol (55).
Very Low Density Lipoprotein (VLDL) An mRNA for a major apoprotein of chicken VLDL, apoVLDL-II (56), has been partially purified and translated in a heterologous cell-free system (57). Chan et al have shown that estrogen stimulation of VLDL synthesis in the cockerel produces an increased amount of translatable mRNA for apoVLDL-II. The number of specific nuclear binding sites for estrogen rapidly increases. This change is associated with enhanced activities of RNA polymerase I and II and with an increase in the number of RNA
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synthesis initation sites (58). In this system, the control of VLDL synthesis by estrogen resides, in part, at the level of gene transcription. Synthesis of immunoprecipitable VLDL and the stimulation by estrogen have also been demonstrated in nonproliferating chicken liver in cell cultures (59). Isolated rat hepatocytes in suspension also synthesize a material which, by immuno logical, flotation, and electrophoretic criteria, appears to be VLDL (60). The site within the hepatic cell at which VLDL is synthesized is the subject of dispute (61, 62). Alexander et al (62) have suggested that both triglycerides and phospholipids are made in the smooth endoplasmic reticu lum, independently of apoprotein synthesis in the rough endoplasmic reticulum. According to Nestruck & Rubinstein (63), apoB combines with the lipid components in the first stage of VLDL assembly. In their view, apoC proteins are acquired subsequently during or after secretion into the space of Disse. Glucosamine incorporation occurs during passage through the Golgi (64). VLDL of intestinal origin contains apoA, while hepatic VLDL reportedly does not (3). The basis for this difference, which exists in spite of apoprotein exchange, has not been studied.
Low Density Lipoprotein (LDL) P lasma LDL is produced primarily by the action of lipoprotein lipase on triglyceride-rich lipoproteins and is discussed later in this review as well as elsewhere (49, 5).
High Density Lipoprotein (HDL) Hamilton et al (65) have isolated from the perfused rat liver newly secreted, nascent HDL that are disc-shaped in negatively stained electron micro graphs, and differ from plasma HDL by a low content of esterified choles terol (4.3 versus 23.8%) and by apoprotein composition; the amount of apoE is much greater than that of apoA-I (66). However, the discs are still highly active as a substrate of LCAT (65). LIPOPROTEIN CATABOLISM
Catabolism of plasma lipoproteins proceeds by three distinct processes: (a) physical processes of transfer and exchange of lipid and apoprotein compo nents; (b) enzymatic changes in composition involving LeAT and LPL, and (c) receptor-mediated cellular uptake and passive endocytosis. The dynamics and the contributions of each process (Table 2) under various nutritional and physiological states are poorly defined, and compli cated further by the appreciable differences in lipoprotein composition and metabolism in various species studied. With new information about lipo protein structure and catabolic mechanisms, rapid progress in the under standing of lipoprotein metabolism should ensue.
PLASMA LIPOPROTEINS Table 2
763
Lipoprotein synthesis and metabolism
Lipoprotein Chylomicron
Process
Major components
assembly, secretion
apoA, apoB, phospholipid, choles-
Location Intestine
terol, cholesteryl ester, triglyc-
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eride Lymph
transfer
apoC, apoEa
Plasma
transfer
apoC, cholesterol
Endothelial
b hydrolysis
triglyceride, phospholipid
transfer
fatty acid, cholesterol, phospho-
assembly, secretion
apoB, apoC, cholesterol, phospho-
cell lipid, apoC, apoA, apoEa VLDL
Liver
lipid, triglyceride, cholesteryl ester Plasma
transfer
apoCa, apoEa, cholesteryl ester
Endothelial
hydrolysisa
triglyceride, phsopholipid
transfer
fatty acid, cholesterol, phospho-
cell lipid, apoC, apoEa LOL
Plasma
Peripheral tissues,
, formation
from VLOL and chylomicrons
exchange
cholesterol, phospholipid
uptake, degradation,
cholesterol, cholesteryl ester
regulation
livera HOL
Liver
assembly, secretion
apoA, apoE, phospholipid,
Plasma
formationa
from chylomicronsa
acyltransferc
phosphatidyJcholine, cholesterol
transfer
cholesteryl ester, apoC, apoEa
exchange
apoC, cholesterol, phospholipid
uptake, degradation
cholesterol, cholesteryl ester
cholesterol
Liver, peripheral tissuesa
a Inferred but not determined, bLipoprotein lipase (triacylglycerol hydrolyase, apoC-II activated). cLecithin cholesterol acyltransferase.
Exchange and Transfer It has been known for many years that phospholipid and cholesterol ex change takes place among lipoproteins and various blood components. However, the mechanisms of these transfers are poorly understood. Pub lished values for the rates of transfer of many lipids are of questionable validity since the times required to isolate the lipoproteins for analysis were
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much greater than the half-times of exchange. The use of high concentra tions of salt (67) for ultracentrifugal separation of reactants,and of heparin MnCh to precipitate lipoproteins (68) creates further ambiguity concerning the interpretation of the experiments, since the effects of these procedures on lipoprotein structure are uncertain. For further discussion the reader is referred to Bruckdorfer (69) and Bell (70). Pyrene transfer between HDL has provided a model of lipid exchange (71). When pyrene-Iabeled HDL are mixed with unlabeled lipoproteins, pyrene excimer fluorescence decreases with a half-time of approximately 3 msec. The fact that changes in the concentration of either pyrene-Iabeled or unlabeled HDL do not affect the half-time of pyrene exchange is compell ing evidence that the limiting step is the dissociation of the lipid into the aqueous solvent. Similar phenomena have been shown with pyrene-contain ing lipids, 1O-(3-pyrenyl)-decanoic acid (72), l -oleyl-2-[4(3-pyrenyl)buta noyl]glycerol, a fluorescent alkylacylglycerol (73), and 3'-pyrenemethyl23,24-dinor-5-cholen-22-oate-3,B-ol, a cholesterol analog (74). Exchange of these lipids between lipoproteins appears to be a physiochemical process that may not involve apoproteins. IH-NMR evidence excludes fusion of phosphatidylcholine vesicles as a mechanism of cholesterol transfer (75). The mechanism of phospholipid exchange between lipoproteins has not been studied (70). However, in an artificial, protein-free system, Martin & MacDonald (76) find that transfer between phosphatidylcholine vesicles occurs by way of a monomolecular species in the aqueous solution. A similar mechanism has been demonstrated for lysophosphatidylcholine, from true or micellar solution, which transfers into LDL or HDL across a dialysis membrane (77). Proteins that catalyze the exchange of phos pholipids between various organelle membranes in vitro have been de scribed (78). A similar function for apoproteins or other plasma proteins in promoting lipid transfer or exchange between lipoproteins remains an inter esting but untested possibility. The report that a high molecular weight plasma protein stimulates cholesteryl ester exchange between VLDL and LDL (79) also merits further attention. Both rapid exchange and net trans fer of apoC between chylomicrons or VLDL and HDL (49,80-83) occur in vivo and in vitro, but no studies of mechanism or kinetics have been conducted. It is not known whether lipids are involved in these transfers of apoproteins. The physiological significance of lipid exchange remains to be deter mined. The equilibrium of lipid components of lipoproteins with the plasma compartment may regulate physical properties such as fluidity of the lipo protein and membrane surfaces which in tum may influence enzymatic degradation. In addition,cholesterol and other lipids may be removed by transfer apart from endocytosis of the carrier lipoprotein. An important
PLASMA LIPOPROTEINS
765
task of future studies will be to define the contribution of the physical, enzymatic, and endocytotic processes of lipid removal from plasma in various tissues.
Enzymatic Modification LCAT is an extracellular enzyme of hepatic (84) and intestinal origin (85) that circulates in the plasma and catalyzes the transfer of the C-2' fatty acid from phos phatidylcholine to cholesterol (86). LCAT is activated by apoA-I (87) and by apoC-I (88). Activation by apoA-I is greatest with unsaturated PC, and exceeds by fourfold the stimulation by apoC-I, which is equally effective with both saturated and unsaturated PC (88). One report claims that apoD activates LeAT (89). The transfer of apoE from HDL to VLDL occurs concomitantly with LeAT action (90). The relationship of these apo proteins to LCAT catalysis awaits clarification by quantitative studies with LeAT preparations which have no contaminating apoproteins. The reac tion catalyzed by LeAT is poorly understood because there is no stable homogeneous enzyme preparation; various attempts yielded only a partially purified enzyme (89, 91, 92). In a complex set of procedures, Albers et al (93) prepared an enzyme of high purity, the stability of which was not reported. They removed contaminating apoD with an immobilized anti body to this protein. The apparent molecular weight of the purified LCAT is 68,000. One approach with great potential for studying apoprotein activation of LCAT is that of solid-phase peptide synthesis of activators. ApoC-I, synthe sized independently by Sparrow and associates (94) and by Harding et al (95), spontaneously forms a lipid-protein complex when mixed with syn thetic PC (94). Synthetic apoC-I activates LCAT indistinguishably from the naturally occurring apoprotein. A synthetic fragment composed of residues 17-57 is as active as the entire apoprotein in enhancing LeAT activity, while synthetic fragment 24-57 is only 60% as active (96). These findings suggest that the residues 17-57 contain the determinants for LeAT activa tion. Thus by peptide synthesis of native and model apoproteins, function ally important regions of apoproteins can be localized. The exact role of LeAT in lipoprotein metabolism remains to be defined. The presence of apoA-I in intestinal chylomicrons (50, 54) suggests that LCAT has an important role in the processing of the HDL components PC, cholesterol, and apoA-I-originating from chylomicrons during cata bolism (98). A lipoprotein deficient in cholesteryl ester appears in the HDL density range in the postprandial plasma of LCAT-deficient patients (97). LCAT may remove cholesterol and PC in excess of the amount assimilated
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LECITHIN CHOLESTEROL ACYLTRANSFERASE (LCAT)
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by cellular membranes.LCAT action may follow transfer of substrates and apoA to preexisting HDL. Alternatively, by analogy with the studies in perfused liver systems, there may be coordinate release of a nascent lipo protein from which HDL (66) is formed byLCAT action. Characterization and thorough kinetic studies of the various lipoprotein substrates will lead to an understanding of the physiological role of this enzyme. The transfer of cholesteryl ester from HDL to VLDL has been reported (99); the quanti tative importance is not known. The major pathway for removal of choleste ryl ester formed by LCAT action appears to be uptake of the intact HDL in the liver (1�102). The hydrolysis of di- and triglyceride in chylomi crons and VLDL in extrahepatic tissues is accomplished by an exoenzyme, lipoprotein lipase, at the luminal surface of the endothelial cell (103, 104). For maximal activity, this enzyme requires apoC-II, a component of the surface film of the lipoprotein substrate ( l05, 106). Interaction ofLPL with apoC-II produces an extremely stable complex. The calculated equilibrium dissociation constant for the enzyme:apoC-II complex is
