Apolipoprotein B and Low-Density Lipoprotein Structure: Implications for Biosynthesis of Triglyceride-Rich Lipoproteins

APOLIPOPROTEIN B AND LOW-DENSITY LIPOPROTEIN STRUCTURE: IMPLICATIONS FOR BIOSYNTHESIS OF TRIGLYCERIDE-RICH LIPOPROTEINS By VERNE N. SCHUMAKER, MARTIN L. PHILLIPS, and JON E. CHATTERTON Department of Chomlrty and Blochemlstry,and the Molecular Biology Instltute, Unlverrlty of Calltornla, Lor Angeler, Lor Angeler, Callfornla BOO24

I. Introduction

.......................................................

11. Apolipoprotein B Structure .......................................... A. Gene for Human Apolipoprotein B ............................... 111.

IV.

V.

V1.

B. Apolipoprotein B Message ....................................... C. Primary Sequence of Apolipoprotein B ........................... Low-Density Lipoprotein Structure ................ A. Emulsion Particle Model for Lo ins ............. B. Quantitative Molecular Model for Low-Density Lipoproteins ........ Structural Studies of Apolipoprotein B on Low-Density Lipoprotein Surfaces ........................................................... A. Lipid Extraction after Attachment of Low-Density Lipoproteins to Electron Microscope Grids ....................................... B. Mapping of Apolipoprotein with Monoclonal Antibodies C. Relating Low-Density Lipoprotein Core Circumferences to Apolipoprotein B Fragment Sizes ................................. Lipoprotein Assembly ............................................... A. Gotranslational Lipoprotein Formation in Rough Endoplasmic Reticulum ...................................................... B. Two-step Model for Assembly of Triglyceride-Rich Lipoproteins .... Summary .......................................................... References .........................................................

205 207 207 209 210 213 215 217 226 227 227 235 240 240 243 243 244

I. INTRODUCTION In this article the structure and function of apoliprotein B (apoB) will be described and an attempt will be made to relate its structure to its function. Apolipoprotein B is a moderately hydrophobic, extraordinarily large (-550 kDa) glycoprotein intimately involved in the packaging, transport, and utilization of apolar lipids, particularly cholesteryl ester and triglyceride. An apoB-like protein may be found on plasma lipoproteins from most, if not all, vertebrate species, including a “primitive” vertebrate, the hagfish [Mill and Taylaur, 1978; Goldstein et al., (1977)l. Outside of the vertebrates, apoB has not been found, although there are intriguing reports of a large (-600 kDa) apolipoprotein associated with triglyceride-rich lipoproteins present in sea urchin eggs (Marsh, 1968; Ichio et al., 1978). Apolipoprotein B plays a unique role in lipid transport ADVANCES IN PROTEIN CHEMISTRY, Vol. 45

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Copyright Q1994 by Academic Press, Inc. All rights of reproduction in any form reserved.

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APOLIPROTEIN B AND LDL STRUCTURE

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in the vertebrates, directing the biosynthesis of the triglyceride-richlipoproteins. In humans genetically deficient in apoB, chylomicron and very low-density lipoprotein (VLDL)levels are unmeasurable, and the two cell types that synthesize these lipoproteins, the enterocytes and the hepatocytes, respectively, accumulate droplets of unsecreted lipids. A shortened version of apoB, apoB48, directs chlomicron formation, whereas fulllength apoBlOO directs the assembly of human VLDL. ApoB plays a second metabolic role in the conversion of a portion of the VLDL remnants to low-density lipoproteins (LDLs). During this process a conformational change in apoBlOO results in the formation of a receptor-binding site (Lund-Katz et al., 1988, 1991). The recognition of this binding site on apoB by the hepatic LDL receptor is a critical step in the maintenace of a low serum LDL level and, in consequence, the delay or prevention of atherosclerosis (Goldstein and Brown, 1989). 11. APOLIPOPROTEIN B STRUCTURE A. Gene for Human Apolipopotein B

The gene for human apolipoprotein B is located on chromosome 2, and regional mapping and in situ hybridization have positioned the gene to the short arm in the p23 to p24 region (Mehrabian et al., 1986; Knott et al., 1985; Cann and Guyer, 1992). Thus, the apoB gene is unlinked to members of the gene family encoding the other major apolipoprotein species, which are dispersed on chromosomes 1, 11, and 19 (Bruns et al., 1984; Fojo et al., 1984;Jackson et al., 1984;Jeanpierre et al., 1984; Knott et al., 1985). Twenty-eight introns interrupt 29 exons (Fig. 1A) and the gene spans 43 kbp of genomic DNA, a modest stretch for a gene encodFIG. 1. The locations of significant features along the apoB polypeptide. Each line represents apoB100, with the N terminus on the left and the C terminus on the right. The horizontal axis corresponds to amino acid number. (A) Intron boundaries. (B) Sites of N-linked carbohydrate attachment. (C) Disulfide bonding pattern. The diamonds are known free cysteines; the squares are cysteines whose status is unknown. (D) The midpoints of extremely hydrophobic segments in apoB. (E)ApoB internal repeats; The taller segments are the 52-residue proline-rich repeats; the shorter segments are the 22-residue amphipathic helix repeats. (F) The domain structure proposed for apoB based on differential trypsin releasability (Yang el al., 1989). TR, Trypsin releasible; TN, trypsin nonreleasible; M, both. (G) The two sites cleaved on limited proteolysis of apoB with 12 different proteases of various specificities (Chen et al., 1989) and the location of the thrombin fragments generated by limited digestion. (H) Binding sites of monoclonal antibodies used in immunoelectron microscopy of LDLs. (I) Approximate positions of the C termini of apoB fragments generated on puromycin treatment of HepC2 cells.

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ing a protein the size of apoB. The first exon contains the 5‘-untranslated region and also codes for the signal sequence. The mature protein sequence starts with exon 2. Most of the exons range in size from 150 to 250 nucleotides, and 24 of these encode the first one-third of apoB. Exon 26 is composed of 7572 base pairs and is “by far the longest reported for a vertebrate gene” (Blackhart et al., 1986). T h e last exon, exon 29, is also very long, and contains 1906 bp. Complete sequences of all 29 exons and all but the middle regions of about a dozen large introns have been reported. Th e intron/exon boundaries have all been sequenced and conform to the standard pattern for these junctions (Blackhart et al., 1986; Carlsson el al., 1986; Higuchi et al., 1987; Wagener et al., 1987; Ludwig et al., 1987). Regulatory sequences located in the 5‘ region of the gene include a classical TATA box located 29 nucleotides 5’ of the transcriptional start site and a CAAT box 31 nucleotides 5’ of the TATA box. Two GC boxes occur on the 3’ side of the transcriptional start site within the untranslated portion of the mRNA, and therefore are “of dubious functional significance” (Blackhart et ad., 1986). Liver-specific expression is controlled by two positive elements located from -128 to -85 and -84 to -70 (Das et al., 1988). Rat liver nuclear proteins bind to these elements; thus, BRF-2 binds to the -128 to -85 region, and BRF-1 and C/EBP bind to overlapping sites at -84 to -61 and -70 to -50 (Zhuang et al., 1992). In addition, Brooks and Levy-Wilson (1992) have identified a tissue-specific transcriptional enhancer containing four distinct proteinbinding sites in the second intron of the apoB gene from +806 to +952. A C/EBP-related protein and multiple hepatocyte nuclear factors appear to compete for these four sites within the second intron. Negative regulatory sites in the 5’ region of the apoB gene have also been reported (Das et al., 1988; Paulweber et al., 1991; Paulweber and Levy-Wilson, 1991). Th e length of the chromatin loop containing the human apoB gene was determined by locating three nuclear matrix attachment sites, two at the 5’ end and one at the 3‘ end. At the 5’ end, the distal site was located between nucleotides -5262 and -4048, and the proximal site, between -2765 and -1801. At the 3’ end, a single site was found between +43,186 and +43,850. HepG2 cells, which express the apoB gene, contained all three sites whereas HeLa cells, which do not express apoB, lacked the 5’ distal site (Levy-Wilson and Fortier, 1989). A substantial number of common polymorphisms are associated with the human apoB gene, and Table I lists those which have been reported more than once. These have proved valuable in genetic (Young el al., 1986), anthropological (Rapacz et al., 1991). forensic (Butler, 1990), and medical studies (Soria et al., 1989).

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TABLE I Common Polymorphism of Apolipoprotein B

Common coding sequence polymorphisms (observed more than once) Restriction Exon Common name Amino acids Residue no. endonuclease 1 3 14 14 23 26 26 26 26 26 26 26 27 27 29 29

Signal Seq Ag(c/g) Ag(al1d)

Leu-Ala-Leu ThrlIle Val1Ala Leullle GluIGln LeuILeu ThrlThr LeuIPro AlalAla HisIAsp GlnlArg IleIThr PhelLeu TrylPhe Serl Asn GlulLys

--14 to --16 73 59 1 618 1191 206 1 2488 2712 2822 3292 361 1 3705 3922 3937 431 1 4514

BSP12861; ApLl AluI

XbaI

Mae1

MspI Secl

EcoRI

Common polymorphism located outside the coding sequence Intron Location Restriction endonuclease 5' end 2 3 4 4 20 3' end

4 kb upstream of exon 1 Nucleotide #722 92 bp 3' to exon 3 171 bp 3' to exon 4 523 bp 5' to exon 5 146 bp 5' to exon 21 Microsatellite 490 bp 3' to exon 29

A d 1

Ball HincII Pm II BalI MspI

Ref." 1 2, 3 4 5 5 5.6 7 8.9 5 5 10 597 5 5 9 7, 11, 12

Ref." 13 14 13 13 13 13

15, 16

"Key to references: (1) Boerwinkle and Chan, 1989; (2) Ma et al., 1989; (3) Young and Hubl, 1989; (4) Wang et al., 1988; (5) Ludwig et al., 1987; (6) Olofsson et al., 1987; (7) Blackhart et al., 1986; (8) Wu el al., 1991; (9) Dunning el al., 1992; (10) Xu et al., 1989; (11) Maetal., 1987; (12) Huangetal., 1986; (13) Huangetal., 1990; (14)Levy-Wilsonetal., 1991; (15) Boerwinkle et al., 1989; (16) Ludwig el al., 1989.

B . Apolipoprotein B Message

The processed mRNA transcribed from the human apoB gene contains 14,121 and 14,112 nucleotides (Cladaras etal., 1986),dependingon the presence or absence of a 9-nucleotide insertion/deletion polymorphism in the signal sequence (Boerwinkle and Chan, 1989). Messenger

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RNA is abundant in rat liver and intestine, the two organs that synthesize triglyceride-rich lipoproteins; moreover, a small amount of message is found in rat adrenal tissue and a “20-fold increase in placental apoB mRNA concentrations during the last 48 hr of pregnancy. . . suggests a specific role for this organ in maternal-fetal lipid transport immediately prior to parturition” (Demmer et al., 1986). Two different versions of the apoB protein are employed in the biosynthesis of the triglyceride-rich lipoproteins (Kane et al., 1980). T h e smaller version, called apoB48, is used for chylomicron biosynthesis by the intestine. The full-length apoB, called apoBl00, is used for VLDL biosynthesis by the liver. These two different apoB proteins are encoded by the single apoB gene. Processing occurs at the RNA level to create the message encoding apoB48 from the full-length apoB 100 message. In the human and rabbit, this processing event is tissue specific, occurring only in the enterocytes of the intestine (Powell et al., 1987), whereas in the rat, both messages are produced and expressed in the liver (Davidson et al., 1988). Processing of apoB message involves a unique mechanism consisting of the enzymatic deamination of a single cytidine at human nucleotide 6666, converting the glutamine codon 2153 (CAA) to a stop codon (UAA) (Powell et al., 1987; Chen et al., 1987; Hospattankar etal., 1987). With the formation of this stop codon, the altered message is translated to express the apoB48 polypeptide in the rat. Further processing occurs in humans and rabbits to truncate and polyadenylate the message at cryptic polyadenlyation sites located downstream from the newly formed stop codon (Powell et al., 1987). The editing reaction can be duplicated in a cell-free extract, and no metal ion cofactors, DNA or RNA cofactors, or energy requirements have been found (Garcia et al., 1992).

C. Primary Sequence of Apolipoprotein B Complete cDNA sequences of human apoB 100 have been reported by several laboratories (Knott et al., 1986; Yang et al., 1986; Cladaras et al., 1986; Law et al., 1986; Olofsson et al., 1987). Partial sequences are also available for apoBlOO from rats (Reuben et al., 1988; Matsumoto el al., 1987),swine (Maeda et al., 1988) and chickens (Kirchgessner et al., 1987). From estimation of substitution rates, it appears that apoB evolves at about twice the rate of ordinary mammalian proteins (O’hUigin et al., 1990). These authors found that the rate of substitution is not uniform across the molecule, however. There is a general increase in substitution rates going from the 5’ to the 3’ end; for example, in a comparison of the human and the rat, the 5‘-most 1155 nucleotides evolve at one-fourth the

APOLIPROTEIN B AND LDL STRUCTURE

21 1

rate of the 3’-most 1089 nucleotides. This suggests that the amino terminus of apoB may be structurally or functionally more constrained than the carboxyl terminus. In addition to a gradient in substitution rates, there are several regions, two of which have been implicated in binding to the LDL receptor, that are much more conserved than surrounding sequences (O’hUigin et al., 1990). Portions of apoB may have evolved from internal duplications, because it contains some internally homologous sequences. At least two families of related sequences are present; there are six similar 52-residue hydrophobic proline-rich sequences, unique to apoB (Fig. lE), and eight similar 22-residue sequences, which are also homologous to the peripheral apolipoproteins (Knott el al., 1986; Yang et al., 1986; De Loof et al., 1987). In addition to this self-homology, residues 3352-337 1 of apoB are similar to residues 136-155 of apoE, which contain the LDL receptor-binding site of apoE. The amino-terminal 1000 amino acids of apoB contain several long sequences homologous to the vitellogenins of vertebrates and nematodes, precursors to the egg yolk lipoproteins, the lipovitellins (Baker, 1988; Perez et al., 1991). Homologous regions to vitellogenin in apoB span approximately residues 20-280,530-600, and 800-970 (Banaszak et al., 1991). The apoB 100 sequence, deduced from cDNA sequences, consists of a signal sequence 27 or 24 amino acids long, depending on the presence or a deletion of amino acids - 14 to - 16, and the mature protein sequence of 4536 amino acids. Peptides covering approximately 90% of the sequence of the serum protein have also been sequenced directly (Yang et al., 1989). Aside from the signal sequence, no other amino acids are lost during the maturation of the protein. Ignoring the signal sequence, apoB48 consists of the N-terminal2 152 amino acids of apoB 100. Protein sequencing of the apoB48 C terminus from chylous ascites fluid indicates that in this system, Ile-2 152 has been removed, presumably by a carboxypeptidase A type activity, leaving Met-215 1 as the C-terminal amino acid (Chen et al., 1987). The protein portion of apoBlOO has a predicted molecular weight of 513,000 and that of apoB48 is 243,000. ApoBlOO contains 5 4 % carbohydrate (Lee and Breckenridge, 1976; Shireman and Fisher, 1979; Vauhkonen, 1986),so the total molecular weight of the apoB 100 glycoprotein is 540,000-550,000; that of the apoB48 glycoprotein is about 260,000. Of the 19 potential N-glycosylation sites in apoB100, 16 are actually glycosylated (Yang et al., 1989); 5 of these sites are in apoB48 (see Fig. 1B). Both high-mannose and complex forms of oligosaccharides are present. In apoB 100 there are 8-10 mol of complex type and 5-6 mol of high-mannose form (Taniguchi et al., 1989); in apoB48 there are about 4

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mol of complex oligosaccharides and 1 mol of the high-mannose form (Sasak et al., 1991). It is not known whether there are O-linked carbohydrates in apoB; however, they are not present in large amounts. There are 25 cysteines in apoB; 16 of these are known to be involved in disulfide bonds, 7 are known to be free, and the remaining two, Cys-2906 and Cys-4326, are unknown (Yang et al., 1990). Only two of the free cysteines of apoB, Cys-3734 and Cys-4190, are labeled in LDL by the fluorescent probe 5-iodoacetamidofluorescein,possibly because they are exposed on the surface of the lipoprotein (Coleman et al., 1990). Of the 8 disulfide bonds in apoB100, 7 are within 1000 amino acids of the N terminus of the protein (6 in the first 500 amino acids). T h e first two disulfides, CysL2-Cys6'and C y ~ ~ ' - C ycross; s ~ ~ ,the remainder of the disulfides are between nearest neighbors. T h e number of amino acids separating the cysteines in each disulfide is relatively small, 5-49 amino acids; the exception is C y ~ ~ ' ~ ' - C wherein y s ~ ~ ~the ~ , cysteines are separated by 130 amino acids. This disulfide is apparently unique to the human sequence, because cysteines are not found in homologous positions in rat, mouse, Syrian hamster, pig, rabbit, o r chicken apoB (Law and Scott, 1990). The disulfide pattern of apoBlOO is shown schematically in Fig. 1C. The average hydrophobicity of apoB 100 is 0.916 kcallresidue, a value intermediate between that of the peripheral apolipoproteins and that of intrinsic membrane proteins (Chen et al., 1986). Aside from the signal sequence, there are no stretches of 20 or more hydrophobic amino acids corresponding to typical bilayer membrane-spanning helices. There are, however, other features of the sequence that may be related to the lipid-binding characteristics of apoB. Thus, there are 39 short sequences (5-13 amino acids, with only two sequences being more than 10 amino acids long) that have hydrophobicities typical of membrane-spanning domains (Olofsson et al., 1987). These sequences are distributed throughout the apoB sequence (see Fig. 1D). They are not uniformly hydrophobic, but are occasionally interrupted by uncharged polar or, frequently in the longer sequences, charged residues. These segments are usually predicted to have P-sheet character. T h e folding of apoB may bring some of these hydrophobic sequences together. Indeed, the first two of these sequences, residues 8-12 and 60-65, are linked by the C y ~ ' ~ - C y disulfide s~' bond. The amphipathic helix, in which residues are spaced so that the helical periodicity places hydrophobic side chains on one side of the helix and hydrophilic side chains on the other, is a common structural motif used by the peripheral apolipoproteins to bind lipid (Segrest et al., 1992); it is also a structural element present in globular proteins (Perutz et al., 1965).

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There are many predicted amphipathic a helices in apoB. Of particular interest is a family of related sequences 22 amino acids long, which are homologous to the other apolipoproteins (De Loof et al., 1987).These are found in two regions, residues 2000-2500 and 4000-4500 (see Fig. 1E). Also noteworthy are residues 3352-337 1 of apoB, which are homologous to the LDL receptor-binding region of apoE (residues 136-155) and conform to an amphipathic a helix. The homologous sequence in apoE has been shown to be an amphipathic a helix by crystallographicanalysis of the amino-terminal domain of apoE (Wilson et al., 1991). Sequences corresponding to arnphipathic @ strands, in which predicted @ structure is combined with alternation of hydrophobic and hydrophilic amino acids, are also present in apoB. These sequences are spread throughout apoB. Although there are a few long amphipathic @-strand segments containing 10-20 residues, most are short, no more than five amino acids long with 3 hydrophobic residues. There is some clustering of amphipathic @ strands in the proline-rich repeats (see Fig. lE),with a five-amino acid consensus sequence of acidic-aromatic-polaraliphatic-proline (Knott et al., 1986).In the moderate-resolution crystal structure of lamprey lipovitellin (Raag et al., 1988),the lipid hydrocarbon chains, localized by neutron diffraction in varying D20/H20 mixtures (Timmins et al., 1992),are in a cavity lined by @ sheet. Lamprey vitellogenin (lipovitellin)displays homology with other vertebrate and nematode vitellogenins (Banaszak et al., 1991) to which apoB is homologous, and it is suggestive that homologous amphipathic @-strandsequences are present. A good example of such a segment is shown below.

apoB 48-58

R

E

Iv/

Vitellogenins Xenopus A 1 54-64

K

N K E S \I/ \c/ \v/ \I/

\A/Y

C. Elegans Vit-2 54-64

R

S R R Q V \I/ \A/ \A/ \I/ \A/

\/

N

K

E

\=/ jV/ \L/

P

I1 I. LOW-DENSITY LIPOPROTEIN STRUCTURE

Low-density lipoproteins contain a single molecule of apoB 100 (Knott

et al., 1986) and almost no other protein; therefore, they are uniquely

suited to the study of the interactions between apoB and lipids. Lipoproteins have densities that are lower than the densities of plasma proteins, which do not contain lipids. This characteristic is used to purify and fractionate lipoproteins by sequential flotation centrifugation to

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yield the familiar lipoprotein categories, which include the chylomicrons, the very low-density lipoproteins, the intermediate-density lipoproteins (IDLs), the low-density lipoproteins, and the high-density lipoproteins (HDLs) (Schumaker and Puppione, 1986). Traditionally, human LDLs have been defined as those lipoproteins isolated in the density interval between 1.063g/ml > LDL > 1.019g/ml. This density interval may be too broad, however, because a substantial contamination with apolipoproteins other than apoB may be present on those lipoproteins at the two extremes of this density range; the contaminating apolipoproteins are principally apoE (present on IDLs and HDLs at the low- and high-density extremes of this density interval, respectively) and apo(a) [present on Lp(a) at the high-density extreme]. This contamination was almost absent in the more tightly defined LDL fraction lying between 1.024 and 1.050 g/ml (Chapman et al., 1988). However, it is possible that loosely associated apolipoproteins are normally bound to LDLs in plasma and are subsequently lost during the multiple flotation steps involved in lipoprotein isolation (Mahley and Holcombe, 1977). The average weight percent composition of pooled human LDL is about 19% protein, 1% carbohydrate, 43% cholesteryl ester, 11% unesterified cholesterol, 4% triglyceride, and 22% phospholipid (see later, Table 11). Because there is a single molecule of apoBlOO on the LDL, a simple method of obtaining the number-average molecular weight of a lipoprotein preparation is to divide the molecular weight of the apoB 100 by the weight percent protein on the LDL. An exact value for the molecular weight of the protein portion of the apoB 100 glycoprotein was calculated from its amino acid sequence by Yang et al. (1986)to be 512,937. Thus, the number-average molecular weight of typical LDL, calculated from its protein content, is M , = 512,937/0.19= 2.7 x lo6. Moreover, assuming a spherical shape and a density of 1.030 g/ml, the anhydrous lipoprotein radius may be calculated from Eq. (1):

where d is the density and N is Avogadro's number. Substituting the values for density and molecular weight, given above, Eq. (1)yields R = 101 A. By definition, the Stokes radius is obtained as the product of the anhydrous radius and the translational frictional ratio; the translational frictional ratio has been measured for LDLs and found to be 1.1 1 (Fisher

APOLIPROTEIN B AND LDL STRUCTURE

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et al., 1971). Thus, the average Stokes radius of pooled human LDL, as calculated from the number-average molecular weight, is 112 A.

A. Emulsion Particle Model f o r Low-Density Lapoproteins Structurally, LDLs are well described as emulsion particles. An emulsion may be defined as one liquid embedded in another and kept in solution by an emulsifying agent. For LDLs, the first liquid is a droplet of oil, largely cholesteryl ester but containing some triglyceride; the second liquid is the aqueous plasma and the emulsifying agent is a monolayer of phospholipid, unesterified cholesterol, and protein. This monolayer forms an amphipathic surface coat surrounding the oil droplet and separating the hydrophobic, liquid core of the LDL from the aqueous plasma (Bradley and Gotto, 1978). What is the evidence supporting the emulsion particle model for LDLs? One feature that may be used to distinguish an emulsion particle from a closed bilayer vesicle is that, for an emulsion particle, all of the phospholipid should be exposed to the external medium, whereas for a bilayer vesicle, somewhat more than one-half should be exposed. Enzymatic hydrolysis of LDLs by phospholipase Ag converted all of the phosphatidylcholine and phosphatidylethanolamine to their corresponding lysophospholipids (Aggerbeck et al., 1976), indicating that all of the phospholipid was located at the aqueous interface at the LDL surface. In addition, 31P NMR studies of LDLs have demonstrated that all of the phosphate was accessible to small amounts of Pr3+, a rare earth probe that should not cross a bilayer because of its ionic nature (Yeagle et al., 1978). These results agree with the emulsion particle model. The emulsion particle model also places apoB at the surface of the LDL, consistent with proteolysis studies, and with studies of the binding of anti-apoB monoclonal antibodies to LDLs. Thus, trypsin removed about 30% of the protein (Triplet and Fisher, 1978; Chapman et al., 1987; Margolis and Langdon, 1966a), leaving a collection of peptide fragments of variable lengths associated with the lipoprotein; both the trypsin-removable and trypsin-nonremovable peptides have been sequenced and found to be distributed along the length of apoB (Yang et al., 1990) (see Fig. lF), showing that trypsin cleavage was not restricted to a few domains. Many monoclonal antibodies have been generated to epitopes located along the length of apoB using LDL as an immunogen; these monoclonals also bound to LDLs, demonstrating that much of the protein must be exposed to the solvent (Pease et al., 1990). Variablecontrast neutron scattering studies have also indicated that the protein is

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located far from the center of the LDL (Laggner et al., 1981). Again, the results are consistent with the emulsion particle model. The emulsion particle model places the nonpolar lipids in an oil droplet at the center of the LDL. The nonpolar lipids are observed to undergo a liquid-to-liquid crystalline transition at physiological temperatures, characteristic of cholesteryl esters (Deckelbaum et al., 1977). These workers showed that the midpoint of the temperature transition varied with the triglyceride content of the LDL. Detailed analysis of the lowangle X-ray scattering pattern strongly suggested that oscillations in the radial distribution function, with a periodicity of 30 to 40 81, and the 36-81 fringe at 10°C were “explained by two layers of cholesteryl ester molecules oriented radially in a smectic-like phase within the core of a single LDL” (Deckelbaum et al., 1977). The thickness of the surface monolayer of phospholipid, cholesterol, and protein has been estimated from the variation in lipoprotein composition with lipoprotein size, and values of 21.5 81 (Sata et al., 1972) and 20.2 81 have been estimated (Shen et al., 1977). These values seem reasonable, although they are somewhat less than one-half the thickness observed for egg lecithin bilayers. For such bilayers, the thickness of the lipid layer varies inversely with the degree of hydration, from about 36 81 for the least hydrated form to about 30 81 for the most hydrated. To these values must be added twice the distance “extending from the glycerol-3carbon to the edge of the phosphorylcholine group, when lying parallel to the plane of the bilayer, [which] gives a distance of about 8 81 perpendicular to the plane of the bilayer” (Small, 1986). Thus 8 81 + 881 + 30 81 = 46 81 for the bilayer, and one-half of this value, or 23 81, would be estimated for the thickness of a hydrated monolayer. The average LDL size and density are found to vary between individuals (Adams and Schumaker, 1969; Fisher et al., 1975; Krauss and Burke, 1982), and these studies suggest that the variation is due to both genetic and dietary factors. Figure 2 shows hydrodynamic data taken from three studies of subfractionated LDLs and IDLs isolated from different individuals, as summarized by Schumaker (1973). Subfractionation of LDLs by density has yielded particles differing substantially in molecular weights, as determined from their flotation coefficients. To compare values measured under different solvent conditions in these three studies, the flotation coefficients for the particles shown in Fig. 2 have all been corrected for solvent density and viscosity to the values they would exhibit in a KBr solvent with a density of 1.20 g/ml, and the viscosity of KBr at 25°C (Schumaker, 1973). A quantitative molecular model for LDLs consistent with these data will be developed next.

APOLIPROTEIN B AND LDL STRUCTURE

217

BUOYANT DENSIM (g/rnl)

FIG. 2. Density dependence of the flotation coefficient for LDLs and IDLs. The experimental values (0)were compiled by Schumaker (1973) and represent fractionated lipoproteins from both normal and abnormal lipidemic individuals. The calculated values, represented by the solid line, are a plot of column 2, Table 11, of this review, and demonstrate that the emulsion particle model is compatible with the observed hydrodynamic properties of these lipoproteins.

B . Quantitative Molecular Model for Low-Density Lipoproteins For molecular modeling of the lipoproteins, values for the partial specific volumes of the lipoprotein components are required. The partial specific volume of an aqueous egg yolk lecithin suspension is 0.984 ml/g (Hauser and Irons, 1972), and this provides a reasonable approximation for the partial specific volume of the phospholipid occupying the surface monolayer of a lipoprotein. The reciprocal of the density of liquid triolein (Small, 1986) yields its partial specific volume, 1.102 mVg, and provides a reasonable approximation for triglyceride dissolved in the cholesteryl ester-filled core of the LDL. For cholesterol, the partial specific volume of 1.021 ml/g measured in benzene (Haberland and Reynolds, 1973) has been employed. The value of 0.740 ml/g employed for the partial specific volume of apoBlOO was determined from its amino acid composition (Lee et al., 1987). A value of 0.60 ml/g was used for the partial specific volume of the carbohydrate moiety. One important parameter, the partial specific volume of cholesteryl ester, remains to be determined. As will be shown below, its value is estimated to be 1.058 ml/g. The emulsion particle model for LDLs developed here assumes that the particles have a spherical core consisting of cholesteryl esters and

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triglyceride. The triglyceride content as a percentage of total core lipid was allowed to vary according to a least-squares quadratic fit between the lipoprotein radius and the compositional data of Chapman et al. (1988); this variation in the triglyceride content resulted in only a very small change in lipoprotein density, size, and shape, while bringing calculated lipoprotein composition into agreement with experimental observation. According to the emulsion particle model, the core is surrounded by a spherical shell of 20.2 A (Shen et al., 1977) consisting of apoBlOO (M 5 12,937), phospholipid, and cholesterol. T h e phospholipid and cholesterol were assumed to be present in a 1 : 1 molar ratio, close to that observed for LDLs (Chapman et al., 1988).T h e single molecule of apoB was assumed to be located at the surface of the LDL but embedded within the surface shell surrounding the core. This is reasonable, because the volume of phospholipid and cholesterol is sufficient to fill only about 70% of the volume of a 20.2-A surface shell around an LDL of average size. Conveniently, the volume of a single apoB molecule almost exactly fills the remaining 30% of this volume. From this emulsion particle model, it was possible to predict the lipoprotein composition, buoyant density, and hydrodynamic properties of the LDL as a function of lipoprotein size, given the partial specific volumes of the lipid, protein, and carbohydrate components. A crosssectional slice through the model is shown in Fig. 3 as two concentric circles, representing the hydrophobic core surrounded by a monolayer of phospholipid, cholesterol, and protein. The model parameters are given in the footnote to Table I1 and include the thickness of the shell, the

ApoB FIG.3. The emulsion particle model for LDLs. This cross section of the spherical emulsion particle shows a core of radius, r, surrounded by a shell of thickness, t, in which is embedded a single molecule of apoB. The external radius R = r + t.

APOLIPROTEIN B AND LDL STRUCTURE

219

partial specific volumes of each component, and the molecular weight of the protein. The carbohydrate, which is attached to the surface of the protein, was allowed to extend into the aqueous phase. It contributes to the molecular weight, volume, and density of the lipoprotein and to the frictional ratio of 1.1 1, but does not displace the shell lipids. Table I1 lists predicted values for the lipoprotein size, hydrodynamic properties, and composition, calculated according to this model. In order to calculate the parameters listed in Table 11, a radius was selected and the lipoprotein volume was calculated. The radius of the core was determined by subtracting the shell thickness from the lipoprotein radius; then the volume of the core was computed. From the calculated ratio of the two core lipids (from the quadratic fit between radius and the percentage of core triglycerides) and their partial specific volumes, the density of the core lipid was determined; then, given the core volume, the core weight was calculated. Once the weight of the core was known, then the weights of cholesteryl ester and triglyceride were calculated from their weight ratio. The difference between the lipoprotein volume and the core volume yielded the shell volume. From the shell volume was subtracted the volume of a single apoBlOO protein molecule, to yield the volume occupied by the phospholipid and cholesterol. From the 1 : 1 molar ratio assumed for these two surface lipids and their partial specific volumes, the density of the surface lipid was determined; then, given the surface lipid volume, its weight was calculated; the weights of phospholipid and cholesterol then followed from their ratio. The weight and volume of the carbohydrate were added to the weight and volume of the remainder of the LDL components before calculating the lipoprotein density. Finally, from the weights of all the components, the percentage of each component was calculated and listed in Table 11. For simplicity of calculation, the core was assumed to contain all of the triglyceride and cholesteryl ester, although it is known that small amounts of the core lipids are dissolved in the surface monolayer, where they represent about 3 mol% of the surface lipids, and a larger fraction, about one ninth of the cholesterol, is dissolved in the core (Miller and Small, 1987).The presence of core lipids in the lipoprotein surface is very important metabolically, for the lipases and transfer proteins have access to these core lipids without having to penetrate the surface monolayer. For the calculation of composition, density, and size, however, the effects of component transfer between surface and core affect these quantities about one part in the fourth significant figure, and have been neglected in Table 11. Once the composition and partial specific volume are known for each lipid, protein, and carbohydrate component of the LDL, then the LDL

TABLE I1 Predicted Physical Properties and Composition of LDL( According to Emulsion Particle ModeP

Composition in weight (percent)

w

0

Radius

Densityb

Molecular weight'

SZ5,,.20d

90.0 91.0 92.0 93.0 94.0 95.0 96.0 97.0 98.0 99.0 100.0 101.0 102.0 103.0 104.0 105.0 106.0 107.0 108.0 109.0 110.0 111.0 112.0

1.0555 1.0527 1.0499 1.0473 1.0447 1& 23 I 1.0399 1.0376 1.0354 1.0333 1.0312 1.0292 1.0273 1.0254 1.0236 1.0219 1.0202 1.0186 1.0170 1.0154 1.0139 1.0124 1.0110

1.96 2.02 2.08 2.14 2.21 2.27 2.34 2.41 2.48 2.55 2.62 2.69 2.77 2.84 2.92 3.00 3.08 3.16 3.25 3.33 3.42 3.51 3.60

-26.42 -27.54 -28.66 -29.79 -30.94 -32.09 -33.25 -34.42 -35.60 -36.79 -38.00 -39.2 1 -40.43 -41.66 -42.91 -44.16 -45.42 -46.70 -47.99 -49.28 -50.59 -51.92 -53.25

s; 1.19 1.68 2.18 2.68 3.17 3.68 4.18 4.69 5.20 5.72 6.24 6.76 7.29 7.82 8.35 8.89 9.43 9.98 10.53 11.08 11.64 12.21 12.78

CE

TG

PL

C

Protein

Carbohydrate/

39.2 39.6 39.9 40.2 40.5 40.8 41.1 41.4 41.6 41.9 42.1 42.3 42.5 42.7 42.9 43.0 43.2 43.3 43.4 43.5 43.6 43.7 43.8

2.1 2.3 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.5 4.7 5.0 5.2 5.5 5.8 6.0 6.3 6.6 6.9 7.2

20.6 20.9 21.1 21.2 21.4 21.5 21.7 21.8 21.9 22.0 22.1 22.2 22.3 22.4 22.4 22.5 22.5 22.6 22.6 22.6 22.6 22.7 22.7

10.2 10.3 10.4 10.5 10.5 10.6 10.7 10.7 10.8 10.8 10.9 10.9 11.0 11.0 11.0 11.1 11.1 11.1 11.1 11.1 11.2 11.2 11.2

26.2 25.4 24.7 23.9 23.3 22.6 21.9 21.3 20.7 20.1 19.6 19.1 18.5 18.0 17.6 17.1 16.7 16.2 15.8 15.4 15.0 14.6 14.3

1.7 1.7 1.6 1.6 1.5 1.5 1.4 1.4 1.3 1.3 1.3 1.2 1.2 1.2 1.1 1.1 1.1 1.1 1.o 1.o 1.0 1.o 0.9

r4

2

113.0 114.0 115.0 116.0 117.0 118.0 119.0 120.0 121.0 122.0 123.0 124.0 125.0 126.0 127.0 128.0 129.0

1.0096 1.0083 1.0070 1.0057 1.0044 1.0032 1.0020 1.0009 0.9997 0.9986 0.9976 0.9965 0.9955 0.9944 0.9935 0.9925 0.99 15

3.69 3.78 3.88 3.97 4.07 4.17 4.27 4.38 4.48 4.59 4.69 4.80 4.92 5.03 5.14 5.26 5.38

-54.59 -55.95 -57.32 -58.70 -60.09 -61.49 -62.91 -64.34 -65.78 -67.23 -68.70 -70.18 -71.67 -73.18 - 74.69 -76.23 -77.77

13.35 13.93 14.51 15.10 15.69 16.29 16.90 17.51 18.12 18.74 19.37 20.00 20.64 2 1.29 21.94 22.59 23.26

43.8 43.9 43.9 43.9 43.9 43.9 43.9 43.9 43.8 43.8 43.7 43.6 43.5 43.4 43.3 43.2 43.0

7.5 7.8 8.2 8.5 8.8 9.2 9.5 9.9 10.3 10.6 11.0 11.4 11.8 12.2 12.6 13.0 13.5

22.7 22.7 22.7 22.7 22.7 22.7 22.6 22.6 22.6 22.6 22.5 22.5 22.5 22.5 22.4 22.4 22.3

11.2 11.2 11.2 11.2 11.2 11.2 11.2 11.1 11.1 11.1 11.1 11.1 11.1 11.1 11.0 11.0 11.0

13.9 13.6 13.2 12.9 12.6 12.3 12.0 11.7 11.4 11.2 10.9 10.7 10.4 10.2 10.0 9.7 9.5

0.9 0.9 0.9 0.8 0.8 0.8 0.8 0.8 0.7 0.7 0.7 0.7 0.7 0.7

0.6 0.6 0.6

~________

"Shell thickness, 20.2 A. Partial specific volumes of cholesteryl ester (CE), triglyceride (TG), phospholipid (PL), cholesterol (C), protein, and carbohydrate are 1.058, 1.102, 0.984, 1.021, 0.740, and 0.60 g/ml, respectively. A 1 : 1 molar ratio of phospholipid to cholesterol is assumed in the surface. Core triglyceride, as the percentage of total core lipids, is given by core T G = 9.191 - 0.4132R + 0.00408364R2,where R is the lipoprotein radius in angstroms. T h e buoyant densities in this column = (Hc,v,) + 0.0016 g/ml, where the last additive term is to adjust for the differential compressibilities of water and lipoproteins at 52,640 rpm. 'LDL molecular weight X lo6. "Sedimentation coefficient in a KBr solvent with a density of 1.20 glml, in a solvent with the viscosity of 0.8534 centipoise (25°C).T h e negative sign indicates flotation. T h e flotation coefficient in a NaCl solvent with a density of 1.063 g/ml and viscosity of 1.0260 centipoise (26°C). 'The carbohydrate composition was assumed to be 6.5% by weight of the protein concentration.

222

VERNE N . SCHUMAKER ET AL.

density may be computed. Given the density and radius, the sedimentation coefficient, s, also may be computed from the following expression:

where M is molecular weight, qsOlv is the solvent viscosity, dl, is the lipoprotein density, N is Avogadro's number, dsolvis the solvent density, R is the lipoprotein radius, and f/fi is the translational frictional ratio. For a spherical, anhydrous particle, the frictional ratio is 1.00; however, LDLs are hydrated, and the frictional ratio, determined through diffusion measurements, is 1 . 1 1 (Fisher et al., 1971). This value was used in calculating the sedimentation coefficients listed in Table 11. The lipoprotein density that should be employed in Eq. (2) is the buoyant density, which is the density at which the sedimentation coefficient is equal to zero. T h e buoyant density was obtained experimentally by measuring the sedimentation coefficient at several solvent densities and extrapolating to zero. Kahlon et al. (1982) have shown that the buoyant densities, which they call the u densities, vary with the rotor speed of the centrifuge, reflecting the different compressibilities of water and lipid. In order to convert the lipoprotein density, as determined from its composition, to the buoyant density at a rotor speed of 52,640, the data of Kahlon et al. (1982) were used calculate a correction factor of 0.0016 g/ml, which was added to the compositional densities. T h e values of buoyant densities listed in Table I1 have been calculated by adding 0.0016 g/ml to the density values determined from their compositions. Utilizing the buoyant densities listed in Table I1 and the frictional ratio of 1 . 1 1, values for the sedimentation coefficient have been calculated at two different solvent densities: the standard S,"value routinely used to characterize human serum lipoproteins is defined as value of the flotation coefficient in Svedberg units (the negative sedimentation coefficient x l o p i 3 sec) in an aqueous NaCl solvent with a density of 1.063 g/ml and a viscosity of 1.021 centipoise (the viscosity of a 1.063 g/ml sodium chloride solution at 26°C). These values are listed in Table 11. The S," value is very sensitive to small variations in lipoprotein density because the solvent density is close to the lipoprotein density. T o compare the particle sizes or molecular weights, values of the sedimentation coefficient (s)in a solvent with a density of 1.20 g/ml and the viscosity of KBr at 25°C are preferred, and the computed values are listed in Table 11.

TABLE I11 Experimentul Physical Properties and Composition of Pooled Human LDL" Composition in weight percent Fraction 4 5 6 7 8 9 10 11

12 13 14

Gradient density ( g W

Buoyant densityb ( g W

1.0234 1.0260 1.0286 1.0314 1.0343 1.0372 1.0409 1.0451 I .0502 I .0580 1.OM0

1.0172 1.0196 1.0224 1.0256 1.0289 1.0318 1.0358 1.0404 1.0460 1.0545 1.0632

Sf 9.30 8.50 7.60 6.70 5.90 5.20

CE

TG

PL'

C

Proteind

Carbohydrate'

41.50 41.81 41.61 43.06 43.62 43.43 42.98 4 1.06 39.5 1 40.08 40.95

6.3 1 5.49 4.90 3.77 3.57 3.78 3.89 3.30 3.10 2.27 2.59

22.75 22.64 23.14 22.1 1 22.24 20.88 2 1.35 20.63 22.08 2 1.32 20.57

10.99 11.60 10.71 11.81 11.21 10.93 9.72 9.70 8.77 9.40 9.20

17.33 17.33 18.44 18.08 18.18 19.70 20.71 23.77 24.93 25.28 25.07

1.13 1.13 1.20 1.18 1.18 1.28 1.35 1.54 1.62 1.64 1.63

"Experimental values were taken from Chapman et al., (1988) and adjusted as described in footnotes c and d . 'Buoyant densities were determined from a plot of gradient density versus buoyant densities (udensities), constructed using the appropriate values from Kahlon et al. (1982). 'PL values listed by Chapman et al. (1988)were divided by 0.93 to adjust for the phospholipid not accounted for by the phosphocholine-specific assay used by these workers. dLowry values listed by Chapman et al.(1988)were multiplied by a color correction factor of 0.80 wt apoB/wt BSA, to adjust their Lowry values for the BSA standard. 'The carbohydrate composition was assumed to be 6.5% by weight of the protein.

-2 +

50 4

48--

v

v, l-

z

W

Z 0

46--

0

44-

a @a

0 0

0

42--

0

5 W

0 K

a L

2

W

--

40-

381

r

n *

25 1-

6 IY

0

@ a

O

231

a

a a0

1

ma

21 19/

a

1157 ; Y ( 1.01 0 1.020

1.030

1.040

1.050

1.060

BUOYANT DENSIlY (g/rnl)

FIG.4. A comparison of the observed and calculated sedimentation and compositional were taken from Table 111; the calculated properties of LDLs. The observed properties (0) properties ( 0 )are from Table 11. (A) A comparison of core lipids, defined as the sum of the percentages of cholesteryl esters and triglycerides. (B) A comparison of the percentages of protein. (C) A comparison of the flotation coefficients in a solvent of density 1.063 g/ml (SP values).

225

APOLIPROTEIN B AND LDL STRUCTURE

C

" E I-

z w 0 L LL

w

0 0 Z

0

F

s

1.010

1.020 1.030 BUOYANT DENSITY (g/rnl)

1.040

FIG. 4. (Continued)

How well do the sedimentation coefficients and densities predicted by the model match the values actually observed for LDL? Excellent agreement with the experimental points is shown by the solid curve of Fig. 2, which is a plot of the values for ~ 2 5 , 1 . 2 0given in Table 11. However, this agreement was achieved by selecting a value for the partial specific volume of the cholesteryl esters to make the best fit, yielding the value of 1.058 ml/g for this this quantity. [If a value of 1.044 ml/g were employed for the partial specific volume of the cholesteryl esters, as was used by Sata et al. (1972), the values of ~ 2 5 . 1 . 2 0 listed in Table I1 would have decreased by about 3.5%. The values of Sf in Table I1 would have dropped by 1 to 2 Svedbergs.] How well do the compositional values for the individual lipid components and protein and carbohydrate values predicted by Table I1 agree with experimental values? For this comparison, we have selected the experimental values for fractionated LDL published by Chapman et al. (1988). Before these values could be compared, it was necessary to make three adjustments to the experimental values. These workers employed an enzymatic phospholipid assay (Takayama et al., 1977) specific for phosphocholine-containing lipids, which included phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine, making u p about 93% of LDL lipids (Skipskiet al., 1967). Therefore, the weight of phospholipid has been increased by a divisor of 0.93 in calculating the adjusted experimental values listed in Table 111. Second, Chapman and co-workers

226

VERNE N. SCHUMAKER ET AL.

(1988) employed the Lowry procedure to determine the apoB content of LDL, using bovine serum albumin as a standard. Such values must be multiplied by a color conversion factor to convert bovine serum albumin weights to apoB weights. Therefore, the weight of protein has been decreased by a factor of 0.80 g apoB polypeptide/l g serum albumin (Fisher and Schumaker, 1986; Margolis and Langdon, 1966b) to adjust the values reported by Chapman et al. (1988) to those reported in Table 111. Finally, Chapman el al. (1988) did not list the carbohydrate content of their LDL; we have assumed that the apoB 100 polypeptide contains 6.5% carbohydrate by weight, the average of the reported values, which vary from about 5 to 8%. The adjusted experimental values, converted to percentages of the total LDL weight, are listed in Table 111. Differences between the adjusted observed values of Table 111 and those calculated from the emulsion particle model in Table I1 fall within the experimental error limits reported by Chapman et al. (1988). This comparison is also provided in graphical form for total core lipids (triglycerides plus cholesteryl esters) (Fig. 4A), for protein (Fig. 4B), and for the values of the flotation coefficient, S,“(Fig. 4C). The agreement is seen to be excellent in all cases. We conclude that the emulsion particle model provides a satisfactory description of the composition, size, density, and hydrodynamic properties of normal human LDL isolated by density gradient centrifugation. IV. STRUCTURAL STUDIES OF APOLIPOPROTEIN B ON LOW-DENSITY LIPOPROTEIN SURFACES What is the configuration of apoB 100 on the surface of the LDL? Is it folded into a single domain, as suggested by the electron microscopy (EM) studies of Lee et al. (1987)? Is it composed of three domains, as suggested by the proteolysis studies of Chen et al. (1989), (Fig. lG); five domains, as suggested by the trypsin releasability studies of Yang el al. (1989) (Fig. 1F);or four domains, as suggested by low-angle X-ray studies (Luzzatti et al., 1979) and freeze-fracture electron microscopy (GulikKryzwicki el al., 1979)? Other EM studies have interpreted the structure of apoB on LDLs to be composed of 20 domains (Pollard et al., 1969) or “strand-like substructures, possibly forming a surface network” (Forte and Nichols, 1972). T o explore this question, three different approaches have been employed to map apoB on the surface of the LDL: (1) lipid extraction after attachment of the LDL to an EM grid, (2) mapping of apoB on the LDL surface with monoclonal antibodies, and (3)determination of how core circumference is related to apoB size during lipoprotein biosynthesis in cell culture.

APOLIPROTEIN B A N D LDL STRUCTURE

227

A . Lipid Extraction after Attachment of Low-Density Lipoproteins to Electron Microscope Grids The first of these techniques (Phillips and Schumaker, 1989) involved adsorbing the LDL to a carbon-coated electron microscope grid, extracting the lipid with 4: 1 ethanokether, which solubilized both polar and nonpolar lipids, and negative staining of the proteinaceous residue, which remained attached to the grid. After negative staining with uranyl acetate, the grid was examined in the electron microscope. Figure 5A shows the intact LDL absorbed to the grid prior to lipid extraction, and Fig. 5B shows the apoB 100 remaining after extraction. The protein (Fig. 5B) is seen to be a long, flexible structure, usually bent into a circular or semicircular configuration, with a diameter approximately equal to that of the LDL (Fig. 5A) from which is was obtained. If the LDLs were first treated with a cross-linking reagent such as glutaraldehyde (Fig. 5C) or water-soluble carbodiimide, then the apoB 100 remaining after extraction appeared as solid circles (Fig. 5D) with diameters equal to those of the LDLs from which they were derived. How were these results to be interpreted in terms of the configuration of apoB 100 on the surface of the LDL? The obvious interpretation was that apoB surrounds the LDL like a belt; moreover, it was also suggested that chemical cross-linking buckles the belt (Fig. 6) (Phillips and Schumaker, 1989). Other interpretations might be advanced; for example, if apoB covered the surface of the LDL like a hair net, then removal of the lipid with the organic solvent might cause a netlike apoB to settle into acircular pool, with much of the mass at the periphery, artifactually creating a circular belt from a structure that was not beltlike at all. Therefore, a second approach was required to test the beltlike model. For this purpose monoclonal antibodies were employed to map apoB 100, in the presence of lipid, on the surface of the LDL.

B . Mapping of Apolipoprotein B on Low-Density Lipoprotein Sulfaces with Monoclonal Antibodies

Electron microscopy of negative-stained native LDL samples does not resolve the distribution of apoB 100 on the lipoprotein surface. However, in electron micrographs of LDL-antibody complexes, antibodies, recognized as small, Y-shaped objects, have been observed protruding from the LDL particles (Chatterton et al., 1991). Therefore, monoclonal antibodies directed against apoB can be used to locate specific regions of apoB on the LDL surface. Because each LDL contains only a single copy

FIG. 5. LDL before and after lipid extraction when bound to the EM grid. After adsorption to the carbon-coated grid, lipid was extracted by brief immersion in ice-cold (4 : 1) ethanol :ether. Negative-stained images include (A) untreated LDL, (B) lipidextracted LDL, (C) glutaraldehyde-treated LDL, (D) lipid-extracted, glutaraldehydetreated LDL, (E) glutaraldehyde-treated LDL, (F) lipid-extracted, glutaraldehyde-treated IDL. Bar = O.1pm for A-F. (Phillips and Schumaker. 1989.)

AFQLIPROTEIN B AND LDL STRUCTURE

229

FIG. 6. ApoB100 is modeled as a belt surroundingthe LDL. The protein is assumed to be embedded in the monolayer, where the inner surface of the belt makes contact with the core lipids and the edges of the belt interact with the phospholipid andlor cholesterol. To fulfill volume requirements, the cross section of the belt would be about 20 x 54 8, and the average length about 585 8,. Cross-linkingwith a chemical reagent “buckles”the belt.

of apoB, and because apoB lacks any repetitive sequences, each LDL should possess only one site complementary to a given monoclonal antibody. As expected, when a single monoclonal antibody was added to LDL, a maximum of one antibody was observed attached to a single LDL. When two different monoclonal antibodies recognizing separate apoB epitopes were simultaneously added to the LDLs, occasionally both types of antibody were seen attached to the same LDL particle (Fig. 7). Visualization of bound antibody was a fortuitous occurrence, requiring that the LDL-antibody complex adsorb to the carbon-coated grid surface with an orientation such that both bound antibodies lay flat on the grid, displacing the thin layer of negative stain (Fig. 8). For a small percentage of the LDLs (1- 10%)this fortuitous orientation did occur, allowing both monoclonals to be visualized and permitting measurement of the angle, at the LDL center, separating the two recognized apoB epitopes (Fig. 7). In a few cases, LDL dimers were seen with two LDLs joined by two different monoclonal antibodies to form a circular complex. Again, the angles could be measured for both members of the pair. Accurate determination of the average angle between a given pair of monoclonal antibodies bound to the same LDL required multiple measurements. In each case, a wide spread of angles was obtained from which an average value could be calculated (Fig. 9). Why was such a large variation in the measured angle observed? Repeating the measurements on the same set of electron micrographs showed that the reading error

FIG. 7. Negative-stained images of monoclonal antibodies binding to apoBlOO on LDL. (A) An equimolar mixture of LDL and each of two anti-apoB100 monoclonal antibodies, MB47 and MB19, were adsorbed to the carbon-coated grid, stained with 1% uranyl acetate, and examined in the electron microscope. The insert shows both open (0) and closed (c) complexes. Bar = 0.1 pm. (B) Complexes formed between LDL and monoclonal antibodies MB47 and MB24. (From Chatterton etal., 1991.)

rI

FIG.8. Illustration of two monoclonal antibodies bound to a flattened LDL. If a sphere of diameter 208 8, was uniformly flattened, holding volume constant, until it had an apparent diameter of 285 8, in projection, it might resemble half of an oblate ellipsoid. A truncated oblate ellipsoid with a major axis of 285 8, and a semiminor axis of 11 1 A would have the same volume as a 208-8, sphere. Diameters of this size or larger were frequently observed. Monoclonal antibody 8, has bound to the LDL above the layer of negative stain, and would not be observed. Monoclonal antibody B has bound to the LDL in the plane of the EM grid, and has displaced the layer of negative stain, SO that it would be observed in the electron micrograph.

APOLIPROTEIN B AND LDL STRUCTURE

23 1

30 20 10

0

60 120 ANGLE (deg)

180

ANGLE (deg)

FIG. 9. Observed angular distributions of pairs of monoclonal antibodies binding to LDL. (A) The distribution of 124 angles measured between 4G3 and B3 yield an average angle of 104". (B) The distribution of 139 angles measured 4G3 and B4 yield an average angle of 60".

was small, about 23". Parallax could not explain this variation because the two antibodies both must have lain in the plane of the grid to be visualized. It was concluded that the spread of measured angles was real, and probably reflected an actual variation in the angle between epitopes caused by flexibility of the apoB embedded in a fluid surface monolayer. To determine the configuration of apoBlOO on the LDL surface, 10 different monoclonal antibodies have been employed (Table IV). In the original model (Chatterton et al., 1991), three antibodies (MBl 1, MB44, and anti-B,,, 16) were placed on a three-dimensional map relative to MB47, MB24, and MB19, which defined the North Pole, the prime meridian, and the handedness, respectively (Fig. 10). In the current model, angles between the first three antibodies (MB19, MB24, and MB 1 1) were used to establish a spherical coordinate system on the LDL surface. To minimize errors inherent in measuring angles between epitopes separated by extremely long distances along the apoB sequence, the next five antibodies (2D8, B4, B3, 4G3, and MB47) were placed on the map by triangulation relative to the three sites that immediately preceded them in the sequence (Chatterton et al., 1993). For example, monoclonal antibody B3 was placed on the map relative to MB 11, 2D8, and B4. The results are shown in Fig. 11. The monoclonal antibodies recognized epitopes that were welldistributed along the first 3500 amino acids of apoBl00. In the threedimensional model (Fig. 1l), the flags, which locate the sites recognized by the eight monoclonal antibodies, are connected by a string in the same order as they appear along the apoB 100 primary sequence. The model was superimposed on a globe and rotated such that antibodies MB19,

232

VERNE N. SCHUMAKER ET AL.

TABLE IV Location of Epitopes Recognired by Monoclonal Antibodies against Apolipoprotein'

Antibody

Sourceb

MB19 MB24 MBI 1 2D8 B4 B3 4G3 MB47 Anti-B,, 16

1 1

1 2 3 3 2 1 2

Binding site location (apoB amino acid residue) 71 399-580 995-1082 1438- 1480 1827- 1943 2330-2376 2980-3084 344 1-3568 4 154-4189

"Determination of epitope positions along the apoB sequence has been reported previously by Knott et al. (1986), Milne et al. (l989), Young and Hub1 (1989), and Pease et al. (1990). bMonoclonalantibodies were obtained from (1) Dr. L. K. Curtiss. Scripps Research Institute, California; (2) Dr. Y. L. Marcel and Dr. R. W.Milne, Clinical Research Institute, Montreal; and (3) Dr. J.-C. Fruchart, The Institute Pasteur, Lille, France.

MB24, and MB 11 occupied approximately the same locations as they did in the original model (Chatterton et al., 1991). Thus, apoB runs from the N terminus near MB19 located on the coast of Colombia, 4" west of Bogota (5"N, 78"W); to MB24 above the Atlantic Ridge, 10" north of the equator (10"N, 36"W); to M B l l off the coast of Uruguay, 6" east of Buenos Aires (35"S, 52"W); to 2D8 off the coast of Antarctica near Enderby Land (62"S, 50"E); to B4 in Queensland, Australia (25"S, 142"E); to B3 in the Indian Ocean off the southern tip of India, near the Maldive Islands (2"N, 75"E); to 4G3 in the Pacific Ocean, near Johnston Island southwest of Hawaii (19"N, 172"W); and to MB47 in the Pacific Ocean, near the Island of Clarion off the western coast of Mexico (2 1"N, 119"W) (J. E. Chatterton, et al. 1994.) T h e most significant difference between this map and the original involves the position of MB47. In the original map, MB47 was placed at the North Pole 80" north of MB19. In the present map, MB47 is only 45.6" from MB19. This difference is believed to be due to flattening of the LDL spheres on the electron microscope grid, leading to an increase

APOLIPROTEIN B A N D LDL STRUCTURE

A

m h l (N. Pole)

B

mAbl

233

Prime Meridian

mAbl

Longitudes

FIG. 10. lnterpretation of mapping measurements. (A) The position where the first monoclonal antibody bound (mAbl) was defined as the North Pole. Therefore, the angle between mAbl and a second monoclonal antibody gave the latitude of the second antibody. (B) The position where mAb2 bound was defined as "Greenwich." Therefore, the great circle that passes through the North Pole (mAbl) and Greenwich (mAb2) was 0" longitude. (C) From the two angles measured between mAbl and mAb2 and any other monoclonal antibody, mAb3, both the latitude and longitude of the other monoclonal antibody were calculated. An ambiguity arose, because the third monoclonal antibody may be placed either in the Eastern or Western Hemisphere. This ambiguity was resolved by arbitrarily placing the third monoclonal antibody in the Western Hemisphere. Thereafter, additional monoclonals were located unambiguously by triangulation. The final map, however, may be a mirror image of the correct map.

in the radius of curvature of the lipid surface, causing the two ends of apoB to drift apart on the surface. This conformational change may reflect a similar change that apoB must undergo in order to exist on the surface of the much larger VLDL particles. In addition, MB44 was

234

VERNE N. SCHUMAKER ET AL.

FIG.1 1 . Mapping apoB100 on the surface of the LDL. The string between toothpicks connects the epitopes in order as they occur along the primary sequence of apoB (Table IV).

removed from the map due to the extremely broad distributions in measured angle always found with this antibody. Finally, anti-B,,116 has been temporarily removed from the map pending acquisition of additional data. Clearly, the first 3500 amino acids of apoB 100 nearly circumnavigate the globe. T h e gap of 45.6"separating MB47 and MB 19 could easily be spanned by the remaining 1000 amino acids between the site recognized by MB47 and the C terminus of apoB. Making the assumption that apoB is relatively uniform along its length locally, only 400 of the remaining

APOLIPROTEIN B AND LDL STRUCTURE

235

1000 amino acids would be required to complete the circumnavigation, placing amino acid residue 3900 somewhere near the N terminus of apoB. This leaves open the question of where the remaining approximately 600 C-terminal amino acids are located. Does apoB continue from residue 3900 in the same general direction past the N terminus, essentially retracing its own steps? Or does apoB double back, thus placing the C terminus somewhere near the site recognized by MB47 and the LDL receptor? These questions will be resolved as additional monoclonal antibodies between MB47 and the C terminus are placed on the map. Thus, the results of these mapping studies support the cross-linking and lipid extraction studies, and are consistent with a beltlike model for the structure of apoB 100 on the LDL surface. This proposed structure is further supported by a preliminary analysis of LDL in vitreous ice at 34 A resolution by electron microscopy (Atkinson, 1989, 1993)-480 LDL particles were examined to yield five independent sets containing 4- 12 images. “The independent views indicate that LDL is a semi-spherical, 200-220 A diameter particle, with an area of low density (lipid) surrounded by a ring (in projection) of high density believed to represent apolipoprotein B-100. This ring is seen to be composed of four or five (depending on view) regions of high density material that may represent protein superdomains linked by areas of somewhat lower density.” (Atkinson, 1993). Another independent approach to the mapping of apoB on the lipoprotein surface arose unexpectedly during studies on the mechanism of lipoprotein assembly in hepatocyte cell lines, and this will be described next. C . Relating Low-Density Lipoprotein Core Circumferences to Apolipoprotein B Fragment Sizes

A remarkable observation was made by Yao et al. (1991), who expressed a homologous series of C-terminal truncated apoB molecules in stably transfected McArdle 7777 cells, a rat hepatocyte cell line. Plotting the buoyant density of the secreted lipoproteins as a function of the logarithm of the number of amino acid residues, they noticed a linear relationship between these two quantities, implying that the lipoprotein density was inversely proportional to the apoB size. Evidently some fundamental architectural principle was being explored in their studies. A homologous series of C-terminal truncated apoBs in transiently transfected HepG2 cells was prepared by Spring et al. (1992a,b). These data, together with the data taken from Yao et a/. (1991), are presented in Table V, which lists the sizes of the newly expressed apoB molecules using the centile nomenclature of Kane et al. (1980), the observed

236

VERNE N . SCHUMAKER ET AL.

TABLE V Expression and Secretion of a Series of C-Tenninnlly Truncuted Fragmenfs in Hep-G2 and McArdlc 7777 Cells Cell Type HepG2

McArdle 7777

ApoB size

Densitv (g/ml)

Radiusb(A)

ApoB26 ApoB31 ApoB37 ApoB42 ApoB49 ApoB18 ApoB23 ApoB28 ApoB31 ApoB37 ApoB48 ApoB53 ApoB 100

1.163 1.143 1.136 1.103 1.101 1.23 1.23 1.17 1.17 1.14 1.10 1.06 1.006

43.7 48.7 51.3 57.3 59.5 35.0 37.9 44.2 45.6 50.9 60.0 69.7 104.0

"Experimentally observed density values for HepG2 (Spring et al., 1992b) and McArdle 7777 cells (Yao et al., 1991). "Calculated from the emulsion particle model, given the size of the apoB, the observed density, and the partial specific volumes of the constituent lipid, protein and carbohydrate, a 1 : 1 molar ratio of C to PL,a 25 :6 wt ratio of TG :CE (Thrift et al., 1986), and a shell thickness of 20.2 A.

buoyant density of the secreted lipoproteins, and the predicted lipoprotein radii as calculated assuming the emulsion particle model, as explained in the footnote to Table V. The values listed in Table V are plotted in Fig. 12. The lipoprotein radius is plotted as a function of apoB size, and the points, which include all of the data of Yao et al. (1991) and all of ours, define a single straight line. The existence of this linear relationship strongly suggests that these lipoproteins form a homologous series of emulsion particles; moreover, it provides an additional insight into the nature of the fundamental relationship underlying the structure of these lipoproteins, that is, lipoprotein radius is a linear function of apoB size. Two parameters may be obtained from a straight line, the slope and the intercept. The slope of the line shown in Fig. 12 is 0.166 A/kDa. The intercept is approximately 20 A, which is close to the thickness of the surface monolayer of phospholipid, cholesterol, and protein surrounding the core of the lipoprotein particle. This interesting relationship between lipoprotein radius and apoB size has been confirmed by a study of the sizes of the secreted lipoproteins formed after puromycin treatment of HepG2 cells. This antibiotic causes

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237

1

0

0

2E5

4E5

6E5

MOLECULAR MASS OF APOB FRAGMENT FIG. 12. Lipoprotein radii as a function of apoB molecular size. Lipoprotein radii were determined from the measured buoyant densities and apoB fragment molecular masses according to the emulsion particle model (Table V). The straight line through the experimental points has a slope of 0.166hkDa apoB, and a vertical intercept of 19.7 A. A, From Yao el al. (1991); 0,from Spring el al. (1992b).

premature release of the growing polypeptide chain, creating a homologous series of C-terminally truncated apoB polypeptides translated from endogenous message. These truncated apoB polypeptides are used by the HepC2 cells to synthesize small lipoproteins, which they secrete into the medium and which may be isolated from the medium by flotation. Hydrodynamic characterization of these particles yields both flotation coefficients and densities (Table 11), from which the radii may be calculated (Spring et al., 1992a,b). Radii determined in this manner are listed in column 4 of Table VI, and can be seen to increase with the size of the apoB. These radii were obtained without assuming the emulsion particle model. The data of Table VI were plotted to yield the relationship between lipoprotein radius and apoB size (Fig. 13A). Here again, the relationship between the lipoprotein radius and apoB size was linear, with the points defining a straight line intercepting the vertical axis at a radial value of about 20 A, as seen previously for the case of the C-terminally truncated apoBs expressed by the transfected hepatocytes (Fig. 12). The vertical intercept of 20 A (Figs. 12 and 13A) was suggestive of the thickness of the monolayer in the emulsion particle model. In fact, if

238

VERNE N. SCHUMAKER ET AL. TABLE Vl Radll of Puromycin-Generated Particles Calculated from Sedimentation Coefficients and Buoyant Densities"

ApoB sizeb(centile)

Observed density (g/ml)

80 70 55 51 49 45 42 38 36 33 31 29 25

1.034 1.048 1.058 1.071 1.080 1.09I 1.094 1.104 1.109 1.125 1.144 1.150 1.170

(Svedberg)

5~0,1.2,1

-30.4 -20.1 -14.1 - 12.2 - 10.6 -7.78 - 7.07 -5.82 -5.34 -3.68 -2.51 - 1.83 -0.77

Radius'

(8)

96.1 81.6 70.8 69.1 66.6 60.0 58.0 55.3 54.3 49.7 47.5 42.9 36.0

"Springel al. (1992b). *ApoBsize is expressed in the centile nomenclature,which indicates the apparent molecular weight, as determined by SDS-PAGE as a percentage of the molecular weight of' the full-length apoB100. 'Calculated from the observed buoyant densities and sedimentation coefficients by Eq. (2).

the monolayer thickness, assumed to be 20.2 A, was subtracted from each radial value, the differences became the radii of the lipoprotein cores. In order to replot the data, core radii were multiplied by 27r, to yield core circumferences. It was also convenient to multiply the apoB sizes on the horizontal axis by 513 kDa, the approximate size of the full-length apoB 100 polypeptide. When lipoprotein core circumference was plotted as a function of apoB molecular weight, (Fig. 13B), the points defined a straight line, which now intercepted the vertical axis close to the origin. Th e slope of the line was 1.14 8, of core circumferencelkilodalton of apoB. These data demonstrate that lipoprotein core circumference is directly proportional to apoB molecular weight (Fig. 13B). What is the interpretation of this relationship in terms of molecular structure? Spring et al. (1992a,b) suggested that it is best explained by a beltlike model for apoB, because the circumference of any object is directly proportional to the mass of the belt that surrounds it. Thus, a third, independent approach has yielded a beltlike model for apoB on the surface of small emulsion particles.

A 1 100-

.,.

0

~

20

40

60

80

100

SIZE OF TRUNCATED APOB (PERCENT OF APOBl00)

B

MOLECULAR WEIGHT OF TRUNCATED APO~B(MW x 10-3)

FIG. 13. Lipoprotein size as a function of apoB size. (A) Lipoprotein radius, in di units, is plotted as a function of the length of the apoB polypeptide, in centile units. These data, taken from Table VI, were obtained from the spectra of different sizes of lipoproteins and apoBs secreted by pulse-labeled HepG2 cells after puromycin treatment and analyzed as shown in the previous figure. The straight line through the points has an intercept of 18.8 di and a slope of 0.946 k e n t i l e . (B)Lipoprotein core circumference, in A units, is plotted as a function of the molecular weight of the C-terminally truncated apoB polypeptide. These are the same data as plotted in A, lipoprotein core circumference was calculated as 2a(R - t), where R is lipoprotein radius, and t = 20.2 A, the thickness of the shell.

240

VERNE N. SCHUMAKER ET AL

Why does apo B form a beltlike structure on the surface of these small lipoproteins? What is the functional significance of this architectural design? To answer these questions, it has been suggested that during lipoprotein formation, apoB functions by nucleating and determining the size of the oil droplet that pinches off from the bilayer (Spring et al., 1992b).

V. LIPOPROTEIN ASSEMBLY A.

Cotranslational Lipoprotein Formation in Rough Endoplasmac Reticulum ApoB is synthesized in the rough endoplasmic reticulum (RER), where it was first detected by immunoelectron microscopy (Alexander et al., 1976). In vitro translation experiments utilizing rabbit reticulocytes or wheat germ extracts and dog pancreatic microsomes have shown that the apoB is inserted into the lumen of the ER (Chuck and Lingappa, 1992), and that once the insertion is completed, the apoB appears to be associated with the inner leaflet of the bilayer (Pease et al., 1991). In the in vitro translation systems employed in these experiments, the dog pancreatic microsomes were probably not synthesizing appreciable quantities of phospholipid or triglycerides, and therefore did not incorporate apoB into lipoproteins. In contrast to the in vitro results, Boren et al. (1992) have shown that lipoprotein biosynthesis occurred cotranslationally in HepG2 cells, that is, while the C-terminal portion of apoB was still being synthesized on the ribosome, the N-terminal portion was already incorporated into a small lipoprotein. Figure 14 illustrates the formation of a small lipoprotein particle on the luminal surface of the endoplasmic reticulum. The growing polypeptide chain extends from the ribosome through a membrane channel, and is incorporated into the inner leaflet of the ER membrane. Here, the apoB folds into a continuous strip about 50 8, wide and many hundreds of angstroms long, displacing the phospholipid monolayer to each side. We speculate that the embedded hydrophobic surface of the apoB nucleates the formation of an oil droplet from the supersaturated bilayer, which bulges out into the luminal space because of the presence of the apoB in the inner leaflet of the bilayer. This process is imagined to continue, with more triglyceride added to the growing oil droplet and the apoB growing in length until translation is completed. At this point, the C-terminal end of the apoB polypeptide is released from the ribosome and is free to surround the oil droplet.

APOLIPROTEIN B AND LDL STRUCTURE

24 1

FIG. 14. Cotranslational assembly of a lipoprotein from the inner leaflet of the ER bilayer and apoB. In this model, translation of the C-terminal portion of apoB proceeds on a membrane-bound ribosome, while translocation and lipoprotein assembly occur on the luminal side of the ER. The N-terminal portion of apoB is believed to be embedded in the inner leaflet of the bilayer, where it nucleates the formation of an oil droplet from the supersaturated ER membranes. As the hydrophobic inner surface of apoB attempts to surround the oil droplet, it bulges into the lumen, as depicted here. On completion of translation, the two ends of apoB become free to meet, which would automatically result in the detachment of the lipoprotein from the bilayer. (Not drawn to scale.)

242

VERNE N . SCHUMAKER ET AL.

How is the nascent lipoprotein released from the surface of the ER? This probably would occur spontaneously if the two ends of the apoB were to meet, or come close together. Thus, the phospholipid monolayers that form both hemispheres of the surface coat of the nascent lipoprotein particle are attached by noncovalent forces to the sides of apoB molecule. When the two ends of the apoB touch, continuity be-

SER Lipids

Frc. 15. One version of the two-step model for lipoprotein biosynthesis. This model (Alexander et al., 1976) proposes that VLDL-sized emulsion particles lacking apoB were synthesized in the smooth ER (SER) and that these particles subsequently migrated to the junction between the smooth and the rough ER (RER), where the apoB was incorporated into the surface monolayer of the nascent VLDL. In this review it is suggested that the primary lipoprotein, shown in Fig. 14, is the vehicle that transports the apoB to the emulsion particle and merges with it to complete the assembly of the VLDL. Reproduced from theJouml of Cell Biology (Alexander et a/., 1976), by copyright permission of the Rockefeller University Press.

APOLIPROTEIN B AND LDL STRUCTURE

243

tween the bilayer of the ER and the monolayer surrounding the lipoprotein is automatically broken, and the particle becomes detached from the bilayer. It should be emphasized that according to this model, apoB does not choke the lipoprotein like a noose around the neck of a hanged man, but rather surrounds the lipoprotein along a great circle running from the North Pole to the South Pole. The circle becomes complete when the growing polypeptide is released from the ribosome. Completing the circle automatically separates the lipoprotein from the inner leaflet of the bilayer forming the membrane of the endoplasmic reticulum. B . Two-step Model for Assembly of Triglyceride-Rich Lipoproteins The permanent hepatocyte cell lines that have been studied, HepG2 and McArdle 7777 cells, are defective and do not make the large VLDLs characteristicof normal liver. Instead, they secrete the small, triglyceriderich lipoproteins described in the previous section of this review. We will call these prhury lipoprotein particles. We have suggested (Spring et al., 1992b)that lipoprotein formation is a two-step process, and that the first step is the elaboration of these small primary lipoprotein particles. The second step probably occurs in the smooth ER, where the large, VLDL-sized particle formed does not contain apoB. We will call these the secondar lipoprotein particles. Evidence for the second step was described by Alexander et al. (1976)in an electron microscope study following immunocytochemical staining to localize apoB within various cellular organelles of rat liver hepatocytes. They proposed that VLDL-sized emulsion particles lacking apoB were synthesized in the smooth ER, and that these particles subsequently migrated to the junction between the rough and the smooth ER where they acquired apoB (Fig. 15).A plausible mechanism for the acquisition of apoB would be through coalescence of the primary and secondary lipoproteins to form the nascent VLDL, which would now contain the apoB required for their transfer to the Golgi apparatus and subsequent secretion.

VI. SUMMARY ApoB 100 is a very large glycoprotein essential for triglyceride transport in vertebrates. It plays functional roles in lipoprotein biosynthesis in liver and intestine, and is the ligand recognized by the LDL receptor during receptor-mediated endocytosis. ApoB 100 is encoded by a single gene on chromosome 2, and the message undergoes a unique processing event to form apoB48 message in the human intestine, and, in some

244

VERNE N. SCHUMAKER ET AL.

species, in liver as well. The primary sequence is relatively unique and appears unrelated to the sequences of other serum apolipoproteins, except for some possible homology with the receptor recognition sequence of apolipoprotein E. From its sequence, structure prediction shows the presence of both sheet and helix scattered along its length, but no transmembrane domains apart from the signal sequence. The multiple carbohydrate attachment sites have been identified, as well as the locations of most of its disulfides. ApoB is the single protein found on LDL. These lipoproteins are emulsion particles, containing a core of nonpolar cholesteryl ester and triglyceride oil, surrounded by an emulsifying agent, a monolayer of phospholipid, cholesterol, and a single molecule of apoBl00. An emulsion particle model is developed to predict accurately the physical and compositional properties of an LDL of any given size. A variety of techniques have been employed to map apoB 100 on the surface of the LDL, and all yield a model in which apoB surrounds the LDL like a belt. Moreover, it is concluded that apoBlOO folds into a long, flexible structure with a cross-section of about 20 X 54 A' and a length of about 585 A. This structure is embedded in the surface coat of the LDL and makes contact with the core. During lipoprotein biosynthesis in tissue culture, truncated fragments of apoBlOO are secreted on lipoproteins. Here, it was found that the lipoprotein core circumference was directly proportional to the apoB fragment size. A cotranslational model has been porposed for the lipoprotein assembly, which includes these structural features, and it is concluded that in permanent hepatocyte cell lines, apoB size determines lipoprotein core circumference. ACKNOWLEDGMENTS Supported by Research Grants GM 13914 and HL 28481 from the National Institutes of Health.

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