Mapping Apolipoprotein B on the Low Density Lipoprotein Surface ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY (cl 1991 hy The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 9. Issue of March 25, pp. 5955-5962,1991 Printed in U. S. A.

Mapping Apolipoprotein B on the Low Density Lipoprotein Surface by Immunoelectron Microscopy*

(Received for publication, May 10, 1990)

Jon E. Chatterton$, Martin L. Phillips$, Linda K. Curtissg, Ross W. Milnenl), Yves L. MarcelllII**, and Verne N. SchumakerS $$ From the $Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90024-1569, the §Research Institute of Scripps Clinic, La Jolla, California 92037, and the lClinica1 Research Institute of Montreal, Montreal, Quebec H2W lR7, Canada

Apolipoprotein B (apoB) was mapped using electron microscopy to visualize pairs of monoclonal antibodies binding to the low density lipoprotein (LDL) surface. The sites at which these monoclonals bind the apoB polypeptide sequence had already been established. The angular distances between all possible pairs of binding sites except one allowed the relative placement of six epitopes on the LDL sphere. We conclude that apoB extends over at least a hemisphere of the LDL surface since four epitopes are located in the Northern Hemisphere at sites arbitrarily designated as the North Pole, the Aleutian Islands, Bogota, and in the Atlantic Ocean, while two are found in the Southern Hemi- sphere at Buenos Aires and at Madagascar. ApoB ap- pears to possess a restricted flexibility, since these relative epitope locations show a substantial standard deviation in latitude and longitude. Mapping of additional epitopes may provide an answer to the question of whether apoB circumnavigates the LDL sphere.

A widely accepted structural model for human plasma LDL’ (1, 2) describes a spherical particle containing a hydrophobic core of cholesteryl esters and triglycerides surrounded by an amphipathic monolayer of phospholipid and cholesterol in which resides a single molecule of apoB. This model is supported by compositional (3), electron microscopic (4), hydrodynamic ( 5 , 6), sequencing (7, 8), calorimetric (9), neutron and small angle x-ray diffraction (lo), nuclear magnetic res- onance (ll), and reassembly (12) evidence. One of the remaining structural questions concerns the disposition of the

* This work was supported in part by National Institutes of Health Research Grant GM 13914 (to V. N. S.), United States Public Health Service National Research Service Award GM 07185 (to J. E. C.), Investigative Group Fellowship Award 764 IG6 (to M. L. P.) from the American Heart Association (Greater Los Angeles Affiliate), and Atherosclerosis Score Grant HL 14197 (to L. C. K.). Funds for the electron microscope facility were provided by National Institutes of Health Grant USPHS 1 P41 GM 27556. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

11 Supported by Research Grant PG27 from the Medical Research Council of Canada.

** A Scientist of the Medical Research Council of Canada. $$ To whom correspondence should be addressed. ’ The abbreviations used are: LDL, low density lipoprotein; IDL,

intermediate density lipoprotein; VLDL, very low density lipoprotein; apoB, apolipoprotein B; EDAC, l-ethyl-3-(3-dimethylaminopro- py1)carbodiimide; EM, electron microscopy; SDS-PAGE, sodium do- decyl sulfate-polyacrylamide gel electrophoresis; mAb, monoclonal antibody.

apoB, a glycoprotein containing 4536 amino acid residues (7, 8) and about 8% carbohydrate (13, 14). On the intact LDL much of the protein is susceptible to proteolytic attack (151, and over 40 epitopes along the length of apoB are exposed to monoclonal antibodies (16); clearly, much of the apoB must residue at the surface. It has been suggested that apoB may be represented as a single, compact globular domain (1, 171, 3 domains (18), 4 domains (19,20), 20 domains (21), or a long string (22). In the present paper, we explore this question using electron microscopy to visualize pairs of monoclonal antibodies bound to surface epitopes on apoB.

A large number of monoclonal antibodies are available for these studies. The locations of the epitopes recognized by these antibodies along the apoB polypeptide have been established (7, 23, 24). By a judicious selection of pairs of monoclonals, it should eventually be possible to construct a “road map” of apoB on the LDL surface, assuming for the moment that apoB is spread out over the surface and is not so flexible that long-range structure fails to exist. In addition, some of these monoclonal antibodies block binding to the LDL receptor, and, therefore, may serve as markers for the site on LDL complementary to the LDL receptor (16).

Immunoelectron microscopy is not a new technique. It has been used to explore the surface topology of the ribosome and establish the locations of ribosomal proteins (25), and to examine the structure of a2-macroglobulin (26). The technique has also provided a wealth of detail concerning the structure and flexibility of antibody molecules (27-30). Here we introduce immunoelectron microscopy to the study of the low density lipoprotein, and provide a rudimentary road map describing the three-dimensional distribution of apoB on the LDL surface.

MATERIALS AND METHODS

LDL Isolation and Fractionation-LDL was isolated by a modified sequential flotation procedure (31). Approximately 50 ml of blood was drawn from a normolipemic subject into Vacu-tainers (Becton- Dickinson, Rutherford, NJ) containing EDTA as an anticoagulant. Polybrene, henzamidine, e-aminocaproic acid, and soybean trypsin inhibitor (Sigma) were immediately added to final concentrations of 24 pg/ml, 2 m M , 5 m M , and 20 pg/ml, respectively, by addition of a 100 X stock solution containing 0.195 M NaCI. Plasma was isolated by centrifugation at top speed in a clinical centrifuge for 10 min. EDTA, sodium azide, and gentamicin sulfate were added to final concentrations of‘ 0.04% (w/v), 0.05% (w/v), and 0.005% (w/v), respectively, which were then maintained throughout the isolation.

The density (20 “C) of the solution was adjusted to 1.063 g/ml by addition of a 25% NaCl solution (w/v), making the assumption that plasma is approximately 6% protein (by volume) and has the same density as aqueous 0.195 M NaCI. LDL, IDL, and VLDL were isolated by centrifugation a t 44,000 rpm for 24 h a t 20 “C in a Beckman Ti- 70.1 rotor and Beckman L5-65 ultracentrifuge. The top l ml of each t,ube (containing VLDL, IDL, and LDL) was collected and pooled,

5955

5956 Mapping ApoB on the LDL Surface

the density of the background salt solution was adjusted to 1.019 g/ ml (20 “C), and the run was repeated. IDL and VLDL were then removed in the top 1 ml of each tube, and the next 6.5 ml of each tube was discarded. The LDL “pellet” was gently resuspended using a Pasteur pipette.

All solvents were degassed under house vacuum and saturated with nitrogen prior to addition to lipoprotein-containing solutions. Poly- carbonate bottles were used for each run in the Ti-70.1 rotor; each bottle was layered with nitrogen before sealing. Lipoproteins were collected from the top of the centrifuge bottles using a 2OO-pl Pipet- man (Rainin Instr. Co., Inc., Emeryville, CA) to avoid bubbling air through the solution.

The purified LDL were fractionated into different density classes by a modification of the isopycnic gradient procedure reported by Shen et al. (32). The resuspended LDL were dialyzed against an aqueous NaCl solution of density 1.040 g/ml (1.037 M NaCl) at 4 “C under nitrogen. Dialyzed LDL (3.42 ml) was gently layered above 4.29 ml of 1.396 M NaCl (density 1.054 g/ml) in Beckman Ultra-Clear centrifuge tubes for the SW 41 rotor. Finally, 4.29 ml of 0.720 M NaCl (density 1.0275 g/ml) was gently layered above the LDL. All solutions contained EDTA, azide, and gentamicin sulfate as above. After centrifugation for 42 h at 20 “C, the tubes were fractionated by piercing the tube bottom and injecting an inert, dense fluorocarbon, permitting the removal of fractions from the top using the ISCO (Lincoln, NE) fraction collector. Densities were monitored, and the lipoprotein fractions were dialyzed into Mock (an aqueous NaCl solution with the same density as plasma and containing 0.05% NaN3, 0.04% EDTA, and 0.005% gentamicin sulfate) a t 4 “C. Lipoproteins were stored under nitrogen at 4 “C.

Analytical Ultracentrifugation-Analytical ultracentrifugation was performed on fractionated LDL in a Beckman model E analytical ultracentrifuge equipped with a xenon light source, monochromator, mirror optical system, and Beckman photoelectric scanner and mul- tiplexer. The signal was taken directly off the chart recorder, sent to the A/D converter of a DEC PDP 11/03 computer, stored on flexible disks, and analyzed on a VAX 780 computer.

Runs were made at 48,000 rpm at approximately 26 “C (temperature monitored during the run) in aqueous NaCl solvents of density 1.063 g/ml (26 “C) or 1.006 g/ml (20 “C) containing 0.04% EDTA, 0.05% azide, and 0.005% gentamicin sulfate. Scans were taken at 4- min intervals. Sedimentation rates were determined from In r uersus t plots; buoyant density, pb, was determined from an r~ s versus p plot; and LDL radius was determined using Equation 1.

Radius = [ (9 /2) (~ 7 f / f o ) / ( p b - ps)l” (1)

where s is the sedimentation coefficient, p s and r~ are the density and viscosity of the solvent, respectively, Pb is the buoyant density of the LDL, and f / fo is the translational frictional ratio of LDL, which has been reported to be 1.11 (5,6).

Monoclo~al Antibodies-Monoclonal antibodies MB47 and MB44 (33); MB19, MB24, MBll (34); and anti-B,116 (35) have been described, and the approximate locations of their binding sites along the apoB polypeptide have been determined (7, 16, 23, 24).

Sample and Grid Preparation for EM-LDL (in Mock) was mixed with monoclonal antibody at an antibody concentration to give binding site equivalence. The sample was diluted with 0.195 M NaCl to a final apoB concentration of 50-100 nM, and incubated at room temperature for 1 h. The sample was then diluted 1 : l O with 0.195 M NaCl, and immediately spread on the grid, thus limiting the dissocia- tion of the LDL-antibody complexes at the lower concentration.

Grid preparation was performed using the single carbon technique (36). Carbon was evaporated onto cleaved mica and aged for at least a month. The mica was gently inserted into the sample-containing solution at about a 45” angle, carbon side up, allowing the end of the carbon film to detach and float on the aqueous surface. After 30 s, the mica was withdrawn, which removed the carbon from the aqueous surface as well. Next, the mica was inserted into double distilled water for 5 s and withdrawn. Finally, the carbon was floated off the mica strip onto aqueous 1% uranyl acetate (Eastman Kodak). A 500- mesh grid (Ted Pella, Inc., Redding, CA) was placed shiny side down on the carbon, immediately picked up with forceps, and blotted from the edge with premoistened Whatman No. 1 filter paper. The grid was allowed to dry on a piece of dry filter paper for 1 h and examined in the electron microscope within 3 h.

Cross-linking and Extraction of LDL-LDL (50-100 nM apoB) in phosphate-buffered saline (pH 7.4) was cross-linked for 1 h with 10 mM EDAC (Sigma), spread on carbon-coated grids, and extracted

with 4:l ethanol/ether as described previously (22). Grids were then stained with 1% uranyl acetate and observed in the electron microscope.

Electron Microscopy-Electron microscopy was performed on a JEOL JEM 1200 EX electron microscope at 80 kV, using magnifica- tions between 30,000 and 40,000 (calibrated using a diffraction grating replica).

Mapping of Antibody Binding Sites on LDL-The EM negatives were projected onto an HP 9864A digitizer screen connected to an HP 9825A calculator (Hewlett-Packard). LDL-antibody complexes were selected for analysis if the following criteria were met: the LDL

that both antibodies were visible at the edge of the LDL particle, and appeared nearly circular in projection, the complex was oriented such

the antibodies were clearly in contact with the LDL surface. The two-dimensional LDL projections were assumed to be perfectly circular, an assumption which does not imply that the LDL particles were actually spherical on the EM grid. Coordinates were taken at three points along the LDL perimeter: at each of the two antibody binding sites and at an opposite third site close to the imaginary line that bisected the angle between the two binding sites. A computer program was written in BASIC for a personal computer to solve the three simultaneous equations for the center and diameter of the circle that contained all three of these points on the LDL perimeter (Equa- tion 2). The linear distance between the two binding sites was determined using the formula for the distance between two points (Equation 3). The angle between the binding sites was determined from the distance and the particle diameter using the Law of Cosines (Equation 4). Deviations from circularity in the projected LDL image caused only small angular errors in this analysis.

(xl - h)’ + (yl - k)’ = r2

(x2 - h)’ + (y2 - k)’ = r2 (2)

(x3 - h)’ + (y3 - k)’ = r2

where (x1, y,), (x2, y2) , and (xg, y3) are points on the surface of a circle with center (h, k ) and radius, r.

distance = [(x2 - x# + (y2 - yl)’]l” (3)

where (x1, yl) and (x2 , y2) are the coordinates at the edge of the LDL particle where the antibodies are attached.

a’ = b2 + C2 - 2bc COS A (4)

where a, b, and c are the lengths of the sides of a triangle, and A is the angle (in radians) opposite side a. In this application, a is the distance between the antibody binding sites, and b = c is the radius of the LDL particle.

Other Methods-Protein was determined by a modified Lowry assay (37) . SDS-PAGE was performed using 5% acrylamide, 0.1% bisacrylamide (w/v) gels and the Laemmli buffer system (38). Gels were stained with Coomassie Brilliant Blue, and scanned using a model SLR-lD/PD scanning densitometer (Biomed Instruments, Inc., Fullerton, CA).

RESULTS

Characterization of LDL-The LDL fraction used in the electron microscope study (1.0320-1.0366 g/ml) was charac- terized by analytical ultracentrifugation. These LDL had a peak-& value of 6.77 S and a buoy9nt density of 1.031 g/ml, corresponding to a diameter of 208 A (Equation 1). The range of particle sizes was estimated from the width of the sym- metrical boundary, corrected for diffusion. Ninety-five per- cent of the particles had sizes between 201 and 215 A.

LDL are subject to proteolytic and oxidative damage leading to degradation of the apoB (39, 40). To retard such damage, a mixture of inhibitors was added to the blood, as described under “Materials and Methods.” LDL which had been stored for two months at 4 “C following isolation were analyzed by SDS-PAGE. Densitometer scans of Coomassie-stained gels showed that well over 90% of the apoB remained intact as apoB-100. All of the samples employed for the electron microscope measurements were prepared for examination within 2 months of LDL isolation.

Observation of LDL and LDL-Antibody Complexes-In the

Mapping ApoB on the LDL Surface 5957

electron microscope, fractionated LDL appeared round with no protrusions and had ?pparent particle diameters which ranged from 240 to 340 A (Fig. l), considerably larger than the diameters measured hydrodynamically. We interpret these larger apparent diameters to reflect a flattening of the LDL on the electron microscope grid; the extent of this distortion and its effect on our measurements will be discussed later.

When these LDL were treated with a zero-length protein cross-linking reagent (EDAC) and extracted on an EM grid, the proteinaceous residue consisted of ring-shaped structures of approximately the same diameter as LDL (Fig. 2). These structures were very similar to those observed following glu- taraldehyde cross-linking and extraction of LDL in a previous study (22).

When native LDL were complexed with two monoclonal antibodies which recognized distinct, nonoverlapping epitopes on apoB, about 10-20% of the LDL were seen bound to a t least one antibody (Fig. 3). Antibodies were identified, by their size and shape (28), as the small, Y-shaped objects making contact with the LDL periphery, and extending into the negative stain. A few LDL (1-4%) were visualized with two peripherally bound antibodies; in some instances, two LDL were bridged by two antibodies, forming a closed, circular complex. Under these conditions, no LDL have been observed to bind more than two antibodies. In control experiments in which LDL were complexed with a single monoclonal antibody against apoB (data not shown), no LDL bound more than one antibody, consistent with the presence of a single molecule of apoB on LDL (7). These results remained un- changed even when the antibodies were present a t a concentration 10 times that required for binding site equivalence (data not shown).

Angular Measurements Used for Mapping of ApoB on the LDL Surface-Fractionated LDL were complexed with all but one possible pairs of six monoclonal antibodies (MB47, MB44, MB24, MB19, MB11, and anti-BsO116), and observed in the electron microscope (Fig. 4). As described under “Materials and Methods,” for each LDL which bound a pair of antibody molecules, the x,y coordinates of the two apoB epitopes located at the tips of the Fab arms were recorded, as well as a third point on the opposite side of the LDL. These measurements allowed calculation of the apparent LDL diameter, the length of the chord separating the two epitopes, and the angle between the two epitopes (i.e. the central angle, as illustrated in the inset to Fig. 3A).

For each pair of antibodies, 30-100 such measurements were obtained. For most antibody pairs, the measured dis-

30m

APPARENT DIAMETER (A) FIG. 1. LDL size distribution by electron microscopy. Ap-

parent LDL diameter was determined from electron micrographs as described under “Materials and Methods.” Grids were prepared by the single carbon technique and stained with 1% uranyl acetate.

FIG. 2. EM of cross-linked LDI, extracted with organic solvents. LDI, were cross-linked with a water-soluble carhodiimide, adsorbed to carbon-coated grids, extracted with 4:l ethanol/ether, and stained with 1% uranyl acetate. The ring-shaped objects are apoB in the absence of lipid. The inset shows two rings linked by a monoclonal antibody, obtained by cross-linking an LDL-monoclonal antibody (MR47) mixture, and then extracting on the grid. The bar indicates 0.1 pm.

tances between epitopes increased as a function of apparent LDL diameter, however the central angles did not (Fig. 5). Despite being independent of apparent LDL diameter, the angle separating the apoB epitopes recognized by these antibodies did not have a discrete value; a distribution of angles was found for each antibody pair (Fig. 6). Most distributions had well defined maxima; only those distributions in which one of the antibodies was MB44 lacked an identifiable peak (Fig. 6, panels G, H, I , and J). The average angular distances between LDL surface epitopes were calculated for each pair of antibodies (Table I), including those whose distributions did not show well defined peaks. In the discussion section, it will be demonstrated that these measurements may be used to map apoB on the LDL surface.

DISCUSSION

The very large discrepancy between the average di!meter of the LDL measured hydrodynamically (about 208 A) and that determined by EM (about 285 A) probably reflects flattening of the LDL on the EM grid, due to surface tension forces generated during drying. The hydrodynamic values for t)e diameter are almost certainly correct; a diameter of 208 A yields molecular weight, compositional, and density data corresponding closely with the observed values for a spherical LDL containing? single apoB glycoprotein of 550,000 Da. A diameter of 285 A is greatly inconsistent with these values. It is well recognized that LDL are “soft”; deformation is often seen in the region of contact between two adjacent LDL (4). Furthermore, other authors have reported LDL diameters


FIG. 3. E M of LDL binding two mAbs. Fractionated LDL (50-100 nM apoB) were mixed with two mAbs (at binding site equivalence) which recognized distinct sites on apoB, and did not compete for LDL binding. Grids were prepared by the single carbon technique, and stained with 1% uranyl acetate. Arrows indicate LDL with two bound antibodies. 0 and C refer to open and closed complexes as shown in the inset. Also shown in the inset is the central angle, a, which subtends the chord connecting the two epitopes bound by mAbs. A, mAbs MB47 and MB19; B, mAbs MB47 and MB24. The bar indicates 0.1 pm.

W d- .- d- h 1 - c\r 7 + m m m C U I

+ .A - I I I. I urn

d o

MB19

M 824

MB11

M 844

MB47

FIG. 4. Representat ive electron micrographs of all possible pairs of six mAbs (except one) bound to LDL. Arrows point to mAbs attached to LDL. The bar indicates 0.1 pm.

LDL Diameter (A) LDL Diameter (A)

FIG. 5. Relationship between chord length, central angle, and appa ren t LDL diameter. A, the length of the chord separating the two epitopes to which mAbs MB47 and MI319 bind, is plotted as a function of apparent LDL diameter. The least squares line had a slope of 0.80 and a correlation coefficient of r = 0.43. B, the central angle between the same two epitopes (panel A ) is plotted as a function of apparent LDL diameter. The least squares line had a slope of 0.07 degrees/A and a correlation coefficient of r = 0.085. The inset to panel A illustrates how the central angle may remain constant as the chord length increases with LDL diameter; the same central angle subtends the longer chord on the larger circle.

measured by EM which are inconsistent with their reported hydrodynamic data (22, 32). Clearly, LDL are susceptible to significant distortion when observed in the electron microscope. The extent of this distortion was estimated by assuming that flattened LDL resemble truncated spheres with the same volume as native LDL. Thus, a 26% increase in apparent diameter would correspond to an LDL deformed into a hemisphere. The average increase in diameter was even greater than this, about 37%, indicating considerable deformation of these LDL. Thus, the circular projections of LDL observed


- 20- o G W

n

15

10

L 5

'0 60 120 1800 60 120 1.300 60 120 1800 60 120 180 A N G L E ( d e g )

FIG. 6. Distributions of measured central angle between pairs of mAbs bound to LDL. The angle between epitopes on the LDL surface was determined for at least 30 complexes between LDL and two mAbs as described under "Materials and Methods." A, MB47/MB19; B, MB47/anti-B,,116; C, MB24/MB47; D, MB19/anti- B.,~16; E, MB19/MB24; F , MB24/anti-BS,116; G, MB44/MB47; H , MB44/MB24; I, MB44/MB19; J, MB44/anti-Bs,116; K, MBll/MB47; L, MBll/MB24; M , MB11/MB19; N , MB11/anti-B,,116.

TABLE I Angular distances between apoB epitopes on LDL

Pairs of monoclonal antibodies bound to apoB on the LDL surface were visualized by electron microscopy. Measured angles between the antibody binding sites were determined as described under "Materials and Methods." Values presented are the means of 30-110 measurements. Calculated angles are a least squares fit of the measured angles on a suhere (see "Discussion").

Monoclonal antibody Measured Calculated

MB47/MB19 85" 84 MB47/MB24 80" 82 MB47/MBll 132" 126" MB47/MB44 113" 109" MB47/anti-B8,,116 45" 41" MB24/MB19 43 38" MB24/MBll 45" 51" MB24/MB44 89" 87" MB24/anti-BS,J6 117" 119" MB19/MB11 45" 43" MB19/MB44 115" 122" MB19/anti-B8,116 101" 103" MB11/MB44 ND 95" MBll/anti-B.,116 135" 140" MB44/anti-BRO116 118" 124"

pair angle angle

by EM are not produced by spherical particles, but by dis- torted spherical particles with larger apparent diameters.

In the electron micrographs presented in Fig. 3, few of the LDL appeared to bind antibody. However, in these experiments, antibodies were present in a stoichiometric ratio at an apoB concentration of >50 nM prior to final dilution and immediate spreading on the EM grid. The Kd for binding of MB47 (one of the antibodies in Fig. 3) has been estimated to be 2.5 X 10"' M (33). Under these conditions, 90% of the LDL should have a bound antibody. At a much higher concentration (1.4 PM apoB), all of the absorbing material in a stoichiometric mixture of LDL and antibody floated with the LDL in the analytical ultracentrifuge (data not shown); about one-third of this absorbance was due to antibody. Thus, both the requisite antibody binding sites and the corresponding epitopes on the LDL appeared to be present on all particles, suggesting that many of the LDL have antibodies bound

which are not visible in the EM. The most likely explanation is that only antibodies bound at the LDL periphery displaced stain and could be visualized. Antibodies binding to the upper or lower surfaces of the LDL could not be seen probably because they were not outlined in stain and lacked contrast.

Measurements of a substantial number of antibody pairs attached to epitopes on the LDL yielded a broad distribution of angles (Fig. 6). Possible reasons for these broad distributions could be (i) human error in measurement of epitope positions, (ii) parallax, (iii) accidental location of unbound antibodies at the LDL periphery, (iv) proteolysis, (v) deformation of the LDL resulting in deformation of the apoB, and (vi) intrinsic flexibility of the apoB polypeptide on the surface of the LDL. However, human error was small, since repeated measurements made on the same photographs yielded the same angles almost exactly (to within f3"). Likewise, parallax was probably not a significant factor, since the antibodies which were visualized must have displaced stain and, therefore, all lay in the plane of the carbon grid. Accidental location would have resulted in a uniform distribution of angles, not a central peak. To minimize this source of error, great pains were taken to select only images for which the Fab tips made contact with the LDL sphere. Occasional outlier measurements probably resulted from accidental location; most of these were ignored in calculating the average angle, although all measurements are shown in Fig. 6.

Proteolysis or oxidative scission of apoB might permit the separated epitopes to drift to random locations on the LDL surface. SDS-PAGE revealed that no more than 10% of the apoB was degraded after 2 months of storage. Since all experiments were performed within 2 months of LDL isolation, proteolysis and oxidative scission of the apoB probably con- tributed only an occasional outlier measurement, and had little effect on the average angle.

LDL distortion on the EM grid was substantial, as discussed above. The data of Fig. 5A show that the distance between epitopes increased significantly with the apparent LDL diameter, indicating that the conformation of apoB changed in response to the increased radius of curvature on the flattened LDL particle. It is presently unclear whether such a change in apoB conformation is purely artifactual or a reflection of different conformations apoB can assume on larger LDL, IDL, and VLDL.

In contrast, the data of Fig. 5B show that the central angle separating these same apoB epitopes remained essentially constant with increasing LDL diameter. This is portrayed in the insert to Fig. 5A, where the same central angle subtends both the longer chord on the larger circle and the smaller chord on the smaller circle. For this to be true, the increase in length of the chord (the distortion in apoB) must be directly proportional to the increase in diameter (the distortion of the LDL). The value of the square of the correlation coefficient, r2, may be used to estimate that fraction of the observed variation attributable to the independent variable. The variation of central angle with LDL diameter shown in Fig. 5B gave a value of r2 = 0.007. This value may be interpreted to mean that the remaining 99.3% of the observed variation in angle is attributable to causes other than LDL deformation.

Therefore, since no artifactual cause can account for the breadth of the distributions of measured angle, this breadth must be due to an innate flexibility in apoB. In spite of this flexibility, however, most of these distributions have well defined peaks, and the distributions for two different pairs of antibodies are clearly distinct. Therefore, they probably reflect some aspect of a long-range order in the disposition of


apoB on the surface of the LDL which exists together with its innate flexibility.

A model was constructed, using a polystyrene ball and toothpicks to represent an LDL and surface-bound antibodies, to build up a three-dimensional apoB map consistent with the average angular measurements presented in Table I. The binding site for MB47 was defined as the North Pole. The angle between this monoclonal and any other monoclonal defined the latitude at which the second monoclonal antibody binds on the sphere. Thus, MB24 binds on a line of latitude 10" north of the equator. Its location was selected by arbitrarily placing a toothpick at this latitude; this toothpick then defined a "Prime Meridian." MB19 binds 85' from MB47, and 43" from MB24. Circles were traced on the model 85" and 43" from the toothpicks defining the MB47 and MB24 binding sites. These circles intersected at two points: one in the Western hemisphere, and one in the Eastern hemisphere. The point in the Western hemisphere was selected arbitrarily, leaving the "handedness" of the subsequent model in question. The remaining three antibodies were placed on the model by triangulation from the locations of the fvst three, generating a three-dimensional map (Fig. 7).

A map of the earth was superimposed on the model and

FIG. 7. Three-dimensional map of apoB on LDL. Using the angles listed in Table I, the six epitopes were arranged on the surface of a sphere relative to MB47 and MB24, which defined the North Pole and a prime meridian, respectively. A map of the earth was superimposed on the sphere, and rotated 36' about the polar axis to align the antibody binding sites with geographical reference points. MB47, North Pole; MB19, Bogoti at 6'N, 74'W MB11, Buenos Aires at 3673, 64"W, MB44, Madagascar at lYS, 48"E antiB,ll6, Aleutian Islands at 49"N, 169"E MB24, in the Atlantic Ocean at 8"N, 36'W. MB24, which was used as a prime meridian in constructing the map, actually lies 36" west of the Greenwich Meridian. The handedness of this model is in question; the "true" map may be the mirror image of the one shown above. The purple string connects the toothpicks in the same order as the epitopes lie on the polypeptide chain.

rotated 36" about the polar axis to align a cluster of cities and other recognizable geographical features with the antibody binding sites. It was found that if ME47 binds at the North Pole, and MB19 binds at Bogoki (Colombia), then MBll binds at Buenos Aires, anti-B.,J6 binds in the Aleutian Islands, MB44 binds at Madagascar, and MB24 binds in the Atlantic Ocean, on the Atlantic Ridge, 8" north of the equator. The new "Prime Meridian" defined by MB24 does not pass through Greenwich, but instead coincides with a great circle at 36" west longitude.

The three-dimensional map was constructed using angular distances relative to only three reference points: the first three antibodies. To estimate the error in placing additional antibodies on the map, angular distances between all antibody pairs were calculated from the map, and compared to those determined by electron microscopy. Positions on the map were adjusted to minimize deviations from the experimental data using a least squares approach. Calculated angular distances differed from experimental values by a maximum of 7" (Table I), indicating the world map was internally consistent. Any three antibodies would place the other three at the same global locations within M".

The binding site positions of these five monoclonal anti-

1


TABLE I1 Location of epitopes recognized by monoclonal

antibodies against apoB Determination of epitope positions along the apoB sequence has

been reported previously (7, 16, 23, 24).

Antibody Binding site location (apoB amino acid residue)

MB19 71 MB24 399-580 MBll 995-1082 MB44 2488-2658 MB47 3441-3568 Anti-B,,116 4154-4189

bodies along the apoB sequence have been reported (Table 11). Thus, the N terminus of apoB is located near the binding site for MB19 at Bogota. From there, apoB travels due east to MB24, located in the Atlantic, and then southwest to MB11, at Buenos Aires. From there it wraps around the backside of the particle, passing through MB44 at Madagas- car, up to the North Pole (MB47), the south toward the Aleutians (anti-B,,116) and the C terminus, nearly circumna- vigating the globe. This is illustrated by the purple string in Fig. 7, which describes the numerical order of the epitopes along the apoB polypeptide. The length of string between the toothpicks does not reflect the relative numbers of amino acid residues separating the epitopes; rather, the string is stretchfd tautly to connect the epitopes by great circles. At 1.5 A/ residue, an a-heiix ?f 4536 residues could wrap around the LDL (diameter 200 A) almost 11 times. Since, as shown here, it actually seems to wrap around only once, apoB is probably composed of a chain of domains connected by flexible chain. As more monoclonals are placed on this three dimensional map in future studies, this numerical order of epitopes will provide a road map of the course of the apoB polypeptide.

In addition, placement of more epitopes on the map may provide information on the domain structure of apoB. On our three-dimensional map, the first 1000 amino acids, 22% of apoB, are within 45” of each other, only 12.5% of the LDL circumference. This may represent an N-terminal structural domain in apoB, which may be connected to the next domain by a more extended, flexible segment of polypeptide chain. Limited proteolysis of LDL has revealed “three principle structural domains” in apoB that are relatively resistant to this treatment (18). Amino acids 1-1280 comprised the first of these three domains, which was followed by a highly susceptible region, believed to be a more flexible region of apoB. Flexibility of the polypeptide chain in regions connecting apoB domains may explain why, in the present study, certain pairs of antibodies (those binding in two different domains) displayed significantly greater variation in angular separation than others (those binding within the same domain). For example, the MB19, MB24, and MBll epitopes all reside in the first domain defined by limited proteolysis; the MB44 epitope resides in the second (Table 11). The distributions of angles for all combinations of MB19, MB24, and MBll were considerably narrower (Fig. 6, panels E, L, and M ) than those for combinations involving MB19 and MB24 with MB44 (Fig. 6, panels H and I ) , which lacked well defined peaks.

It is interesting that treatment with a zero-length cross- linking reagent and lipid extraction yields a circular structure on the electron microscope grid (Fig. 2). The size and shape of this delipidated apoB structure suggest that it could wrap about the perimeter of an LDL particle (22). A likely inter- pretation is that domains located close to the N and C termini of apoB are sufficiently close on the native LDL particle to

be cross-linked with a zero-length cross-linking reagent. On the preliminary road map generated by the monoclonal

antibodies (Fig. 7 ) , several of the apoB epitopes are within a few degrees of the LDL perimeter, forming a ring which extends around 72% of the LDL circumference. The presence of epitopes more than a few degrees from the LDL perimeter may correspond to larger apoB domains. The most N-terminal antibody (MB19) and the most C-terminal antibody (anti- B,,,16) are 101“ apart on the LDL surface, corresponding to an arc length of 180 A. However, the anti-BJ6 binding site is between 347 and 482 amino acids from the C terminus; the MB19 binding site is 71 amino acids from the N terminus. This stretch of 418 to 533 amino acids is more than adequate to fill the distance between these two points on the map, allowing the N and C termini to be near each other on the LDL surface, and close the ring.

Therefore, we again suggest a previously proposed model (22) in which apoB is a belt, composed of domains linked by flexible chain, which wraps once about the LDL sphere, with overlapping ends which may be “buckled” with a zero-length cross-linking reagent. The flexible chain connecting the domains allows for considerable flexibility in the overall protein.

Acknowledgments-We thank Larry Levine, for performing the least squares analysis of the mAb binding site positions on the globe, and Robert Hemmerling, for his assistance in developing our data collection techniques.

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Mapping Apolipoprotein B on the Low Density Lipoprotein Surface ...

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