Apolipoprotein A-I from normal human plasma: definition of three distinct antigenic determinants

390 Biochimicu et Biophysics Acre 835 (1985) 390-401

Elsevier

BBA 51942

Apolipoprotein A-I from normal human plasma: definition of three distinct

antigenic determinants

Philip K. Weech, Ross W. Milne, Peter Milthorp and Yves L. Marcel

Laboratoire du Metabolisme des Lipoproteines, Institut de Recherches Cliniques de Montreal, I IO Avenue des Pins, ouest.

Montreal, Quebec (Canada)

(Received December 27th, 1984)

Key words: Apolipoprotein A-I; HDL; Monoclonal antibody; CNBr fingerprint; (Human plasma)

We have prepared, selected and cloned four mouse hybridomas that secreted monoclonal antibodies against human plasma apolipoprotein A-I. These antibodies are all of the IgGI subclass, and were named anti-A-I 6B8, 5G6, 3D4 and 5A6. We characterized the specificity of the antibodies, finding that all four of them reacted similarly, and with only the major proteins having the molecular weight and isoelectric focusing characteristics of apolipoprotein A-I. The antibodies reacted with all known charge-polymorphs of apolipoprotein A-I and pro apolipoprotein A-I. Thus, the polymorphs of apolipoprotein A-I are alike in that they all contain the antigenic sites of these four antibodies. In a solid-phase, antibody competition radioimmunoassay we found inhibition or enhancement of antibody binding to apolipoprotein A-I, according to the pair of antibodies tested. Antibodies 6B8, XX and 3D4 were different from one another and reacted with different antigenic determinants, but 5A6 was similar to 3D4 and reacted at the same site. We compared the reactions of the four antibodies with CNBr-cleaved fragments of apolipoprotein A-I separated by polyacrylamide gel electrophoresis. We found three different patterns of reaction with the apolipoprotein A-I fragments; 6B8,5G6 and 3D4 were different, but 5A6 resembled 3D4. Thus, the four antibodies reacted with at least three different antigenic sites in apolipoprotein A-I, which were present in different CNBr fragments of apolipoprotein A-I, but not on fragment 4 which forms the carboxy-terminal segment.

Introduction

Apolipoprotein A-I is the major apolipoprotein of normal human plasma (about 140 mg/dl) and

is found in the high-density lipoproteins (HDL),

accounting for at least 55% of the HDL protein. Apolipoprotein A-I structure and its function as a

cofactor in the reaction of lecithin : cholesterol acyltransferase are well established (for reviews see Refs. 1, 2), however its exact role in cholesterol transport remains in part speculative [l]. Serum

Abbreviations: HDL, high-density lipoproteins of density

1.063-1.21 g/ml; VLDL, very-low-density lipoproteins of den-

sity < 1.006 g/ml.

concentrations of apolipoprotein A-I and HDL cholesterol are both negative predictors for cardiovascular disease but apolipoprotein A-I ap-

pears to add to the predictive value of HDL cholesterol [3-51, a finding which has further increased the interest shown for this protein. Vari- ous immunoassays for apolipoprotein A-I have been reported which shared, to different degrees, the difficulties inherent in the determinations of serum apolipoproteins [6,7]. Self-association of apolipoprotein A-I [8,9] has made difficult the development of appropriate standards, while apolipoprotein A-I in lipoproteins does not always express all its potential antigenic sites (lO,ll], im- plying that some sites are masked by lipids in

0005-2760/85/$03.30 0 1985 Elsevier Science Publishers B.V. (Biomedical Division)

391

intact lipoproteins or that their ability to bind an antibody depends on the conformation of A-I as affected by lipids or other proteins.

A valid approach to the problems of standardiz- ing apolipoprotein A-I immunoassays is to use well-characterized monoclonal antibodies to study the expression of antigenic sites in A-I, without the confounding effects of uncharacterized antibodies from polyclonal antisera. This will enable us to develop assays that measure apolipoprotein A-I antigenic sites that are always expressed in all isomorphs of A-I [12-171, whether lipids are present or absent. Furthermore, monoclonal antibodies will provide precise markers to study the structure-function relationship in apolipoprotein A-I. We report here the production and characteriza- tion of four monoclonal antibodies to apolipoprotein A-I which recognize at least three distinct antigenic determinants on apolipoprotein A-I, each of which reacts with all the known polymorphic forms of apolipoprotein A-I and pro apolipoprotein A-I.

Materials and Methods

Chemicals for electrophoresis were obtained from Bio-Rad (~chmond, CA). ~pholytes and Ultrodex for isoelectric focusing were from LKB (Bromma, Sweden), 0.45 pm nitrocellulose sheet from Millipore (Bedford, MA), Immulon II Re- movawell Strips (Dynatech Laboratories, Inc., Alexandria, VA) and all other chemicals from Fisher, Ltd (Montreal, Canada). Affinity-pu~fied rabbit anti-mouse IgG was purchased from Kirk- egaard and Perry Laboratories, Inc. (Gaithersburg, MD).

Production of monoclo~a~ antibodies Female, &week-old Balb/c mice (Jackson

Laboratories, Bar Harbor, ME) were injected intraperitoneally with 50 pg of apolipoprotein A-I emulsified in complete Freund’s Adjuvant. Identi- cal injections were given three weeks later. After a further three weeks the mice received an in- travenous injection of 10 Erg apolipoprotein A-I, and four days later their spleens were excised for the cell fusion. Hybridomas were produced using a mixture of 30 * lo6 SP2-0 cells and 120 - lo6 cells from two spleens [ 181. The cells were distributed in

1000 microculture wells (Costar, Cambridge, MA) and growth was later seen in 300 wells. Ten wells were positive for the presence of antibodies specific for apolipoprotein A-I on screening by radioimmunoassay using a solid-phase technique with Re- movawells coated with A-I (10 pg/ml in 5 mM glycine, pH 9.2) overnight at room temperature. The cells from the positive wells were cloned by limiting dilutions in 96 well microculture plates, yielding four stable clones that were recloned three times by limiting dilutions. To identify the immunoglobulin class and subclass, a sample of hy- bridoma supematant was concentrated by precipi- tation in 50% saturated ammonium sulphate and tested by double diffusion against antisera specific for mouse immunoglobulin (Ig) classes and sub- classes [19]. Selected subclones were injected intraperitoneally into Balb/c mice and their ascitic fluid was used directly as the source of antibodies for most experiments.

~solafjo~ of lipoproteins and apo~~pop~otei~ A-l Either fresh, normal human serum, following an

overnight fast, or fresh plasma from the Red Cross was used to isolate HDL, by sequential ultra- centrifugation between the densities 1.125 and 1.210 g/ml for 40 h at 12°C 45 000 rpm in a 50.2 rotor and L2-65 ultracentrifuge (Beckman Instru- ments Inc., Palo Alto, CA). After dialysis against 10 mM NH,HCO,/l mM EDTA/O.OZS NaN,, the HDL, were lyophilized, then delipidated using chloroform/methanol [20]. Apolipoprotein A-I was isolated from HDL, by preparative isoelectric focusing [12] for the immunization of mice, screening of hybridomas by radioimmunoassay for specific antibody secretion, and studies of the fragments of A-I cleaved by CNBr.

The technique described by Brewer et al. [21], with a 500-fold excess of CNBr, was followed to prepare fragments of apolipoprotein A-I, typically using 1.65 mg lyophilized A-I redissolved in 165 pl formic acid 70% containing 9.38 mg CNBr. This was allowed to react under N,, in the dark, at about 22’C for either 2 or 24 h. In some experiments, the apolipoprotein A-I was treated with 5% 2-mercaptoethanol before lyophilization and CNBr treatment, and a &fold molar excess of

392

tryptophan (over apolipoprotein A-I) was added

to the CNBr solution. At the end of the CNBr treatment, the samples were diluted with water

and lyophilized.

Po!vacrylumide gel electrophoresis, isoelectric focus-

ing and transfer to nitrocellulose sheets

Polyacrylamide gels were cast as vertical slabs

(14 X 18 X 0.15 cm) for electrophoresis in the Pro- tean cell (Bio-Rad, CA). The gels were made to the recipes of (i) Kane [22] for electrophoresis in al-

kaline, urea-containing buffer, (ii) Neville [23]

using 15% acrylamide and sodium dodecyl sulphate

(SDS), or using 20% acrylamide and substituting 18% glycerol for water (v/v) in the recipe, to

improve resolution of low molecular weight poly-

peptides as recommended by Connelly and Kuksis

[24], (iii) Warnick et al. [25] for isoelectric focusing

using Ampholytes pH 4-6 or pH 3.5510. Urea-

containing gels were stained with Coomassie blue

G250 0.04% in 3.5% perchloric acid [26] and in-

tensified in 5% acetic acid [27]; SDS-containing gels were stained in Coomassie blue R250 0.25%

dissolved in 45% methanol/9% acetic acid, and destained in the same solvent; replicas of electrophoresis on nitrocellulose sheet were stained with

Amido black 0.1% dissolved in 45% methanol/ 10% acetic acid and destained in 90% methanol/2%

acetic acid [28].

Immediately after electrophoresis. a replica of

the separated proteins was transferred onto a

nitrocellulose sheet by the method of Towbin et al. [28] using the methanol/Tris/glycine buffer for

SDS-containing gels and 0.7% acetic acid as the

electrolyte for urea-containing gels. A constant

current of 100 mA, 50-20 V, for about 16 h was

used in the Transblot cell (Bio-Rad, Richmond,

CA) to effect the transfer, at about 22’C. To detect reactions of the monoclonal antibodies with

the protein replicas, the nitrocellulose sheets were then treated as described by Theolis et al. [29].

lodinution of monoclonul antibodies Monoclonal antibodies (IgG-I) were purified by

adsorption to Protein A-Sepharose (Pharmacia, Uppsala, Sweden) at pH 8.0, and elution with 0.1 M citric acid (pH 6.0). Iodination of these affinity purified, mouse, anti-A-I monoclonal antibodies was carried out by the method of Mellman and

Unkeless [30] as described by Milne, et al. [31].

Briefly, 24 pg of antibody were labelled with 0.5 mCi of “‘1 (Amersham). Free “‘1 was removed on

an AGIX8 resin (Bio-Rad) and the labelled anti-

body (eluant) stored in phosphate-buffered saline

(0.154 M NaCl/lO mM Na2HP0,. 7H,0/5 mM NaH2P0,. H,O (pH 7.2)) containing 0.1% bovine serum albumin and 0.02% sodium azide.

Competitive antibody binding USSUJS Solubilized apolipoprotein A-I was diluted in

coating buffer (15 mM NazCO,/ 35 mM

NaHCOJ3 mM NaN, (pH 9.6)) to a final concentration of 2 pg/ml. Aliquots (50 ~1) were distributed in Immulon 2 Removawell strips and

left refrigerated overnight to allow the binding of

the antigen to the wells. The wells were emptied.

washed one time with phosphate-buffered saline, and excess liquid was removed by inversion of the

wells on paper towelling. To saturate non-specific

binding sites, the wells were incubated with 250 ~1

of 10%’ bovine serum albumin in phosphate-

buffered saline for 1 h at room temperature, then emptied, washed with phosphate-buffered saline as described above, and drained. Serial two-fold dilutions of the ascites fluids were prepared in phosphate-buffered saline containing 0.05% Tween-20,

1% bovine serum albumin, 1.5 mM MgCl Z and 2.5 mM mercaptoethanol using round-bottomed, mul-

tiwell, microtitre plates (Linbro) and a multichannel pipettor (Titertek). These dilutions were im-

mediately transfered with the multichannel pipet-

tor to Removawells containing apolipoprotein A-I, and incubated for 2 h at room temperature. The

wells were then emptied and washed three times

with washing buffer (phosphate-buffered saline

containing 0.05% Tween-20, 1.5 mM MgCl, and 2.5 mM mercaptoethanol). Excess liquid was re-

moved as described above. At this point we con-

tinued the experiment in one of two ways. (i) Each well was incubated with the same

amount of ‘251-labelled, goat anti-mouse immunoglobulin, washed again, and finally the ra- dioactivity was measured. The amount of labelled. anti-immunoglobulin bound to the wells increased, with increasing monoclonal antibody concentration, up to a maximum. The lowest concentration of monoclonal antibody necessary to achieve this maximum binding was the saturating concentra-

393

tion for the conditions of that experiment. In subsequent antibody competition experiments, serial, two-fold dilutions of monoclonal antibodies were made from a starting concentration that was

4-times more concentrated than this saturating

concentration. (ii) In antibody competition experiments a 50

~1 aliquot of ‘251-labelled, affinity-purified, mono-

clonal anti-A-I was added to each well. This aliquot

contained lo-20 ng of protein and from 5 . lo4 to 1 . lo5 cpm, depending on the antibody used. The

wells were then incubated for 2 h at room temper-

ature. In some experiments using combinations of the monoclonal antibodies 6B8 and 5G6, the ascites

and radiolabelled affinity-purified antibody were added to the removawells together, eliminating the washing step between the addition of the ascites

and the radiolabelled antibody. After incubation with the radiolabelled IgG, the wells were emptied,

rinsed with washing buffer three times and excess

liquid was removed as described above. The wells

were then counted in an LKB 1271 Riagamma gamma counter. The results were expressed as (B/Be) x 100, where B = 1251 counts per minute

(cpm) bound mins non-specific binding, and Bo = 125I cpm bound in the absence of competing

antibody minus non-specific binding.

Results and Discussion

From the cell fusion and screening by radioim-

munoassay we selected and cloned four stable hybridomas, which were injected intraperitoneally

into mice and allowed to grow as ascites tumours. The intraperitoneal fluid from these mice served as

the source of antibodies for subsequent experi-

ments. The antibodies produced by the four clones

of cells were all of the IgG-I subclass and were

named 3D4,5A6,6B8 and 5G6. We made two kinds of experiments: (i) to iden-

tify whether the antibodies reacted with an antigen

having the known properties of apolipoprotein A-I upon polyacrylamide gel electrophoresis and isoelectric focusing, and (ii) to determine if the indi-

vidual antigenic sites, recognized by each antibody, were close together, far apart or at least on the same molecule, based on immunoreactivities of CNBr-cleaved fragments of apolipoprotein A-I, and also on competitive cotitrations of apolipoprotein A-I with each possible pair of antibodies.

SDS-polyacrylamide gel electrophoresis After SDS-polyacrylamide gel electrophoresis

each of the antibodies reacted with the major protein band in HDL and purified apolipoprotein

A-I, and with only that band. Fig. 1 shows the

stained strips of polyacrylamide gel cut from slab

gels containing purified apolipoprotein A-I (strip

1) or HDL, (strip lo), with the autoradiographs alongside of strips cut from the nitrocellulose rep-

licas of the same gels reacted with 3D4, 5A6, 6B8 and 5G6. Even though the gels were overloaded

with both purified apolipoprotein A-I (40 pg per cm width of gel) and HDL, (40 pg per cm width

of gel), none of the minor bands seen on the stained gels were detected on the autoradiographs.

Apolipoprotein A-I is the major protein found in

HDL, and the antibody reactions and stained bands corresponded closely in electrophoretic mo-

bility with the molecular weight of apolipoprotein

A-I [21].

Polymorphism of apolipoprotein A-I by isoelectric focusing

Apolipoprotein A-I was first seen to be poly-

Fig. 1. Autoradiography of monoclonal antibody reaction with replicas of SDS-polyacrylamide gel electrophoresis. A sample of apolipoprotein A-I (40 gg/cm width of gel) was applied to one slab gel (strips 1-5) and HDL, (40 pg/cm) was applied to a second gel (strips 6-10). After electrophoresis ([23] 15% acrylamide) a slice of each gel was stained with Coomassie blue R250: apolipoprotein A-I shown in strip 1, HDL, strip 10. Replicas of the electrophoreses of the remaining gels were transferred to nitrocellulose [28] and reacted with monoclonal antibodies 5G6 (strips 2 and 6). 3D4 (strips 3, 7), 6B8 (strips 4, 8) and 5A6 (strips 5, 9). Migration was toward the anode at the bottom of the figure.

394

morphic using chromatography on DEAE-cel-

lulose [13], and since then the intraindividual het-

erogeneity has been confirmed in many laboratories using isoelectric focusing [12,14-171. With

ampholytes of pH 4-6, at least four forms of

apolipoprotein A-I and two forms of pro apoli-

poprotein A-I have been identified in normal

plasma and lymph [16,32]. A minor basic form of

apolipoprotein A-I was described in patients with

cerebrotendinous xanthomatosis [33] and a claim

was made, but not substantiated, for a relatively

basic form (pZ 6.5) in normal plasma HDL [14].

Fig. 2 shows two experiments to determine the specificity of our antibodies for the polymorphs of

apolipoprotein A-I in plasma HDL. A sample of delipidated HDL was applied to one isoelectric

focusing gel (36 pg protein per cm width of gel,

ampholytes pH 4-6) and a mixture of equal amounts of delipidated VLDL and HDL (18 pg

HDL protein per cm gel) was applied to a second gel. Fig. 2, strips 1 to 7, shows the replica of the

VLDL plus HDL gel; strips 8 to 10 show part of

Fig. 2. Autoradiography of monoclonal antibody reaction with

replicas of isoelectric focusing. A sample of equal amounts of

delipidated HDL (18 /.~g protein/cm width of gel) and VLDL

were applied to one slab gel (strips l-7), and twice the quantity of HDL (36 pg/cm) was applied to a second gel (strips 8-10).

Following isoelectric focusing [25] with ampholytes pH 4-6.

replicas of the two gels were transferred [28] to nitrocellulose.

Strips 1 and 10 show the nitrocellulose replicas stained with

Amido black. Strips 2-9 show autoradiography of the replicas

reacted with monoclonal anti-E 6C5 (strip 2), monoclonal

anti-E 3B7 (strip 3), monoclonal anti-A-l 5G6 (strips 4, 8). 5A6

(strips 5, 9), 6B8 (strip 6) and 3D4 (strip 7). The reactions of

the anti-A-1 are indicated by arrows, using the nomenclature of

Ghiselli et al. [16].

the replica of the HDL gel. Fig. 2, strips 1 and 10.

show the edges of the nitrocellulose replica stained with Amido black immediately after transfer. The

three major and three minor stained bands in the

top half of the replicas (Fig. 2, strip 10) are the pro

A-I and A-I isomorphs as identified by comparison with standards and according to pl values.

The additional stained bands in the lower half of

Fig. 2, strip 10, were probably apolipoproteins A-II, C-II and C-III, visible at the higher HDL

protein concentration applied to that gel. Fig. 2 strips 4 to 7, show the autoradiograph of

the replica reacted with antibodies 5G6, 5A6, 6B8 and 3D4, respectively. Using the numbering de-

scribed by Ghiselli et al. [16] we have indicated on Fig. 2 the positions of seven, resolved, radio-

graphic bands that showed the reactions of the antibodies. In this, and in a replicate experiment,

all four antibodies reacted with all seven bands.

Bands 1 and 2 correspond in pl to pro A-I, bands

3 and 4 as the major isomorphs of apolipoprotein

A-I in plasma, and bands 4 to 7 as minor iso-

morphs of A-I. Band 7 was detected im-

munochemically but could not be seen on the stained replicas (Fig. 2, strips 1 and 10). Although

Fig. 2, strips 4 and 5 showed less intense bands than strips 6 and 7. the antibodies 5A6 did react with bands identical to those positive for 6B8 and 3D4, as seen when the film was overexposed and

also on the autoradiograph of the gel containing only HDL, at a higher protein concentration (Fig. 2. strips 8 and 9: antibodies 5A6 and 5G6, respec-

tively, on the HDL isoelectric focusing replica).

The autoradiograph of 6B8 and 3D4 reactions on the replica of this second gel (HDL only, 36 ,rtg

protein per cm) were identical to those shown in Fig. 2, strips 6 and 7. and so are not shown.

Although there appeared to be differences in

the sensitivity with which each antibody reaction could be detected on the replica’s autoradiograph we conclude that antibodies 5G6, 5A6, 6B8 and 3D4 each reacted with all known charge polymorphs of pro A-I and apolipoprotein A-I, and in addition with a minor form of lower pl (A-I, in Fig. 2). Further, heterogeneity was absent from the autoradiographed replica of SDS-polyacrylamide gel electrophoresis (Fig. l), which showed that the antigen had the molecular weight properties of apolipoprotein A-I. With the similarity between

the molecular weights of apolipoprotein A-I and pro A-I [32] we did not expect to separate them

easily by SDS-polyacrylamide gel electrophoresis, and show in Fig. 1 that the antibodies did not

detect either grossly lower or higher molecular

weight proteins than normal apolipoprotein A-I. In contrast to the reactions of the four anti-A-I

monoclonal antibodies (Fig. 2, strips 4 to 9) two other monoclonal antibodies, anti-apolipoprotein-

E (anti-E) 3B7 and KS previously described [18] did not react with the seven polymorphs of apolipoprotein A-I on the replicas. Instead, both 3B7 and 6C5 (Fig. 2, strips 2 and 3) reacted similarly

with four charge isoforms of apolipoprotein E, three of which were intercalated with the isomorphs pro apolipoprotein A-I, and pro A-I,, and

apolipoproteins A-I, and A-I,. The major band

on the autoradiograph of these anti-E, however,

was of pZ immediately higher than that of pro A-I, (Fig. 2, strips 2, 3) and could be identified

with a faintly stained band on the replica of

VLDL plus HDL isoelectric focusing (Fig. 2, strip

1). These differences between the autoradiographs of anti-A-I and anti-E reaction on isoelectric

focusing replicas indicated not only that these two sets of antibodies were each specific for their own antigen, but also the great danger that is inherent in identifying stained bands on isoelectric focusing of apolipoproteins, unless immunochemical data is simultaneously available.

Competition between antibodies for binding to

apolipoprotein A-Z

The technique for the competitive radioim-

munoassays was described under Methods. Each of the unlabelled antibodies was capable of

specifically blocking its ‘251-labelled homologue,

indicating the specificity of the binding (Figs. 3 and 4). Antibody lD7, specific for apolipoprotein

E [19], did not inhibit the binding of any of the ‘251-labelled anti-A-I antibodies (data not shown).

Antibodies 3D4 and 5A6 showed the same con-

centration-dependent inhibition as each other that was characteristic of both antibodies being the same, whether binding of labelled 3D4 (Fig. 3b) or labelled 5A6 (Fig. 3c) was tested. Thus, these two antibodies seemed to sterically hinder the binding of one another, no matter which of them bound first to apolipoprotein A-I, possibly because their

120- A

1 I I I I

256 64 16 4 I 256 64 16 4 I

REClPROCAl OF FIRST ANTIEODY DILUTION

Fig. 3. Competition of monoclonal antibodies. Competition

was performed sequentially as described in the Methods sec-

tion. (a) Initial dilutions of the first antibodies 6B8 (0). 3D4

(0) and 5A6 (A) were l/1000, and 5G6 (0) was l/500. The labelled second antibody was 6B8 (0.16 ag/ml). (b) Initial

dilutions of the first antibodies 6B8,3D4 and 5A6 were l/1000.

The labelled second antibody was 3D4 (0.32 ag/ml). (c) Initial

dilutions of the first antibodies 6B8,3D4 and 5A6 were l/1000.

The labelled second antibody was 5A6 (0.16 ug/ml). (d) Initial

dilutions of the first antibodies, 6B8, 3D4 and 5A6 were

l/1000, 5G6 was l/200. The labelled second antibody was

5G6 (0.32 ug/ml).

antigenic sites were identical or close together in the primary structure, or in the tertiary structure of apolipoprotein A-I.

The binding of ‘251-labelled 5G6 was inversely proportional to the concentration of 6B8 (Figs. 3

and 4). At the highest concentration of 6B8 that was tested there was still some binding of labelled 5G6 (about 40% of the binding in the absence of any other antibody). However, in the inverse experiment, the binding of labelled 6B8 was unaf- fected by the presence of 5G6, except at the highest 5G6 concentration. Although 6B8 was an inhibitor

396

256 64 16 4 i

RECIPROCAL OF FIRST ANTIBODY DILUTION

5G6 a

Fig. 4. Competition of monoclonal antibodies. Both antibodies

were added at the same time, as described in the Methods

section. Initial dilutions of the unlabelled antibody 6B8 (0) was

l/125 and 5G6 (0) was l/62.5. (a) Labelled antibody 6B8

(0.16 pg/ml). (b) labelled antibody 5G6 (0.32 pg/ml).

of 5G6 binding, the reverse was not evident and so the relationship between the binding of 6B8 and 5G6 was different from the mutual inhibition of 3D4 and 5A6. The 6B8 and 5G6 antigenic sites

appeared from the previous experiments to be in different CNBr fragments of apolipoprotein A-l. These two antigenic sites may be close together in

one of the tertiary structures of apolipoprotein A-l, such that antigen-bound 6B8 sterically hinders

further binding of 5G6, but not vice versa. How-

ever, other explanations of this observation are

tenable. The binding of 6B8 was independent of

the concentration of any other non-labelled anti-

body (Figs. 3a and 4a), indicating that 6B8 bound

at a different site than either 5A6 or 3D4. The binding of antibodies 3D4 and 5A6 was

proportionately increased by prior binding of 6B8 (Fig. 3b, c). Similarly, the binding of 5G6 was

directly concentration-dependent on 3D4 and 5A6 (Fig. 3d). Enhanced binding of one monoclonal antibody, following binding of a different monoclonal antibody to the same antigen, has been observed in published studies of antigens other than apolipoprotein A-l [34-361. The authors ex-

plained the enhancement by one of two mechanisms: (i) an increased affinity of the antigen for the second antibody, allowing the binding of the second antibody to a set of antigen molecules that were otherwise unreactive, and (ii) the formation of tetrameric complexes of two antigens and two

different antibody molecules that were more stable

than complexes containing only one kind of antibody. In our experiments we did not try to dis- tinguish between these two possible mechanisms of apparent enhancement of ‘2sl-labelled antibody binding. However, finding that 6B8 enhanced

‘25 I-labelled 3D4 binding to apolipoprotein A-l. which was fixed on the Removawells, suggests that

6B8 induced a change in conformation of some apolipoprotein A-l molecules, permitting 1251-

labelled 3D4 to bind to a set of A-l molecules that were otherwise unreactive with 3D4. Although

other explanations of the enhancement mechanism

are tenable, the observation demonstrates some

variability in the expression of 3D4 and 5G6 anti- genie sites.

The observations that all antibodies interacted

with each other, either by inhibition or enhance-

ment during competition, suggested that all four antigenic sites were present together on

apolipoprotein A-l molecules, rather than being present separately on different A-l molecules. To- gether, the radioimmunoassay results support further the classification of our four anti-A-l antibodies into three groups, i.e., 6B8, 5G6 and the similar

pair, 3D4 and 5A6.

Location of antigenic determinants on apolipoprotein

A-I fragments from CNBr cleavage

We used three techniques of gel electrophoresis

to study the fragments from CNBr cleavage of

apolipoprotein A-l: isoelectric focusing with

ampholytes pH 3.5510, polyacrylamide gel elec-

trophoresis in alkaline, urea-containing buffer, or SDS-containing buffer. We compared the stained

patterns of the separated fragments from apolipoprotein A-l cleaved for 2 and 24 h (Fig. 5, gels 1, 2) and A-l treated with mercaptoethanol before cleavage, and then cleaved in the presence of ex-

cess tryptophan (Fig. 5, gel 5). In each experiment there was cleavage of apolipoprotein A-l, yielding a pattern of bands that was not noticeably in- fluenced by the different conditions of cleavage that we tested (Fig. 5, compare gels 1 with 2, and 4 with 5). The cleavage appeared to be incomplete, some apolipoprotein A-l remained intact and there were many bands of incompletely cleaved apolipoprotein A-l fragments. This has been a common finding for apolipoprotein A-l in other laborato-

397

Fig. 5. Electrophoresis of apolipoprotein A-I cleaved with

CNBr. Electrophoresis in alkaline, urea-containing buffer [22]

was used for gels 1-8; gels from three separate experiments are

shown (gels 1-3, 4 and 5, 6-8). Isoelectric focusing with

ampholytes pH 3.5-10 [25] was used for gels 9-11. The sam-

ples were 1, apolipoprotein A-I (A-I) cleaved with CNBr for 2

h, 30 pg; 2, A-l cleaved for 24 h. 30 gg; 3, HDL, 50 pg; 4, A-I

cleaved for 2 h, 36 pg/cm width of gel; 5, A-I treated with

2-mercaptoethanol and cleaved with CNBr for 2 h, in the

presence of excess tryptophan, 36 pg/cm; 6, A-I CNBr frag-

ment 1, about 19 pg; 7, A-I CNBr fragment 4, about 6 pg; 8,

A-I cleaved with CNBr, 2 h, 30 pg; 9, A-I cleaved for 2 h,

isoelectric focusing, 30 pg; 10, A-I cleaved for 24 h, isoelectric

focusing, 30 pg; 11, A-I 20 pg.

ries [17]. Methionine-serine bonds are often re-

sistant to cleavage by CNBr [37] and such a bond exists between CNBr fragments 1 and 2 in apoli-

poprotein A-I [21], possibly explaining some of the resistance of apolipoprotein A-I to cleavage with

CNBr.

The separation of fragments by isoelectric

focusing (Fig. 5, gels 9, 10) resembled that pub-

lished recently by Menzel et al. [17]. Stained fragments were focused close to both extremities of the

gel and were not well separated from one another at the acidic end which was evident by two-dimen-

sional isoelectric focusing - SDS-polyacrylamide gel electrophoresis. Menzel et al. [17] suggested that the fragments of lowest molecular weight were not fixed and stained in the gel, and our experi- ence agrees with that. A better separation of the fragments of low pZ was obtained by electrophoresis in alkaline, urea-containing buffer [22]. The band of least mobility was identified by this technique as fragment 4 by comparison with authentic

fragment 4 supplied by Dr. H.B. Brewer Jr. (Fig.

5, gels 8 and 7, respectively). It barely entered the separating gel, suggesting that its pZ value was

close to the pH of the stacking and cathode buffers (pH 9.1 and 8.9, respectively), and was the band of

highest pZ seen on isoelectric focusing. We were able to make a nitrocellulose sheet

replica of alkaline - urea polyacrylamide gel elec-

trophoresis and isoelectric focusing, but not of the fragments separated by SDS-polyacrylamide gel

electrophoresis. It seemed that all of the apolipoprotein fragments that were stained in the gels following alkaline - urea polyacrylamide gel elec-

trophoresis and isoelectric focusing could also be stained with Amido black on the nitrocellulose

replicas. However, only the uncleaved apolipopro-

tein A-I was detected on the replica of SDS-poly-

acrylamide gel electrophoresis of apolipoprotein fragments when stained with Amido black, and no

fragments were detected immunochemically on the

SDS-polyacrylamide gel electrophoresis replicas. All four antibodies reacted with the replicas of

alkaline-urea polyacrylamide gel electrophoresis

and isoelectric focusing of CNBr-cleaved apolipoprotein A-I (Fig. 6a, b, respectively). The same patterns of immunoreactive bands were found in

repeated experiments. Three of the four antibodies (6B8, 3D4, 5G6) gave three different patterns of immunoreactive bands (Fig. 6, strips, 3, 4, 5, respectively). Antibodies 5A6 and 3D4 gave identi-

cal patterns of immunoreactive bands (Fig. 6, strips

2, 4). This suggested that antibodies 6B8, 3D4 and 5G6 reacted at different sites within apoli-

poprotein A-I, i.e., at sites that occurred in differ-

ent CNBr fragments, but that antibodies 3D4 and

5A6 reacted either at the same site, or at nearby

sites that occurred within the same CNBr frag-

ment of apolipoprotein A-I. Similar results were

seen after isoelectric focusing of CNBr fragments of apolipoprotein A-I: antibodies 6B8, 3D4 and 5G6 gave three different patterns of immunoreac-

tivity (Fig. 6, strips 8, 9, 10) but antibodies 3D4 and 5A6 gave identical patterns (Fig. 6, strips 9, 7). We identified fragments of apolipoprotein A-I using the data of Menzel et al. [17] and their apparent molecular weights on two-dimensional, alkaline urea and SDS-polyacrylamide gel electrophoresis (Fig. 7). Although the pattern of immunoreactive bands was different for each of three

398

Fig. 6. Autoradiography of monoclonal antibody reaction with CNBr-cleaved fragments of apolipoprotein A-l. A sample of

apolipoprotein A-I cleaved for 2 h was applied to two polyacrylamide gels: panel a, for alkaline electrophoresis 1221, 30 pg/cm, strips

1-5; panel b, for isoelectric focusing [25] with ampholytes pH 3.5-10, 40 pg/cm, strips 6-10. Strips 1 and 6 show the stained

polyacrylamide gels. Strips 2-5 and 7-10 show autoradiography of the monoclonal antibody reactions: 5A6 (strips 2, 7). 6B8 (strips 3,

8). 3D4 (strips 4, 9) and 5G6 (strips 5, 10). The immunoreactive fragments are identified by arrows, using the nomenclature of Brewer

et al. [5]. All of the immunoreactive fragments were polymorphic in charge.

Fig. 7. Two-dimensional electrophoresis of apolipoprotein A-I cleaved with CNBr. Electrophoresis in alkaline, urea-containing buffer

[22] was used for separation in the first dimension of 200 pg apolipoprotein A-I cleaved for 2 h. Separation in the second dimension

used a slab gel of 20% acrylamide with 18% glycerol, and electrophoresis in the presence of SDS [23]. Panel A shows a gel stained after

the first dimension and superimposed over the stained gel of the second dimension. Panel B shows the same second-dimension gel; arrows indicate the CNBr fragments of apolipoprotein A-I, using the nomenclature of Ghiselli et al. [14].

antibodies (Fig. 6, strips 2-5) some fragments or bands reacted with all four of the antibodies, e.g., uncleaved apolipoprotein A-I and fragment l-2-3.

A pair of bands (fragment 2-3-4) reacted with antibodies 6B8, 5A6 and 3D4 (Fig. 6, strips 3, 2, 4), thus containing two different antigenic sites (6B8 and 5A6-3D4). Antibody 6B8 was the only antibody that reacted with three bands (fragment 3-4) which migrated between fragment 4 and apolipoprotein A-I. Thus, fragment 3-4 contained the antigenic site of only 6B8, but fragment 2-3-4 contained the site of 3D4-5A6 as well, and fragment l-2-3 contained all three antigenic sites (6B8, 5G6, 3D4-5A6). Antibody 5G6 was the only antibody which reacted with a pair of bands (fragment l-2 or 1; Fig. 6, strip 5) which we tentatively identified as fragment l-2 or 1, by comparison with authentic fragment 1 from Dr. H.B. Brewer Jr. and from the data of Menzel et al. [17] and apparent molecular weights (Fig. 7). Fig. 8 sum- marizes the CNBr cleavage sites of apolipoprotein A-I, and Table I lists the patterns of reactions of the antibodies and fragments. For each antibody we could see that the immunoreactive fragments contained a specific part of the apolipoprotein A-I structure, and that fragments missing that structure did not react with the antibody. The results therefore indicate that the antigenic site for 6B8 is in fragment 3, and that the 3D4-5A6 site is in fragment 2. The 5G6 reactions were not as intense on autoradiography as those of the other antibodies. Nevertheless, 5G6 gave its strongest reactions when fragments 1 and 2 were present, and we suggest that the 5G6 site is either in fragment 1, or spans the CNBr cleavage site between fragments 1

TABLE I

399

“2fJm l Coon

RESIDUE I 86 112 14.9 243

AV Met Met Met Gill ‘--k

CNBr I CNEr 2 CNBr 3 CNBr 4

Mr 9880 3190 4250 10700

Fig. 8. Simplified diagram of the primary structure of apolipoprotein A-I. The position of the methionine residues at

which CNBr can potentially cleave the peptide chain are shown.

The fragments are indicated, as CNBr 1 to CNBr 4, using the

nomenclature of Brewer et al. (51, and their calculated molecu-

lar weights are shown.

and 2, or requires the presence of intact fragments 1 and. 2 for a protein conformation that expresses antigenic activity.

All of the charge polymorphs of apolipoproteins A-I reacted with all antibodies (Fig. 2, strips 4-9), indicating that the charge heterogeneity of apolipoprotein A-I is qualitatively unrelated to the presence of antigenic sites recognized by these four antibodies. It appeared that the CNBr fragments of apolipoprotein A-I were themselves polymorphic in charge, both from their patterns of immunoreaction (Fig. 6) and from the series of two or three spots of identical molecular weight but of a different charge than we saw on two-dimensional polyacrylamide gel electrophoresis (Fig. 7). The charge heterogeneity is most evident for fragments 4 and 3-4, but is also present for fragments 1, 1-2, and l-2-3 (Fig. 7). Thus, it may be that several parts of apolipoprotein A-I are polymorphic in charge, and that the polymorphism of whole A-I is the average of these potentially varia- ble sites within the molecule. This charge polymorphism was seen in both fragments 1 and 4 supplied by Dr. H.B. Brewer Jr. (Fig. 5, gels 6, 7).

IMMUNOREACTIVITY OF CNBKLEAVED FRAGMENTS OF APOLIPOPROTEIN A-I

Reactions were found on replicas of alkaline electrophoresis or isoelectric focusing (Fig. 6).

Apolipoprotein

Common fragments

Fragment

A-I 2-3-4

3-4 l-2-3 l-2orl

Antibodies

3D4 5A6 6B8 5G6

+ + + + + + + - - + -

+ + + + - _ - + 2 or 2-3 2 or 2-3 3 I,2 or 1-2

400

In conclusion, we have characterized four monoclonal antibodies which react with a protein which has the molecular weight and charge characteristics of apolipoprotein A-I and also with purified apo~poprotein A-I. Noteworthy was the observation that these antibodies react with all known charge polymorphs of apolipoprotein A-I and pro A-I and that therefore the antigenic determinants recognized by these antibodies are un- affected by this polymo~~sm. The finding that different CNBr-fragments of apolipoprotein A-I are also heterogeneous in charge is important. since it indicates that apolipoprotein A-I charge polymorphism is the sum of several charge varia- tions at different sites within the molecule. Com- petitive radioi~unoassays using different pairs of antibodies have shown that the antibodies recognized antigenic sites present together on apolipoprotein A-I molecules rather than on alter- nate molecules and that three different sites are expressed and identified by 6B8, 5G6 and by the pair, 3D4 and 5A6. The location of these sites on the apolipoprotein‘A-I molecule has been further defined by the CNBr-cleavage experiments which indicate that 5G6 is on fragment 1 or l-2,3D4-5A6 on fragment 2, and 6B8 on fragment 3. While these antibodies recognize antigenic deter~nants that are far apart on apolipoprotein A-I molecule, it is interesting to note that none of them could inhibit the apolipoprotein A-I-mediated activation of lecithin : cholesterol acyltransferase reaction in a system using a purified enzyme and an apolipoprotein A-I-phosphatidylcholine-cholesterol substrate (unpublished results). These results are consistent with the data of several groups, showing that the apolipoprotein A-I fragment responsible for activation of the enzyme is located within CNBr fragment 4 [2], a fragment not recognized by our antibodies. Further experiments with these and other antibodies specific for different sites will be needed to help elucidate the mechanism which mediates the apolipoprotein A-I activation of lecithin : cholesterol acyltransferase. Finally, we have observed that the four ~tibodies described here are specific for sites on apolipoprotein A-I which are expressed equally well in native as in delipidated HDL and which should thus be appli- cable to quantitative immunoassays of apolipoprotein A-I.

Acknowledgements

We are grateful to Dr. H.B. Brewer Jr., Molecu- lar Disease Branch, National Heart, Lung and Blood Institute, National Institutes of Health, U.S.A. for the generous gift of purified A-I CNBr fragments 1 and 4. Louise Lalonde gave excellent secretarial help. We are indebted to C. Vezina and T.D. N’guyen for their assistance in several parts of this work. The studies were supported by grants from the Medical Research Council of Canada (PG-27), the Quebec Heart Foundation and the Fondation de Recherche en Sante du Quebec. R.W.M. is a scholar of the Fondation de Re- cherche en Sante du Quebec. P.M. is a recipient of an Industrial Research Fellowship {Natural Scien- ces and Engineering Research Council of Canada) and under contract with A-B Biological Supplies Inc., Hamilton, Ontario.

References

I

2

3

4

5

6

7

8

9

10

II

12

13

14

15

16

Eisenberg, S. (1984) J. Lipid Res. 25, 1017-1058

Marcel, Y.L. (1982) Adv. Lipid Res. 19. 85-136

Avogaro. P., Cazzolato, G., Bittolo Bon, G. and Quinzi.

G.B. (1979) Lancet i. 901-903

Weisweiler, P., Sperland. B. and Schwandt, P. (1981) Clin.

Chem. 27, 348

Maciejko, J.J., Holmes, D.R., Kottke, B.A., Zinsmeister,

A.R., Dinh, D.M. and Mao, S.J.T. (1983) N. Engl. J. Me.d

309, 385-389

Lippel, K. (ed.) (1983) Proceedings of the Workshop on

apolipoproteinquantification. In U.S. Department of Health

and Human Services, National Institutes of Health. NIH

Publication 83-1266, pp. 7-22. 443-449. 450-458

Steinberg, K.K., Cooper, G.R.. Graiser. S.R. and Rosseneu.

M. (1983) Chin. Chem. 29, 415-426

Vittelo, L.B. and Scanu. A. (1976) J. Biol. Chem. 251.

1131.-1136

Formisano, S., Brewer. H.B., Jr. and Osborne, J.C. (1978) J.

Biol. Chem. 253, 354-359

Schonfeld. G., Bradshaw, R.A. and Chen. J.S. (1976) J.

Biol. Chem. 251, 3921-3926

Mao, S.J.T.. Miller, J.P., Gotto, A.M.. Jr. and Sparrow. J.T. (1980) J. Biol. Chem. 255. 3448-3453

Nestruck. A.C., Suzue, G. and Marcel. Y.L. (1980) Biochim.

Biophys. Acta 677, 110-l 21

Shore. G. and Shore, V. (1969) Biochemistry 8, 4510-4516

Zannis, V.I.. Breslow, J.L. and Katz. A.J. (1980) J. Biol.

Chem. 255. 8612-8617

Utermann, G.. Feussner, G.. Franceschini, G., Haas. J. and

Steinmetz, A. (1982) J. Biol. Chem. 257, Sol-507

Ghiselli, G., Schaefer, E.J., Light, J.A. and Brewer, H.B., Jr.

(1983) J. Lipid Res. 24, 731-736

401

17 Menzel, H.J., Assman, G., Rall, SC., Weisgraber, K.H. and

Mahley, R.W. (1984) J. Biol. Chem. 259, 3070-3076

18 Milne, R.W., Douste-Blazy, p., Retegui, L. and Marcel,

Y.L. (1981) J. Clin. Invest. 68, 111-117

19 Chaion, M.-P., Milne, R.W. and Vaerman, J.-P. (1979) Eur.

J. Immunol. 9, 747-751

20 Olofsson, S.-O., McConathy, W.J. and Alaupovic, P. (1978)

Biochemistry 17, 1032-1036

21 Brewer, H.B., Jr., Fairwell, T., LaRue, A., Ronan, R.,

Houser, A. and Bronzert, T.J. (1978) B&hem. Biophys.

Res. Commun. 80, 623-630

22 Kane, J.P. (1973) Anal. B&hem. 53, 350-364

23 Neviile, D.M. (1971) J. Biol. Chem. 246, 6328-6334

24 Connelly, P.W. and Kuksis, A. (1982) B&him. Biophys.

Acta 711, 245-251

25 Wamick, G.R., Mayfield, C., Albers, J.J. and Hazzard,

W.R. (1979) Clin. Chem. 25, 279-284

26 Reisner, A.H., Nemes, P. and Bucholtz, C. (1975) Anal.

B&hem. 64, 509-516

27 Holbrook, LB. and Leaver, A.G. (1976) Anal. Biochem. 75,

634-636

28 Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Nat].

Acad. Sci. USA 76, 4350-4354

29 Theolis, R., Jr., Weech, P.K., Marcel, Y.L. and Mime, R.W.

(1984) Arteriosclerosis 4, 498-509

30 Mellman, I.S. and Unkeless, J.C. (1980) J. Exp. Med. 152,

1048-1069

31 Milne, R.W., Theohs, R., Jr., Verdery, R.B. and Marcel,

Y.L. (1983) Arteriosclerosis 3, 23-30

32 Brewer, H.B., Jr., Fairwell, T., Kay, L., Meng, M., Ronan,

R., Law, S. and Light, J.A. (1983) Biochem. Biophys. Res.

Commun. 113, 626-632

33 Shore, V., Salen, G., Cheng, F.W., Forte, T., Shefer, S.,

Tint, G.S. and Lindgren, F.T. (1981) J. Chn. Invest. 68,

1295-1304

34 Tosi, R., Tanigaki, N., Sorrentino, R., Accolla, R. and

Corte, G. (1981) Eur. J. Immunol. 11, 721-726

35 Ehrhch, P.H., Moyle, W.R., Moustafa, Z.A. and Canfield,

R.E. (1982) J. Immunol. 128, 2709-2713

36 Holmes, N.J. and Parham, P. (1983) J. Biol. Chem. 258,

1580-1586

37 Schroeder, W.A. Shelton, J.B. and Shelton, J.R. (1969)

Arch. Biochem. Biophys. 130, 551-556

Documents

Apolipoprotein A-I from normal human plasma: definition of three distinct antigenic determinants