THE JOURNAL 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc

OF BIOLOGICAL CHEMISTRY Vol. 267, No. 19, Issue of July 5, pp. 13257-13261,1992 Prmted in U.S.A.

Antibodies to the Carboxyl Terminus of Human Apolipoprotein A-I THE PUTATIVE CELLULAR BINDING DOMAIN OF HIGH DENSITY LIPOPROTEIN 3 AND CARBOXYL- TERMINAL STRUCTURAL HOMOLOGY BETWEEN APOLIPOPROTEINS A-I AND A-11*

(Received for publication, September 12, 1991)

Charles M. Allan, Noel H. Fidge, and Jerry Kanellos From the Protein Chemistry and Molecular Biology Unit, Baker Medical Research Institute, Prahran, Victoria 3181, Australia

We have studied the binding of ‘2SI-labeled high den- sity lipoproteins (HDLJ) to liver plasma membranes, which are thought to contain specific HDL receptor sites, using anti-peptide antibodies directed against two sites in the carboxyl-terminal region of human apoA-I. Two distinct antibody populations raised to peptides corresponding to amino acid residues 205- 220 and 230-243, respectively, recognized regions of apoA-I that are exposed in the lipid environment of HDL3. However, anti-AI[230-2431 IgG, but not anti- AI[205-2201 IgG, recognized HDL2, suggesting that residues 205-220 of apoA-I are expressed differently in the two HDL populations. In addition, anti-AIr230- 2431 IgG showed strong cross-reactivity toward apoA- 11. Epitope mapping studies showed that anti-AIr230- 2431 binds to an epitope located in the carboxyl-ter- minus of apoA-11, demonstrating significant structural homology between the carboxyl-terminal of apoA-11, demonstrating significant structural homology be- tween the carboxyl-terminal regions of apoA-I and A- 11, two candidate proteins for mediating the specific cellular interaction of HDL3. Fab fragments from anti- AI[205-2201 and anti-AI[230-2431 inhibited the bind- ing of ’“I-HDL3 to liver plasma membranes by approx- imately 80% and 60%, respectively. These findings are in agreement with our recent work using isolated CNBr fragments of apoA-I (Morrison, J., Fidge, N. H., and Tozuka, M. (1991) J. Biol. Chem. 266, 18780- 18785), which suggest that the carboxyl-terminal re- gion of apoA-I contains a binding domain which me- diates the specific interaction of HDL3 with liver plasma membranes, possibly through the involvement of specific HDL receptors.

Apolipoprotein A-I (apoA-I),’ the major protein constituent of human plasma high density lipoprotein (HDL), plays an important role in lipid transport and metabolism. It is an activator of 1ecithin:cholesterol acyltransferase (I), and evi- dence from several studies have also implicated a role for apoA-I as a ligand capable of recognizing specific HDL cellular binding sites (2-6). Following the recognition of an inverse

*This research was supported in part by the National Heart Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: apo, apolipoprotein; ELISA, enzyme- linked immunosorbent assay; HDL, high density lipoprotein; KLH, keyhole limpet hemocyanin; OVA, ovalbumin; PBS, phosphate-buff- ered saline; RP-HPLC, reversed-phase high pressure liquid chroma- tography; SDS, sodium dodecyl sulfate; DMPC, L-a-dimyristoylphos- phatidylcholine.

correlation between plasma HDL, as well as apoA-I levels (7), and the incidence of atherosclerosis, apoA-I has become the subject of numerous immunochemical investigations. The ap- plication of apoA-I-specific antibodies include immunoassays for quantification of serum apoA-I or HDL levels (8-11), studies of genetic variants (12, 13), and epitope expression of apoA-I within different lipoprotein subclasses (8, 14-20). They have also been used to probe functional domains in- volved in binding of HDL to cellular receptors (3-6).

Synthetic peptides representing selected regions of a pro- tein sequence can elicit antibodies capable of reacting with the whole protein (21,22). We have generated two populations of anti-peptide antibodies recognizing residues 205-220 and 230-243 of human apoA-I, respectively. This report describes the ability of these site-specific antibodies to inhibit the binding of HDLs to liver plasma membranes, which are thought to contain specific receptors for HDL3 (2, 23). These studies extend our recent findings using purified cyanogen bromide digest fragments of apoA-I (24, 25) and suggest that residues 205-243 contain, or form part of, the binding domain of HDL3. We propose that the interaction of HDL3 with liver plasma membranes is mediated by HDL receptor sites which are specific for a region in the carboxyl-terminal portion of apoA-I.

EXPERIMENTAL PROCEDURES

Lipoprotein and Apolipoprotein Isolation-Human HDL (d 1.063- 1.210 g/ml), HDL2 (d 1.063-1.125 g/ml), and HDL, (d 1.125-1.210 g/ ml) were obtained from human plasma (Red Cross) by ultracentrifu- gation as previously described (26). ApoA-I and apoA-I1 were isolated from total HDL treated with 6 M guanidine HC1 (8), followed by ultracentrifugation at d 1.210 g/ml for 20 h at 60,000 rpm and 4 “C. The infranatant containing apoA-I dissociated from HDL, was di- alyzed against 5 mM ammonium acetate, pH 6.8, lyophilized, dissolved in urea buffer (6 M urea, 0.02 M Tris, pH 8.0), and subjected to chromatography on DEAE-Sephacel (Pharmacia LKB Biotechnology Inc.) as described previously (27). ApoA-I1 was isolated from the supernatant following ethano1:ether (3:1, v/v) delipidation and chro- matography on DEAE-Sephacel in 6 M urea buffer, as described (28). The purity of the isolated apolipoproteins was verified by SDS, 10- 15% polyacrylamide gradient gels (Phastgel system, Pharmacia) stained with Coomassie Brilliant Blue R-250. Purified apolipopro- teins were stored lyophilized a t -20 “C. ApoA-I-phospholipid com- plexes were prepared by incubation of apoA-I with dimyristoylphos- phatidylcholine (DMPC; Calbiochem) liposomes (29) at a ratio of 1:4 (w/w, protein to lipid), for 20-24 h at 23 “C (30). ApoA-I concentra- tions were determined by immunoturbidometric analysis using human apoA-I antisera (Boehringer Mannheim) and apoA-I calibration serum (Boehringer Mannheim) on a COBAS-BIO (Roche), according to the recommendations of the manufacturer.

Peptide Synthesis and Conjugation-Two peptides, denoted AI[205-2201 and AI[230-2431, were synthesized using the human apoA-I sequence reported by Brewer et al. (31) and correspond to the amino acid residues 205-220 and 230-243, respectively. Both peptides represent regions of apoA-I that are predicted to be exposed in a lipid environment (32). The peptides were synthesized using t-Boc chem-

13257

13258 Carboxyl-terminal-specific Antibodies to Human apoA-1

istry on an Applied Biosystems Model 430A peptide synthesizer. To facilitate conjugation of the peptides to carrier proteins, Cys-Gly spacers were added to the amino-terminal ends. Cleavage and depro- tection were performed using hydrogen fluoride. The crude peptides were then purified by reversed-phase (RP)-HPLC, and the amino acid sequences were validated using an Applied Biosystems Model 470A protein Sequencer, equipped with an on-line Model 120A PTH analyzer. For the immunization procedures described below, peptides were coupled to ovalbumin (OVA) with maleimidobenzoyl-n-hydrox- ysuccinimide ester (Pierce). The specificity of each antisera for AI[205-2201 and AI[230-243] were determined by ELISA (described below) using the peptides coupled to keyhole limpet hemocyanin (KLH) as antigens. To avoid cross-reaction with maleimidobenzoyl- n-hydroxysuccinimide ester, the coupling agent succinimidyl 3-(2- pyridy1dithio)propionate (Pierce) was used to prepare the KLH con- jugates.

Production and Purification of Anti-peptide Antibodies-Antisera against the peptides AI[205-2201 and AI[230-2431 (conjugated to OVA) were produced in 5-6-month-old rabbits which received 0.5 mg of conjugate emulsified in 1 ml of phosphate-buffered saline (PBS), pH 7.4, and Freund's complete adjuvant, by subcutaneous injection at six different sites. The animals were boosted 3 weeks after the first immunization and bled 2 weeks thereafter. Each antiserum was subjected to affinity chromatography on Protein A-Sepharose (Phar- macia) (33). Antibody eluted from Protein A was dialyzed against PBS, then passed down an OVA-Sepharose 4B column to remove anti-OVA reactivity (determined by ELISA). Specific antibody was then eluted from a human apoA-I-Sepharose 4B column using 0.1 M glycine HC1, pH 2.8, and neutralized immediately with 1 M Tris buffer, pH 8.0. Purified human apoA-I and OVA (Sigma) were coupled to CNBr-activated Sepharose 4B as recommended by Pharmacia. Antibody specific for apoA-I (designated anti-apoA-I) was also ob- tained from rabbits immunized with purified human apoA-I, as pre- viously described (3). For the production of Fab fragments, affinity- purified antibody was subjected to papain digestion according to the procedures of Gorini et al. (34). Fab fragments were removed from undigested IgG and Fc fragments by passage through Protein A- Sepharose. The activity and purity of isolated Fabs were assessed by ELISA and SDS, 10-15% polyacrylamide gradient gel electrophoresis (Phastgel system, Pharmacia), respectively. Fab fragments were also prepared from two apoA-I-specific monoclonal antibodies, denoted AI-1 and AI-3, which recognize epitopes exposed on the surface of HDL:, particles (35) that have recently been localized to residues 28- 47 and 140-147 of apoA-I, respectively (36).

Enzyme-linhd Zmmunosorbent Assays-Titration curves of the anti-peptide antibodies toward different antigens were determined by ELISA. Briefly, 96-well plates (Immulon 11) were coated with 10 pg/ ml antigen in 0.05 M sodium carbonate buffer, pH 9.6, for 1 h, a t room temperature (100 pl/well). The wells were then washed three times in PBS containing 0.05% Tween 20, followed by the addition of 100 pl of serially diluted (1:2, from 50 pg/ml IgG), affinity-purified antibody. After 1 h at room temperature, the wells were washed as before, then incubated for another 1 h with 100 pl of goat anti-rabbit IgG (H + L) horseradish peroxidase conjugate (Bio-Rad) diluted 1 in 2000 in PBS/O.O5% Tween 20. After three washes with PBS-Tween, 100 pl/well of 0.1% ABTS (2',2-azinobis(3-ethylbenzothiazoline-6- sulfonic acid)), 0.02% H202 in 0.1 M citrate buffer, pH 4.0, was added for 30 min, and the color which developed was quantitated using a Titertek Multiscan (Flow Laboratories) with a filter setting of 414 nm. To compare antibody reactivities toward isolated apoA-I, HDLZ, or HDL:!, a competitive ELISA system was employed that followed the same coating and washing procedures as described above; how- ever, the incubation buffer included l % skim milk rather than Tween 20, which has been shown to alter the immunoreactivity of apoA-I in HDL (18, 19, 35). These duplicate wells of plates coated with apoA-I received 50 pl of serially diluted (1:2) antigen, followed by 50 pl of anti-peptide antibody (diluted 1/1000), for 1 h at room temperature. Specifically bound antibody was then detected as described above.

Tryptic Peptides of ApoA-ZZ in a Competitive ELISA-The tryptic peptides of apoA-I1 used in a competitive ELISA were prepared using modifications of the method described by Lux et al. (38). Briefly, purified apoA-I1 was reduced using 100-fold excess of dithiothreitol, then alkylated with 110-fold excess iodoacetic acid. Following reduc- tion of S-carboxymethylation, apoA-I1 monomers were cleaved using trypsin (~-l-tosylamido-2-phenylethylchloromethyl ketone-treated, Sigma) at 1:30 (w/w; trypsin:apoA-11) in 20 mM Tris-HC1, pH 8.0, for 45 min at 37 "C. Tryptic peptides were purified by RP-HPLC on an RP300 column (1.0 X 50.0 mm inside diameter, Brownlee) using

a 30-min linear gradient from 0-60% acetonitrile containing 0.1% trifluoroacetic acid. The peptides were identified by amino acid analy- sis (27) and amino-terminal sequencing. For the competitive ELISA, 96-well plates were coated with apoA-I1 (10 pg/ml) for 1 h at room temperature (100 pl/well). After three washes with PBS/0.05% Tween 20, duplicate wells then received serial dilutions (1:2) of purified apoA-I1 or tryptic peptides (50 pl/well), followed by affinity-purified anti-AI[230-2431 IgG, diluted 1/1000 (50 pl/well). After 1 h at room temperature, specifically bound antibody was then detected using the procedure described above.

Binding Studies-Binding assays were performed in triplicate. Assay tubes contained 200 pg of rat liver plasma membrane protein, 0.2 pg of 1251-labeled HDL3, and varying amounts of purified Fab fragments in a final volume of 200 pl. Incubations were performed in buffer containing 100 mM NaCI, 50 mM Tris-HCI, 0.01% EDTA, and 0.1% casein, pH 7.4. Rat liver plasma membranes were prepared with modifications (23) of the method described by Fleischer and Kervina (39). HDL3 was radiolabeled with '''I by the McFarlane procedure (40), as described previously (41), to a specific activity of 300-400 cpm/ng. Fab fragments were preincubated with lZ5I-HDL3 for 2 h at 37 "C in 1.5-ml Microfuge tubes (Eppendorf); following the addition of liver plasma membrane, the tubes were incubated for a further 4 h at 37 "C. 170 pl of each incubation mixture was transferred onto a vacuum filter manifold fitted with GF/C glass fiber filters (Whatman) presoaked for 3 h in 0.1% casein. The membranes were washed under vacuum with 6 X 1 ml incubation buffer and transferred to tubes for counting. Nonspecific binding to GF/C filters (measured in the ab- sence of plasma membranes) represented 5-8% of the total counts bound to the filters. Results were expressed as a percentage of the total lZ5I-labeled HDL3 bound in the absence of antibody.

Other Procedures-Isoelectric focusing was performed in 7.5% polyacrylamide gels containing 6.8 M urea and 2% ampholine pH 4- 6.5 (LKB-Pharmacia, Sweden), using a Bio-Rad Mini Protean I1 Dual Slab Gel apparatus. Protein bands were stained with 0.1% Coomassie Brilliant Blue R-250 in 45% ethanol, 10% acetic acid. Lipoprotein and protein concentrations were determined according to the method described by Lowry et al. (42) using bovine serum albumin as standard; peptide concentrations were determined by amino acid analysis.

RESULTS

Specificity of the Anti-peptide Antibodies-To select anti- bodies immunoreactive toward the parent protein, human apoA-I, anti-peptide antibodies were purified by affinity chro- matography on apoA-I-Sepharose 4B as described under "Ex- perimental Procedures." The recovery of apoA-I-specific im- munoglobulin from antisera raised to peptide AI[230-2431 was 5-fold greater than the amounts recovered from anti- AI[205-2201 sera (data not shown). The antigenic reactivities of antisera and affinity-purified antibody were determined by ELISA.

The purified anti-peptide antibodies bound only to their respective peptides, with no cross-reactivity observed toward the other peptide (Fig. 1). Although both antibody populations recognized the parent protein (apoA-I), of particular interest was the ability of anti-AI[230-2431 IgG to recognize human apoA-I1 with an unusually high cross-reactivity (Fig. 1, panel B ) . In contrast, anti-AI[205-2201 IgG was unable to bind apoA-11. To determine whether the anti-AI[205-2201 and anti-AI[230-2431 antibodies recognized the prominent apoA- I isoforms found in HDL, immunoblotting was performed following isoelectric focusing of apoA-I (and apoA-11) derived from human HDL (d 1.063-1.210 g/ml). The immunoblot patterns for each antibody were identical, both clearly detect- ing the major apoA-I isoforms (Fig. 2). Furthermore, the reactivity of anti-AI[230-2431 toward the apoA-I1 isoforms confirmed the cross-reactivity identified by the ELISA method.

Both anti-peptide antibody preparations reacted with iso- lated apoA-I-DMPC and HDLs particles (Fig. 3), demonstrat- ing that apoA-I residues 205-220 and 230-243 contain epi- topes that are exposed in the lipid environment of these

Carboxyl-terminal-specific Antibodies to Human apoA-I 13259

E C * * -

0 0

0.4

0.2

0.0

0.8 E

0.6

8 0.4

0.2

C

F *

0.0 10” IO-* IO-’ 10”

Antibody Dilution FIG. 1. Titration curves of affinity-purified anti-A1[205-

2201 (panel A ) and anti-AI[230-2431 IgG (panel B ) , as de- termined by ELISA. Serially diluted antibody was added to 96-well plates coated with either AII230-2431-KLH (O), AI[205-220]-KLH (V), apoA-I (O), or apoA-I1 (V). Specific binding was determined as described under “Experimental Procedures.”

A B C

1 2 3 4 1 2 3 4 FIG. 2. Immunoblots of purified human apoA-I and apoA-I1

using the anti-peptide antibodies. Panel A shows Coomassie Blue R-250 staining of apoA-I (lanes I and 3 ) and apoA-I1 (lanes 2 and 4 ) following isoelectric focusing (pH 4-6.5). as described under “Exper- imental Procedures.” Following isoelectric focusing, proteins were passively transferred onto nitrocellulose over 1 h a t 37 “C. Nitrocel- lulose strips were then incubated for 1 h with a blocking buffer containing 2% (w/v) skim milk, followed by incubation with either anti-AI[230-2431 (panel B ) or anti-AI[205-2201 (panel C ) , both diluted 1/1000, for 1 h a t room temperature. Antigenic species were then visualized using horseradish peroxidase-conjugated goat anti- rabbit immunoglobulin (diluted 1/500) followed by a substrate solu- tion containing 0.5 mg/ml 4-chloronapthol and 0.05% H202.

particles. However, different reactivities of the anti-peptide antibodies toward isolated apoA-I, HDLz, and HDLB sug- gested structural differences in the expression of these two regions of apoA-I. Anti-AI[230-2431 showed reactivity toward all three lipid-bound forms of apoA-I, whereas anti-A1[205- 2201 had little or no reactivity toward apoA-I in HDL2 (Fig. 3). Both anti-peptide populations were also compared for their ability to immunoprecipitate “‘I-labeled HDLB, relative to anti-apoA-I IgG. Assigning an arbitrary 100% for the maxi- mum immunoprecipitation produced by anti-apoA-I IgG, the

0

w 40 m

* 20

0

1 10 100

Concentration of ApoA-l (rg/ml)

FIG. 3. Competition curves of anti-A1[205-220] (panel A ) and anti-AI[230-243] (panel B ) I& binding to isolated apoA- I (O), apoA-I-DMPC (V), HDLz (O), HDLs ( V , and apoA-I1 (O), as determined by ELISA. 96-well plntes coated with npoA-I received increasing amounts of competing antigen, followed I y the addition of diluted (1/1000) antibodv. Specifically hound antibody was detected as described under “Experimental I’rocedures.” Results are shown as the ahsorhance obtained in the presence of competing antigen ( R ) , expressed as a percent of the maximum ahsorhance I h J , obtained in the absence of competing antigen.

AIIMU3I

spo*-ll a~-+-fl~--a, FIG. 4. Comparison between the carboxyl-terminal amino

acid sequences of human apoA-I and apoA-11. Amino acid resi- dues a t identical positions within the carbnxvl-terminus are indicated by the boxes.

affinity-purified anti-AI[205-220] and anti-AI(230-2431 I& precipitated 82% and 96% of “’I-HDL, respectively.

Epitope Mapping of ApoA-II Using Anti-AI/230-243]-As described above, anti-AI[230-2431, although raised to a pep- tide representing a portion of human apoA-I, displayed strong reactivity toward purified apoA-I1 using both ELISA and immunoblotting techniques. Close inspection of the primary structures of apoA-I (31) and apoA-I1 (38) reveals a 41% sequence homology between the last 17 carboxyl-terminal residues of both apolipoproteins (Fig. 4). Most of the region showing this homology is included in the peptide AI12.30- 2431. To confirm that the carboxyl-terminus of apoA-I1 con- tains the epitope(s) recognized by anti-AI[230-243] IgC, tryp- tic fragments of apoA-I1 were prepared and used in a compet- itive ELISA. The RP-HPLC chromatograph of the generated peptides is shown in Fig. 5. The identities of the peaks, determined by amino acid analysis and amino-terminal se- quencing, agreed with the expected peptides previously re- ported by Lux et al. (38). Selected peptides were then com- pared for their abilities to inhibit the binding of anti-AIl230- 2431 to apoA-11, as described under “Experimental Proce- dures.” Only peptide AII[56-771 could reduce the binding of anti-AI[230-2431 to immobilized apoA-I1 (Fig. 6). The inhi- bition produced by peptide AII[56-77] was identical with that produced by whole apoA-11, confirming that the AI/AII cross-

13260 Carboxyl-terminal-specific Antibodies to Human apoA-I

- z eeabeap t ide 2 1.2 v 1 A11[4-23] al c 0

2 All[29-39]

e 0.6 4 A11[45-54] 3 A11[40-44]

w 5 A11[56-7i'l 2 6 A11[55-77]

0.0 0 10 20

Time (rnin)

FIG. 5. RP-HPLC chromatograph showing the separation of peptides generated by trypsin digestion of apoA-11. The purified peptides, corresponding to the numbered peaks, were identi- fied by amino acid and amino-terminal sequencing analysis, as sum- marized in the right-hand table.

120"

100" n g 80"

2 60-

x 40.-

20"

v

0- 1 10 100 1000

Concentration of Antigen (pmol)

FIG. 6. Competition curves of anti-AI[230-2431 IgG bind- ing to apoA-I1 in the presence of apoA-I1 tryptic peptides. 96- well plates coated with apoA-I1 received increasing amounts of the purified peptides AII[4-231 (O), AII[29-391 (A), AII[40-441 (B), AI1[45-541 (O), AII[56-771 (A), and intact apoA-I1 (O), followed by the addition of anti-AI[230-2431 IgG. Results are shown as the absorbance obtained in the presence of competing antigen ( B ) , ex- pressed as a percent of the maximum absorbance (Bo) , obtained without competing antigen.

reactivity is due to similarities which reside in the carboxyl- terminal regions.

Inhibition of "'I-HDL3 Binding to Rat Liver Plasma Mem- branes-To determine the effects of the antibodies on the binding of HDL3 to rat liver plasma membranes, lZ5I-labeled- HDL, particles were preincubated with each antibody prior to the addition of plasma membranes. Initial studies using whole antibody molecules resulted in enhanced levels of lZ5I- HDLs binding to membranes, presumably due to the forma- tion of antibody-HDL3 aggregates (3). T o avoid this effect, and to minimize the possibility of steric inhibition resulting from the use of whole IgG molecules, Fab fragments were prepared. Fabs of anti-apoA-I, anti-AI[205-2201, and anti- AI[230-2431, with similar reactivities toward apoA-I, all pro- duced inhibition of binding, whereas Fabs from an unrelated anti-peptide antibody with no specificity toward apoA-I, had little or no effect (Fig. 7). Anti-AI[205-2201 and anti-apoA-I at concentrations of 300 pg/ml Fabs reduced the binding of '"I-HDL3 to less than 80% of control values. At similar concentrations, anti-AI[230-2431 inhibited binding by ap- proximately 60%. Under the same conditions, Fab fragments from the two monoclonal antibodies, AI-1 and AI-3, had no significant effect on the binding of lZ5I-HDL3 to the hepatic membranes (Fig. 7).

DISCUSSION

Anti-peptide antibodies generated to two peptides, synthe- sized from two distinct regions of the carboxyl-terminal region of human apoA-I, were found to inhibit the binding of HDL3

Y

9

=Q + 0 - 6

0 100 200 300 600

Concentrotion of Fob fragments (pg/ml)

FIG. 7. Inhibition of lZ6I-HDL, binding to rat liver plasma membranes. 1251-labeled HDLB (1 pg/ml) was preincubated with increasing amounts of Fab fragments derived from the monoclonal antibodies AI-1 (B) and AI-3 (0) and from anti-apoA-I (0), unrelated anti-peptide (V), anti-AI[205-2201 (O), and anti-AI[230-2431 IgG (V), for 4 h at 37 "C, followed by the addition of liver plasma membranes for 4-h binding studies at 37 "C. Each point represents the mean of triplicate (or duplicate for AI-1 and AI-3) determinations.

to liver plasma membranes. In addition to supporting the proposal that apoA-I can act as a specific ligand for HDL3 cellular binding sites (2-6), the present studies further suggest that a specific region in the carboxyl-terminus of apoA-I is responsible for the cellular binding of HDL3. Furthermore, characterization of the specificities displayed by the anti- peptide antibodies has identified a region of structural ho- mology between human apoA-I and apoA-11, two proteins previously implicated in mediating the binding of HDL to human (6) and rat (2) liver plasma membranes.

Peptides AI[205-220] and AI[230-2431, corresponding to residues 205-220 and 230-243 of apoA-I, respectively, gener- ated two distinct populations of anti-peptide antibodies which could recognize the major isoforms of apoA-I from HDL (Fig. 2). Anti-AI[205-2201 and anti-AI[230-2431 also recognized apoA-I associated with apoA-I-DMPC and HDL3 particles (Fig. 3), which is consistent with the proposed orientation of apoA-I in the lipid environment (32), in which both regions are thought to contain sites exposed on the lipoprotein sur- face. Such sites are potentially available for interactions with enzymes, receptors, or other blood components involved in lipid metabolism; therefore, these antibodies may provide useful tools for further probing the structural-functional prop- erties of apoA-I. However, apparent structural differences between the expression of apoA-I in HDL, and HDL3 were identified by the inability of anti-AI[205-2201 to recognize HDLz particles. I t is possible that residues 205-220 become hidden in the lipid environment of the larger HDLz particles due to conformational changes of apoA-I, or, alternatively, these residues may be masked by other protein moieties on the particle surfaces.

Anti-AI[205-2201 and anti-AI[230-2431 Fab fragments sig- nificantly reduced the interaction between HDL3 and rat liver plasma membranes (Fig. 7), whereas two monoclonal antibod- ies, AI-1 and AI-3, recognizing epitopes positioned toward the amino-terminal (residues 28-47) and middle (residues 140- 147) portions of apoA-I, respectively (37), had little or no effect. Thus, both anti-peptide antibodies may recognize epi- topes which contain, or lie close to, a cellular binding domain located in the carboxyl-terminal region of apoA-I. The higher levels of inhibition observed with anti-AI[205-2201 may in- dicate that residues within 205-220 are more specifically involved in, or lie closer to, the actual binding region. The region of apoA-I recognized by anti-AI[205-2201 IgG is thought to include a p-conformation between two amphi- pathic a-helical regions (32). Recent studies involving chem- ical modification of lysine or arginine residues of HDLB (43,

Carboxyl-terminal-specific Antibodies to Human apoA-I 13261

45), or tetranitromethane treatment of HDL3 (6, 44-46), suggested that the amphipathic a-helical regions of apoA-I mediate the cellular binding of HDL. The present findings cannot rule out this possibility as anti-AI[205-2201 may hinder the adjacent amphipathic regions from interacting with the cellular binding sites, although the inhibition observed suggests that the cellular binding of HDL3 does not require complete accessibility of all the apoA-I a-helical repeats. Following our previous identification of HDL-binding pro- teins (putative receptors), present in human and rat liver plasma membranes that recognize HDL, and purified apoA-I (23), and a more recent demonstration that these HDL- binding proteins are specific for cyanogen bromide fragment 4 (residues 149-243) of apoA-I (24, 25), we postulated that a binding domain within this region mediates the interaction between apoA-I and specific HDL receptor sites (24). The ability of the anti-peptide antibodies to inhibit HDL, binding to liver plasma membranes is consistent with this proposal.

One unexpected finding from this study was the high level of cross-reactivity shown by anti-AI[230-2431 to apoA-11, detected by means of ELISA and immunoblotting (Figs. 1 and 2). Immunological cross-reactivity between apoA-I and apoA- I1 has been previously reported by Silberman et al. (18), where two monoclonal antibodies showed high reactivity with apoA- I1 and slight cross-reactivity with apoA-I. Comparison be- tween the amino acid sequences of both apolipoproteins shows sequence homology over several amino acid residues at the carboxyl-terminal regions (Fig. 4). By epitope mapping, we then confirmed that the antigenic site of apoA-I1 recognized by anti-AI[230-2431 IgG is contained in the carboxyl-terminal portion (residues 56-77) (Figs. 4 and 5). Previous studies have shown that this region of apoA-I1 corresponds with the most antigenic part of the molecule (47), which is not masked when associated with lipids in HDL. The significance of such struc- tural homology between apoA-I and apoA-I1 is unclear, al- though it is interesting to speculate that similar structural conformations residing in the carboxyl-terminal portions may account for the ability of apoA-I and apoA-I1 to recognize the HDL-binding proteins identified in both human and rat liver plasma membranes (23).

In summary, the finding that two anti-peptide antibodies, both directed toward the carboxyl-terminus of apoA-I, can inhibit the interaction between HDL, and liver plasma mem- branes provides strong evidence that the carboxyl-terminus of apoA-I mediates the cellular binding of HDL, possibly through specific plasma membrane receptors. Further, we anticipate that these new antibodies will provide useful tools for immunochemical characterization of previously unknown structural and functional domains of apoA-I involved in lipid metabolism.

Acknowledgments-We thank Brendan Murray and John Morrison for help in preparingpeptides and Jim Andreou for excellent technical assistance.

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