THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Vol. 261, No. 5, Issue of February 15, pp. 2068-2076 1986 Printed in L h A .

Human Apolipoprotein E DETERMINATION OF THE HEPARIN BINDING SITES OF APOLIPOPROTEIN E3*

(Received for publication, June 17,1985)

Karl H. WeisgraberSS, Stanley C. Rall, Jr.& Robert W. MahleyS, Ross W. MilneTi, Yves L. Marcelll, and James T. Sparrow (1 ** From the $Gladstone Foundation Laboratories for Cardiovascular Disease, Cardiovascular Research Institute, Departments of Pathology and Medicine, University of California, San Francisco, California 94140-0608, the 9Laboratory of Lipoprotein Metabolism. Clinical Research Institute of Montreal. Montreal H2W lR7, Quebec, Canada, and the IlDepartment of Medicine, Baylor College of Medicine, Houston, Tez& 77030 ‘

The interaction of human apolipoprotein (apo-) E3 with heparin was examined using heparin-Sepharose as a model system. The approach taken to determine the region of apo-E that is responsible for binding to heparin was to identify apo-E monoclonal antibodies that inhibited heparin binding, to determine the epi- topes of the inhibiting antibodies, and finally to ex- amine the heparin binding of fragments containing the inhibiting antibody epitopes. Three antibodies, desig- nated 1D7,6C5, and 3H1, were found to inhibit bind- ing, suggesting that multiple heparin binding sites were present on apo-E. The epitopes of the inhibiting antibodies were determined by immunoblot analysis of synthetic or proteolytic fragments of apo-E. Measure- ment of the heparin binding activity of fragments aon- taining epitopes of the inhibiting antibodies demon- strated that apo-E3 contains two heparin’binding sites. The first site is located in the vicinity of residues 142- 147 and coincides with the 1D7 epitope. The second binding site is contained in the carboxyl-terminal re- gion of apo-E and is inhibited by 3H1, the epitope of which is located between residues 243 and 272. The epitope of the third inhibiting antibody, 6C5, is located at the amino terminus of apo-E; however, this antibody inhibits the second heparin binding site located in the carboxyl-terminal region. A head-to-tail association of apo-E, in which the 6C5 epitope and the second heparin binding site would be in close proximity, is proposed to account for this observation. In the lipid-free state both heparin binding sites on apo-E are expressed; however, when apo-E is complexed to phospholipid or on the surface of a lipoprotein particle, only the first binding site (residues 142-147) is expressed.

Apolipoprotein (apo-) E is an M, = 34,000 protein of known sequence (1) and is a component of several classes of plasma lipoproteins (2,3). The heterogeneity exhibited by this protein is the result of a genetically determined polymorphism (4, 5) combined with the post-translational addition of sialic acid

* The peptide synthesis work was supported by Grant HL30064 from the United States Public Health Service. The costs of publica- tion of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 To whom correspondence and reprint requests should be ad- dressed Gladstone Foundation Laboratories, P. 0. Box 40608, San Francisco, CA 94140-0608.

** Established Investigator of the American Heart Association.

(5). Several variants of apo-E have been identified by isoelec- tric focusing, with the most common isoforms focusing in the E4, E3, or E2 positions (6). Genetic control is exerted at a single gene locus, resulting in the expression of six common phenotypes: E4/4, E3/3, E2/2, E4/3, E4/2, and E3/2. The E3 form occurs with the highest frequency in the population (5, 7-9). The structures of several of the apo-E variants have been determined and several sites of amino acid interchange have been identified (1, 10-14).

Functionally, apo-E plays a major role in the metabolism of plasma lipoproteins because of its ability to interact with lipoprotein receptors on the surface of a variety of cell types (for review, see Ref. 15). Several apo-E variants are defective in their ability to interact with receptors, and this dysfunction has been shown to be an underlying cause of the lipoprotein disorder type 111 hyperlipoproteinemia (16,17). We have used a variety of approaches, including structure-function studies of the various apo-E mutants (11-13, 18), chemical and en- zymatic fragmentation of the protein (19), and use of apo-E monoclonal antibodies (20), ts) localize the lipoprotein recep- tor binding domain to the vicinity of residues 140-160.

Another property of apo-E, which is also characteristic of apo-B (the major apolipoprotein of low density lipoproteins), is its ability to interact with various proteoglycans, including heparin (21-23). It has been suggested that the interaction of lipoproteins with the proteoglycans of the arterial wall may be involved in the cholesterol deposition associated with ath- erosclerosis (24). A second potentially important aspect of apolipoprotein-proteoglycan interactions involves the enzyme lipoprotein lipase. This enzyme, which hydrolyzes triglycer- ide-rich lipoproteins, occurs on the surface of endothelial cells lining capillary beds and is known to be associated with heparan-like structures that are present on the surface of these cells. The lipase can be released from the cell surface with an intravenous injection of heparin. It has been suggested that the interaction of triglyceride-rich lipoproteins with the heparan structures might serve to anchor the lipoprotein particles to the capillary wall, thus allowing the lipase access to the particle to hydrolyze the triglyceride core (25). Poten- tially, apo-E and apo-B, both present in triglyceride-rich lipoproteins, could be involved in the anchoring process. In support of this hypothesis, it has been demonstrated that addition of apo-E to triglyceride emulsions resulted in en- hanced lipolysis of these emulsions by purified milk lipopro- tein lipase that was noncovalently bound to heparin-Sepha- rose. This enhanced lipolytic effect of apo-E correlated with an increased binding of triglyceride to the heparin-Sepharose column (26).

Previously, the nature of the interaction of apo-E and apo-

2068

Heparin Binding Sites of Apolipoprotein E 2069

4.0 t

10 20 30 40 Apo-E3 Bound (p/lOrng Heparin-Sepharose Gel)

FIG. 3. Scatchard analysis of ‘2SI-apo-E3 binding to hepa- rin-Sepharose. Increasing concentrations of lZ5I-apo-E3 in 300 p1 of 50 mM NaCl, 5 mM Tris-HC1, pH 7.4, were incubated with 10 mg of heparin-Sepharose for 4 h a t 4 ”C. After incubation, the gel was sedimented by centrifugation, and the supernatant was removed. The gel was washed twice with 300 p1 of buffer. The nonspecific binding was determined in parallel incubations with a non-heparin-containing Sepharose.

B with heparin has been studied by selectively modifying specific amino acid residues of these apolipoproteins (21). These studies have demonstrated the importance of lysyl and arginyl residues in the binding of apo-E and apo-B to heparin and suggested that the positive charges carried by these basic amino acids are interacting with negatively charged groups on heparin. This is consistent with an ionic mechanism for the interaction of low density lipoproteins with heparin as proposed by Iverius (27) . Because of the potential importance of apolipoprotein-proteoglycan interactions in atherosclerosis and Iipoprotein metabolism, together with our continuing interest in mapping functional domains of apo-E, we have determined the sites on apo-E3 that bind to heparin.

EXPERIMENTAL PROCEDURES~

RESULTS

To study the properties of apo-E binding to heparin, hep- arin coupled to Sepharose gel was chosen as a model system. Several different preparations of heparin-Sepharose were found to have similar capacities to bind apo-E. One prepara- tion was chosen for use in the following studies, and in one experiment, the binding of apo-E to this preparation was characterized in more detail by Scatchard analysis (28). As shown in Fig. 3, the binding of lZ5I-apo-E to the gel was saturable and gave a linear plot with a correlation coefficient of 0.99. The apo-E bound with a Kd of 6.2 X lo-’ M and

~ ~~~~

Portions of this paper (including “Experimental Procedures,” part of “Results,” Tables I and 11, Figs. 1 and 2, additional references, and Footnote 2) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Doc- ument No. 85M-1985, cite the authors, and include a check or money order for $3.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

saturated the gel at a level of 29.2 pg of apo-E/10 mg of gel, wet weight. The K d observed for apo-E binding to heparin is in the range (10-6-10-s M) reported for heparin binding to antithrombin 111 and various hemostatic enzymes (29).

The approach taken to determine the region of apo-E that bound to heparin was: 1) to identify apo-E monoclonal anti- bodies that inhibited heparin binding; 2) to determine the epitopes of the inhibiting antibodies; and 3) to examine the heparin binding of fragments of apo-E that contained the inhibiting antibody epitopes. Previously, this approach was used to delineate the lipoprotein receptor binding domain of apo-E (19,20). In those studies, it was demonstrated that the antibody 1D7 was capable of totally inhibiting binding of apo- E to lipoprotein receptors and that fragments of apo-E con- taining the 1D7 epitope bound to receptors. The four antibod- ies used in the present study were designated 1D7, 3H1, 6C5, and 7C9.

The individual antibodies were preincubated at increasing concentrations with 1251-apo-E3, and the mixtures were added to heparin-Sepharose. As shown in Fig. 4, three of the four antibodies (1D7, 3H1, and 6C5) inhibited the binding of apo- E to the gel. Inhibition by any one of the three antibodies was less than 100% even at very high concentrations of added antibody. The 1D7 antibody had the greatest effect, inhibiting approximately 60% of apo-E binding to heparin. The 3H1 and 6C5 antibodies inhibited approximately 30-40% of apo-E binding. When a mixture of 1D7 and 6C5 was added at ratios of 201 (antibody:apo-E), an inhibition of approximately 80%

0 U - 80

60 6C5 0 m

I I I I I I I I I

50:l 1004 150:l 2004

Antibody: Apo-E3 (wt:wt)

FIG. 4. Comparison of the abilities of apo-E monoclonal antibodies to inhibit the binding of apo-E to heparin. Increasing concentrations of apo-E monoclonal antibodies (0, 7C9; m, 6C5; 0, 3H1; and A, 1D7) were pteincubated for 1 h at room temperature with 20 pg of lZ51-apo-E3 in 300 pl of 50 mM NaCl, 5 mM Tris-HCI, pH 7.4. After the preincubation, the mixture was added to 125 mg (wet weight) of heparin-Sepharose gel or control gel that did not contain heparin. The gel was incubated for 3 h at 4 “C on a rocking platform and then sedimented by centrifugation; the radioactivity in the supernatant was measured. The amount bound to the gel was determined as the difference in concentration of 9 - a p o - E in the control gel supernatant uersus the heparin-Sepharose supernatant.

TABLE 111 Epitopes of apolipoprotein E monoclonal antibodies

Effect on apo-E Antibody Epitope binding to

heparin

1D7 Vicinity of residues 140-150 Inhibits 6C5 Partially or completely contained Inhibits

7C9 Partially or completely contained No effect

3H1 Residues 243-272

within residues 1-13

within residues 1-13 Inhibits -

2070 Heparin Binding Sites of Apolipoprotein E was observed (A in Fig. 4). Because the individual effects of each antibody were approximately additive, these results sug- gested that multiple heparin binding sites are present on apo- E. As will be presented below, two heparin binding sites were demonstrated.

The first step in determining the heparin binding sites was to map the epitopes of the inhibiting (1D7, 3H1, and 6C5) and noninhibiting (7C9) antibodies. As presented under "Re- sults" in the Miniprint and summarized in Table 111, the epitopes of the apo-E monoclonal antibodies were mapped to three separate locations on apo-E. These results were con- sistent with the suggestion that multiple heparin binding sites were present on apo-E.

Although the 6C5 and 7C9 epitopes were mapped to the same location, only 6C5 inhibited heparin binding. In an attempt to resolve this apparent paradox, the relative spatial relationship of the 6C5 and 7C9 antibodies was examined in a solid-phase competition assay. The relative abilities of the antibodies to compete with lZ5I-6C5 for binding to immobilized apo-E3 revealed that 7C9 competed for binding, but not as effectively as unlabeled 6C5, and that 1D7 did not compete (Fig. 5). In the reciprocal experiment using lZ5I-7C9, an iden- tical difference between the two antibodies was also observed (data not shown), except that in this case 7C9 was a more effective competitor than 6C5. These results indicated that the 6C5 and 7C9 epitopes were not identical even though epitope mapping failed to differentiate them, thus suggesting that the 6C5 epitope is spatially oriented such that antibody interaction inhibits heparin binding, whereas the 7C9 epitope does not share this spatial relationship. Addition of 1D7 had no effect on the ability of lZ5I-6C5 to bind to apo-E, suggesting that both antibodies might be able to bind to apo-E simulta- neously.

With the location of the epitopes of the three antibodies that inhibited heparin binding established, the next step was to examine the heparin binding of apo-E fragments containing these epitopes and to determine the effect that these antibod- ies had on the ability of the fragments to bind. Because the M, = 22,000 (residues 1-191) and the M, = 12,000 (residues 192-299) thrombolytic fragments included the entire apo-E sequence, the binding of these fragments was examined first. As shown in Table IV, both the M, = 22,000 and 12,000 fragments bound well to heparin, confirming that multiple heparin binding sites existed. However, an unexpected obser- vation was made when the effect of antibody addition on the ability of these fragments to bind to heparin was examined.

0.8 - A 6C5 A x9

107 m" 0.6 - . m

OA - a2 -

10 1 0 2 103 104

ng Protein/POO pl

FIG. 5. Ability of various apo-E monoclonal antibodies to compete with lesI-6C5 immunoglobulin G for binding to apo- E3. Apolipoprotein E was immobilized in microtiter wells, and the abilities of increasing concentrations of unlabeled antibodies to com- pete with lZ5I-6C5 for binding were determined.

TABLE IV Ability of apolipoprotein E and thrombolytic fragments of

apolipoprotein E to bind to heparin-Sepharose and the ability of apolipoprotein E monoclonal antibodies to inhibit binding

~ ~~

Fragment Bound" Binding

1D7 6C5 3H1 __ % %

Intact apo-E3 100 50 70.1 66.0 M, = 22,000 (residues 90.2 7.4 130.4 ND'

M, = 12,000 (residues 82.2 ND 90.1 50.0 1-191)

192-299) "Results are the average of duplicate experiments and are ex-

pressed relative to apo-E3 binding; results are normalized to 100% for intact apo-E3 binding.

* Results are the average of duplicate experiments at an antibody to fragment ratio of 15:l (w/w) and are expressed as a per cent of fragment binding in the presence of added antibody compared to that in the absence of antibody, which was set a t 100%.

ND, not determined.

In contrast to intact apo-E, 1D7 essentially completely inhib- ited binding of the M, = 22,000 fragment, and 6C5 did not inhibit binding of this fragment. These results indicated that the heparin binding site sensitive to 1D7 inhibition was the only one present in the amino-terminal M, = 22,000 throm- bolytic fragment. Furthermore, this indicated that the heparin binding site that was inhibited by 6C5 is located in the carboxyl-terminal M, = 12,000 fragment, even though its epitope is located at the amino terminus. A potential expla- nation for this observation will be discussed later.

The effect of 6C5 and 3Hl on the heparin binding activity of the M, = 12,000 fragment was determined (Table IV). As was expected from the location of its epitope at the amino terminus, 6C5 had no significant effect on the heparin binding of the carboxyl-terminal M, = 12,000 fragment. The 3H1 antibody, whose epitope is located in the M, = 12,000 frag- ment, partially inhibited the binding activity of this fragment.

In an attempt to localize the heparin binding site in the carboxyl-terminal M , = 12,000 fragment, the relative affinities of the thrombolytic MI = 12,000 (residues 192-299) and M, = 10,000 (residues 216-299) fragments for heparin were com- pared. This was accomplished by passing a thrombolytic digest of apo-E through a heparin-Sepharose column, eluting the bound fragments with an NH4HC03 gradient, and then identifying the fragments by sodium dodecyl sulfate gel elec- trophoresis. As shown in Fig. 6, the order of elution of the thrombolytic fragments was M, = 10,000, 12,000, and 22,000. These results indicated that the M, = 10,000 fragment has a lower affinity for heparin than the MI = 12,000 fragment. This result suggested that a portion of the binding site was amino-terminal to residue 216 and was contained between residues 191 and 216 and that a second portion extended beyond residue 216 toward the carboxyl terminus. It was also apparent that each of the thrombolytic fragments had a lower affinity for heparin than did apo-E, since they all eluted at lower ionic strengths than did intact apo-E (arrow in Fig. 6).

To locate the heparin binding site present in the M, = 22,000 fragment that is sensitive to inhibition by 1D7, a series of synthetic fragments spanning residues 139-169 was used. These fragments were used previously to locate the 1D7 epitope (20). As shown in Table V, under the conditions of the binding assay, the 139-169 synthetic fragment bound well to the gel. Significantly less of the shorter 144-169 fragment bound, and the 148-169 fragment did not bind. These results demonstrated that residues 139-147 constitute the heparin binding site contained in the M, = 22,000 fragment. Further-

FIG. 6. Elution profile of a throm- bolytic digest of apo-E3 on heparin- Sepharose. A digest that contained 3 mg of apo-E3 in 25 mM NH4HC03 and 0.1% P-mercaptoethanol was applied to a heparin-Sepharose column and eluted with an NH4HC03 gradient. Fraction I represented the unbound material from the digestion mixture, and Fractions II- IV represented the bound material. Ar- row indicates elution position of intact apo-E3 (18.0 mMHO). Inset, sodium do- decyl sulfate-polyacrylamide gel electro- phoresis of various apo-E fragments and heparin-Sepharose fractions. Lane 1, in- tact apo-E lane 2, residues 1-191; lune 3, residues 192-299; lune 4, residues 216- 299; lane 5, thrombin-digested apo-E lane 6, Fraction 11; lune 7, Fraction 111; and lune 8, Fraction IV.

2071 Heparin Binding Sites of Apolipoprotein E

- -

34K--

22K- - - " 0.05- 12K- -

10K- "

C

0)

0

cv

E m

1 2 3 4 5 6 7 8 3

0.025 s n a

1 3 5 7 20 30 40 50 60 Tube Number

TABLE V Comparison of the ability of apolipoprotein E3 and various apolipoprotein E synthetic fragments to bind to heparin-

Seuharose

Fragment activitP Binding Partial fragment sequence

I n t a c t a p o - E 3 100 139 144 148 150 169

Residues 139-169 84.0 Ser-His-Leu-Arg-Lys-Leu-Arg-Lys-Arg-Leu-Leu-Arg . . . G l y 144 148 150 169

Residues 144-169 54.0 Leu-Arg-Lys-Arg-Leu-Leu-Arg . . . G l y 148 150 169

Residues 148-169 0 Leu-Leu-Arg . . . G l y

a Values are the average of two determinations; results are normalized to 100% for intact apo-E3 binding.

more, the data suggested that the cluster of arginyl and lysyl residues between residues 142 and 147 were likely to be directly involved. This study also demonstrates that this heparin binding site coincides with the 1D7 epitope.

Unlike the binding of apo-E to lipoprotein receptors (30), apo-E binding to heparin did not require that the apo-E be complexed to lipid. All of the heparin binding data presented to this point are from studies that used apo-E in the absence of lipid. Because apo-E is associated with lipoprotein particles in vivo and is, therefore, in a lipid environment, and because lipid association is known to affect the secondary structure of apo-E (31), it was important to determine the heparin binding ability of apo-E complexed with lipid. Thus, the artificial complex apo-E-DMPC3 and a native lipoprotein, canine apo-E HDL,, were used to measure the heparin binding ability of complexed apo-E. Canine apo-E HDL, contain apo-E as their only apolipoprotein component and are induced by cholesterol feeding in dogs (32).

Under the conditions of the binding assay, apo-E3 - DMPC and canine apo-E HDL, bound as effectively as the uncom- plexed protein. The effect that the 1D7 and 6C5 antibodies had on the ability of these lipoprotein particles to bind to heparin was then determined. As shown in Table VI, 1D7 essentially blocked all of the heparin binding of both apo-E3. DMPC and apo-E HDL,. This is in marked contrast to the

The abbreviations used are: DMPC, dimyristoylphosphatidylcho- line; HDL,, high density lipoproteins induced by cholesterol feeding and containing apo-E as their major apolipoprotein component; 'BOC, t-butoxycarbonyl.

TABLE VI Effect of monoclonal antibodies on the ability of apolipoprotein E3. DMPC complexes and apolipoprotein E HDL, to bind to heparin

Complex and antibody Binding" %

apo-E3. DMPC 100 apo-E3. DMPC + 1D7 9.8 apo-E3. DMPC + 6C5 93.8 apo-E HDL, + 1D7 0

"The antibody to protein ratio was 15:l (w/w) for 1D7 and 6C5 with apo-ES.DMPC and 25:l for 1D7 with apo-E HDL,. Results are expressed as a per cent of binding in the presence of added antibody compared to that in the absence of antibody, which was set a t 100%.

incomplete inhibition in the case of lipid-free apo-E3 (Fig. 4, Table IV). Furthermore, 6C5 had no significant inhibitory effect on the ability of apo-E3. DMPC complexes to bind to heparin (Table VI). Previously, we determined that 6C5 was capable of interacting with apo-E3. DMPC complexes, indi- cating that the 6C5 epitope was expressed in a DMPC recom- binant (20).

The reason for the lack of inhibition by 6C5 became appar- ent when the ability of the M , = 12,000 thrombolytic frag- ment, complexed with DMPC, to bind to heparin was exam- ined. The lipid-complexed M, = 12,000 fragment did not bind to heparin, demonstrating that the carboxyl-terminal heparin binding site was not expressed when apo-E was complexed to DMPC. These results are consistent with the observation that 1D7 inhibits essentially all of the heparin binding of intact human apo-E. DMPC recombinants or the canine apo-E HDL,. Therefore, only one heparin binding site (residues 142-

2072 Heparin Binding Sites of Apolipoprotein E

147) appears to be expressed in the native lipoprotein and the apo-E.phospholipid complex.

DISCUSSION

The presence and identification of two heparin binding sites on apo-E3 were established with apo-E monoclonal antibodies, synthetic fragments of apo-E, and enzymatically derived fragments of the protein. The first heparin binding site contains a cluster of five basic amino acids that is located near the center of the protein (residues 142-147): Arg-Lys- Leu-Arg-Lys-Arg. This heparin binding site is located in the same region of apo-E that has been demonstrated to contain the lipoprotein receptor binding domain of the protein (19, 20). Residues 142-147 are contained within the epitope for the apo-E monoclonal antibody 1D7, an antibody capable of interfering with both lipoprotein receptor binding activity (20) and heparin binding to this site. This heparin binding site is the only one expressed when apo-E is complexed with lipid.

The second heparin binding site of apo-E3, expressed only in the lipid-free protein, is located in the carboxyl-terminal region of the protein. Although its precise location was not established, it appears that residues 192-215 contain at least a portion of the binding site. This was suggested by the demonstration that the M, = 10,000 thrombolytic fragment (residues 216-299) has a lower affinity for heparin than does the M, = 12,000 fragment (residues 192-299) (Fig. 6). The 3H1 antibody, the epitope of which is located in the carboxyl- terminal region of the protein (residues 243-272), also inhibits heparin binding, confirming the presence of a second site. However, the inhibition of the binding of the M, = 12,000 fragment is not complete (Table IV). This raises the possibil- ity that there are two binding sites in this region of the protein, one contained between residues 191 and 216 and the other closer to or coinciding with the 3H1 epitope. An alter- nate explanation for the incomplete inhibition is that the 3H1 epitope may not exactly coincide with the heparin binding site and may be capable of only partial inhibition. These two possibilities cannot be distinguished at this point, but if there are two distinct binding sites in the carboxyl-terminal region, then both are masked by lipid. The data presented in the present studies indicate that both heparin binding sites of apo-E contribute to heparin binding in the lipid-free protein. The presence of multiple heparin binding sites in a heparin- binding protein has also been observed in fibronectin in which three heparin binding sites, each with differing affinities for heparin, have been described (33).

The importance of basic amino acids in the amino-terminal heparin binding site of apo-E (residues 142-147) is consistent

'with data from previous chemical modification studies that established the importance of arginyl and lysyl residues in the binding of both apo-E and apo-B to heparin (21). Lysyl residues have also been suggested to be involved in the inter- action of antithrombin I11 with heparin. Pyridoxylation of lysyl residues of antithrombin 111 demonstrated that modifi- cation of 1 or 2 residues is sufficient to inhibit heparin binding (34). Although the location of these critical lysyl residues in antithrombin I11 is not known, fluorescence energy transfer exper>ments indicate that they may be located near trypto- phan 49, as selective modification of this tryptophan also blocks heparin binding of antithrombin 111 (35). Recently, a mutant form of antithrombin 111, in which arginine 47 is replaced by cysteine, has been demonstrated to lack the ability to bind to heparin (36). This suggests that the positive charge at arginine 47 may also be essential for heparin binding. The sequence of antithrombin I11 in the vicinity of arginine 47 is

of interest because it contains the critical tryptophan at residue 49 and lysine at residue 53: Arg-Arg-Val-Trp-Glu- Leu-Ser-Lys (residues 46-53). Lysine 53 may be the critical residue implicated in the pyridoxylation studies.

Platelet Factor 4, another heparin-binding protein, also contains a cluster of lysyl residues in the peptide fragment that has been demonstrated to contain the heparin binding site (37): Lys-Lys-Ile-Ile-Lys-Lys (residues 61-66). Inspection of the sequence of another heparin-binding protein, ,6-throm- boglobulin, reveals the presence of two clusters of basic resi- dues (38): Lys-Asp-Gly-Arg-Lys (residues 56-60) and Arg-Ile- Lys-Lys-Ile-Val-Gln-Lys-Lys (residues 69-77). Although structural data on heparin binding sites are limited, it appears unlikely that there is a unique sequence shared by heparin- binding proteins.

The importance of positively charged amino acids as a component of heparin binding sites is consistent with the postulated ionic interaction between these basic amino acids and negatively charged sulfate or carboxylate groups of hep- arin or other sulfated glycosaminoglycans (27). The structural component of heparin that binds to antithrombin I11 has been investigated (39, 40). It is composed of a linear sequence of four to eight disaccharide units that are enriched in negatively charged groups. The binding site does not appear to be a unique oligosaccharide structure but does have some nonvar- iable features that, presumably, confer specificity to the bind- ing (39). Consistent with an ionic interaction between basic amino acids and negatively charged groups on glycosamino- glycans is our observation that the binding affinity of apo-E increases directly with the level of sulfation of heparan prep- arations:

Our studies have allowed us to propose a model for apo-E- heparin interaction (summarized schematically in Fig. 7). The partial inhibition of lipid-free apo-E binding to heparin by 1D7 is explained by the fact that the 1D7 epitope (residues 140-150) coincides with the first heparin binding site (vicinity of residues 142-147). This results in a complete blocking of the first site but does not affect the heparin binding of the carboxyl-terminal region of apo-E (Fig. 7). In a similar man- ner, 3H1, with its epitope located between residues 243 and 272, is capable of interfering with the second heparin binding site, and we would propose that it does not affect the first heparin binding site.

However, the reason for the partial inhibition of 6C5 and lack of inhibition of 7C9 is less obvious. The 6C5 epitope is located at the amino terminus of apo-E, and yet, this antibody inhibits heparin binding to the second site that is located in the carboxyl-terminal region of apo-E. This suggests that in the lipid-free form, the amino-terminal region of apo-E is in close proximity with the second heparin binding site located in the carboxyl-terminal region of apo-E. Furthermore, im- munoblots (Fig. 1, in Miniprint) and antibody competition studies (Fig. 5) indicate that the 6C5 and 7C9 antibodies recognize similar but not identical epitopes in the vicinity of the amino-terminal 13 residues of apo-E. These results sug- ge-st that there are rather specific spatial requirements for an antibody that interacts with the amino terminus and that interferes with the heparin binding site located in the car- boxyl-terminal region.

According to the model depicted in Fig. 7, the 6C5 epitope in lipid-free apo-E is in close spatial proximity to the second heparin binding site located in the carboxyl-terminal portion of the protein (left side of Fig. 7). As a result of this close spatial arrangement, antibody binding to the 6C5 epitope

K. H. Weisgraber and M. Hook, unpublished observations.

Heparin Binding Sites of Apolipoprotein E 2073

n

0 Heparin Binding

Antibody Epitope

FIG. 7. Schematic model depicting the location of the two apo-E heparin binding sites and their spatial relationship to the ID7 and 7C9/6C5 epitopes. Left, it is proposed that one heparin binding site is located in the center of the apo-E molecule and that this binding site coincides with the 1D7 epitope. A second heparin binding site is located in the carboxyl-terminal region of the protein. In the free protein, it is proposed that the second heparin binding site is in a close spatial relationship with the 6C5 epitope such that antibody interaction with this epitope inhibits heparin binding to the second site. The epitope for 7C9 is also located at the amino terminus of apo-E; however, it differs from the 6C5 epitope in that antibody’interaction with this site does not interfere with heparin binding at the carboxyl-terminal heparin binding site. Right, when apo-E is complexed with DMPC, a recombinant is produced in which the first heparin binding site and the 6C5 and 7C9 epitopes are available for heparin binding or antibody interaction, respectively, but the second heparin binding site is “masked” by the DMPC and does not bind to heparin.

inhibits heparin binding to the site in the carboxyl-terminal region through steric interference. In the simplest case, this head-to-tail association would be intramolecular, as shown in the model. However, there are no data at present to preclude that the association is intermolecular, as several of the apo- lipoproteins are known to self-associate in solution. Although the 7C9 epitope appears to be located in close proximity to the 6C5 epitope, this antibody does not inhibit heparin bind- ing, suggesting that the 7C9 epitope must be situated such that it cannot sterically interfere with heparin interaction at the second heparin binding site.

In the absence of lipid, both of the heparin binding sites on apo-E are capable of binding to heparin, and the three anti- body epitopes are available for interaction with their respec- tive antibodies. However, when apo-E is complexed with phospholipid, only one binding site is expressed, i.e. the site in the vicinity of residues 142-147. In a similar manner, apo-E contained in the native lipoprotein particle, apo-E HDL,, is completely inhibited from binding to heparin by the 1D7 antibody. Furthermore, the data suggest that the pro- posed apo-E head-to-tail association is altered by the inter- action with lipid, such that the 6C5 epitope remains available for antibody interaction but that the second heparin binding site is no longer capable of binding to heparin (right side of Fig. 7). This lipid “masking” of the second binding site could be the result of the site being “buried” in the lipid bilayer of the recombinant particle or the result of a conformational change in the site induced by the lipid interaction. The carboxyl-terminal portion of apo-E is predicted to contain several a-helical structures with amphipathic character and appears to be one of the major lipid binding regions of apo-E (1). The a-helical content of rabbit apo-E is known to increase when the protein becomes associated with DMPC (31). This change in secondary structure may render the second site unavailable for binding to heparin. Because apo-E is normally associated with lipid on the surface of circulating plasma lipoproteins, the second heparin binding site in the carboxyl- terminal region may not be expressed and available for hep-

arin interaction under normal physiological conditions and, therefore, may be of no physiological importance.

Acknowledgments-We wish to thank Jana Seymour, Yvonne New- house, Martha Kuehneman, and Harold Goldstein for their excellent technical assistance. Appreciation is also extended to Kerry Hum- phrey for manuscript preparation, James X. Warger, Norma Jean Gargasz, and Karen Leung for graphic arts, and Barbara Allen and Sally Gullatt Seehafer for editorial assistance.

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SUPPLEMENTARY MATERIAL FOR

HUMAN APOLIPOPROTEIN E: DETERMINATION OF THE HEPARIN BINDING SITES OF APOLIPOPROTEIN E3

K a r l H. Weisgraber, Stanley C . R a l l , Jr., Robert W. Mahley, Ross W . Milne, Yves L. Marcel, and James T. Sparrow

EXPERIMENTAL PROCEDURES

t i o n - Ihe p roduc t lon and c h a r a c t e r ~ z a f i o n o f t h r e e a n t i - a p o l l p o p r o t e i n A p o l i p o p r o t e i n E Monoclonal Ant ibodies and A p o l i p o p r o t e i n E3 I s o l a -

(apo-) E monoclonal an t i bod ies (6C5, 107, and 7C9) have been desc r ibed p r e v i o u s l y ( 1 ) . The 3H1 an t ibody has no t been desc r ibed and was produced i n m i c e t h a t had been immunized w i t h human chv lomicron remnants. A l l o f the an t ibod ies a re o f the immunog lobu l in G ( IgGj subc lass and were ob ta ined f r o m t h e a s c i t i c f l u i d o f h y b r i d o m a - b e a r i n g m i c e b y P r o t e i n A-Sepharose 48 (Pharmacia) chromatography (2).

The apo-E3 was i s o l a t e d f r o m t h e d <1;02 plasm.? l i p o p r o t e i n s o f a s u b j e c t w i t h t h e E313 phenotype by Sephacryl 5-300 column chromatography w i t h a 4 M guan id ine co lumn bu f fe r , con ta in ing 0 .1 M Tris-HC1 and 0.01% EDTA, pH 7.4, as d e s c r i b e d p r e v i o u s l y ( 3 ) . The l i p o p r o t e i n s were d e l i p i t i - a t e d w i t h CHCl .MeOH (2:1, v l v ) and s o l u b i l i z e d i n column b u f f e r p r i o r t o a p p l i c a t i o n t o 3 i h e S-300 column. The t h r e e apo-E3 th romboly t i c f ragments were i s o l a t e d b v column chromatosraohv on Seohadex 6-100 SF as desc r ibed ( 4 ) . Can ine a io -E HDL w e r e i Z o l a t e d frm t h e plasma o f cho les tero l - :a t fed dogs, as p r e v i o u s l y & s c r i b e d ( 5 ) .

H i g h l y p u r i f i e d t h r o m b i n ( s p e c i f i c i t y a c t i v i t y , 2800 u n i t s l m l ) was a

A1 bany, NY. generous g i f t from Dr. J. Fenton I1 of t h e New York Department of Health,

E n z y m a t i c D i q e s t i o n o f o l i p o p r o t e i n E3 Bound t o Heparin-Sepharose - n - m e r c a n t o e t h a n o l ( f i n a l c o n c e n t r a t i o n l and add2d t d 5 ml of heoarin-Seoh- A p o l i p o p r o t e i n E (5 mg) i n h Z mM NH HCO was reduced wi th 1% . . ~. r~~~~ ~ - . ~~

arose. The s l u r r y was i n c u b a t e d f o r ' 2 h a t 4 'C on a r o c k i n g p l a t f o r m . The unbound apo-E was removed by washing the ge l w i th 25 mM NH HCO ; 50 ug of Staphylococcus aureus V8 p r o t e a s e ( M i l e s L a b o r p - i e s ) i n 5 0 4 u l af 25 mM NtlKUCO. were added t o t h e s l u r r y , and t h e s l u r r y was incubated overn ight on a ;ock?ng p l a t f o r m a t room temperature. A second enzyme a d d i t i o n , i d e n t i - c a l t o t h e f i r s t a d d i t i o n , was made the next morn ing, and t h e d i g e s t i o n was c o n t i n u e d f o r 6 h, a f te r wh ich the ge l was washed w i t h 100 ml o f 25 mM NYHC03 t o renove the re leased f ragments and t h e n w i t h 20 ml o f 1.0 M NYHC03 t o renove the bound f ragments. The bound f ragments were lyophi l -

EDTA: and 0.1% E-mercaptoethanol (pH 7.4), and separated by column chroma- i z e d s o l u b i l i z e d i n 4 M guan id ine hyd roch lo r i de , 0 .1 M Tris-HC1, 0.01%

toqraphy (2 .2 x 180 cm, h i c o n ) on Sephadex 6-100 SF (Pharmac ia) equ i l i - b r a t e d w i t h t h e g u a n i d i n e b u f f e r .

Amino ac id and anino-terminal analyses were performed as p r e v i o u s l y desc r ibed ( 5 ) . Cyanogen bromide d igest ion o f apo-E and separa t i on o f t he f ragments were carr ied out as desc r ibed ( 6 ) .

m e n t s . o r a o o - t HUL- were i o d i n a t e d w i t h t h e b l t o n - H u n t e r r e a a e n t f h ~ r - I o d i n a t i o n o f P r o t e i n s - A p o l i p o p r o t e i n E3, t h e t h r o m b o l y t i c f rag -

sham) ' f o r t h e h e p a r i k b i n d i n q assays as p r e v i o u s l y d e s c r i b e d (7;. ~ T k e 6C5 IgG and synthet ic f raqments were l a b e l e d w i t h 1 mCi o f 1 2 5 1 (he rsham) by an adapta t ion o f the Hunter and Greenwood method ( 8 ) . The r e a c t i o n was

con t inue f o r 10-20 s , and then s topped wi th t h e a d d i t i o n o f 860 "1 sodium i n i t i a t e d w i t h 1 0 u l o f ch loramine-T reagent (5 u g / u l ; Bakes), a l l o w e d t o

m e t a b i s u l f i t e s o l u t i o n ( 0 . 3 u q l u l ; B a k e r ) . Nonbound 1251 was removed from the p ro te in o r pep t ides by pass ing t he reac t i on m ix tu re t h rough a 1 .0 -m l column of AG1-X8 (Bio-Rad) ion exchange res in wi th phosphate-buf fered

was l a b e l e d as desc r ibed above, excep t t ha t phospha te -bu f fe red sa l i ne t ha t s a l i n e (pH 7.2). Rabbit anti-mouse IgG (1.4 mg o f t o t a l p r o t e i n ; C a p p e l )

con ta ined 1% bov ine serum albumin and 0.01% th imerosal was used as t h e i o n exchange column b u f f e r . A p o l i p o p r o t e i n EeOMPC complexes were prepared and i s o l a t e d as p r e v i o u s l y d e s c r i b e d ( 7 ) .

p o r c i n e l n t e s t l n a l mucosal heparin (Hynson, Westcott and Ounning) t o Sepha- Hepar in B ind ing Assays - Heparin-Sepharose was p repared by coup l i ng

rose 68 CL (Pharmacia l that had been act ivated wi th cvanoqen bromide as p r e v i o u s l y d e s c r i b e d ( 9 ) . A c o n t r o l g e l was p r e p a r e d b y - c a r r y i n g a p o r t i o n o f Sepharose 68 CL t h r o u g h t h e a c t i v a t i o n , c o u p l i n g , and b l o c k i n g s t e p s i n t h e absence of heparin. The b i n d i n g assays were performed a t 4 'C in 50 mM NaCl, 5 mM Tris-HC1, pH 7.4. To e i t h e r 125 mg o f packed heparin-Sepharose q e l o r c o n t r o l g e l i n 0.5-1111 p l a s t i c Eppendorf microfuge tubes, 250 t o 350 u l of 1 2 s I - l a b e l e d p r o t e i n ( 1 5 t o 2 0 u g ) were added and the tubes mixed on a rock ing p la t fo rm fo r 3 h. A f t e r i n c u b a t i o n , t h e g e l was sedimented by

f o r c o u n t i n g . D u p l i c a t e samples were assayed, and t h e r e s u l t s c a l c u l a t e d c e n t r i f u g a t i o n , and an a l i q u o t , u s u a l l y 50 ul, o f t h e s u p e r n a t a n t was taken

by:

N. (1984) Proc. Natl. Acad. Sci. U. S. A. 8 1 , 289-293 37. Walz, D. A., Wu, V. Y., de Lamo, R., Dene, H., and McCoy, L

E. (1977) Thromb. Res. 11,893-898 38. Begg, G. S., Pepper, D. S., Chesterman, C. N., and Morgan, F. J

(1978) Biochemistry 17,1739-1744 39. Lindahl, U., Backstrom, G., Hook, M., Thunberg, L., Fransson

L.-A., and Linker, A. (1979) Proc. Natl. Acad. Sci. U. S. A. 76 3198-3202

40. Rosenberg, R. D. (1985) Fed. Proc. 44,404-409

A p p r o x i m a t e l y 7 0 t o 85% of t h e apo-E a p p l i e d was hound t o t h e g e l u n d e r t h e c o n d i t i o n s of t h e assay.

o f apo-E t o b i n d t o h e p a r i n , t h e a n t i b o d y was p r e i n c u b a t e d w i t h i n t a c t o r a To de te rm ine t he e f fec t o f apo-E monoclonal ant ibodies on t h e a b i l i t y

t h rombo ly t i c f ragmen t o f apo-E f o r 1 h a t room t e m p e r a t u r e p r i o r t o a d d i -

b i n d i n g d a t a f o r S c a t c h a r d a n a l y s i s ( 1 0 ) was ob ta ined by i ncuba t ing t i o n of t h e m i x t u r e t o t h e h e p a r i n - S e p h a r o s e o r c o n t r o l g e l . The d i r e c t

i n c r e a s i n g m o u n t s o f l 2 s 1 - a p o - E 3 i n 300 "1 o f 50 mM NaCl, 5 mM Tris-HC1, pH 7.4, w i t h e i t h e r 10 mg o f hepar in -Sepharose o r con t ro l ge l fo r 4 h a t

washed t w i c e w i t h 300 u l o f buf fer . The tube con ta in ins t he ae l was then 4 'C. A f t e r i n c u b a t i o n , t h e g e l was sedimented by cent r i fugat ion and

c o u n t e d . P a r a l l e l i n c u b a t i o n s w i t h c o n t r o l g e l were use; t o c a l c u l a t e t h e n o n s p e c i f i c b i n d i n q . The s p e c i f i c a c t i v i t e s o f t h e apo-E3 used fo r t hese

b i n d i n g i n d i c a t e d t h a t maximum b i n d i n g was o b t a i n e d a f t e r 2.5 h o f i ncuba- s tud ies ranged f rom 100 to 200 cpmlng o f p ro te in . A t ime course o f the

t i o n .

An t ibody B ind inq S tud ies - The a n t i b o d y c m p e t i t i o n s t u d i e s Were pe r - formed by d i l u t i n g apo-E3 t o 0.5 ug/ml i n 5 mM g l y c i n e b u f f e r , pH 9.2, and

were incubated overnight at room temperature and then were washed w i t h a app ly ing 200 u l t o each well of a Removawell plate (Oynatech). The w e l l s

m i x t u r e o f 0.15 M NaCl and 0.025% Tween 20 . I nc reas ing concen t ra t i ons o f compet ing un labeled monoclonal ant ibodies in phosphate-buf fered sa l ine and 5% f e t a l c a l f serum, pH 7.2, were added t o 1251-6C5 i n t h e same b u f f e r , and 200 u l o f t h e m i x t u r e were added t o each w e l l . The c o n c e n t r a t i o n o f '251-6C5 was ad jus ted so t h a t a p p r o x i m a t e l y 50% o f the added ant ibody bound i n t h e absence of any compe t i t o r . The w e l l s were i n c u b a t e d o v e r n i g h t a t room temperature, washed w i t h 0.15 M NaCl t h a t c o n t a i n e d 0.025% Tween 20, and a n a l y z e d f o r r a d i o a c t i v i t y . N o n s p e c i f i c b i n d i n g was de te rm ined to be l e s s t h a n 1% of the maximm mount bound.

performed on 1 0 t o 20% g rad ien t s lab ge l s acco rd ing t o t h e method of Sodium dodecyl su l fa te (SDS) p o l y a c r y l m i d e g e l e l e c t r o p h o r e s i s was

Laemmli 1111. and the ae l s were s ta ined w i th Coomassie b l u e R250. Samoles fo r immunob ib t t ing were t rans fer red f rom SOS g e l s i o n i t r o c e l l u l o s e p a p e r (Bio-Rad). The n i t r o c e l l u l o s e r e p l i c a t e s were b locked for 6 h w i t h 3% ge la t in (B io -Rad) , 500 mM NaCl, and 20 mM Tris-HC1, pH 7.5. Monoclonal a n t i b o d i e s 107 and 6C5 ( -1 mg iml ) were d i lu ted 1:lOO w i t h 1% g e l a t i n , 5% Tween 20, 500 mM NaCl, and 20~mM Tris-HC1, pH 7.5, and incuba ted ove rn igh t w i t h t h e r e p l i c a t e s a t room temperature on a r o c k i n g p l a t f o r m . The r e p l i - c a t e s were then washed (IO t i m e s ) w i t h t h e a n t i b o d y d i l u t i o n b u f f e r , i n c u b a t e d f o r 3 h w i t h 1 2 5 I - l a b e l e d r a b b i t a n t i - m o u s e I g G i n t h e d i l u t i o n b u f f e r , washed ( 1 0 t i m e s ) w i t h t h e d i l u t i o n b u f f e r , and s u b j e c t e d t o a u t o - rad iog raphy .

a s o l i d phase procedure on a SchwarzlEann Bioresearch S y n t h e s i z e r m o d i f i e d P e p t i d e a n d P u r i f i c a t i o n - The pep t ides were syn thes i zed by

fo r canputer con t ro l (12 , 13) us ing a p rev ious ly descr ibed p rogram (14) . The var ious length f ragments, encoyass ing res idues 139-169, were synthe- s i z e d b y b e g i n n i n g w i t h 0 . 1 5 mM BOC g l y c i n e l g o f r e s i n and removing a

were a t tached (15) . The frapment encompassing residues 202-243 was synthe- p o r t i o n of t h e r e s i n f r o m t h e s y n t h e s i z e r a f t e r r e s i d u e s 148, 144, and 139

t h e s y n t h e s i s and p u r i f i c a t i o n o f t h i s p e p t i d e will be pub l i shed e lse- s j z e d b e g i n n i n g w i t h 0.15 M BOC l e u c i n e f g o f r e s i n . A d d i t i o n a l d e t a i l s o f

where.2 The pept ides were deprotected and c l e a v e d f r o m t h e r e s i n b y t r e a t - ment wi th anhydrous HF. The pept ides were pur i f ied by ion exchange chroma- tog raphy on SP-Sephadex. h i n o a c i d a n a l y s i s o f t h e p u r i f i e d p e p t i d e s i n d i c a t e d t h a t t h e y had the expected composi t ion. The pept ides were judged t o be >95% pure by h igh-per formance l iqu id chromatography on a r a d i a l l y compressed G B reversed-phase column.

RESULTS

Prev ious l y , we have used a ser ies o f syn the t ic f ragments and thrombo-

t h e r e g i o n o f res idues 140-150 (16) . L imi ted th rombin d iges t ion o f apo-E l y t i c fragments of apo-E t o demons t ra te t ha t t he 107 e p i t o p e i s l o c a t e d i n

produces three major f ragments: a M, = 22,000 f ragment ( res idues 1-191) . a M = 12 DO0 fragment (residues 192-299), and a Mt. = 10,000 fragment ( r e s i - dCes 21;-299). The i s o l a t i o n and c h a r a c t e r i z a t l o n o f t h e M = 22 000 and 10 ,000 f ragments have been p rev ious ly descr ibed (4, 17 ) . T6e M, 1 12,000 fragment can be produced by a short-term thrombin digestion (181, 'and amino a c i d c m p o s i t i o n and m i n o t e r m i n a l sequence da ta con f i rmed the iden t i f i ca- t i o n o f t h i s fragment (data not shown).

A second s e r i e s of p r o t e o l y t i c f r a g m e n t s t h a t was used t o map an t ibody e p i t o p e s was ob ta ined f rom 5 . aureus V8 p r o t e a s e d i g e s t i o n o f apo-E t h a t

f i v e fragments were produced tha t remained bound to the ge l , and these was bound t o h e p a r i n - S e p h a r o T e . m e t e r m i n e d by SDS g e l e l e c t r o p h o r e s i s ,

ranged i n mo lecu la r we igh t f rom 32,000 t o 10,000 ( d a t a n o t shown). The fragments were separated by column chromatography on Sephadex 6-100 SF and t h e i r s t r u c t u r e s deduced from t h e i r m i n o - t e r m i n a l sequences (data not o resen ted l and amino ac id canoos i t i ons (Tab le I l . These d a t a were consis- t e n t w i t h t h e s t r u c t u r e s o f ' t h e f i v e ' f r a g m e n t s b e i n g r e s i d u e s 14-299, 14-179, 14-168, 187-299, and 206-299. I n add i t ion , a syn the t ic f ragment t h a t spanned residues 202-243 was ava i lab le a long w i th var ious cyanogen bromide fragments o f apo-E (6 ) . A summary o f t h e apo-E f ragments that were used i n t h e e p i t o p e m a p p i n g s t u d i e s i s p r e s e n t e d i n T a b l e 11.

2 J. T. Sparrow, unpubl ished observat ion.

Heparin Binding Sites of Apolipoprotein E 2075

Amino ac id

TABLE I TADLE I 1

composi t ions of Staphylococcus aureus V8 generated apol ipoprotern t3 f rXgEi iEs

protease- P r o t e o l y t i c , s y n t h e t i c , and cyanogen bromide fragments o f a p o l i p o p r o t e i n E used i n monoclonal antibody mapping

Residues/molea - React ion w i th an t ibodya

h i n o Pept ide Pept ide Pept ide Pe t i d e Pept ide a c i d 14-299 14-179 14-168 18?-299 206-299

Fraqnent 107 6C5 IC9 3H1

P r o t e o l y t i c 6.9 Residues 1-191 + t +

Residues 192-299 Residues 216-299 Residues 14-299 n.d. Residues 14-179 n.d. Residues 14-168 n.d.

+ + Arq 32.2 (34) 19.4 (20) 17.8 (18) 12.3 (13) 10.6 (11)

Asp 12.8 (12) 7.3 ( 7 ) 7.3 ( 7 ) 5.2 ( 5 ) 5.1 ( 5 ) Thr 9.8 (10) 6.6 ( 7 ) 6.5 ( 7 ) 2.8 ( 3 ) 1.9 (2 ) Ser 13.9 (14) 8.2 ( 9 ) 7.4 ( 8 ) 5.2 ( 5 ) 3.7 ( 4 ) Glu 63.3 ( 6 5 ) 38.8 (38) 36.5 (36) 24.9 (26) 21.4 (22)

G ly 19.9 (17) 9.3 ( 9 ) 7.7 (7) 8 .3 (7 ) 4.8 (4 ) Pro 6.6 ( 6 ) 1.2 (1) 0.6 (1) 4.1 (4 ) 3.2 ( 3 ) Cyanoqen bromide

A la 32.7 (34) 17.4 (17) 15.7 (15) 16.0 (17) 13.7 (14) Val 19.6 (20) 9.0 (9) 8 .9 (9 ) 9.7 (10) 8.2 ( 8 ) Met 6 . 0 (7) 3.7 (4) 3.7 (4 ) 2.7 ( 3 ) 2.7 (3) I l e 2.3 ( 2 0.9 (1) 0.1 (0 Leu 36.7 (371 26.1 (26) 24.8 (251 1;:; ::$ Residues 139-169 n.d. n.d. n.d. Tyr 4.2 ( 4 ) 3.9 ( 4 ) 3.7 ( 4 ) 0 (0) 0 (0) Phe 3.4 ( 3 ) 1.0 (1) 1.0 ( 1 ) 1.9 ( 2 ) 2.0 (2 )

n.d. n.d. n.d. n.d. n.d. n.d.

t t t

t Residues 126-218 Residues 222-272 n.d. n.d. n.d. Residues 273-299 n.d. n.d. n.d.

t

Synthet ic t

Residues 144-169 Residues 140-169 Residues 202-243 n.d. n.d. n.d.

aResults are from dup l i ca te de te rm ina t ions . Numbers i n pa ren theses re fe r to theore t ica l compos i t ion . aReSul tS a re p resented as + o r - r e a c t i o n as determined f rom so l id

phase assay (16) or immunoblots (F igs. 1 and 2); n.d. = not determined.

b i n d i n q and 7C9, w h i c h d i d n o t , r e c o q n i z e d ep i topes i n t h e M = 22,000 Prev ious ly , we determined t h a t an t ibod ies 6C5, which i n h i b i t e d hepar in

thrombol;t ic fraqment (residues 1-191) (16). In an a t t e m p t t o !o:aliZe the 6C5 ep i tope more p rec ise ly , we examined the a b i l i t y of 6C5 t o b m d t o v a r l - ous fraqments o f apo-E3 u s i n g i m m u n o b l o t t i n o f n i t r o c e l l u l o s e r e p l i c a t e s o f SDS qe ls (F ig . 1) . E l o t t i n q w i t h ID7 !Fig. 1, panel E) served as a c o n t r o l . As expected, 107 i n t e r a c t e d w i t h r n t a c t a p r res idues 14-299 ( l a n e I ) , 14-179 ( lane 2). 14-168 ( lane 3). and 126-218 ( lane 4). The 6C5 a X i 5 o i y interacte-nry w i t h intac5po:E o r t h e M, = 2 m t h r o m b o l y t i c f raqment ( res idues 1-191) (F ig . 1, panel C). It d i d n o t b i n d t o any o f the f raqments that were lack ing the t l i i tn res idues o f the amino terminus ( l a n e s 1, 3, and 4) . An i d e n t i c a l b l o t t i n g p a t t e r n was ob ta ined w i th 7C9 (=n?it Ihown).- These r e s u l t s i n d i c a t e t h a t t h e e p i t o p e s f o r b o t h 6C5 and 7C9 a r e e i t h e r t o t a l l y o r p a r t i a l l y c o n t a i n e d i n t h e f i r s t 13 res idues o f t he o ro te in mo lecu le .

B. ._ . .

1 D7

c9 n

I g 35- X 22- 5

c.

12 -

Intact 1 2 3 4 5 Intact 1 2 3 4 5 Intact 1 2 3 4 5 Apo-E3 Apo-E3 ApO-E3

FIG. 1. Sodium dodecy l su l fa te -po lyac ry lam ide g rad ien t ge l e lec t rophores i s and innnunoblots o f v a r i o u s apo-E3 f ragments wi th 1D7 and 6C5 monoclonal ant ibodies. (A) Coomassie-s ta ined ge l : in tact aPO-E3 was used as a standard; lane' I , residues 14-299: lane 2, res idues 1-191; lane 3, m i x t u r e o f res idueT lZ "T79 and 14-168; lane-sidues 126-218; ana"EnF 5, r e s i - dues 192-299. ( E ) Autoradiogram %f innnunoblot probed w i t h o c l o n a l

r e p l i c a t e probed w i t h m o n o c l o f i n t i b o d y - 6 C 5 : l a n e s as i n panel A. Po ly - an t ibody 107: l%es as i n panel A. (C) Au to rad iog ram o f n i t roce l l u la r

a c r y l a n i d e g e l e l e c t r o p h o r e s i s was performed on 10 t o 2 m d E n t s lab ge ls . Samples (2 t o 5 ug) were incubated for 1 h a t 37 'C i n a 3% SDS b u f f e r c o n t a i n i n q 1% R-mercaptoethanol . Af ter the rep l icates were t rans- f e r r e d t o n i t r o c e l l u l o s e s h e e t s , t h e y were b locked w i th a 1% g e l a t i n s o l u - t i o n , washed, and incubated wi th monoclonal ant ibody. Ant ibody b ind ing was detected by incubat ion wi th 1251-ant imur ine IgG fo l lowed by autoradio- graphy.

2076 Heparin Binding Sites of Apolipoprotein E

A. B.

35 - n ?

22- X

i U

12 -

FIG 2. Sodium dodecy l su l fa te -po lyac ry lam ide g rad ien t ge l e lec t rophores i s and imnunob lo ts o f va r ious aoo-E f raoments wi th the 3H1 monoclonal ant i - ~~

body. (A) Coomassie-s ta ined ge l : ln tact apo-E3 was used as a standard; l a n e 1, res idues 1-191; lane 2, res idues 126-218; lane 3, res idues 192-299; ianeil, r e s i d u e s 2 1 6 - 2 9 m a i i e 5, res idues 2 0 2 - 2 4 3 T l % e 6, res idues 222-

and lane 7, r e s i d u e s m - 2 3 9 . ( 6 ) A u t o r a d i o g r a m o f n i t r o c e l l u l o s e r e p l i c a t e p r o b e 7 w i t h m o n o c l o n a l a n t i b o a y 3H1: lanes as i n panel!.

u s i n a i m u n o b l o t t i n o of the var ious am-E f raoments. This ant ibodv bound The eu i tope of t h e t h i r d i n h i b i t i n g a n t i b o d y , 3H1, was a l s o l o c a l i z e d

t o f;aqments f rom the carboxy l - te rmina l por t ion o f apo-E; i t bound-to the M = 1 2 000 and 10.000 thrombolyt ic f raqments (Fig. 2, lanes 4 and 5 ) . In adrdition: i t bound t o t h e cyanogen bromide fragment CBl(resi3ues 272-272,

cyanogen bromide fragment CB8 ( res idues 273-299, lane 8 ) . T G s F r e s u l t s lane 7 ) but no t to the syn the t ic f ragment ( res idues 202-243, lane 6) o r t h e

demonstrated that the ep i tope for 3H1 was loca ted m e e n res idues 243 and 272.

"

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