THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 5, Issue of March 10, pp. 2518-2521, 1982 Printed in U. S. A .

Abnormal Lipoprotein Receptor-binding Activity of the Human E Apoprotein due to Cysteine-Arginine Interchange at a Single Site*

(Received for publication, September 14, 1981)

Karl H. Weisgraber3, Thomas L. Innerarity, and Robert W. Mahley From the Gladstone Foundation Laboratories for Cardiovascular Disease, University of California, Sun Francisco, Sun Francisco, California 94140

Previously, we demonstrated that the human E apo- protein existed in three forms (E-2, E-3, and E-4), and that the three forms differed from one another by cys- teine-arginine interchanges at two substitution sites (A and B). The E-2, E-3, and E-4 apo-E contain cysteine/ cysteine, cysteine/arginine, and arginine/arginine at sites A/B, respectively. Subjects with Type I11 hyper- lipoproteinemia, a genetic disease associated with de- fective plasma lipoprotein clearance, possess the E-2 form of apo-E. It was postulated that the substitution of cysteine for arginine at site B in the E-2 might be responsible for an impaired interaction of Type I11 apo- E with cell surface receptors. To test this possibility, the binding activities of the various forms of apo-E to the receptors on human fibroblasts were compared. The E-3 and E-4 apo-E readily bound to the receptors; however, the E-2 apo-E-binding activity was defective. Consideration was given to the possibility that a posi- tively charged residue at site B, as occurs in both E-3 and E-4, was important for normal binding activity. To investigate this, the cysteine residues of the E-2 apo-E were converted by cysteamine treatment to a positively charged lysine analogue. This resulted in a marked increase in the binding activity of the E-2 apo-E. These studies demonstrated that the defective binding of the E-2 apo-E from Type I11 hyperlipoproteinemic subjects was due, at least in part, to the cysteine-arginine inter- change at site B, and they suggested the importance of a positively charged residue at this position in the sequence to mediate normal apolipoprotein-receptor interaction.

We recently demonstrated that three forms of the human E apoprotein exist (E-2, E-3, and E-4), each with a distinct amino acid sequence (1). The three distinct forms of apo-E’ result from differences in their amino acid sequences, involv- ing an interchange of arginine and cysteine a t two sites, A and B, in the protein. The E-3 apo-E has a cysteine residue at site A and an arginine residue at site B, whereas the E-2 apo-E has cysteine residues at both sites. The E-4 apo-E, on the other hand, is characterized by an absence of cysteine and the apparent existence of arginine residues at both sites A and B (1). The interchange of the arginine and cysteine residues is sufficient to explain the known charge differences observed

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. + To whom requests for reprints should be addressed at: Gladstone Foundation Laboratories, P. 0. Box 40608, San Francisco, CA 94140.

’ The abbreviations used are: apo, apoprotein; LDL, low density lipoproteins; DMPC, dimyristoylphosphatidylcholine.

for the E-2, E-3, and E-4 isoforms by isoelectric focusing polyacrylamide gel electrophoresis.

The demonstration of these forms of apo-E is consistent with the genetic model of Zannis and Breslow (2,3). According to this model, the biosynthesis of the human E apoprotein is under the control of three different alleles at a single genetic iocus. The fact that the three forms differ in their primary structure establishes that the genetic influence is at the level of gene coding for apo-E (1). Furthermore, the minor isoforms observed by isoelectric focusing of the human E apoprotein are related structurally to the three primary forms of the protein. We have shown that the minor isoforms of apo-E from a subject homozygous for the E-3 have an identical amino acid analysis, i.e. the parent E-3 band and the minor forms contain a single residue of cysteine (1). In addition, in a subject homozygous for E-2 apo-E, the major E-2 protein and the minor isoforms contain 2 residues of cysteine. This observation is consistent with the hypothesis of Zannis and Breslow that the minor isoforms, which occur in a given individual, are the result of post-translational glycosylation of the parent form of the E apoprotein (2, 3).

Type 111 hyperlipoproteinemia (primary dysbetalipopro- teinemia) is a pathological lipid disorder associated with the E-2 apo-E homozygous state (4, 5). The hyperlipidemia of affected subjects is thought to occur because of a defective clearance of remnant lipoproteins from the plasma (6, 7), and it has been postulated that the E-2 apo-E may be associated with this defect (1). Furthermore, the E-2 apo-E-phospho- lipid complexes from certain patients with Type I11 hyperlip- oproteinemia interact poorly with lipoprotein receptors (8). In light of the demonstrated cysteine-arginine interchanges in the three types of apo-E, it is reasonable to speculate that these substitutions may be the cause of the abnormality in receptor recognition of E-2 apo-E-containing lipoproteins in man. In the present study, we assess the effects of these demonstrated cysteine-arginine interchanges on the binding of apo-E- phospholipid complexes to LDL (apo-B,E) receptors on human fibroblasts. Moreover, we demonstrate the impor- tance of the arginine residue at one interchange site for apo-E binding to lipoprotein receptors.

EXPERIMENTAL PROCEDURES

Plasma Lipoprotein Preparation and Apoprotein Isolation-The d < 1.02 plasma lipoproteins were prepared by ultracentrifugation of plasma from fasting subjects as previously described (1). The lipopro- teins were exhaustively dialyzed and 50-mg aliquots of lipoprotein protein were frozen and lyophilized. The dried lipoproteins were delipidated with three extractions of CHCL:MeOH (2:l v/v) at 4 “C. The apoproteins were pelleted by low speed centrifugation (loo0 X g) after each extraction. After 0.5 h, additional MeOH was added to each extraction to ensure complete pelleting of the apoprotein. The three CHCl,?:MeOH extractions were followed by a MeOH wash. The moist apoprotein pellet was solubilized in 6 M guanidine containing 0.1 M

2518

by guest on February 13, 2018http://w

ww

.jbc.org/D

ownloaded from

Abnormality of Type 111 Apo-E Binding 2519

Tris, pH 7.4, and the protein was reduced with P-mercaptoethanol. The apo-E was isolated from the mixture by Sephadex G-200 column chromatography as previously described (1). The purity of each preparation was determined by sodium dodecyl sulfate gel electro- phoresis (9).

Human LDL (d = 1.02 to 1.05) were isolated from the plasma of a young fasted male subject by centrifugation for 18 h at 59,000 rpm in a 60 Ti rotor (Beckman) and washed by recentrifugation for 16 h at 59,000 rpm. Human "'I-labeled LDL were prepared as described (IO) and had a specific activity range of 180 to 350 cpm/ng. Human lipoprotein-deficient serum was prepared by centrifugation at d = 1.215 for 48 h at 59,000 rpm.

Isoelectric Focusing-Analytical isoelectric focusing was per- formed on 6-cm, 5% polyacrylamide gels containing 8 M urea and 2% Pharmalyte, pH 4 to 6 (Pharmacia Fine Chemicals), according to the method of Pagnan et al. (5), with modification previously described (9). For the purposes of isoelectric focusing, the d < 1.02 lipoproteins were chemically modified with P-mercaptoethylamine (cysteamine, Sigma) by adding 1.0 mg of cysteamine to 150 pg of d < 1.02 lipoprotein protein in 0.1 M NH4HCO:r and incubating the mixture for 4 h a t 37 "C. The mixture was then lyophilized and delipidated before isoelectric focusing. Control samples were incubated in parallel with modified samples and were reduced with P-mercaptoethanol before isoelectric focusing.

Chemical Modification of Apo-E-Apo-E was reduced and car- boxamidomethylated or carboxymethylated by solubilizing the lyoph- ilized apoprotein in 6 M guanidine, 0.1 M sodium phosphate, 1 mM EDTA, pH 8.2, at a concentration of 5 mg/ml. The tube was flushed with Nz and the dithiothreitol (Baker) was added in a 10-fold molar excess and the capped solution was allowed to stand overnight at room temperature. An 11-fold molar excess of iodoacetamide or iodoacetic acid (Sigma) was added and the reaction allowed to proceed in the dark at 30 min. Prior to lyophilization, the solution was dialyzed exhaustively against 5 mM NH,HCOa in the dark at 4 "C.

Cysteamine modification was performed by treating 200 pg (1.0 to 1.5 mg of protein/ml) of apo-E in 0.1 M NH4HCO:, with 20 p1 of a cysteamine solution (100 mg/ml). The mixture was incubated over- night at room temperature or for 4 h at 37 OC. Control samples were treated with 20 pl of /3-mercaptoethanol solution (1:lO dilution with H20) and incubated in parallel with the cysteamine-treated samples.

Preparation of Apo-E. DMPC Complexes-The apo-E. dimyris- toylphosphatidylcholine complexes were prepared as previously de- scribed (10, ll), with the following modifications. Apo-EeDMPC complexes were isolated by centrifugation on a KBr gradient (d = 1.006 to 1.21) which was spun at 55,000 rpm (SW 55 rotor) for 20 h at 15 "C.

Cells and Assays-Human skin fibroblasts were maintained in culture, and competitive binding assays, using I2'II-LDL, were per- formed at 4 "C as described (IO).

Amino Acid Analysis-Samples for analyses were hydrolyzed for 20 h at 110 "C with 6 N HCl in sealed, evacuated acid-washed tubes. Analyses were performed on a Beckman 121MB Analyzer.

RESULTS AND DISCUSSION

To determine the effect of the cysteine-arginine inter- changes on the ability of apo-E to bind to the apo-B,E receptor on fibroblasts, we isolated the apo-E from three subjects, each homozygous for one of the three forms of apo-E and compared their binding activities. The homozygous apo-E pattern was characterized by the presence of one major E isoform, either the E-2, E-3, or E-4 isoform band (Fig. 1). The E-3 and E-2 apo-E were from the same subjects used for sequence analysis (1). The cysteine-arginine interchanges as they occurred in the E-2, E-3, and E-4 apo-E are summarized in Table I.

As shown in Fig. 2, both the E-4 and E-3 apo-E had essentially identical abilities to compete with '*'I-LDL for binding to the cell surface receptors on the fibroblasts. In contrast, the E-2 apo-E used in this study was much less effective in binding than were the E-3 or E-4 apo-E. In all cases, the apo-E was complexed with DMPC prior to incuba- tion with cells. We have shown previously that apo-E - phos- pholipid complexes are necessary for receptor-apoprotein in- teraction (10). All these forms of apo-E produced similar protein -phospholipid complexes, as previously described (8).

From the results of-the competitive binding study, it can be suggested that at site A, the cysteine-arginine interchanges were not critical to the binding of the apo-E to the fibroblast receptors. The E-3 and E-4 apo-E differ structurally at site A

" "

" " - " " " cJ= =

T T T

€-2/E-2 E-3/€-3 E-4/E-4 FIG. 1. Isoelectric focusing on polyacrylamide gels (pH 4 to

6) of control and cysteamine-treated (T) d < 1.02 lipoproteins from subjects homozygous for the E-2, E-3, and E-4 isoforms. Brackets indicate position of the E isoforms and position of the C apoproteins.

TABLE I Cysteine-arginine interchanges a t substitution sites A and B in E-

2, E-3, and E-4 ADO-E ADO-E twe Site A Site B

E-2" Cysteine E-3 Cysteine E-4 Arginine

Cysteine Arginine Arginine

" E-2 apo-E isolated from subject D. R.

m I I t I 1 Qs 10 15 21) Apo-E DMPC Complex ( pg proteln/ml)

FIG. 2. Ability of apo-E-DMPC complexes from subjects homozygous for E-2 (A; subject D. R.), E-3 (A), and E-4 (0) to compete with human lZ5I-LDL for binding to normal human fibroblasts. Cells previously incubated for 48 h in Dulbecco's modi- fied Eagle's medium containing 10% human lipoprotein-deficient se- rum received 1 ml of the same medium with 2 pg/ml of lZ5I-LDL and the indicated concentrations of apo-E.DMPC complexes. After a 2-h incubation on ice at 4 "C, the cells in 35-mm Petri dishes were extensively washed and the "'I-LDL bound to the cells was deter- mined (IO). The 100% control value was 111 ng of '"I-LDL protein bound/mg of cellular protein.

by guest on February 13, 2018http://w

ww

.jbc.org/D

ownloaded from

2520 Abnormality of Type 111 Apo-E Binding

(E-3 has cysteine and E-4 has arginine), but they are identical at site 8, both possessing arginine at this site. The E-2 apo-E, which was defective in binding, differs from both E-3 and E-4 in that it contains cysteine at both sites A and B. To correlate the structural data with the receptor-binding activity, two possibilties were considered. First, consideration was given to the possibility that the defective binding of the E-2 apo-E was secondary to an intramolecular disulfide linkage made possible by the presence of 2 cysteine residues in this particular form of apo-E. Second, it was possible that the defective binding of E-2 apo-E was directly related to the presence of cysteine at site B. Both the E-3 and E-4 apo-E have arginine residues at this site. The importance of arginine and lysine residues in the interaction of apo-E with the apo-B,E receptors has been previously established (12, 13).

To test the possibility that the E-2 defective binding might be due to an intramolecular disulfide bond formation, the binding abilities of E-2 and E-3 apo-E were compared follow- ing reduction or reduction and alkylation of the protein with iodoacetamide. As shown in Fig. 3, the binding activities of the E-2 and E-3 apo-E were not significantly changed by these treatments. Amino acid analysis of the reduced and alkylated apo-E indicated that both cysteine residues of the E-2 apo-E had been successfully alkylated. Thus, the decreased ability of E-2 apo-E to bind to the fibroblast receptors did not appear to be due to the presence of an intramolecular disulfide linkage. Essentially identical results were obtained when iodoacetic acid, which introduced a negative charge at the site of cysteine residues, was used as the alkylating agent. This indicated that the insertion of a negatively charged residue at

I

L E - 3 apo-E

1 2 3 4 Apo-EeDMPC Complex (rg proteidml)

FIG. 3. Comparison of the effect of reduction or reduction and alkylation with iodoacetamide on the ability of E-2 apo-E or E-3 apo-E to compete with human la61-LDL for binding to normal human fibroblasts. E-2 apo-E.DMPC, (A); E-2 apo-E- DMPC reduced with /3-mercaptoethanol, (0); E-2 apo-E. DMPC re- duced and alkylated with iodoacetamide, (0); E-3.DMPC, (A); E-3 apo-E. DMPC reduced with /3-mercaptoethanol, (0); and E-3 apo-E. DMPC reduced and alkylated with iodoacetamide (U). The binding experiments were performed as described in Fig. 2. The 100% control value was 139 ng of Iz5I-LDL protein bound/mg of cellular protein.

I 1 1 1 I I , I

0.4 0.8 1.2 ” 25 Apo-E- DMPC Complex ( p g protein/ml)

FIG. 4. Comparison of the ability of control (8-mercapto- ethanol-treated) and cysteamine-modified apo-E DMPC com- plexes to compete with “‘I-LDL for binding to normal human fibroblasts. Control E-2 apo-E. DMPC (U); cysteamine-treated E-2 apo-E-DMPC (0); control E-3 apo-E. DMPC (0); cysteamine-treated E-3 apo-E. DMPC (0). The E apoproteins were treated with p- mercaptoethanol or cysteamine as described under “Experimental Procedures” before they were complexed with DMPC. The binding experiments were performed as described in Fig. 2. The 100% control value was 143 ng of IZ5I-LDL protein bound/mg of cellular protein.

site A in the E-3 apo-E did not interfere with binding activity and that insertion of a negative charge at sites A and B did not affect the binding activity of the E-2 apo-E.

The importance of this arginine-cysteine substitution at site B was examined by taking advantage of the reagent cystea- mine which converts cysteine to a lysine analogue. Cysteamine treatment of E-2 apo-E converted the cysteine residue at site B to a positively charged residue (a positively charged arginine residue exists at this site in E-3 and E-4 apo-E). The effect of treating the E apoproteins with cysteamine was monitored by observing the charge shifts of the proteins on isoelectric focusing. A positive charge was introduced into the protein for each cysteine residue modified. Therefore, the E-3 and E- 2 apo-E, which contain l and 2 cysteine residues/mol, respec- tively, were shifted one and two charge positions, respectively, on isoelectric focusing gels (Fig. 1). TheE-4 apo-E, which lacks cysteine, was not affected by cysteamine treatment (Fig. 1).

The abilities of cysteamine-treated E-2 and E-3 apo-E to compete with 1251-LDL for binding sites are compared in Fig. 4. The cysteamine-treated E-2 apo-E from subject D. R. exhibited a marked increase in binding activity, whereas the binding activity of the E-3 apo-E remained essentially un- changed. The treated E-2 apo-E displaced 50% of the ‘251-LDL at a protein concentration of 1.2 pg/ml. This represented a 7.7-fold increase in binding activity. Cysteamine-treated E-3 apo-E had essentially the same activity as unmodified E-3 apo-E. This indicated that introducing a positive charge at site A had no effect on binding. However, introducing a positive charge at site B, as occurred with cysteamine modi- fication of E-2 apo-E, significantly increased the binding abil- ity of E-2 apo-E. This suggested the importance of a positively charged residue at this site.

Recently, it has been reported that there is a heterogeneity in the ability of apo-E from different Type 111 subjects (E-2 homozygotes) to be bound to lipoprotein receptors from var-

by guest on February 13, 2018http://w

ww

.jbc.org/D

ownloaded from

Abnormality of Type III Apo-E Binding 2521

TABLE I1 Effect of cysteamine treatment of Apo-E from E-3 and E-2

homozygotes on receptor- binding activity - Concentration of Apo-E at which 50% lZ5I-

LDL was comDeted from fibroblasts" Control/ Subject cystea-

Control Cystea- mine

mine

pgprotein/ml E-3/E-3b 0.058 f 0.027 0.046 * 0.018 1.3

D.R.' 10.4 1.11 9.4 S.B.' 10.7 1.75 6.2

R.R.' 9.6 0.75 12.7 G.P.' 2.1 0.63 3.3 J.G.' 8.8 0.50 17.6

a These data were compiled from competitive binding experiments performed as described in legends of Figs. 2 to 4. The binding data were analyzed by a logit-log plot to determine the 50% competition point.

E-2/E-2

J .T.~ 0.45 f 0.27 0.20 k 0.1 2.2

* Average f S.D. of four preparations from two subjects. e Average of two preparations.

e Single determination. Average & S.D. of three preparations.

ious tissues and to be taken up by perfused rat livers (8). The binding activity of Type 111 apo-E ranged from relatively inactive, like that shown for the E-2 apo-E in Fig. 2, to nearly normal. It was of interest to determine the effect of cysteamine treatment on the E-2 apo-E of other subjects, in addition to subject D. R., with Type I11 hyperlipoproteinemia. Each subject was classified clinically as having Type 111 hyperlipo- proteinemia and, although sequence information was not available on these subjects, isoelectric focusing patterns of their d < 1.02 lipoproteins showed the E isoform patterns to be identical with that of subject D. R. (all lacked detectable E-3 or E-4 isoforms). In addition, in all the subjects, cystea- mine treatment produced the same two-charge shift, as shown in Fig. 1 for subject D. R., which indicated that 2 cysteine residues were present in the apo-E of all of these Type I11 subjects.

Cysteamine treatment of the E-2 apo-E from the various subjects with Type 111 hyperlipoproteinemia significantly in- creased the binding activity of the apo-E in all cases (Table 11). However, the cysteamine treatment did not restore the binding activity to the normal values observed for the E-3 apo-E. Although cysteamine treatment did convert the cys- teine to a lysine analogue and introduced a positive charge, it was apparently not equivalent to the insertion of an arginine residue such as exists in the E-3 apo-E. Despite the consistent increase in binding activity of the E-2 apo-E following cys- teamine treatment, the marked heterogeneity in activity and in degree of activation of the various E-2 apo-E was obvious (Table 11). It is likely that there are additional differences

between the E-2 and E-3 apo-E. These differences may be in the primary structure of the protein or in its carbohydrate moieties.

In summary, the influence of specific cysteine-arginine sub- stitutions in the primary structure of the human E apoprotein on the ability of the E-2, E-3, and E-4 apoproteins to bind to the apo-B,E receptors of fibroblasts has been studied. The cysteine-arginine interchange at site A has no influence on receptor-binding activity. However, the importance of a posi- tively charged residue at site B has been demonstrated. The E-2 apo-E of patients with Type I11 hyperlipoproteinemia has defective receptor binding and has the uncharged amino acid residue cysteine at site B. By comparison, arginine occurs at this site in the E-3 and E-4 apo-E. Cysteamine treatment of the E-2 converts the cysteine at site B to a positively charged analogue of lysine and markedly increases the binding of the E-2 apo-E to the apo-B,E receptors.

Acknowledgments-We are indebted to Dr. Thomas P. Bersot, Gladstone Foundation Laboratories, and Dr. Jean Davignon, Clinical Research Institute of Montreal, Montreal, Canada, for obtaining plasma from patients and volunteers. We also wish to thank Dr. Stanley C. Rall, Jr., for amino acid analyses and Ms. Kay Arnold and Mr. Gregory Perrone for excellent technicai assistance. In addition, we wish to thank Mr. Joe F. Andres for editorial assistance and Mr. Richard A. Wolfe for typing the manuscript.

REFERENCES 1. Weisgraber, K. H., Rall, S. C., Jr., and Mahley, R. W. (1981) J.

2. Zannis, V. I., and Breslow, J. L. (1980) J. Biol. Chem. 255, 1759-

3. Zannis, V. I., and Breslow, J. L. (1981) Biochemistry 20, 1033-

4. Utermann, G., Jaeschke, M., and Menzel, J. (1975) FEBS Lett.

5. Pavan, A., H a d , R. J., Kane, J. P., and Kotite, L. (1977) J. Lipid Res. 18,613-622

6. Havel, R. J., Chao, Y.-S., Windler, E. E., Kotite, L., and Guo, L. S. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,4349-4353

7. Gregg, R. E., Zech, L. A., Schaefer, E. J., and Brewer, H. B., Jr. (1981) Science 211,584-585

8. Schneider, W. J., Kovanen, P. T., Brown, M. S., Goldstein, J. L., Utermann, G., Weber, W., Havel, R. J., Kotite, L., Kane, J. P., Innerarity, T. L., and Mahley, R. W. (1981) J. Clzn. Inuest. 68,

9. Weisgraber, K. H., and Mahley, R. W. (1978) J. Biol. Chem. 253, 6281-6288

10. Innerarity, T. L., Pitas, R. E., and Mahley, R. W., (1979) J. Biol. Chem. 254,4186-4190

11. Pitas, R. E., Innerarity, T. L., and Mahley, R. W. (1980) J. Biol. Chem. 255, 5454-5460

12. Mahley, R. W., Innerarity, T. L., Pitas, R. E., Weisgraber, K. H., Brown, J. H., and Gross, E. (1977) J. Biol. Chem. 252, 7279- 7287

13. Weisgraber, K. H., Innerarity, T. L., and Mahley, R. W. (1978) J. Biol. Chem. 253.9053-9062

Biol. Chem. 256,9077-9083

1762

1041

56,352-355

1075-1085

by guest on February 13, 2018http://w

ww

.jbc.org/D

ownloaded from

K H Weisgraber, T L Innerarity and R W Mahleycysteine-arginine interchange at a single site.

Abnormal lipoprotein receptor-binding activity of the human E apoprotein due to

1982, 257:2518-2521.J. Biol. Chem. 

  http://www.jbc.org/content/257/5/2518.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/257/5/2518.citation.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on February 13, 2018http://w

ww

.jbc.org/D

ownloaded from