Differential transfer of C and E apolipoproteins from very low-density lipoprotein to lysophosphatidylcholine-phosphatidylcholine micelles

Biochimica et Biophysics

371

Acta, 752 (1983) 371-382

Elsevier

BBA 51438

DIFFERENTIAL TRANSFER OF C AND E APOLIPOPROTEINS FROM VERY-LOW-DENSITY LIPOPROTEIN TO LYSOPHOSPHATIDYLCHOLINE-PHOSPHATIDYLCHOLINE MICELLES PHILIP University

W. CONNELLY

and ARNIS

KUKSIS

of Toronto, Department of Biochemistv,

C.H. Best Institute,

II2 College Street, Toronto, Ontarro MSG IL6 (Canada)

(Received January 5th, 1983) (Revised manuscript received April 26th, 1983)

Key words: Apolipoprotein

C; Apolipoprotein

E; Lysophosphatidylcholine;

Lipid micelle; VLDL;

Lipoprotem

transfer

Rat plasma VLDL was incubated with lysoPC/PC micelles consisting of 45-90 mol% IysoPC at micelle/ VLDL phospholipid ratios of 0.33-6. Following incubation, the VLDL and micellar particles were reisolated by ultracentrifugation and the lipid and apolipoprotein composition determined by high-temperature gas chromatography and polyacrylamide gel electrophoresis, respectively. Lysophosphatidylcholine was found to equilibrate between the very-low-density lipoproteins and micellar fraction without transfer of phosphatidylcholine or sphingomyelin between these fractions. This produced reisolated VLDL and micellar particles with nearly identical lysoPC/PC ratios. Only apolipoprotein E transferred to reisolated micellar fractions with less than 35 mol% IysoPC. The C apolipoproteins were also transferred to the micellar fraction when the reisolated micelles contained more than 35 mol% IysoPC. It is concluded that apolipoproteins E and C can bind to HDL-size micellar particles of appropriate composition. The differential transfer of apolipoproteins E and C indicates that fundamental differences exist between these apolipoproteins in their interaction with lipid interfaces.

Introduction Recent studies of nascent and circulating lipoproteins have clearly demonstrated the dynamic nature of apolipoprotein-lipid association [ 1,2]. Despite a facile redistribution of apolipoproteins A, C and E, isolated lipoproteins are characterized by a unique apolipoprotein composition [3]. The rules which govern the lipid protein interaction have not been established. We have shown both in vivo and in vitro [4,5] that particle size is one of the physicochemical parameters which influence the binding of the C apolipoproteins. The observed preference for particles of chylomicron and Abbreviations: VLDL, high-density lipoprotein; lysophosphatidylcholine.

0005-2760/83/$03.00

very-low-density lipoprotein; PC, phosphatidylcholine;

HDL, IysoPC;

0 1983 Elsevier Science Publishers

B.V.

very-low-density lipoprotein diameter over those of low-density lipoprotein diameter containing identical lipid cores is consistent with the known apolipoprotein composition of VLDL subfractions [6,7]. The particle diameters which influence apolipoprotein adsorption correspond to the range recently reported to influence phospholipid transition temperature [8]. The fact that the C apolipoproteins are also found in association with HDL-size particles [3] implies that their surface properties are modified so as to be similar to those of VLDL-size rather than LDL-size particles. Synthetic phosphatidylcholine emulsions are limited to a minimum diameter of 18-20 nm [9,10], making them unsuited for a study of particles of HDL size and necessitating an approach different from that used in our previous work. In the present investigation we have taken advantage of the small

372

diameter characteristic of lysophosphatidylcholine mixed micefles f 11,121 and have prepared 6-8 nm diameter particles by combining lysophosphatidylcholine with phosphatidylcholine. To facilitate the monitoring of apolipoprotein transfer to the small synthetic particles, we have carried out the present study in vitro using VLDL as a source of C and E apolipoproteins in contrast to the previously used in vivo protocol [4]. It is concluded that both s&e and surface composition affect the differential transfer of C and E apolipoproteins to micellar particles. The marked effects of lysophosphatidylcholine point to the interfacial surface tension as a common regulatory factor in apolipoprotein binding.

Experimental procedures Materials. Egg yolk phosphatidylcholine (PC) was a product of British Drug Houses Ltd. (Toronto, Canada). It contained, by weight, approximately 1.8% sphingomyelin and 6.5% lysophosphatidylcholine (1ysoPC). LysoPC produced from egg PC by phospholipase A, digestion was purchased from Sigma Chemical Co. (St. Louis, MO) and was found to be chromatographitally pure. Radioactive 1ysoPC was produced by phospholipase A, digestion [ 131 of [N-m&$hatidylcholine (New Eng“C]dipalmitoylphosp land Nuclear, Amherst, NJ). Lipoprotein prepuration. The chylomicron plus VLDL fraction (d < 1.006 g/ml) was isolated from male Wistar-strain rats (Canadian Breeding Laboratories, Montreal, Canada) weighing 400500 g and receiving laboratory chow ad libitum, by centrifugation of EDTA/plasma as described previously [14]. This preparation is hereafter refurred to as the VLDL fraction. Mixed micelle preparation. Standard solutions of egg PC and 1ysoPC were prepared in chloroform, Aliquots of each phospholipid solution were added to graduated conical 15ml screw top centrifuge tubes, vortexed, and the solvent removed at 35°C under a stream of nitrogen. Super Q distilled water, pH 7.0, was added to give a final concentration of 1% phospholipid. The solution was alternately vortexed and incubated at 37°C until the lipid residue was completely suspended. Solutions of egg PC were slightly turbid, while the

mixed micellar

solutions

were optically

clear. The

egg PC liposomes could be quantiatatively isolated between d = 1.006- 1.040 g/ml, and the lysoPC-PC micelles between d = 1.040- 1.080 g/ml. Incubations of VLDL and mixed micelies. VLDL and lysoPC-PC micelles were combined in varying proportions, as indicated in the legends to tables, and incubated at 37°C for 10 min in cellulose nitrate ultracentrifuge tubes. Each sample was then overlayed with saline (d = 1.006 g/ml) and the VLDL isolated using a 40.3 rotor (Beckman Instruments, Palo Alto, CA) at 35000 rpm for 18 h. In those experiments where the mass of starting material was sufficient, the d = 1.006 g/ml infranate was adjusted to d = 1.21 g/ml using solid KBr and the mixed micelle-protein complex isolated by centrifugation at 35 000 rpm for 48 h. Analytical ultracentrifugation was performed at 20°C using a Beckman model E ultracentrifuge with an ANH Ti rotor and double-sector epoxyaluminum cells [ 151. Protein was measured according to the method of Markwell et al. [16] using bovine serum albumin as the standard (fraction V, Sigma Chemical Co., St. Louis, MO). Lipid analysis. Lipids were extracted according to the method of Folch et al. [ 171 using one wash of the aqueous phase with equilibrated Folch lower phase. This procedure recovered greater than 95% of the 1ysoPC as determined by inclusion of a known aliquot of [ N-methyf-‘4C]lysoPC. Phospholipids were digested by incubation with phospholipase C (Clostridium we/&ii) for 2 h and quantitated by high-temperature gas chromatography using a Hewlett-Packard 5880 gas chromatograph and a 1% QV- 1 column, which was temperature programmed from 175 to 350 “C as previously described [ 181. Phospholipase C digestion was performed after appropriate extraction of all samples. Samples with a volume greater than 0.5 ml were lyophilized and suspended in 0.5 ml of distilled water prior to extraction. Under these conditions standard aliquots of radioactive IysoPC were digested to 95% completion after a 2-h incubation with phospholipase C. Some samples were chromatographed on silica gel H in chloroform/ methanol/acetic acid/water (100 : 45 : 20 : 4) to separate the phospholipid classes [ 191. The individual bands were visualized with dichlorofluorescein, eluted from the silica gel according to the method

373

of Arvidson [20], and digested with phospholipase C as above. Since the monoacylglycerols and ceramides produced by the digestion of 1ysoPC and sphingomyelin could be resolved by gas chromatography the silica gel fractions containing these phospholipids were combined prior to elution to simplify analysis. Apolipoprotein analysis Samples were prepared for electrophoresis by dialysis against 4 mM Na,EDTA, pH 8.6, and subsequently lyophilized and solubilized in 0.2 ml of 2% SDS by incubation at 100°C for 2 min. An equal volume of glycerol (Analar grade, BDH Chemicals, Toronto, Canada) was added to 50-~1 aliquots which were made 40 mM in dithiothreitol by the addition of 0.1 vol. of a 400 mM stock solution prepared in distilled water immediately before use. SDS-electrophoresis was carried out in 15% acrylamide/l8% glycerol gels as previously described [21]. Gels were stained in 0.025% Coomassie brilliant blue G-250 for 16 h and destained using a Bio-Rad Model 172A Gel Electrophoresis Diffusion Destainer (Bio-Rad Laboratories, Richmond, CA). All samples from a single experiment were stained and destained simultaneously, thus minimizing variation in subsequent quantitation. The C and E apolipoproteins were quantitated by absorbance using a Joyce-Loebl Chromoscan Mark II Densitometer (Joyce, Loebl and Co.. Gateshead, U.K.). The distribution of the C and E apolipoproteins between the post-incubation VLDL and micellar fraction was determined by calculating the area% of each apoprotein in a given gel scan as follows: % apolipoprotein in micelles = (area % micelle apolipoprotein/area % micelle apolipoprotein + area % VLDL apolipoprotein) . 100, where apolipoprotein refers to apoproteins E or C. No correction was made for the different chromogenicity of the C and E apolipoproteins. Electron microscopy Electron microscopy was carried out after dialysis against 0.04% EDTA, pH 7.4, using 1.5% uranyl oxalate negative stain, pH 7.4 [22]. Freshly prepared samples were examined in a Phillips 300 EM at 60 keV accelerating voltage.

Results Fig. 1 shows the total lipid profiles of the VLDL particles before incubation, and after incubation with two types of lysoPC-PC micelles and reisolation by ultracentrifugation. The original VLDL has the characteristic lipid profile of rat VLDL, consisting primarily of triacylglycerols and smaller amounts of cholesteryl esters, free cholesterol, free fatty acids and the phospholipids, as represented by their diacylglycerol and ceramide moieties. There is little, if any, lysophosphatidylcholine, as indicated by the absence of the corresponding monoacylglycerols. Following incubation in a 1 : 1.6 ratio with lysoPC-PC micelles containing 45 mol% lysoPC, the VLDL lipid pro-

Fig. 1. Total lipid profiles of VLDL before and after incubation with IysoPC-PC micellles. Profiles show pre-incubation VLDL (A). post-incubation VLDL containing 25 mol% IysoPC (B). and post-incubation VLDL containing 50 mol% 1ysoPC (C). Compounds identified as follows: 16-18, trimethylsilyl esters of free fatty acifds with 16 and 18 acyl carbons; 20-22, trimethylsilyl ethers of monoacylglycerols with 16- 18 acyl carbons produced by phospholipase C digestion of lysophosphatidylcholine; 27, trimethylsilyl ether of cholesterol; 30, tridecanoylglycerol, internal standard; 34442. trimethylsilyl ethers of diacylglycerols with 32-40 acyl carbons and trimethylsilyl ethers of ceramides with 18 alkyl base carbons and 16-24 acyl carbons produced by phospholipase C digestion of phosphatidylcholine and sphingomyelin; 43-47, cholesteryl esters with fatty acids having a total of 16-20 acyl carbons; 48-56, triacylglycerols with a total of 48-56 acyl carbons.

374

r

Fig. 2. Total lipid profiles of lysoPC-PC micelles before and after incubation with VLDL. 45 mol% 1ysoPC micelles before (A) and after (B) incubation. 75 mol% IysoPC micelles before (C) and after (D) incubation. Compounds identified as in Fig. 1.

file remains 1argeIy unchanged, except for the increase in monoacylglycerols representing the lysophosphatidylcholines. Likewise, an incubation with IyosPC-PC micelles containing 60 mot% 1ysoPC has no significant effect on the lipid profile of the reisolated VLDL, except for the increase in the 1ysoPC content. Fig. 2 gives the total lipid profiles of the lysoPC-PC micelles used in the incubations with the VLDL shown in Fig. 1, before incubation and after incubation and reisolation. The original micelles are seen to contain the egg yolk phosphatidylcholine and lysophosphatidylcholine, as represented by the corresponding diacylglycerol and monoacylglycerol moieties, along with small amounts of free cholesterol and free fatty acids. There is a significant decrease in the lysoPC/PC ratio of the particles following incubation and reisolation, along with a detectable increase in the amount of free fatty acids, free cholesterol and triacylglycerols. The lipid class distribution of the original particles and the particles reisolated following incubation under various conditions is shown in Tables I-III. It can be seen that each incubation resulted in an essentially complete equilibration of 1ysoPC between VLDL and the micellar particles, as judged by the mol% of 1ysoPC relative to total phospholipid, without significant changes in the triacylglycerol, cholesteryl ester or cholesterol con-

tent of the post-incubation VLDL particles. There was, however, a measurable increase in the triacylglycerol and cholesterol content of the post-incubation micellar fraction, which varied with the 1yosPC content of the particles. Incubation of VLDL and micelles at initial phosphohpid mass ratios of 1 : 2 and 1 : 6 (Table II, 2A and 2B) resulted in a transfer of 87-9758 of the VLDL apolipoprotein to the micellar fraction without disruption of the VLDL particles. No lipid was detected in the d > 1.2 1 g/ml fraction, which contained 0.665.0% of the apolipoprotein originally present in VLDL. It was observed that, on a mass basis, negligible transfer of phosphatidylcholine or sphingomyelin occurred in experiments 1 or 2 (Tables I and II). To investigate whether there was any mixing of these phospholipids, the molecular species (as separated by temperature-programmed gas chromatography) of the particles reisolated after in-

Fig. 3. Electron micrographs of negtaively stained VLDL pre(A) and post- (B) incubation and micellar particles pre- (A) and post- (B) incubation from experiment 2B (Table 11). Instrument magnifications were 2970X (A, B) and 17490 X (C,D). Each micrograph was photographically enlarged 5.35 X. Bar in lower left-hand corner represents 100 nm (A, B) or 50 nm (C’. D).

375

TABLE

I

COMPOSITION

OF VLDL AND MICELLAR

FRACTIONS

FROM

EXPERIMENT

1

Values reported are wt% averages of duplicate incubations. Numbers in parentheses are concentrations in mg/ml. For experiments IA-IG. VLDL (0.38 mg phospholipid/ml) was incubated with micelles at the following concentrations: 0.12 mg/ml (A), 0.19 mg/ml (B), 0.38 mg/ml (C), 0.75 mg/ml (D), 1.5 mg/ml (E), 2.25 mg/ml (F) and egg phosphatidylcholine, 0.75 mg/ml (G), Abbreviations: M/VLDL, micellar phospholipid/VLDL phospholipid ratio of particles isolated after incubation; mol% IysoPC, the mol% of IysoPC relative to the total phospholipid of a given fraction. Sample

Preincubation 1-VLDL I -Micelle Post-incubation d i 1.006 IA

M/VLDL

1.9 0.0

0.19

IC

0.45 0.67

IE

1.44

1F

1.75

IG

0.80

IB IC ID 1E IF IG

PC and sphingomyelin

Cholesterol ester

Triacylglycerol

Protein

mol% IysoPC

0.0 50.0

13.4 50.0

3.5

69.5

11.7

0

0.0

0.0

0.0

60

3.6 (0.12)

81.0 (2.64)

n.d.. -

17.9

2.6 (0.08)

80.3 (2.45) 71.7 (1.78) 16. I (2.30) 76.5 (2.59) 68.0 (1.88)

nd..

-

21.5

nd..

-

33.1

n.d., -

33.2

nd..

-

51.1

n.d.. -

56.9

particles

0.34

d > 1.006 IA

LysoPC

particles

1B

ID

Free cholesterol

0.19 0.34 0.45 0.67 1.44 1.75 0.80

2.1 (0.07) 2.5 (0.08) 2.6 (0.07) 1.8 (0.05) 1.6 (0.05) 2.2 (0.06) 2.2 (0.05) 1.7 ( < 0.01) 6.0 (0.01) 3.2 (0.01) 0.7 ( < 0.01) 1.7 ( i 0.01) 1.9 (0.02) 4.5 (0.02)

1.6 (0.05) 2.2 (0.07) 5,.2 (0.13) 4.6

11.7

(0.0)

(0.38) 12.4 (0.38) 16.5 (0.41) 14.6 (0.44) 11.2 (0.38) 13.4 (0.37) 19.6 (0.43)

14.6 ( < 0.01) 21.3 (0.03) 26.1 (0.07) 39.1 (0.16) 36.0 (0.36) 31.2 (0.42) 11.8 (0.04)

80.4 (0.07) 71.7 (0.12) 65.7 (0.17) 57.5 (0.23) 55.4 (0.55) 53.3 (0.70) 80.8 (0.30)

0.14) 7.5 (0.25) 11.3 (0.3 1) 0.0

cubation 2A and 2B were compared (Table IV). The distribution of the molecular species of the ceramides (derived from sphingomyelin) of the original and reisolated VLDL were essentially identical and clearly distinct from that of the original micelles. On the basis of the C34/C42 ratio, there was no exchange of micellar and VLDL

4.0 (0.10) 2.9 (0.09) 3.2 (0.11) 5.1 (0.14) 2.9 (0.06) 0.0 ( < 0.01)

0.0

( c 0.01) 1.7

( < 0.01) 0.9

( c 0.01) 0.2

( < 0.01) 0.2

( -c0.01) 0.0 (0.0)

75.3 (1.65) 3.3 ( < 0.01) 1.0 ( < 0.01) 3.4 (0.01) 1.8 (0.01) 6.6 (0.02) 7.0 (0.09) 2.9 (0.01)

-

0.0

n.d., -

22.0

nd.,

-

31.0

n.d., -

38.2

n.d., -

51.4

n.d., -

50.3

n.d., -

52.3

nd..

18.6

nd..

-

sphingomyelin. Likewise, the molecular species of the diacylglycerol moieties of phosphatidylcholine of the pre- and post-incubation VLDL fractions were identical and distinctly different from that of the corresponding micellar fractions. On the basis of the C,,/C,, ratio no exchange of micellar and VLDL phosphatidylcholine could be discerned.

376 TABLE

II

COMPOSITION Values reported Table I. Sample

OF VLDL AND are wt% average

M/VLDL

Preincubation

of duplicate

FRACTIONS incubations.

FROM

Numbers

EXPERIMENT

in parentheses

2

are concentrations

Free cholesterol

LysoPC

PC and sphingomyelin

Cholesterol ester

3.2 (0.32) (0.14)

0.4 (0.04) (0.02)

13.4 (1.37) (0.60)

1.5 (1.15) (0.07)

71.7

28.3 (1.58)

in mg/ml.

Triacylglycerol

Protein

13.2 (7.47) (3.27)

9.2 (0.95) (0.41) 0

Abbreviation

as tn

mol% IysoPC

particles

2-VLDL 2A 28 2-micelle 2A 2B Post-incubation d < 1.006

MICELLAR

0 (0) (0)

(4.0) (0)

0

0

(7.0)

(0) (0)

(0) (0)

(0) (0)

0

80 _

particles

2A

0.76

1.6 (0.23)

8.4 (1.22)

9.8 (1.43)

3.2 (0.46)

2B

1.17

1.9 (0.11)

24.3 (1.38)

11.4 (0.64)

1.1 (0.06)

0.76

2.7 (0.14)

42.5 (2.19)

32.1 (1.65)

1.17

1.3 (0.04)

51.6 (1.61)

24.1 (0.75)

0.4 (0.02) 0.3 (0.01)

76.2 (11.1)

57.1

61.1 (3.47)

0.45 (0.06) 0.2 (0.01)

6.0 (0.31) 5.9 (0.18)

16.1 (0.83) 16.7 (0.52)

67.9

76.8

d < 1.21

2A 2B

TABLE

76.9

III

COMPOSITION

OF VLDL AND MICELLAR

FRACTIONS

FROM

EXPERIMENT

3

VLDL (0.4 mg phospholipid/ml) was incubated with micelles (0.66 mg phospholipid/ml) varied from 45 mol% (3A) to 100 mol% (3E). Abbreviation as in Table 1. Sample

Preincubation 3-VLDL 3A 3B 3c 3D 3E Post-incubation d < 1.006 3A 3B 3c 3D 3E d-c 1.21 3A 3B 3c 3D 3E

M/VLDL

in which

the IysoPC concentration

Free cholesterol

LysoPC

PC and sphingomyehn

Cholesterol ester

Triacylglycerol

mol% IysoPC

2.3 0.0 0.0 0.0 0.0 0.0

0.0 34.0 50.0 66.0 88.0 100.0

15.5 66.0 50.0 34.0 12.0 0.0

7.5 0.0 0.0 0.0 0.0 0.0

74.6 0.0 0.0 0.0 0.0 0.0

0.0 45.0 60.0 75.0 92.0 100.0

0.58 0.45 0.59 0.68 0.7 1

2.1 2.0 1.9 1.8 1.7

3.9 5.2 7.9 9.8 10.3

14.9 14.6 13.4 11.8 10.7

7.3 6.9 6.6 6.7 6.4

71.9 71.3 71.3 70.0 70.8

28.9 35.9 48.0 56.3 59.x

0.58 0.45 0.59 0.68 0.71

3.6 4.3 3.9 3.4 3.3

15.9 27.8 38.0 54.5 65.1

14.7 62.6 51.5 31.4 21.8

0.0

0.4 1.1 0.4 1.5

3.6 7.9 5.5 5.x 8.3

24.8 40.9 53.4 73.0 x2.3

particles

particles

was

377

TABLE

IV

PHOSPHATIDYLCHOLINE

AND SPHINGOMYELIN

CARBON

NUMBER

PROFILES

The carbon number distribution of diacylglycerols and ceramides produced by phospholipase C digestion of the phosphatidylcholine and sphingomyelin recovered in VLDL and 90 mol% IysoPC micelles prior to incubation and after incubation and reisolation. Sphingomyelin carbon numbers 34-42 represent trimethylsilyl ethers of ceramides with 18 alkyl base carbons and 16-24 acyl chain carbons. Phosphatidylcholine carbon numbers 34-42 represent trimethylsilyl ethers of diacylglycerols with a total of 32-40 acyl chain carbons. VLDL and micelle values are duplicate determinations. Postincubation values are the mean + SD. of four experiments. Carbon

numbers

34

36

38

40

41

42

Sphingomyelin Preincubation VLDL Micelles Postincubation d-c 1.006 d < 1.21 Phosphatidylcholine Preincubation VLDL Micelles Postincubation d < 1.006

d < 1.21

36.3 19.6

4.3 8.0

2.3 0.0

8.0 0.0

6.9 0.0

42.1 12.4

28.7 f 7.6 15.6 + 3.5

9.5 * 3.1 9.9 f 5.9

5.lk4.3 4.5 k 6.3

8.0? 1.6 0.0 * 0.0

7.0+ 1.4 0.0 * 0.0

41.7*7.6 9.9 k 8.1

1.1 1.5

28.1 56.8

37.0 27.7

25.4 10.1

8.5 3.8

0.6 + 0.8 1.5 +0.01

25.2* 1.4 49.1 k4.1

36.6 k 0.3 30.4 f 0.7

27.9 * 1.O 13.8+2.3

8.8+ 1.8 4.8 k 0.5

When the analytical accuracy of the measurements is considered, any transfer or exchange could not have exceeded 10% and was probably less than 5%. Under the same conditions less than 2% of the VLDL-triacylglycerols, cholesteryl esters and free diacylglycerols were transferred to the micellar fraction. Fig. 3 shows representative electron micrographs obtained for the micellar and VLDL fractions from Experiment 2. The VLDL mean particle size was 53 f 27 and 29 f 3 nm pre- and post-incubation, respectively. The pre- and postincubation micellar particles were nearly identical and had mean diameters of 9 + 7 and 10 t_ 2 nm, respectively. These particles had an F,yz, maximum of 2.74 and, using the nomogram of Lindgren et al. [23], corresponded to the 1.12-1.16 g/ml density range. Apolipoprotein transfer to ijuoPC-PC micelles The transfer of the apolipoproteins of VLDL to 60 mol% 1ysoPC micelles was investigated by varying the relative proportions of VLDL and micelles in the incubation medium. Fig. 4 shows the apoli-

poprotein profiles of the VLDL and micellar fractions reisolated after incubation at initial micelle/ VLDL total phospholipid ratios ranging from 0 : 1 to 6 : 1. The control (I-VLDL) shows that a recentrifugation of VLDL by itself or following incubation with PC liposomes (1G) resulted in a minimal non-specific loss of apolipoprotein. In marked contrast, there was a preferential transfer of apolipoprotein E from VLDL to the micellar fraction as the concentration of the latter increased, with a complete transfer of apolipoprotein E without significant loss of the C apolipoproteins at a micelle/VLDL ratio of 1 : 1 (1C). The C apolipoproteins were transferred as the concentration of the micellar fraction was increased further. The range of micelle/VLDL phospholipid ratios in which apolipoprotein E was selectively transferred to the micellar fraction corresponded to conditions which resulted in a final concentration of 1ysoPC of less than 35 mol% of the total phospholipid of the resiolated VLDL and micellar fractions (Table I). The transfer of the C apolipoproteins occurred at 1ysoPC concentrations above this value. The disappearance of

378

apolipoprotein from the VLDL fraction matched in all instances by its corresponding pearance in the micellar fraction.

was ap-

In order to determine the basis for the observed selectivity of apolipoprotein E transfer, alterations were made in the lysoPC/PC ratio of the micellar preparation over the range from 1 : 2 to 1 : 0 (3A-3E, Table III). Fig. 5 shows the apolipoprotein profiles of the VLDL and micellar fractions after reisolation from the incubation mixture. The results indicate that at the initial ratio of

VLDL phospholipid to micellar phospholipid used in experiment 3 apolipoprotein E is quantitatively transferred to the micellar fraction at final concentrations of 1ysoPC between 28 and 35 mol%. At vafues above 35 mol% lysoPC, the C apolipoproteins also transferred. Transfer of the C apolipoproteins was complete when VLDL contained 50 mol% IysoPC. Table V lists the relative proportions of the C and E apolipoproteins in the VLDL and micellar fractions reisolated in experiment 1 as estimated by densitometry of the polyac~lamide gels. Each value was corrected for the non-specific dissociation of apolipoprotein which was observed when

Fig. 4. SDS-glycerol gel electrophoresis of the apolipoproteins of the VLDL and micellar fractions isolated after incubation of 60 mol% IysoPC micelles with VLDL at initial surface area ratios of 1: 3-6: 1 (experiment IA-IG). Samples identified as in Table I. E, apolipoprotein E; C, apolipoproteins C-I. C-II and C-III, 0, 3; apolipoprotein B remains at the top of these gels.

Fig. 5. SDS-glycerol gel electrophoresis of the apolip~~proteins associated with the reisolated VLDL and micellar fractions after incubation with micelles containing 45- 100 mol’% 1ysoPC (experiment 3A-3E). Samples identified as in Table 111. Apolipoprotein identified as in Fig. 4.

Effect of lysoPC/PC

ratio on ~p~I~p~pr~tein trans-

fer

379 TABLE

V

SELECTIVITY

OF APOLIPOPROTEIN

TRANSFER

Partition and selectivity values for the distribution of apolipoproteins C and E between the VLDL and micellar fractions as determined by densitometry of the stained polyacrylamide gels for the respective postincubation fractions from experiment I. The mol% IysoPC and wtX of total phospholipid are derived from the data presented in Table I. nd.. not determined. Particles

mol% VLDL-IysoPC % PL in micelles % E in micelles % C in micelles Partition No. E Partition No. C Selectivity E/C

I-VLDL

IA

IB

IC

ID

IE

IF

0 0 0 0 n.d. n.d.

17.9 17.4 50 0 2.9 n.d.

21.5 25.4 84 0 3.4 n.d.

33.1 28. I 100 23 3.5 0.82 4.35

33.2 34.6 100 67 n.d. 1.94

51.1 59.5 100 100 nd.

56.9 65.4 100 100 n.d. n.d. 1.00

VLDL was incubated and centrifuged in the absence of micelles. Two phenomena can be observed: one is the preference which an individual apolipoprotein shows for VLDL or micellar particles and the other is the selectivity of transfer of apolipoprotein E relative to the C apolipoproteins. The first effect was quantitated by calculating an apolipoprotein partition number, which relates the apolipoprotein proportions to the proportions of the phospholipid in the VLDL and micellar fractions. The partition number is then expressed as follows: (apolipoprotein in micelles/apolipoprotein in micelles + apolipoprotein in VLDL)/(PL of micelles/PL of micelles + PL of VLDL). Apolipoprotein refers to an individual apolipoprotein or the entire complement of apolipoprotein in the micellar and VLDL fractions and PL is the total phospholipid concentration of the respective fractions. According to this equation an apolipoprotein partitioning between VLDL and the micellar particles in direct proportion to their respective phospholipid content would give a value of 1, indicating an equal affinity for both fractions. Any other distribution would indicate a preference for one or the other particle type and would give a number larger than 1 if it favored micelles and less than 1 if it favored VLDL. Apolipoprotein E has a partition number of 2.9-3.5, indicating a significant preference for the micellar fraction. There is essentially no partition of the C apolipoproteins into the micellar fraction below 35 mol% 1ysoPC. At 1ysoPC concentrations

1.49

1.68 1.00

above this value, the C apolipoprotein partition number ranged from 0.8 to 1.9, indicating a slight preference for the micellar fraction under these conditions. The second effect relates the relative partition of different apolipoproteins between VLDL and micellar particles and may be defined as: (apolipoprotein E in micelles/apolipoprotein E in micelles + apolipoprotein E in VLDL)/(apolipoprotein C in micelles/apolipoprotein C in micelles + apolipoprotein C in VLDL). The preceding is essentially the percent of the total apolipoprotein E present in the micelles divided by the percent of the total apolipoprotein C in the micelles. When the selective transfer of apolipoprotein E relative to apoplipoprotein C is expressed in this way values of infinity are obtained for those experiments in which the VLDL and micelles have less than 35 mol% 1ysoPC (IA and lB, Table I), indicating an absolute selectivity for apolipoprotein E. At higher concentrations of 1ysoPC less selectivity is seen with non-selective conditions reached at 50 mol% 1ysoPC. Discussion We have previously shown (41 that the association of the C apolipoproteins was inversely proportional to the diameter of the particles in the chylomicron to low-density lipoprotein size range. These particles can be formed from phosphatidylcholine, cholesterol and neutral lipid. Particles

380

having the diameter of high-density lipoproteins cannot be prepared from the above components without the use of detergent-like molecules. It was possible to form particles of HDL diameter by the inclusion of the physiological molecule 1ysoPC above 35 mol% [ 11,24,25]. ~tubilit~ of VLDL and micelie pictures Lysophosphatidylcholine has been reported to promote fusion of intact cells [26], but not model membrane systems [27]. The analytical methods of the present study would have detected fusion of VLDL and micellar particles by a decrease in the mass of the micellar phosphatidylcholine content as well as an appearance of the egg phosphatidylcholine and sphingomyelin molecular species in the VLDL fraction. There was neither fusion of VLDL and micellar particles nor exchange of phosphatidylcholine and sphingomyelin by these criteria. Thus, the mixed micellar fraction was found to be a stable entity under the conditions of incubation and isolation used in this study. The absence of detectable transfer of phosphatidylcholine and sphingomyelin between the VLDL and micellar particles is consistent with the lack of equilibration between lipoprotein fractions in vivo, as shown by analyses of the molecular species of sphingomyelin and glycolipids of the plasma lipoproteins [28,29].

The lysoPC-PC micelles served as an appropriate surface which could effectively compete with VLDL for its apolipoproteins. A selective transfer of apolipoprotein E occurred in those experiments where the VLDL and micellar fractions contained less than 35 mol% lysophosphatidylcholine. The extent of apolipoprotein transfer was dependent upon the mass of micellar phospholipid. A complete transfer occurred at a micelle/VLDL phospholipid ratio of 0.45. These conditions result in a micellar fraction which is no longer stable on the basis of the lysoPC/PC ratio alone, and suggest that apolipoprotein E serves a structural role in these particles. In contrast, the C apolipoproteins only associate with the micellar fraction when the 1ysoPC content is maintained above 35 mol% following incubation with VLDL. Since VLDL phospholipid has been shown to exist

entirely at the surface of the particle [30,3 1] and the same can also be assumed for the lysoPC-PC micelles, the correlation of apolipoprotein transfer and VLDL/micelle phospholipid ratios is a reflection of the surface area of each fraction. It is known that the interfacial surface tension of a lipid particle increases with decreasing particle diameter [32], and that IysoPC, like detergents in general, acts to decrease the interfacial surface tension [lo]. Furthermore, other workers [33] have shown that the binding of the C apolipoproteins to phospholipid monolayers is pressure dependent. Therefore, the association of the C apolipoproteins with the large diameter particle in previous work [4,5] and with small diameter lysoPC/PC particles in the present study may be reconciled on the basis of similarities in interfacial tension, although no actual measurements of it were made. It could not be established whether apolipoprotein E was transferred to the micellar fraction as the free apolipoprotein or as an apolipoprotein1ysoPC complex. Previous work [34] has shown that apolipoprotein A-I binds IysoPC with high affinity, and the present results suggest that a similar interaction of 1ysoPC with apolipoprotein E to phosphatidylcholine liposomes. When the experimental data on the micellar composition is recalculated as mol lysoPC/mol of apolipoprotein amino acid (assuming 100 g/mol amino acid), a value of 1 : 15 (lysoPC/amino acid) is obtained for the minimum concentration of 1ysoPC at which the total VLDL apolipoprotein is recovered in the micellar fraction and 1 : 6.5 for the minimum concentration at which only apol~poprotein E is recovered in the micellar fraction. Since the former figure includes binding of 1ysoPC by apolipoprotein E, the above considerations indicate that apolipoprotein E associates with more than twice the amount of LysoPC on a per amino acid basis. as do the C apolipoproteins. A unique interaction with 1ysoPC is also suggested by the dependence of the preferential transfer of apolipoprotein E on the 1ysoPC concentration (Table V). It is our hypothesis that apolipoprotein E binds IysoPC, increasing its solubility in aqueous solution and thus allowing dissociation as an apolipoprotein E1ysoPC complex. Such hypothesis would explain a number of reports in the literature indicating that apolipoprotein E was easily dissociated from par-

381

titles during isolation and washing steps [35]. The conditions and time required for isolation would allow the lecithin-cholesterol acyltransferase reaction to proceed, resulting in the production of 1ysoPC [36]. A particularly important observation which is consistent with the above scheme is the reported loss of apolipoprotein E from apolipoprotein E-rich discoidal HDL subsequent to lecithin-cholesterol acyltransferase activity [37]. It was suggested that apolipoprotein E dissociated due to the change in particle structure from a disc to a sphere. This would seem an unlikely cause since apolipoprotein E is routinely isolated as a component of spherical HDL [3]. In view of the present observations, it is much more probable that apolipoprotein E binds 1ysoPC generated by the lecithin-cholesterol acyltransferase reaction, thereby forming a soluble apolipoprotein E-1ysoPC complex. The possibility that the increase in the relative 1ysoPC content of the VLDL particle was the deciding factor in the loss of apolipoprotein from the VLDL surface must also be considered, as it may have modified the surface properties leading to the dissociation of the apolipoproteins from VLDL. However, the fact that the apolipoproteins were isolated as a lipid-protein complex having the identical lysoPC/PC ratio indicates that transfer was not a result of displacement from the VLDL surface, but rather due to a preferred association with the micellar components. Physiological significance

of apolipoprotein E-lysoPC

complexes

The preference for the in vitro formation of apolipoprotein E-lysoPC-PC complexes suggests that they could play a role in lipoprotein metabolism in vivo. Apolipoprotein E was completely transferred to the micellar particles under conditions where 1ysoPC accounted for 15% of the total phospholipid mass. This value is well within the range of 1ysoPC concentrations of 5.8-26.2% (w/w) of total phospholipid reported for normal plasma [ 191 and below the 33% 1ysoPC of veryhigh-density lipoproteins [38]. There is evidence that apolipoproteins can bind significant amounts of 1ysoPC even in the presence of excess albumin. Nishida [39] has demonstarted that after phospholipase A, digestion of LDL

there exists a pool of 1ysoPC which cannot be bound by a several-fold excess of albumin. Similarly, Pattniak et al. [40] have reported that an inaccessible pool of 1yosPC exists after phospholipase A, digestion of HDL,. Portman and Illingworth [41] have reported significant binding of 1ysoPC to lipoproteins, particularly of the hyperlipemic squirrel monkey. Since the apolipoproteins are integral components of the lipoprotein surface it follows that 1ysoPC generated in vivo by various enzymatic activities could be initially bound by the apolipoproteins. Particles rich in 1ysoPC and containing apolipoproteins other than albumin have been isolated by a number of investigators and described as very-high-density lipoproteins [38,42]. These particles have been reported to be particularly effective in promoting cholesterol egrees from cultured skin fibroblasts [42]. The above investigators did not specifically study which apolipoproteins bound 1ysoPC. Further studies are being conducted to establish the nature of the particles formed from human VLDL under similar conditions of incubation and to establish the extent and specificity of binding of 1ysoPC to apolipoprotein E. It is hoped that such studies will contribute to a better understanding of lipoprotein metabolism and interconversion in the plasma compartment. Acknowledgments This work was made possible by grants from the Ontario Heart Foundation and the Medical Research Council of Canada. References Havel, R.J., Kane, J.P. and Kashyap, M.L. (1973) J. Clin. Invest. 52, 32-38 Schaefer, E.J. S. Eisenberg and R.I. Levy (1978) J. Lipid Res. 19, 667-687 Jackson, R.L., Morrisett, J.D. and Gotto, A.M. (1976) Physiol. Rev. 56, 259-316 Connelly, P.W. and Kuksis, A. (1981) Biochim. Biophys. Acta 666, 80-89 Connelly, P.W. and Kuksis, A. (1983) Can. J. Biochem., in the press Kostner, G.M., Patsch, J.R., Sailer, S., Braunsteiner, H. and Holasek, A. (1974) Eur. J. Biochem. 15, 61 I-621 Packard, C.J., Shepherd, J., Joerns, S., Gotto, A.M. and Taunton, O.D. (1979) Biochim. Biophys. Acta 572, 269-282

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