Simultaneous transfer of cholesteryl ester and phospholipid by protein(s) isolated from human lipoprotein-free plasma

Vol.

93, No.

April

29,

BIOCHEMICAL

4, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

1980

SIMULTANEOUS

TRANSFER

BY PROTEIN(S)

OF

ISOLATED

CHOLESTERYL FROM

ESTER

HUMAN

AND

1114-1120

PHOSPHOLIPID

LIPOPROTEIN-FREE

PLASMA

Ihm and Judith A.K. Harmony*, Chemistry Department, Indiana University, Bloomington, Indiana 47405; and Jeff Ellsworth and Richard L. Jackson, Division of Lipoprotein Research, Departments of Pharmacology and Cell Biophysics, Biological Chemistry and Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio 45267

Jahei

Received

March

11,198O

SUMMARY Protein(s) catalyzing the transfer of [3H]cholesteryl ester and [14C]phosphatidylcholine from high density lipoproteins to low density lipoproteins have been purified 4829-fold from human plasma by chromatography of the d > 1.21 g/ml infranatant fraction of human plasma on phenyl-Sepharose, CMcellulose, concanavalin A-Sepharose, and finally by isoelectric focussing. At each step of the purification, The purified both transfer activities coelute. protein(s), molecular weight 150,000, transfer cholesteryl esters and phosphatidylcholine with a 1:l stoichiometry and at equal rates of flux. Rat plasma contains a protein(s) which facilitates the transfer of phosphatidylcholine as effectively as human plasma. However, the rat plasma protein(s) does not facilitate the transfer of cholesteryl esters. These results suggest that human plasma contains one or more proteins which transfer both lipids, possibly as a 1:l complex, whereas rat plasma lacks the cholesteryl ester transfer protein. INTRODUCTION Although fer

from

HDL

transfer et

to

been

al.

the

* Present Cincinnati

VLDL,

only

1.25

exchange

address: Medical

of g/ml

in

1965

(1)

recently

have

of

has

a molecular

5.

Sniderman

plasma

Department Center,

that the

characterized.

fraction

cholesteryl proteins

from

the

CE between

all

weight et

al.

facilitates

of Biological Cincinnati, Ohio

of (5)

et

density of

esters which

Zilversmit

a glycoprotein

protein point

d>

and

the

exchange

isoelectric

reported

isolated

catalyzes

The

was

isolated

(3,4)

which

in

it

al.

plasma

approximately also

Chemistry, 45267.

(2)

0006-291X/80/081114-07$01.00/0 c 1980 by A cudemrc Prms, of reproduction rn an,vform

Inc.. reserved.

1114

infranatant

80,000

of

Pattnaik

lipoproteins. and

that CE from

University

'Abbreviations: VLDL, very low density lipoproteins (d 1.006 low density lipoproteins (d 1.02-1.05 g/ml); HDL, high density (d 1.063-1.21 g/ml); CE, cholesteryl esters; PC, phosphatidylcholine; phospholipids; Tg, triglyceride; LCAT le
Copwighr Afl righIs

this and

g/ml

demonstrated

exchange

trans-

facilitate

>1.25 the

(CE)l

an

a protein HDL

to

of g/ml); LDL, lipoproteins PL, transferase:

LDL.

Vol.

93, No.

4, 1980

contrast

to

In

Fielding HDL

(6)

to

LDL

molecular (6)

the

facilitated

isolated or

and

sugge.st

that protein

lipoprote-in

the

of

35,000

it

may

which

be

transport

and,

based

apoD

(7,8).

the

protein

of on

RESEARCH

described

facilitates

back

the

BIOPHYSICAL

process

which

mediates

classes;

AND

exchange

a protein

VLDL

weight

transfer

of

BIOCHEMICAL

the Tg

to

properties,

Barter

et

is

a glycoprotein

detailed

knowledge

net

Chajek

transfer This

(9)

an

CE from has

and

of

with

of

purified

transfer

and

protein

Chajek

al.

bidirectional

above,

HDL.

its

COMMUNICATIONS

a

Fielding a serum

Tg between

isoelectric

point

9.0. While

litate

it

the

reexamine

molecular

transfer

of

lipid protein

weight

The

phospholipid

and

its

MATERIALS

evident

the This

(10).

is

ability

AND

that CE and

is

specificity

of

is

from

isolated

>lOO,OOO

and

transfer

protein

to

Tg

facilitate

still

lacking,

the

plasma the

transfer

the

proteins seemed

phospholipid

PC and

CE was

faci-

appropriate

to protein

fraction,

bidirectional 4829-fold

which

exchange

infranatant

purified of

the it

dbl.21

facilitates was

of

transfer and

has of

a

PC.

characterized,

assessed,

METHODS

Materials. 1-Stearoyl-2-oleoyl phosphatidylcholine, purchased from ____ Applied Science Laboratories, was determined to be pure by thin-layer chromatography ton silica gel (chloroform:methanol:water, 65:25:4). Egg yolk [choline-methyl14C]phosphatidylcholine (50 mCi/mmol) and [7-3H(N)]cholesterol (11 mCi/mmol) were purchased from New England Nuclear. Fatty acid-free bovine serum albumin was obtained from Sigma Chemical Company. Phenyl-Sepharose CL-4B, conA-Sepharose 4B and Sephadex G200 were purchased from Pharmacia Fine Chemicals; CM-52 was obtained from Whatman. Amphilites (pH 4.0-6.0) were obtained from LKB Instruments. Isolation of human plasma lipoproteins and d>1.21 infranatant. Lipoproteins were isolated from the freshly-collected plasma of normolipemic fasted human volunteers by sequential ultracentrifugal flotation in salt solutions of KBr. Each lipoprotein class was refloated at its respective density and finally dialyzed against 0.9% NaCl, 0.001 M EDTA, 0.001% sodium azide, pH 7.4. The chemical composition of the lipoproteins was determined by the methods of Hui and Harmony (11). The purity of the lipoprotein fractions was assessed by electrophoresis on agarose (l%, pH 8.6) and by immunodiffusion Following the removal of using antibodies raised against LDL, HDL and apoB. HDL, the md.1.21 infranatant, excluding the clear zone immediately below the lipoprotein layer, was employed as the source of the transfer protein. Description of assay. Double-labeled HDL containing [14C]PC and [3H]CE were prepared as follows: HDLwerelabeled with [3H]cholesterol as described by Chung et al. (12). The [3H]cholesterol in HDL was then esterified with partially purified LCAT (13). After incubating with a lo-fold excess of LDL [14C]PC was to remove unesterified cholesterol, the HDL were reisolated. LDL and purified bovine incorporated into [3H]CE-HDL by using [ 14C]PC-labeled

1115

Vol.

93, No.

BIOCHEMICAL

4, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

[14C]PC-labeled LDL was prepared by inculiver PC exchange protein (14,15). bating sonicated vesicles of 1-stearoyl-2-oleoyl phosphatidylcholine containing egg [14C]PC, LDL and liver PC-exchange protein as described previously (15). Each assay contained [3H]CE-[14C]PC-labeled HDL and unlabeled LDLinl.0 ml of Incubations were performed at 0.9% NaCl, 10 mM Tris-Cl, 1 mM EGTA, pH 7.4. After 37°C either in the presence or in the absence of transfer protein(s). the density of incubation mixture was raised a specified period of incubation, to 1.07 g/ml by addition of solid KBr. The d 1.07 solution was then layered with d 1.063 KBr solution, and the mixture was centrifuged for 19 h at 48,000 rpm in a Beckman 50 Ti rotor. LDL which formed a thin surface film and HDL which remained in the bottom of the tube were collected. An aliquot (usually 0.5 ml) of each lipoprotein solution was counted in 10 ml Aquasol (New England Nuclear). The phospholipid or cholesterol content of each lipoprotein solution was determined to calculate the percent recovery of lipoproteins. Calculations. The maximal theoretical transfer of [14C]PC from HDL to LDL was calculated from the formula [Y/(X+Y)]a cpm, where X and Y are the total HDL PC and LDL PC pools, respectively, and a is the HDL [14C]PC radioactivity. The maximal expected transfer of 13H]CE was calculated in the same manner. The extrapolated, equilibrium value for PC or CE transfer was obtained by a double-reciprocal plot of the counts transferred from HDL to LDL versus the incubation time. The rate of exchange of PC and CE exchange only, with no net transfer of single homogeneous pool, all of which was determined using the formula which

SH(t)

where

=

S,(t),

[SH(0)

-

SH(0)

and

in HDL at time t(h), MH denote the CE or denotes the rate of

SEQ]exp

S

EQ

-

was calculated assuming that there is and that each lipid exists in a mass, is available for exchange. The flux rate was derived by Barter and Jones (16).

F

' +

denote

the

specific

‘EQ

activity

of

CE or

PC

(CPM/nmol)

at zero time and at equilibratium, respectively; PC pool sizes (nmol) in LDL and HDL, respectively, exchange (nmol/h) between LDL and HDL.

ML and and F

RESULTS Purification for

procedures

isolation

of

plasma

dbl.21

majority

of

transfer

activities

second

step,

identical tivities are

on to coelute

chromatographed

is

protein

is are

the

containing (data

reported

by

to

0.15

M NaCl

the

resin

conA-Sepharose,

and

The

The and

1116

active the

(3);

elution both

The and

Both

CE and

water.

In

are

combined

and

profile

from is

the

with

CE and

fractions result

those

(3,6,9).

1).

elute

shown). al.

others

(Fig.

activity

et

essentially

phenyl-Sepharose,

transfer

Pattniak

CM-cellulose. on

not

are by

directly with

by

protein(s) described

applied

eluted

CM-cellulose

from

transfer

protein

retcined

fractions

that

the

CE exchange

infranatant the

matographed

the

of

shown

is

PC the chro-

nearly

PC transfer

ac-

CM-cellulose in

Fig.

2;

CE

Vol.

93, No.

4, 1980

r 3.c

BIOCHEMICAL

AND

BIOPHYSICAL

f I

l-

RESEARCH

COMMUNICATIONS

06

12.0

? ; Y z E c * F; 4

100

E iz @J 2.0

00

‘0 60

8 6 D bIn $

4.0

1.0

.G g 0 c

2.0

L r

01

IO Fraction

02

Number

20

Froctlon

30

40

Number

FIGURE

1. Chromatography of transfer protein(s) on phenyl-Sepharose. The (2.5 x 30 cm) was equilibrated with 150 mM NaCl, 0.02% sodium aside, pH 7.4. The sample (5460 mg protein), corresponding to the infranatant fraction of 500 ml of normal plasma at d>l.21 (see Results for details), was applied to the column and eluted with the equilibration buffer. At the arrow, water was applied to the column. The flow rate was 180 ml/h and 8 ml fractions were collected. Absorbance at 280 nm (0); CE transfer (A); PC transfer (0). COlUEnl

FIGURE 2. Chromatography of transfer protein obtained from CM-cellulose on __conA-Sepharose. The column (1.6 x 30 cm) was equilibrated with 10 ti TrisHCl, pH 7.4, 0.1 mM CaC12. The sample (420 mg protein) corresponding to the active pool from CM-cellulose, was applied and eluted (12 ml/h) with the starting buffer. At the arrow, the buffer was changed to 150 mM a-methyl mannoside; 2.8 ml fractions were collected. Absorbance at 280 nm (0); CE transfer (A); PC transfer (0). and

PC transfer

activities

elute

CM-cellulose ration

as

gle

active

fer

activities.

plasma

is The

time,

150 can

determined

pH

are

with

steps

between

of

activities

by

mM a-methyl

be

on

retained

of

the

active

band,

isoelectric

point

The

purification

overall

molecular

is

~150,000.

fraction of

from 5.2,

resin,

The

The G200

4.0-6.0

affinity

mannoside.

reversed.

Sephadex

the

weight

of

and

the

Isoelectric

of PC

the

active

prepa-

focussing

conA-Sepharose

CE and

both

conkSepharose

consisting of

and

both

produces

a sin-

CE and

PC trans-

transfer

activities

assessed

as

fron

4829-fold. exchange and

the

of flux

CE and rates

transfer

protein(s),

there

transfer

protein(s),

the

PC between were is

flux

HDL

determined

essentially rate

and

is

from no

17.5

1117

LDL

was Fig.

exchange

nmol

CE per

3.

In

of

CE in

h.

In

the

a function

absence 6 h.

contrast,

of

With there

Vol.

93, No.

4, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

A

I

I

I

I

B

1.2 -

fl,;

I 0123456

0

I

3

I

1

Ttme

I

I

IO

(h)

20

30

Protein

(mg)

40

FIGURE 3. Exchange of lipids between HDL and LDL facilitated by the transfer protein(s) from human plasma: (A) CE and (B) PC. [3~]~~ ]14c]~c-~~ (80 nmol CE, 280 nmol PC) and LDL (1538 nmol CE, 1867 nmol PC; were incubated in 10 mM Tris-Cl (pH 7.4) containing 0.9% NaCl and 1 mM EGTA at 37'C in the presence (A, o) or absence (0) of transfer protein (0.4 mg). The data are presented as ln[SH(t) - SE ] vs. time, and the flux rate in nmol/h is the slope divided by [-(ML + MH)/ 'i ML . MH)],as described in Methods. FIGURE 4. Exchange of lipids between HDL and LDL facilitated by the transfer protein(s): (A) PC and (B) CE. [3~]~~,[14~]~~-~~ (110 nmol CE, 380 nmol PC) and LDL (1430 nmol CE, 1750 nmol PC) were incubated in 10 mM Tris-Cl (pH 7.4) containing 0.9% NaCl and 1 mM EGTA at 37°C for 6 h in the absence or presence of human transfer protein(s) from human (0) or rat (0) plasma. is

a significant

However,

amount

the

of

CE and

respectively, of

HDL

of and

PC does

rat

data plasma properties

facilitated

nmol

calculated

before

and

shown has

all

after

much of

the

less rat

of

29.2

layer

that

1118

is

nmol/h).

indicating At

all

at

equilibrium

represent

98%

1:l;

exchange.

that

time

67 and

Chemical

net

LDL

97%,

analysis

transfer of

points,

of

CE and

lipids

after

phosphatidylcholine. PC transfer

protein protein(s)

12.1

nmol/h,

nmol/h.

that

as

CE and

human

(F,

chromatography

was

CE transfer

PC

values of

indicate

[ 14C]PC

and

17.1

extent

reaction

suggest

is

These

thin the

of

PC transfer

maximum

of

above

a rate

exchanged.

Furthermore, that

PC flux

CE and

PC have

the

occur.

showed The

transfer

LDL

not

transfer

Since

73

of

at

proceeds

of

transfer

rate

PC transfer

stoichiometry

nmol

non-facilitated

protein-catalyzed

facilitated the

of

than were

are

coupled

human

plasma

compared.

processes (17), The

the rat

Vol.

93, No.

plasma

BIOCHEMICAL

4, 1980

preparation

that

chromatographs

illustrated

the

in

transfer

of

transfer

are

PC as

on

the

1. is

of

Both

CE.

same.

suggest

that

rat

plasma

4.

In

contrast,

not

contain

does

the

rat

plasma

from

of

not

inhibitor

not

fractions

is

facilitated

shown). of

PC

does

the

which

(data

an

rates

protein(s)

isolated

to

facilitate

the

CE transfer

human

identical

protein(s)

Furthermore,

inhibit

from

COMMUNICATIONS

a manner

transfer

proteins,

not

isolated

RESEARCH

in

rat

Fig.

plasma do

protein

and

in

Rat

BIOPHYSICAL

phenyl-Sepharose

human

indicated

phenyl-Sepharose,

transfer

data

on

the

transfer

retained by

Fig.

qualitatively

facilitate

AND

These

CE transfer.

DISCUSSION The and

protein(s)

from

cholesteryl

active

esters

material, to

are

enhances data

adsorb

bound exchange

indicate

that

esterification

of

terol-HDL

and

protein(s) litates of

least

exchange

two of

is

a small

such

as

is

the

experimental a transfer

the lipid

of

The

situation

is

not

facile.

the

with

at

no

preparation so

from

well-defined It

is

possible

for

an

system

of

[3H]choles-

the

transfer

assay The

if:

data

(a)

one

of

1119

CE;

has

not

transfer HDL does

PC transfer

the

of

determined,

and

LDL.

not

mediate

protein

CE,

LCAT.

CE clearly

unfacilitated

active

PC and

plus

been of

mediates

(c)

constituent

faciconsists

which

or

the

protein

preparation

amounts

protein

the

same

the

of

plasma

on

the

(b)

PC exchange;

that

Preliminary catalyzes

between rat

time. it

The

exchanged

protein

LCAT:

preparation (b).

moieties

contains

amounts,

one

sufficiently

transfer

unit

stoichiometric

least

active

CE is

CE per

transfer

alternative

since

purified

equimolar

is

The

carbohydrate of

complex;

facilitates

and

of

support

protein

in

complex

content data

for

PC:CE

which

HDL,

in

accounted (mol/mol)

of

also

phospholipid steps.

it

contains

PC and

wt/wt).

four

amphipathic: it

protein

present

one

case

partially

relatively

a 1:l

of

of

through

preparation

determined

3:5,

lipoprotein

the

is

as

proteins

PC and

unit

Since

preparation

(ratio

of

purified

active

satisfactorily

transfer

at

most

cholesterol

be

is and

amounts

albumin

may

The

exchange

copurify

~150,000,

equimolar the

facilitate

phenyl-Sepharose

conA. of

which

lipoproteins

weight to

by

plasma

between

molecular

hydrophobic which

human

the requires

Furthermore, CE exchange PC transfer is

an

apo-

Vol.

93, No.

protein tates

of HDL.

The fact

PC transfer

the existence data

BIOCHEMICAL

4. 1980

functions

uncoupled

of distinct

suggest

that

a small

in lipid

the

from

for

formation

for

of cholesterol

BIOPHYSICAL

partially

RESEARCH

purified

rat

CE transfer,

though

PC and CE transfer

proteins.

present

complex,

This

homeostasis.

responsible removal

that

AND

in low

complex

and redistribution from

preparation

not

facili-

unequivocal, Taken

may represent

supports

together,

steady-state

of plasma

lipoproteins

COMMUNICATIONS

the

concentration, the catalytic

cholesteryl

unit

esters

and

and tissues.

ACKNOWLEDGMENTS This work was supported by U.S. Public Health Service grants HL 20882, 22619 and 23019, by the American Heart Association, by the Lipid Research Clinic Program (NIH NHLBI 72-2914) and by General Clinical Research Center grant RR-00068-15. J.E. is supported by NIH Training Grant HL 07382. We the manuscript acknowledge the assistance of Ms. Janet Simons in preparing for publication and of Ms. Gwen Kraft in preparing the figures. REFERENCES 1. 2. 3. 4. 5. 6.

Nichols, A.V., and Smith, L. (1965) J. Lipid Res. 6, 206-210. Zilversmit, D.B., Hughes, L.B., and Balmer, J. (1975) Biochim. Biophys. Acta 409, 393-398. Pattnaik, N.M., Montes, A., Hughes, L.B., and Zilversmit, D.B. (1978) Biochim. Biophys. Acta 530, 428-438. Pattnaik, N.M., and Zilversmit, D.B. (1979) J. Biol Chem. 254, 2782-2786. Sniderman, A., Teng, B., Vezina, C., and Marcel, Y.L. (1978) Atherosclerosis 31, 327-333. Chajek, T., and Fielding, C.J. (1978) Proc. Natl. Acad. Sci. USA 75, 3445-3449.

11. 12.

Kostner, G.M. (1974) Biochim. Biophys. Acta 336, 383-395. McConathy, W.J., and Alaupovic, P. (1976) Biochemistry 15, 515-520. Barter, P.J., Rajaram, O.V., and White, G.H. (1979) Circulation 60 (Suppl. II), 117 (Abstract). Brewster, M.E., Ihm, J., Brainard, J.R., and Harmony, J.A.K. (1978) Biochim. Biophys. Acta 529, 147-159. Hui, D.Y., and Harmony, J.A.K. (1979) Biochim. Biophys. Acta 550, 407-424. Chung, J., Abano, D.A., Fless, G.A., and Scanu, A.M. (1979) J. Biol. Chem.

13.

Aron,

7. 8. 9.

10.

254,

7456-7464.

L.,

Jones,

S.,

and Fielding,

C.J.

(1978)

J. Biol.

Chem. 253,

7220-

FEBS Letters

94,

7226.

14.

Jackson,

R.L.,

Westerman,

R.L.,

Wilson,

J.,

and Wirtz,

K.W.A.

(1978)

38-42.

15.

Jackson, 557,

16. 17.

Barter, Barter,

D.,

and Glueck,

C.J.

(1979)

Biochim.

Biophys.

Acta

79-85.

P.J., P.J.,

and Jones, and Lally,

M.E. J.I.

(1979) (1978)

1120

Atherosclerosis 34, 67-74. Biochim. Biophys. Acta 531,

233-236.