127 Biochimica et Biophysics Acta, 1044 (1990) 127-132

Elsevier

BBALIP 53392

Characterisation of the LDL receptor in Epstein-Barr virus transformed lymphocytes

Paola Lombardi ‘, Elly de Wit ‘, Rune R. Frants 2 and Louis M. Havekes 1

’ Gaubius Institute TN0 and ’ Department of Human Genetics, State University Leiden, Leiden (The Netherlands)

(Received 15 November 1989)

Key words: LDL; Lipoprotein receptor; (Human lymphocyte)

Non-dividing human lymphocytes were transformed upon infection with the Epstein-Barr virus (EBV) into lymphoblasts which are capable of continuous growth in culture. We studied the properties of the LDL receptor in EBV-transformed human lymphocytes (EBV-L) by binding experiments and by ligand blotting. EBV-L show a high affinity binding of LDL in the same order of magnitude as found with fibroblasts; EBV-L obtained from a homozygous familial hypercholesterolemic (FH) patient fail to express LDL receptor activity. Similar to that of fibroblasts, the LDL receptor activity in EBV-L is Ca*‘- dependent and is down-regulated by the presence of an exogenous source of cholesterol in the medium. The LDL receptor protein of EBV-L has an apparent molecular weight of 130000. Since our results show that EBV-L display a LDL receptor protein similar to the LDL receptor present in fibroblasts, we conclude that in comparison with other cell types the EBV-L offer a suitable model system to investigate LDL receptor protein abnormalities in FH patients.

Introduction

Cells require cholesterol for membrane synthesis. In most cell types cholesterol is acquired either from en- dogenous synthesis or, preferentially, from circulating LDL through a receptor-mediated pathway. This path- way involves high-affinity surface binding, intemalisa- tion by endocytosis and lysosomal degradation of LDL

[ll. Familial hypercholesterolemia (FH) is an inherited

disorder in which the uptake of LDL by the LDL receptor is impaired, leading to an accumulation of LDL in the plasma and consequently to premature

coronary artery disease [2]. FH originates from mutations within the LDL recep-

tor gene, resulting in a defective receptor synthesis, or

processing, or in an inability of the receptor to bind or internalise LDL [3-91. Restriction fragment length

polymorphism (RFLP) analyses used in these studies revealed that mutations in the LDL receptor gene are widely heterogeneous. Furthermore, by RFLP analysis, only large rearrangements in the gene can be detected.

Abbreviations: LDL, low-density lipoprotein; EBV-L, Epstein Barr

virus transformed lymphocytes; PHA, phytohemagglutinin; FH,

familial hypercholesterolemia.

Correspondence: L. Havekes, Gaubius Institute TNO, P.O. Box 612,

2300 AP Leiden, The Netherlands.

Point mutations or small deletions/ insertions repre- senting more than 90% of all LDL receptor mutations

[9] are difficult to detect by simple DNA techniques, unless more laborious methods, such as DNA sequence analyses, are applied. Therefore, screening for LDL receptor mutations in a large population sample needs complementary approaches. We suggest that additional

(small) mutations could be revealed by studying the LDL receptor polymorphism on the protein level by

isoelectric focusing and/or by two-dimensional electro- phoresis. Furthermore, mutations affecting the rate of

synthesis or the rate of conversion of the LDL receptor protein from the precursor into the mature form could

be characterised by pulse-chase experiments followed

by immunoprecipitation, SDS-polyacrylamide gel elec-

trophoresis and autoradiography [lo]. The study of the LDL receptor protein abnormalities

requires an established cell line from each subject under investigation. Therefore, a suitable cell line should fulfil the following conditions: (i) primary cultures should be easily isolated; (ii) the cell lines, once established, should be maintained in culture for long periods of time or

stored without loss of viability; and (iii) the cell line should be able easily to provide large amounts of LDL receptor protein.

At present, cultured skin fibroblasts represent the most used cell line for the characterisation of LDL receptor mutants. However, because of the number of advantages Epstein-Barr virus transformed lymphocytes (EBV-L) have over fibroblasts (continuous cell culture

0005-2760/90/$03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)

128

for a long period of time without senescence, short doubling time, growth in suspension), we wondered

whether EBV-L could offer an alternative to fibroblasts to study LDL receptor protein abnormalities in FH

patients. Moreover, lymphocytes are readily available and easily transformed upon infection with the EB virus

1111. The aim of the present study was to characterise the

properties of the LDL receptor activity in EBV-L. By LDL binding studies and by ligand blotting experi-

ments we found that the LDL receptor in EBV-L shows the same characteristics as demonstrated in fibroblasts

[12,13].

Materials and Methods

Transformation of lymphocytes with the Epstein-Barr virus

10 ml of heparinised blood were diluted with the

same volume of balanced salts solution (BSS), layered on 15 ml of Ficoll Isopaque and centrifuged for 30 min

at 400 x g at room temperature. White blood cells were

then collected and washed at room temperature with 3 ~01s. of BSS by centrifugation for 10 min at 100 X g. If

residual red blood cells were present, cells were resus- pended in lysis buffer (155 mM NH,Cl, 10 mM KHCO,,

1 mM EDTA (pH 7.4)) and incubated for 2 min to lyse erytrocytes and then diluted at least lo-times with BSS. Lymphocytes were separated from lysing buffer by cen- trifugation, washed twice in 10 ml of BSS and counted.

The final pellet of white blood cells (approx. 1 . 10’

cells) was incubated for 1 h at 37” C with 6 ml of the Epstein-Barr virus solution, derived from the B95-8 Marmoset lymphoblastoid cell line [14]. Cells were then

harvested by centrifugation for 5 min at 100 X g resus- pended in RPM1 1640 medium supplemented with 15%

fetal calf serum (FCS) and 1% phytohemagglutinin (PHA, reagent grade, Wellcome) and plated in Multi-

well tissue culture clusters (Costar) at a density of about

1 . lo6 cells/ml. Cells were maintained at 37” C in 5% CO, atmos-

phere and refed with the medium containing PHA until they began to proliferate (after 2-6 weeks). EBV-L were then transferred to tissue culture flasks (50 ml, Costar) and maintained in RPM1 1640 medium, supplemented

with 15% FCS.

Cell culture Human skin fibroblasts were cultured in 24-well dis-

hes in Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with penicillin (100 IU/ml), streptomycin (0.1 mg/ml) and 10% (v/v) heat-in- activated FCS.

EBV-transformed lymphocytes were grown in 650 ml flasks (Costar) in RPM1 1640 medium containing peni- cillin and streptomycin and supplemented with 15% FCS at a density of approx. 1 . lo6 cells/ml.

Lipoproteins LDL was isolated from serum of normolipidemic

donors by density gradient ultracentrifugation by the method of Redgrave et al. [15]. A narrow density frac-

tion (density 1.04-1.05 g/ml) was used. LDL was im-

mediately labelled with 125I (Amersham) to a specific activity of 80-120 cpm/ng, exactly as described in Ref. 16. After labelling, the LDL sample was dialysed and

stabilised as described previously [17]. The stabilised “‘1 labelled LDL was stored at 4°C and used within 2

weeks. Less than 1% of the radioactivity was soluble in 10% (v/v) trichloroacetic acid (TCA). When not labelled with ‘25 I, LDL was immediately stabilised with 1% HSA and extensively dialysed against subsequently phos- phate-buffered saline (PBS) and DMEM or RPM1 1640

supplemented with penicillin and streptomycin.

Lipoprotein-depleted serum (LPDS) was obtained by ultracentrifugation of serum at a density of 1.21 g/ml followed by extensive dialysis against subsequently PBS

and DMEM or RPM1 1640 supplemented with penicil-

lin and streptomycin.

j3-VLDL was separated by ultracentrifugation from the plasma of cholesterol-fed rabbits (d < 1.006 g/ml)

and labelled with ‘25I [16] to a specific activity of 100-200 cpm/ng. After labelling, P-VLDL was exten- sively dialysed against PBS. More than 99% of the radioactivity was precipitable with 10% TCA. 125I-j3-

VLDL was stored at 4°C and used within 1 week.

Binding studies

24 h prior to the experiments, cells were washed twice with PBS and then incubated with 10% LPDS

medium, or 10% LPDS medium containing different amounts of human LDL, as indicated in the figure legends. Washings of EBV-L were carried out by

harvesting and centrifugation for 5 min at 800 x g. Immediately before the binding experiment, cells were

washed again with the respective medium containing 1% HSA instead of 10% LPDS. Thereafter, the EBV-L were transferred to 1.5 ml Eppendorf tubes. To the 2 cm*

wells (fibroblasts) or Eppendorf tubes (EBV-L), the indicated amounts of 125 I-LDL were added in the pres- ence or absence of 300 pg of unlabelled LDL/ml.

After 3 h of incubation at 37” C, the medium was removed for determination of LDL degradation as de- scribed previously [17]. After removal of the incubation medium, cells were cooled to 0’ C and washed exactly as described by Goldstein et al. [18].

When total cell association of “‘1-LDL was mea- sured, the washed cells were dissolved in 0.2 M NaOH and an aliquot of the cell lysate was counted. Another aliquot of the cell lysate was used for the protein determination by the method of Lowry et al. [19] with bovine serum albumin (BSA) as a standard.

When ‘251-LDL binding and internalisation were measured separately, the washed cells were incubated

129

with 0.05% trypsin in buffer 0.15 M NaCl, 0.01 M

sodium phosphate (pH 7.4) and 0.02% EDTA at 37’ C

[20]. The radioactivity released into the trypsin buffer

reflects the amount of I*?-LDL bound to the cell membrane. The radioactivity remaining cell-associated represents the amount of ‘251-LDL internalized.

When the binding experiment was carried out at

4OC, the total amount of cell-associated ‘*?-LDL rep-

resents the amount of ‘251-LDL bound to the plasma

membrane. The amount of ‘251-LDL that was degraded, bound, or cell-associated after incubation in the absence

of unlabelled LDL, subtracted by the amount of 12?- LDL that became degraded, bound, or cell-associated in the presence of excess of unlabelled LDL, represents the

‘receptor-mediated’ or ‘high-affinity’ degradation, bind- ing or cell-association.

Ligand blotting Fibroblasts were seeded into 75 cm2 flasks (Costar)

and maintained as described above. EBV-transformed

lymphocytes obtained from the normal and the FH subject were maintained as described above in 650 ml

flasks (Costar) at a density of approx. 1 * lo6 cells/ml. After 24 h of pre-incubation with medium containing

10% LPDS, cell extracts were prepared according to the

method of Van Driel [21]. Briefly, cells from different flasks were combined, washed in buffer A (50 mM Hepes (pH 7.4) 100 mM NaCl, 1 mM PMSF, 0.5 mM

leupeptin, 10 mM EDTA, 10 mM EGTA, 10 mM N-ethyl maleimide, 2.2% DMSO) and solubilised in lysis

buffer (buffer A plus 1% Triton X-100). The soluble protein extracts were clarified by centrif-

ugation at 100000 X g for 1 hour and frozen im- mediately. The protein concentration of the extracts was

assayed by the BCA protein assay reagent (Pierce) for non-ionic detergent containing samples.

Cell extracts were subjected to SDS-polyacrylamide gel electrophoresis and ligand blotting was performed as

described by Daniel [22]. The dried blots were autora-

“‘I-LDL added (pg/ml)

2 50

“‘1-LDL added (pg/ml)

$ 2. Saturation curves of the high affinity binding of human

I-LDL to EBV-transformed lymphocytes (0) and fibroblasts (m).

Cells were pre-incubated in medium supplemented with 10% LPDS

for 24 h. Binding was measured after 3 h of incubation with “sI-LDL

at 4” C as described in Materials and Methods. Results are the mean

of triplicate assays+ SD.

diographed for 2-5 days using pre-flashed films and intensifying screens.

Results

Fig. la shows the receptor-mediated binding, inter-

nalisation and degradation of 12?-LDL by EBV-trans- formed lymphocytes form a healthy subject. Saturation is achieved at low LDL concentration (approx. 10

pg/ml), indicating that a high-affinity binding site is present. High-affinity binding, internalisation and de-

gradation of ‘251-LDL are not expressed in EBV-L derived from a homozygous patient (Fig. lb). Further- more, the high affinity uptake and degradation of ‘251-

LDL by EBV-L is totally inhibited by the presence of 3 mM EDTA in the incubation medium (data not shown), indicating that the LDL receptor activity in EBV-L is

‘“I-LDL added (pg/ml) Fig. 1. Receptor-mediated binding, intemalisation and degradation of human “’ I-LDL by EBV-lymphoblasts from a normal subject (a) and from a homozygous FH subject (b). Cells were pre-incubated in medium supplemented with 10% LPDS for 24 h. Binding (m), intemalisation (v) and degradation (0) were measured after incubation at 37 o C with ‘251-LDL-labelled LDL as described in the Materials and Methods section. Results

are the mean of triplicate measurements f S.D.

130

,

100 200 300

preincubation with LDL (W/ml)

Fig. 3. Effect of preincubation of fibroblasts (m) and EBV-L (0) with

increasing concentrations of LDL on LDL receptor activity. Cells

were incubated for 24 h in medium containing 10% LPDS and

different concentrations of LDL for 24 h as indicated. After washing

of the cells, high-affinity degradation was measured after 3 h of

incubation at 37’C with 10 pg/ml of ‘*‘I-LDL as described in

Materials and Methods. Results are expressed as percentage of con-

trol and they represent the mean of triplicate assays + SD.

Ca’+-dependent, as previously reported in fibroblasts

~31. In Fig. 2, the binding of ‘251-LDL to EBV-L is

compared with that of fibroblasts. The binding curves

showed that for both cell lines the binding of “‘1-LDL

reached saturation at lo-15 pg of ‘251-LDL added. In

I OO 5 10 15 20

hours after removal of LDL

addition, the two cell lines showed similar amounts of maximum ‘25 I-LDL binding.

Studies on human skin fibroblasts have shown that the expression of the LDL receptor activity is regulated

by the presence of cholesterol in the medium [23,24]. In order to compare EBV-L with fibroblasts in this re-

spect, we measured the receptor-mediated degradation of LDL after preincubation of the cells for 24 h in

medium supplied with increasing concentrations of

LDL. As shown in Fig. 3, the receptor activity declined with increasing concentrations of LDL during the prein-

cubation period. Maximal suppression occurred at 50 fig/ml of LDL with fibroblasts (80% inhibition) and at

80-100 pg/ml of LDL with EBV-L (approx. 50% in- hibition).

Fig. 4 shows the time-course of the up- and down- regulation of the LDL receptor activity in fibroblasts

and EBV-L upon depletion and addition of LDL, re- spectively. Both cell lines appeared to be similar in this

respect except that both the up- and down-regulation of the LDL receptor in fibroblasts were more pronounced.

Time-course experiments performed during a 48 h period demonstrated that a 20-24 h the maximal effect is achieved in both cell lines (data not shown).

To gain further information on the nature of the

LDL receptor protein in EBV-L as compared to that of fibroblasts, we performed ligand blotting experiments

after SDS-polyacrylamide gel electrophoresis of cellular membrane extracts. As shown in Fig. 5a, in extracts of

b

5 10 15 20

hours after addition of LDL

Fig. 4. Time-course of the up- and down-regulation of the LDL receptor activity in fibroblasts and EBV-L upon depletion and addition of LDL, respectively. For measuring upregulation (a), fibroblasts (W) and EBV-L (0) were incubated for 24 h in medium containing 10% LPDS and 300

pg/ml of human LDL. Thereafter, the cells were washed and incubated for the indicated time periods in the same medium but without LDL. For measuring down-regulation (b), fibroblasts @) and EBV-L (0) were incubated for 24 h in medium containing 10% LPDS. Thereafter, the cells were

washed and incubated for the indicated time periods with the same medium but supplied with 300 pg/ml of human LDL. High-affinity

degradation was measured after 3 h of incubation at 37 o C with 10 pg/ml ‘251-LDL as described in Materials and Methods. Results are expressed as percentage of control (time 0) and they represent the mean of triplicate assays + SD.

131

a

200-

116- 97-

b

116 - 97-

66-

1 2 3 4 5 6 123456 1234 56

116- 97-

66-

Fig. 5. Identification of the LDL receptor in fibroblasts, normal EBV-L and EBV-L obtained from a homozygous FH patient by ligand blotting.

Fibroblasts and EBV-L were preincubated for 24 h in medium containing 10% LPDS; cell extracts were subjected to SDS-PAGE followed by

electrophoretic transfer to nitrocellulose membranes as described in Materials and Methods. The nitrocellulose blots were incubated with: (A)

rabbit ‘Z51-/3VLDL (4 ng/ml); (B) ‘*‘I-/3-VLDL plus 10 mM EDTA; (C) ‘*%fi-VLDL plus a 30-fold excess of unlabelled PVLDL. Lanes 1-2,

fibroblasts (SO-100 ng of protein, respectively). Lanes 3-4, normal EBV-L (50-100 pg of protein, respectively). Lanes 5-6, EBV-L from a

homozygous FH patient (SO-100 pg of protein, respectively). The positions of the molecular mass standards are indicated.

EBV-L we detected a LDL receptor protein with ap- parent molecular weight of approx. 130000 (lanes 3-4) identical to that detected in human fibroblasts (lanes l-2). No bands were visible in the extracts of EBV-L

obtained from a homozygous FH patients (lanes 5-6). The presence of 10 mM EDTA during the incubation

partially inhibited the binding of iz51-rabbit PVLDL (ligand) to the receptor protein (Fig. 5b). When the

ligand blotting was performed in the presence of an excess of unlabelled PVLDL, no bands were detectable

(Fig. 5~).

Discussion

We presented several lines of evidence that EBV- transformed lymphocytes display LDL receptor activity similar to that of fibroblasts: (i) EBV-L show binding

saturation curves in the same order of magnitude as that of fibroblasts; (ii) EBV-L obtained form a homo- zygous FH patient fail to express LDL receptor activ- ity; (iii) the LDL receptor in EBV-L is sensitive to

EDTA and is regulated by the amount of LDL present in the medium; and (iv) the LDL receptor protein of

EBV-L has an apparent molecular weight of 130000, similar to that of fibroblasts. We did not directly com- pare the rate of conversion of the precursor into the mature form, nor the rate of synthesis of the LDL receptor protein, between both cell lines. However, our results show that in EBV-L the time-course of the up- and down-regulation of the LDL receptor activity upon changing the amount of LDL in the medium is almost the same as that found in fibroblasts. This strongly suggests that in both cell lines the turnover rate of the LDL receptor protein is comparable. The fact that the LDL-mediated regulation of the LDL receptor is less

pronounced than that found in fibroblasts is most prob- ably due to the relatively high growth rate of EBV-L as

compared to fibroblasts. To study the LDL receptor protein polymorphisms,

an established cell line from each subject under investi-

gation is required. A number of cell types could be used for this purpose. In addition to cultured fibroblasts

[24,25], LDL receptor activity has been shown to be present in human peripheral blood monocytes [26],

freshly isolated human lymphocytes [27] and mitogen- stimulated human lymphocytes [28]. For selecting the most useful cell line, a number of requirements should be considered. In particular, primary cultures should be

easily isolated; the cell lines, once established, should be maintainable in culture for long periods of time or storable without loss of viability and they should be

able easily to provide large amounts of LDL receptor

protein.

Data from literature [29] show that phytohemag- glutinin activates the proliferation of freshly isolated lymphocytes, transforming them into blast cells. Phyto-

hemagglutinin-stimulated lymphocytes, displaying T cell characteristics, proliferate effectively through a number

of cell cycles. It has been suggested that these cells represent a suitable model system for studying abnormalities in the LDL receptor protein [30]. How- ever, it has been shown that these cells display an optimal proliferation between the 2nd and 5th day after the activation; after that time the rate of proliferation declines rapidly, as the cells enter a lysis phase [31,32].

EBV-transformed lymphocytes, on the contrary, offer all the advantages of an established cell line. The estab- lishment of lymphoid cells is relatively simple and, once irnmortalised by EBV virus infection, EBV-L represent an unlimited and abundant source of material [ll].

132

Large amounts of LDL receptor protein can easily be obtained by EBV-L as these cells show a high growth rate.

Transformation of human lymphocytes with the Ep-

stein-Barr virus is a common technique for establishing lymphocytes for biochemical studies [33-351. The Ep- stein-Barr virus is a herpes-like DNA virus that is

strongly implicated in the pathogenesis of infectious

mononucleosis and is also associated with the Burkitt’s lymphoma [36]. However, EBV-L can be safely cul-

tured, since these cells carry the latent viral genomes

without supporting virus replication. Moreover, 70% of

the adult population develop antibodies against the Epstein-Barr virus before the age of 40 [37].

Since our results show that EBV-transformed lymphocytes display an LDL receptor protein similar to the LDL receptor present in fibroblasts, we conclude

that, in comparison with other cell types, the EBV- transformed lymphocytes offer a suitable model system

for evaluating LDL receptor protein abnormalities in

hypercholesterolemic patients.

Acknowledgements

These investigations are supported by The Nether- lands Heart Foundation (Project No. 89.057). Mrs. Clara

Horsting-Been and Miss Marisa Horsting are gratefully acknowledged for preparing the manuscript.

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