thiolation of low-density lipoproteins and their interaction with l2c leukemic lymphocytes

Biochhnie, 68 (1986)723-730 © Soci~t6 de Chimie biologique/Elsevier, Paris

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Thiolation of low-density lipoproteins and their interaction with L2C leukemic lymphocytes

Michel VIDAL I*, Josette SAINTE-MARIE I, Jean P H I L I P P O T l and Alain BIENVENUE 2

IC.N.R.S. U.A. 530: 1NSERM U 58, 60 rue de Navacelles, Montpellier 34100, 2U.S.T.L. Laboratoire Biologie Physico-Chimique, Place Ettg~ne Bataillon, Montpellier 34060, France

(Received 12-9-1985, accepted after revision 29-1-1986)

S u m m a r y - We present here, a new method for coupling sulfhydryl groups (SH) to low-density lipoprotein (LDL) surface. This method uses homocysteine thiolactone (HCTL) which reacts with lysine residues in a very mild manner, and permits the selection of the number of SH bound per LDL. Under our experi- mental conditions (8 SH/LDL) , the affinity of thiolated LDL for the specific receptors and their further internalization by L2C lymphocytes are preserved.

uptake / LDL / binding / lymphocytes / thiolation

R~sum~ - Fixat ion de groupements su l fhydry ls h la surface de lipoprot~ines de faiblc dcnsit~ et leur interaction avec des i y m p h o c y t e s leuc~mique L2C. Nousprdsentons tme mdthode de fixation de groupements sulfhydryls (SH) ~ la surface de lipoprotdines de faible densitd (LDL). Cette mdthode, uti- lisant 17zomocyst~ine thiolactone (HCTL), est trbs douce, et permet de choisir le nombre de SH fixds par LDL. Pour le nombre de SH introduits par LDL (8 SH/LDL) utilisd dans nos experiences, la capacitd des lipoprotdines ~ selier ft leurs r6cepteurs sp~cifiqttes est prdservde, comme le montre les expdriences effec- tu#es sttr des lymphocytes de cobayes leucdmiques L2C. Ces cellnles possbdent des rdcepteurs spdcifiqttes des LDL et sont capables d'internaliser et de ddgrader de la m6me manibre les LDL natives ott thioldes.

internalisation I LDL I liaison spgcifique I lkwlphoo'tes I thiolation

Introduction

Low-density lipoprotein (LDL), the major choles- terol transport lipoprotein in human plasma, enters certain cell types by binding to specific surface receptors that mediate its cellular uptake and transport to the lysosomes [1]. Low density lipoproteins have been used for drug targeting. High-affinity LDL-specific receptors are good targets on cell sur- faces, because of their affinity (Ka about 10-SM) for LDL [2, 3], their presence on certain cell types

directly exposed to LDL, such as lymphocytes [1], and their numbers on the cell surface (21 000 on leukemic L2C guinea pig lymphocytes [3]). Previous attempts have been made to administer drugs by the LDL receptor pathway [4-6]. Nevertheless, the drugs have to be hydrophobic enough to be incor- porated into the LDL core. Thus, the choice of drug has been limited and the use of macromolecules has not been feasible.

We have developped a method for targeting via the LDL receptor pathway, by covalent bonding

* To whom reprint requests should be sent. Abbreviations: LDL: low-density lipoprotein ; Stl: sulfhydryl; tlCTL: homocysteine thiolactone; DTN3: dithio-nitrobenzonic acid; LDFCS: lipoprotein deficient fetal calf serum; BSA : bovine serum albumin; DTT: dithiothreitol.

724 M. Vidal et al.

between LDL and targeted molecules. We coupled sulfhydryl groups to LDL, taking care not to inter- fere with the affinity of the lipoproteins for their specific receptors; modification o f lysyl [7] or arginyl residues [8] has been reported to be highly injurious to the ability of LDL to bind to their high- affinity receptors, depending upon the method used. The sulfhydryl groups can form disulfide bonds with liposomes [9] or directly bond with targeted molecules, such as A chain toxins.

In this paper, we describe a method for the covalent coupling of sulfhydryl groups to the surface of LDL, using homocysteine thiolactone (HCTL) to modify the lysyl residues of the LDL apopro- teins. The effects o f this modification on the binding, uptake, and degradation of LDL by leukemic lymphocytes are then assessed.

Mater ia ls and m e t h o d s

precipitated by incubation with 10°70 trichloracetic acid and less than 2% was extracted in ethanol/ether at 4°C. The ~25I-labeled LDL, with a specific radioactivity range of 200-300 cpm/ng of protein, was sterilized by Milli- pore filtration, stored at 4°C, and used within two weeks.

LDL thiolation The LDL protein moiety was thiolated using the method of White [13] slightly modified (Fig. 1). LDL was dialyzed for 24 h against a 100 mM nitrogen-saturated phosphate buffer, pI-:I 8.2. Variable amounts of homocysteine thiolactone (HCTL) were added to 8 mg of LDL protein, to obtain increasing HCTL/LDL molar ratios. The solutions were kept under a nitrogen atmosphere at room temperature throughout the reaction period.

HCTL

Fig. 1. Reaction scheme of LDL thiolation by HCTL.

Materials

Na~2SI was obtained from Amersham France (Les Ulis), homocysteine thiolactone (HCTL) and dithio- nitrobenzoic acid (DTNB) were provided by Fluka AG (Buchs, Switzerland), dibutyl phthala[e was purchased from Kodak (Rochester, NY, U.S.A.). Gel filtrations were performed on PD10 Pharmacia columns (Bois d'Arcy, France). Lymphoprep, Hanks' buffer, and RPMI 1640 were obtained from Flobio (Courbevoie, France). Fat-free bovine serum albumin (BSA) came from Sigma Chemical Co. (St. Louis, MO, U.S.A.).

Methods

Isolation o f LDL Human LDL (density 1.019-1.063 g/ml) obtained from the plasma of healthy subjects (the Montpellier blood bank) was prepared by sequential ultracentrifugation according to the method of Havel [10]. The LDL was resuspended and recentrifuged once using an NaC1-KBr solution (density = 1.063 g/ml). It was then dialyzed for 48 h against 150 mM NaCI, I0 mM Tris-HC1 (pH 7.4), containing 0.4°7o EDTA, and 0.01% NAN3, and subse- quently sterilized by filtration through a Millex G.V. fil- ter unit (0.22 tzm) from Millipore S.A. (Molsheim, France), and stored at 4°C. Protein concentration was measured by the method of Lowry et aL [11] using bovine serum albumin as a standard.

Lipoprotein-deficient fetal calf serum (LDFCS) was prepared as previously described [3].

Iodination LDL was iodinated with t25I, using the monochloride method of McFarlane as modified by Bilheimer [12]. In all preparations, more than 95% of the radioactivity was

After reaction for the desired length of time, LDL was isolated by applying the reaction mixture to a Sephadex G-25 column (PD10 from Pharmacia) equilibrated with 100 mM phosphate buffer, pH 8.2. The number of sulfhydryl groups coupled per LDL molecule was calculated on the basis of the protein concentration, and a determination of the SH groups with Ellman's reagent (DTNB) [14] in the fractions eluted in the void volume.

The native and thiolated LDL were characterized by paper electrophoresis and negative staining electron microscopy according to Chapman [15].

In the binding and uptake experiments, the thiolation was performed on LDL, in the presence of [t25I]LDL (final specific radioactivity: 20-30 cpm/ng).

Cells The L2C leukemia affecting the cells used in this study, arose spontaneously in a strain 2 guinea pig [16] and was serially passaged in syngeneic animals. L2C lymphocytes were harvested and purified by Lymphoprep gradient centrifugation, as previously described [17]. The cells were washed (× 6) in Hanks' balanced salt solution, and again washed in RPMI 1640, 30 mM 4-(2-hydroxyethyl)- 1-piperazine ethane-sulfonic acid (Hepes), pH 7.4, containing 100 units of penicillin and 0.1 mg of streptomycin per ml. They were resuspended in this medium at 5 x 106 cells/ml and used immediately or after overnight storage at 4°C. Cell viability, as assessed by trypan blue dye exclusion, was routinely determined for each lymphocyte preparation and was always greater than 96070.

SH-LDL affinity f o r the LDL receptors The binding of lipoproteins at 4°C was performed according to the method of Ho et al. [18] as modified by Sainte-Marie [3]. Lymphocytes were harvested by cen-

In v i t ro nletabolism o f thiolated L D L 725

trifugation (1200 rpm, 5 min, Beckman Microfuge model 11) and washed once with 10 ml of Dulbecco's phosphate- buffered saline (PBS) containing 20 mg/ml of fat-free bovine serum albumin (BSA), 150 tzM CaC! 2 (medium A), and resuspended in the same ice-cold buffer at a final concentration of 2.5 × 10 s cells/ml. Aliquots (20/zl) of this cell suspension were placed in 1 ml polyethylene microfuge tubes. Indicated amounts, 10-80 t~g of protein of ~25I-labeled LDL, thiolated or not, were added with or without an excess of unlabeled native LDL (1 mg of protein), and the final volume was adjusted to 100 gl with medium A.

After I h of incubation at 4°C, the cells were harvested by centrifugation (3000 rpm, 3 rain) and the pellets were washed twice with I ml of PBS containing 5 mg/ml BSA, pH 7.4. The cells were resuspended in 80tzl of the same buffer and passed through a water-impermeable layer of dibutyl phthalate in 16070 sucrose [19] by centrifugation (1300 rpm, 1.5 min). After centrifugation, the tubes were plunged into liquid nitrogen and their bot- toms were cut off, counted in a gamma-counter (Kon- tron MR 252), dissolved in 500/~1 0.1 N NaOH, and the protein content was measured by the method of Lowry et aL The results are expressed as nanograms of 1551- l'abeled LDL protein bound per mg of total cellular protein. The specific binding of LDL corresponds to the total binding (absence of unlabeled LDL) minus the nonspecific binding (incubation in the presence of an excess of unlabeled LDL).

In displacement experiments, indicated concentrations of [125I]LDL (58/~g of protein/ml final) were incubated with 107 cells for 1 h at 4°C, and increasing concentrations (0 .5-2 mg of protein/ml) of thiolated or native unlabeled LDL were added, bringing the final volume to 100/zl. Amounts of [I25I]LDL bound to the cells were determined as described above.

Uptake and degradation The method was identical to that described above, but the incubation medium was: RPMI 1640 containing 10°70 lipoprotein-deficient fetal calf serum (LDFCS), 5 mg BSA, 150/~M CaCI 2, 100 U penicillin, and 0.I mg/ml streptomycin (medium B). The ceils were incubated in this medium with 20/~g of ~5I-labeled native LDL, or 50 ~g of radioactive thiolated LDL, in the presence or absence of an excess of native unlabeled LDL (50- to 100-fold). At various times, the medium was assayed for degradation of ]2SI-labeled LDL according to the method of Goldstein and Brown [20]. Briefly, the medium was removed by centrifugation, and added to 0.25 ml 50070 (w/v) trichloracetic acid, incubated for 30 min at 4°C, and the precipitate was removed by centrifugation. 7.5/~1 of 40070 (w/v) KI and 30 tzl of 30070 H202 were added to a 0.75 ml aliquot of the superna- tant. After incubation at room temperature for 5 min, the mixture was extracted with 1.5 ml of CHCI 3 to remove free iodine. 300 t~l of the aqueous solution was removed for scintillation counting in a gamma counter. In all experiments, parallel incubations without cells were performed to determine the spontaneous degradations of 125I-labeled LDL, and these values were subtracted from

the results. The cellular pellets were washed and isolated as described above, and counted for a determination of ~25I-labeled LDL uptake.

Results and Discussion

Stoichiometry of LDL thiolation

I nc uba t i on o f h u m a n L D L with H C T L resul ted in a cova len t coupl ing o f su l fhydry l g roups to l ipo- p r o t e i n s , w h i c h was p r o p o r t i o n a l to t h e H C T L / L D L m o l a r ra t io (Fig. 2) and var ied with the t ime o f incuba t ion , as shown in Fig. 3. Th io la - t ion occurs by a m i n o lysis o f the th io l ac tone b o n d o f H C T L . The new homocys t e ine res idue the reby becomes a t t ached t h rough a pep t ide b o n d to the ni trogefi o f a lysine res idue (Fig. 1).

The s to ichiometry o f sulfhydryl (SH) groups cou- p led per mole o f L D L was ca lcu la ted assuming a mo lecu l a r weight o f 500 000 for t h e p r o t e i n moie ty

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Fig. 2. Coupling of sulfhydryl groups to human LDL as a function of the HCTL/LDL molar ratio. The reaction mixtures containing human LDL (8 mg of protein per ml) in 100 mM nitrogen-saturated phosphate buffer, pH 8.2, and variable amounts of HCTL were kept in a nitrogen atmosphere for l h at room temperature. The LDL were isolated by applying the incubation mixture to a Sephadex G-25 column. The protein con- tents and the SH concentrations were determined by the methods of Lowry et aL and Ellman, respectively.

726 M. Vidal et al.

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Fig. 3. Kinetics of sylfhydryl group coupling to human LDL. The reaction mixtures containing human LDL (8 mg of protein per ml) and HCTL at an HCTL/LDL molar ratio of 500 were kept at room temperature for the indicated period. The determination of the SH groups coupled to LDL was performed as in Fig. 2.

of LDL [21]. The sample containing 1500 mol of HCTL per mol of LDL and kept for 1 h at room temperature resulted in the coupling of about 40 moI of SH groups per mol of LDL. At a three- fold lower HCTL/LDL molar ratio and for 30 min of reaction, about 5 mol of SH were bound per mol of LDL. A limiting value of approximately 9 SH per LDL was approached at 60 min. This level of thiolation was used in the study of the binding acti- vity of the modified LDL, because some LDL aggregation occurred at high sulfhydryl group concentrations. This cross-linking reaction, reversed by dithiothreitol (DTT), was probably due to a disulfide bond formation.

Characteristics o f thiolated LDL

At this level of thiolation (about 8 SH/LDL) and using nitrogen-saturated buffer, the reaction of human LDL with HCTL did not alter the physical properties of the lipoproteins. On paper electrophoresis, both untreated and thiolated LDL had typical t3 mobilities (data not shown). Thus, the net charge on the LDL was not significantly altered by reaction with HCTL.

As seen by negative-staining electron microscopy, the untreated and thiolatcd LDL had the same size (about 20 nm) and had the same morphological appearance (Fig. 4). Moreover, gel filtration on a Biogel A-15 m column produced the same elution pattern for both LDL (Fig. 4). Thus, no aggregation occurred at this SH/LDL molar ratio.

Specific binding o f thiolated LDL to L2C cell receptors

In a previous study, we had demonstrated that leukemic L2C lymphocytes have specific high-affinity receptors on their cell surface which bind and inter- nalize LDL [3]. The number of sites per cell is greatly increased (about 10-fold) in leukemic lymphocytes (21000+7000) compared to normal cells (2100+400). The affinity constant is of the same order of magnitude for both cell types: K d = 7 × 10 -8 M. The extent of LDL degradation in leukemic Iymphocytes is also increased (× 1.5) compared to normal cells, even though this increase is not proportional to the number of sites per cell. Under the conditions previously described [8], we examined the ability of native or thiolated human LDL to compete with human t25I-labeled LDL for binding to LEC lymphocytes.

As illustrated in Fig. 5, thiolated LDL produced a typical competitive displacement curve for the binding at 4°C, as did untreated LDL. When the concentration of unlabeled LDL was increased, comparably less and less [t2SI]LDL was bound to the cells, regardless of the kind of unlabeled LDL. A 40-fold excess of native or thiolated LDL dis- placed about 70% of the bound [t25I]LDL. This represents the specific binding for the high,affinity receptor sites. Thus, the treatment of human LDL with HCTL (8 tool of SH per mol of LDL) did not inhibit the ability of this lipoprotein to compete with normal LDL, which in turn shows that this lipoprotein can bind to the LDL-specific receptors of leukemic lymphocytes.

These results do not conflict with previous reports. Weisgraber et aL have demonstrated that about 95°-/0 of the binding ability is preserved when 0.8 out of 20 lysines are modified (assuming 250 amino acid residues/mol of protein) [7]. This corresponds to nearly 10 residues per LDL, as in our experiments. Nevertheless, these displacement studies did not provide information on the nonspecific binding of thiolated LDL. Thus, we performed binding studies of [125I]thiolated LDL to quantify the extent of nonspecific binding by treated LDL. The experiments were carried out with increasing

In vitro metabolbrm of thiolated LDL

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728 M. Vidal et al.

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Fig. 5. Ability of untreated LDL (o) and thiolated LDL (o) to compete with native human [~:~I]LDL for binding to leukemic L2C lymphocytes. In each sample there was a final concentration of 58 vg of protein/ml of human [12SI]LDL (192 cpm/ng of protein), and unlabeled lipoproteins at the indicated concentrations. Samples were incubated with 107 cells for 1 h at 4°C.

amounts of []25Ilthiolated LDL [12SI]LDL ( I 0 , 80/~g of protein/ml). The incubations were performed at 4°C.

As illustrated in Fig. 6, the amounts of labeled LDL (native or thiolated) bound to cells were comparable. Moreover, the quantities of thiolated LDL nonspecifically bound were not different from those of native LDL. Thus, thiolation of LDL does not increase the nonspecific binding of the lipoproteins.

Uptake and degradation o f thiolated L D L

The in vitro metabolism of thiolated LDL was examined by measuring the uptake and degradation of [12sI]thiolated LDL by L2C lymphocytes. The experiment in Fig. 7 was carried out with a constant concentration of native or thiolated [nSI]LDL (20 and 50/zg of protein/ml, respectively) and shows the kinetics of the amounts of native (A) or thiolated (B) labeled LDL taken up and degraded by cells.

Uptake at 37°C (i.e., binding plus internalization) increased as a function of time. The efficiency of uptake and degradation were in the same order of magnitude with [125I]thiolated and native LDL, taking into account the concentrations of labeled lipoproteins. The degradation curves show a 2 h lag phase for both LDL types, as described in a previous paper [3]. These results, in combination with the physical properties and binding studies, indi- cate that the thioIation of LDL by HCTL is a mild reaction.

It has previously been established that the interaction of LDL with the receptor requires the presence of both arginine and lysine within the recognition site; the chemical modification of either residue can prevent the binding, and consequently, the internalization of lipoproteins. Moreover, Weis- graber et aL have demonstrated that the affinity of modified LDL depends upon the kind of chemical modification of the amino acid residues [8]. With the same number of modified lysine residues, the best percentage of original binding was observed in the studies of LDL modified by reductive methy- lation, i.e., a reaction that does not alter the net

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Fig. 6. Specific binding of native (e) or thiolated (o) human [~251]LDL to leukemic L2C lymphocytes as a function of labeled LDL concentration. 5× 106 cells were incubated for 1 h at 4°C with the indicated concentrations of labeled LDL (260 cpm/ng of protein for native LDL, 36 cpm/ng of protein for thiolated LDL), in the presence or absence of a 100-fold excess of unlabeled native LDL. Binding was determined as described in Materials in Methods. Each point is the average of a triplicate experiment. Vertical bars represent :i:S.D.

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Time (,hi Time (h ) Fig. 7. Time course of uptake and degradation of native (A) or thiolated (B) [nSl]LDL by leukemic L2C lymphocytes at 37°C. 5 x 106 cells in 1 ml of medium B were incubated with 20.ug of native (240 cpm/ng of protein) or 50 ,ug of thiolated (25 cpm/ng of protein) labeled LDL, in the presence or absence of 100-fold excess of unlabeled native LDL. At the indicated times, the medium was removed from triplicate samples and the degradation (o) measured in terms of the content of [nSI]LDL acid soluble material [20]. The specific uptake (o) was calculated from the cellular content of tz~I radioactivity measured in the absence or presence of an excess of LDL.

charge o f the l ipopro te in . 61% o f the or iginal b in- d ing was recovered when the n u m b e r o f lysines m o d i f i e d was 1.7 ou t o f 20 (assuming 250 a m i n o ac id r e s i d u e s / m o l o f prote in) , which co r re sponds to a b o u t 20 lysyl residues per L D L . The n u m b e r o f lysine residues mod i f i ed by H C T L in our expe- r iments was a b o u t 2 - 3 t imes smal ler . Thus , the mos t f avo rab le cond i t ions o f th io la t ion were used in our s tudy, bo th in terms o f the number o f modi - f ied res idues , and o f the m e t h o d o f mod i f i ca t ion .

The presen t s tudy, which does not d isagree with the d a t a ci ted above , indicates tha t th io la ted L D L (8 S H / L D L ) are me tabo l i zed hz vitro to the same extent as nat ive L D L via the h igh-af f in i ty L D L - specific receptor pa thway. Thus, thiolated LDL can be used as a car r ie r o f cova len t ly b o u n d drugs o r l iposomes to cells possessing L D L receptors . These th io la ted L D L have been used for coupl ing to l iposomes and subsequent ta rget ing o f this complex t o w a r d LzC lymphocy tes [9].

Acknowlegdements

This work was financed by grants from PIRMED (DA No. 175), the Association pour la Recherche sur le Can- cer, the Fondation pour la Recherche M6dicale, the In- stitut National de la Sant6 et de la Recherche M~dicale and the Centre National de la Recherche Scientifique.

References

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2 Brown M.S. & Goldstein J.L. (1974) Proc. Nail Acad. Sci. USA 71 ,788-792

3 Sainte-Marie J., Vidal M., Philippot J. & Bienvenue A. (1985) J. Receptor Res. 5, 171-192

4 Mosley S.I., Goldstein J.L., Brown M.S., Falck J.R. & Anderson R.G.W. (1981) Proc. NatL Acad. Sci. USA 78, 5717-5721

730 M. Vidal et al.

5 Vitols S., Gahrton G. & Peterson C.D. (1984) Can- cer Treat. Rep. 68, 515-520

6 Rudling M.J., Collins V.P. & Peterson C.D. (1983) Cancer Res. 43, 4600-4605

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9 Vidal M., Sainte-Marie J., Philippot J. & Bienvenue A. (1985) EMBO J. 4, 2461-2467

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12 Bilheimer D.W., Eisenberg S. & Levy R.I. (1972) Biochhn. Biophys. Acta 260, 212-221

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14 Habeeb A.F.S.A. (1972)Methods Enz~vmoL XXVB, 457-464

15 Chapman M.J. & Mills G.L. (1977) Biochem. J. 167, 9-21

16 Congdon C.D. & Lorenz E. (1954) Am. J. PathoL 30, 337-339

17 Chuillon-Sainte-Marie J., Authier M.H., Cayzae M. & Philippot J.R. (1981) Eur. J. Bioehem. 117, 219-224

18 Ho Y.K., Brown M.S., Bilheimer D.W. & Goldstein J.L. (1976) J. Clin. Invest. 58, 1465-1474

19 Noel J.G., Hui D.Y., Blankenship D.T. & Harmony J.A.K. (1981) Biochhn. Biophys. Acta 665, 519-530

20 Goldstein J.L. & Brown M.S. (1974) J. Biol. Cllem. 254, 5133-5162

21 Watt R.M. & Reynolds J.A. (1981) Biochemistry 20, 3897-3901

thiolation of low-density lipoproteins and their interaction with l2c leukemic lymphocytes

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