Jan 25, 1980 - Binding of Toxin to Liposomes. Liposomes containing. DMPC, Chol, and DCP were incubated with different quan- tities of DT. As shown in Fig.
Proc. Natl. Acad. Sci. USA Vol. 77, No. 4, pp. 1986-1990, April 1980
Biochemistry
Binding of diphtheria toxin to phospholipids in liposomes (membranes/receptors)
CARL R. ALVING*, BARBARA H. IGLEWSKIt, KATHARINE A. URBAN*, JOEL Mossf, ROBERTA L. RICHARDS*, AND JERALD C. SADOFF§ Departments of *Membrane Biochemistry and §Bacterial Diseases, Walter Reed Army Institute of Research, Washington, D.C. 20012; tDepartment of Microbiology and Immunology, University of Oregon Health Sciences Center, Portland, Oregon 97201; and *Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20205
Communicated by Roscoe 0. Brady, January 25, 1980
Diphtheria toxin bound to the phosphate porABSTRACT tion of some, but not all, phospholipids in liposomes. Liposomes consisting of dimyristoyl phosphatidylcholine and cholesterol did not bind toxin. Addition of 20 mol % (compared to dimyristoyl phosphatidylcholine) of dipalmitoyl phosphatidic acid, dicety phosphate, phosphatidylinositol phosphate, cardiolipin, or phosphatidylserine in the liposomes resulted in substantial binding of toxin. Inclusion of phosphatidylinositol in dimyristol phosphatidylcholine/cholesterol liposomes did not result in toxin binding. The calcium salt of dipalmitoyl phosphatidic acid was more effective than the sodium salt, and the highest level of binding occurred with li osomes consisting only of dipalmitoyl phosphatidic acid (carcium salt) and cholesterol Binding of toxin to liposomes was dependent on pH, and the pattern of pH dependence varied with liposomes having different compositions. Incubation of diphtheria toxin with liposomes containing dicetyl phosphate resulted in maximal binding at pH 3.6, whereas binding to liposomes containing phosphatidylinositol phosphate was maximal above pH 7. Toxin did not bind to liposomes containing 20 mol % of a free fatty acid (palmitic acid) or a sulfated lipid (3-sulfogalactosylceramide). Toxin binding to dicetyl phosphate or phosphatidylinositol phosphate was inhibited by UTP, ATP, phosphocholine, or pnitrophenyl phosphate, but not by uracil. We conclude that (a) diphtheria toxin binds specifically to the phosphate portion ofcertain phospholipids, (b) binding to phospholipids in liposomes is dependent on pH, but is not due only to electrostatic interaction, and (c) binding may be strongly influenced by the composition of adjacent phospholipids that do not bind toxin. We propose that a minor membrane phospholipid (such as phosphatidylinositol phosphate or phosphatidic acid), or that some other phosphorylated membrane molecule (such as a phosphoprotein) mayb important in the initial binding of diphtheria toxin to cells.
Previous attempts to characterize a membrane molecule to which diphtheria toxin (DT) specifically binds have been unsuccessful (1). Estimates of the number of receptors in cells differ (1, 2), and even presence of receptors in resistant cells has been debated (1, 3). Most investigators believe that a receptor does exist, but the alternative suggestion has been made that the toxin binds "nonspecifically" through electrostatic attraction to cells (4). Although the cellular receptor is often presumed to be a protein, evidence for this presumption is not conclusive. Duncan and Groman (4) reported that cytotoxicity caused by DT was not decreased by treating the target cells with various proteolytic, or other, enzymes. Moehring and Crispell reported that toxin-dependent inhibition of protein synthesis was decreased by treating cells with trypsin, Pronase, or phospholipase C (5). Based on indirect evidence with lectins it was proposed recently that the receptor is the oligosaccharide portion of an uncharacterized glycoconjugate (6). At present the membrane glycoconjugate binding properties of DT are hypothetical.
In this paper we demonstrate that DT is a phosphate-binding protein, and we propose that the receptor might include the phosphate portion of a "phosphoconjugate" (phospholipid, glycophospholipid, phospholipoprotein, or glycophospholipoprotein). This hypothesis would explain the previously described findings: (a) that the receptor is sensitive to phospholipase C (5), (b) that the receptor is present on cells resistant to toxin (3), (c) that binding of toxin to cells is inhibited by nucleoside phosphates (2, 7), and (d) that inhibition is related to the number of phosphate units per nucleoside phosphate (2, 7). As examples of membrane molecules to which DT might bind, we demonstrate binding to certain membrane phospholipids, and inhibition of binding by various simple phosphate compounds, including nucleoside phosphates.
MATERIALS AND METHODS Lipids were obtained from the following sources: dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidic acid (DPPA) (calcium salt), and phosphatidylinositol phosphate (PIP) (Sigma); DPPA (sodium salt) (Supelco, Bellefonte, PA); dilauroyl phosphatidylcholine (DLPC), dipalmitoyl phosphatidylcholine (DPPC), and cholesterol (Chol) (Calbiochem); distearoyl phosphatidylcholine (DSPC) and phosphatidylinositol sulfatide (Applied Science Laboratories, State College, PA); sphingomyelin (SM) and cardiolipin (Pierce); phosphatidylserine (PS) (Applied Science or Calbiochem); palmitic acid (Schwarz/Mann); dicetyl phosphate (DCP) (K & K). Inhibitors were obtained as follows: uracil (Schwarz/Mann); UTP (Sigma); ATP (Nutritional Biochemicals); p-nitrophenyl phosphate (Fisher Scientific, Silver Spring, MD); phosphocholine chloride (calcium salt) (Sigma). DT lot D 279 (1600 flocculating units per mg of nitrogen) was purchased from Connaught Medical Research Laboratories, Toronto, ON, Canada, and purified according to the procedure of Cukor et al. (8) as described (9). The toxin was stored at -70°C at 6.5-60 ,tg/dul in 0.01 M Tris buffer, pH 7.4. The purified toxin contained 20 guinea pig lethal doses per ,tg of protein and was homogeneous when examined by Na-
DodSO4/polyacrylamide gel electrophoresis. Multilamellar liposomes were prepared in 0.15 M NaCl by using a Vortex mixer as described (10, 11) and contained phospholipid (phosphatide) and Chol in molar ratio of 2 to 1.5. The phosphatide was 10 mM with respect to the final suspension in 0.15 M NaCl. When more than one phosphatide was present, the mole % of each was based only on the total phosphatide composition. DCP is a lipid phosphate that is not considered a Abbreviations: Chol, cholesterol; DCP, dicetyl phosphate; DLPC, DMPC, DPPC, and DSPC, dilauroyl, dimyristoyl, dipalmitoyl, and distearoyl phosphatidylcholine; DPPA, dipalmitoyl phosphatidic acid; DT, diphtheria toxin; PIP, phosphatidylinositol phosphate; PS, phosphatidylserine; SM, sphingomyelin.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1986
Biochemistry: Alving et al.
Proc. Natl. Acad. Sci. USA 77 (1980)
phosphatide, and calculation of liposomal concentration of-DCP was based on DCP plus total phosphatide. For analysis of protein binding (except as indicated in Fig. 1), excess DT (65-90,gg/mol of phosphatide) was incubated with liposomes (0.1-0.3 ml) for 30 min at room temperature in a total volume of 0.115-0.31 ml at pH 5-6. The liposomes were washed two or three times by centrifuging in ice-cold saline (10 ml) at 27,000 X g for 10 min at 4VC. When buffered conditions were used in the initial incubation (only in Figs. 2 and 3), 150 .ul of an isotonic barbital/acetate/HCI/NaCl buffer was employed, as described (12). Protein bound to liposomes was measured by a modification of the Lowry method, using rabbit gamma globulin as a standard, and specific binding was defined as micrograms of protein bound per umol of phosphate in the washed liposomes (13). RESULTS Binding of Toxin to Liposomes. Liposomes containing DMPC, Chol, and DCP were incubated with different quantities of DT. As shown in Fig. 1, binding did occur, and at low levels of added toxin binding was proportional to the amount of toxin added. At higher levels of added toxin (>10 ,ug/,gmol of DMPC) larger amounts of toxin were bound than were anticipated, suggesting cooperative binding or preferential binding to a subpopulation of liposomes. Specificity of Binding. From the above experiments it cannot be determined whether DT was specifically recognizing DCP or whether the toxin was attracted by the negative zeta potential caused by the presence of DCP in the membrane. Table 1 summarizes the binding of DT to various negatively charged or neutral liposomes. Liposomes lacking a net negative charge (DMPC/Chol) did not bind toxin. Likewise, liposomes containing either a free fatty acid (palmitic acid) or a lipid having a terminal sulfate group [sulfatide (3-sulfogalactosylceramide)] also did not bind toxin. Toxin binding occurred only when a lipid phosphate was present. When the lipid phosphate concentration was 20 mol % (relative to DMPG), there was a correlation between binding and the number, or degree of exposure, of phosphate groups. Thus, phosphatidylinositol and PS, each of which has a phosphate that is "covered" by another moiety (inositol or serine), either did not bind toxin or bound small amounts of toxin. Cardiolipin has two phosphates, both
1987
Table 1. Influence of liposome composition on binding of DT Specific
Liposome composition
binding*
1.4 DMPC/Chol DMPC/Chol plus 20 mol % of: Palmitic acid 2.6 2.6 Sulfatide DCP 33.6 20 Cardiolipin PIP 16 Phosphatidylinositol 0.9 PS 5.9 DPPA, calcium salt 37.6 11.1 DPPA, sodium salt PS/Chol 70.1 DPPA (calcium salt)/Chol 159 * Specific protein binding, fsg of toxin bound per Mumol of liposomal phosphate.
attached to glycerol, and the binding of toxin to cardiolipin was relatively high. Phospholipids that have "exposed" phosphates (mono- or diesterified), including DCP, PIP, and dipalmitoyl phosphatidic acid (DPPA), bound the highest amounts of toxin. There was a marked difference between the calcium and sodium salts of DPPA, the calcium salt resulting in increased binding. The reason for the greater efficacy of the calcium salt is unknown. When certain phospholipids were presented in 100 mol % concentration rather than 20 mol % (PS/Chol or DPPA/Chol), the binding of toxin was substantially increased, but binding was still correlated with the degree of exposure of the phosphate (DPPA > PS). Dependence on pH. The previous experiments were performed at a pH between 5 and 6. Fig. 2 shows that binding of DT to liposomes containing DCP was dependent on pH, with a maximum at pH 3.6 and with minimal, but still easily measurable, binding above pH 6. Control experiments demon-
pH
Toxin added, fg/,umol DMPC FIG.
1.
Dose-dependent binding
of DT to
liposomes. The lipo-
consisted of DMPC, Chol, and DCP (2:1.5:0.4). Specific protein binding, ,g of toxin bound per umol of liposomal phosphate. somes
FIG. 2. Influence of pH on binding of DT to liposomes containing DCP. The molar ratios of the lipid constituents in the liposomes were 2:1.5:0.4. Liposomes (0.1 ml) were incubated with 90 ,gg of DT and 150 ,ul of isotonic buffer in a total volume of 428 ,l. After incubation, the liposomes were washed with ice-cold 0.15 M NaCl. Liposomes:@, DMPC/Chol/DCP; 0, DMPC/Chol; a, DMPC/Chol/palmitic acid;
o, DMPC/Chol/sulfatide.
Proc. Natl. Acad. Sci. USA 77 (1980)
Biochemistry: Alving et al.
1988
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FIG. 3. Influence of pH on binding of DT to liposomes containing PIP. Incubation conditions were the same as described in the legend of Fig. 2. Where indicated, 4 mM inhibitor was present. Liposomes were DMPC/Chol/PIP, 1.6:1.5:0.4. 0, -Inhibitor; 0, +phosphocholine; A, +ATP.
strated that apparent binding of DT at low pH was not due to nonspecific denaturation and precipitation of DT. This was determined by absence of substantial binding of DT at low pH to liposomes composed of DMPC/Chol, DMPC/Chol/palmitic acid, or DMPC/Chol/sulfatide (Fig. 2). The pattern of pH dependence was different with liposomes containing PIP (Fig. 3). Binding of DT to PIP liposomes also showed a maximum at pH 3.6, but the highest levels of binding occurred at pH 7 and above (Fig. 3). Separate experiments showed that binding of DT was influenced by pH only during the initial interaction with liposomes. Thus, for example, when DT was bound to liposomes containing DCP at low pH, it was not removed by subsequently raising the pH. Inhibition of Binding. Fig. 3 demonstrates that binding of DT to liposomes containing PIP was inhibited above pH 6 by phosphocholine or ATP. Binding of toxin to liposomes containing DCP also was inhibited by UTP, ATP, p-nitrophenyl phosphate, or phosphocholine, but not by uracil (Table 2). The polar group of phosphatidylcholine is phosphocholine and, although binding did not occur to liposomes containing only phosphatidylcholine and Chol (DMPC/Chol), other experiments showed that binding of DT to liposomes containing DMPC, Chol, and DCP was almost totally blocked by less than 1.4 mM phosphocholine. Table 2. Inhibition of binding of DT to liposomal DCP Inhibitor None (isotonic saline) Uracil UTP ATP p-Nitrophenyl phosphate
Specific binding 33.6 34.2 0 0 0 3.9
Phosphocholine Liposomes (0.1 ml) consisting of DMPC/Chol/DCP were incubated with 78-98 gg of DT in a total volume of 0.2 ml. The inhibitors were at 4 mM.
0 Phosphatide
FIG. 4. Effect of phosphatide composition on binding of DT to liposomal DCP. The liposomes consisted of phosphatide as indicated,
Chol, and DCP (2:1.5:0.4).
Influence of Neutral Phosphatides. Binding of DT to DCP was inversely related to the fatty chain length of the liposomal phosphatidylcholine (DLPC > DMPC > DPPC > DSPC) (Fig. 4). The smallest amount of binding to DCP (
