Binding of diphtheria toxin to phospholipids in liposomes

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Abstract
Diphtheria toxin bound to the phosphate portion of some, but not all, phospholipids in liposomes. Liposomes consisting of dimyristoyphosphatidylcholine and cholesterol did not bind toxin. Addition of 20 mol% (compared to dimyristoyphosphatidylcholine) of dipalmitoyl phosphatidic acid, dicetyl phosphate, phosphatidylinositol phosphate, cardiolipin or phosphatidylserine in the liposomes resulted in substantial binding of toxin. Inclusion of phosphatidylinositol in dimyristoylphosphatidylcholine/cholesterol liposomes did not result in toxin binding. The Ca salt of dipalmitoyl phosphatidic acid was more effective than the Na salt and the highest level of binding occurred with liposomes consisting only of dipalmitoyl phosphatidic acid (Ca 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 p-nitrophenyl phosphate, but not by uracil. Diphtheria toxin binds specifically to the phosphate portion of certain phospholipids. Binding to phospholipids in liposomes is dependent on pH, but is not due only to electrostatic interaction. Binding may be strongly influenced by the composition of adjacent phospholipids that do not bind toxin. A minor membrane phospholipid (such as phosphatidylinositol phosphate or phosphatidic acid) or some other phosphorylated membrane molecule (such as a phosphoprotein) may be important in the initial binding of diphtheria toxin to cells.