The Binding of Aβ42 Peptide Monomers to Sphingomyelin/Cholesterol/Ganglioside Bilayers Assayed by Density Gradient Ultracentrifugation

Abstract

The binding of Aβ42 peptide monomers to sphingomyelin/cholesterol (1:1 mol ratio) bilayers containing 5 mol% gangliosides (either GM1, or GT1b, or a mixture of brain gangliosides) has been assayed by density gradient ultracentrifugation. This procedure provides a direct method for measuring vesicle-bound peptides after non-bound fraction separation. This centrifugation technique has rarely been used in this context previously. The results show that gangliosides increase by about two-fold the amount of Aβ42 bound to sphingomyelin/cholesterol vesicles. Complementary studies of the same systems using thioflavin T fluorescence, Langmuir monolayers or infrared spectroscopy confirm the ganglioside-dependent increased binding. Furthermore these studies reveal that gangliosides facilitate the aggregation of Aβ42 giving rise to more extended β-sheets. Thus, gangliosides have both a quantitative and a qualitative effect on the binding of Aβ42 to sphingomyelin/cholesterol bilayers.Early data [8,9] supported the idea that β-secretase associates with liquid-ordered (Lo) nanodomains in the membrane and that integrity of those raft-like domains is required for β-cleavage of APP to occur. This explains why many studies on Aβ-membrane interaction have been performed with bilayers enriched in sphingomyelin (SM) and cholesterol (Chol), a lipid mixture that exists usually in the Lo phase [10]. Moreover, various reports [11,12] have shown that anionic lipids promote fibril elongation. In our previous study [13] we explored the initial stages of the interaction of Aβ42 in the monomeric form with lipid monolayers and with bilayers composed mainly of SM and Chol, i.e., in the liquid-ordered (Lo) state, in the absence and presence of negatively charged phospholipids. In the absence of negatively charged lipids, the interaction was weak. However, in the presence of phosphatidic acid, or of cardiolipin, an interaction could be detected by different methods, including isothermal calorimetry, thioflavin T (ThT) fluorescence and infrared spectroscopy, as well as molecular dynamics simulations. Low (2.5-5 mol %) concentrations of phosphatidic acid or cardiolipin allowed better interaction than 20 mol % of the same lipids.For many years gangliosides have been associated to plaque formation [14], perhaps because of their presumed implication in raft structure [15]. Numerous studies have explored model membranes composed of SM, Chol and gangliosides. Selected examples include those based on molecular dynamics [16][17][18], atomic force microscopy (AFM) [19,20], infrared spectroscopy [21], and other biophysical techniques [22,23]. To our knowledge Aβ42 binding to bilayers after separation of the free and lipid-bound forms of the peptide by density gradient centrifugation have rarely been performed. Vesicle-bound peptide floats in the density gradient whereas the non-bound protein/peptide, after aggregation, sediments [24,25]. The centrifugation method is quite unique in providing direct, quantitative equilibrium measurements of binding, in the abs...

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