Quantitative Measurements of Recombinant HIV Surface Glycoprotein 120 Binding to Several Glycosphingolipids Expressed in Planar Supported Lipid Bilayers

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Abstract

The interaction of recombinant HIV-1 surface glycoprotein gp120 (rgp120) with natural isolates of lactosylceramide (LacCer), glucosylceramide (GlcCer), and galactosylceramide (GalCer) has been quantitatively measured under equilibrium conditions using total internal reflection fluorescence (TIRF) spectroscopy. The binding affinity (K(a)) of rgp120 to these glycosphingolipids (GSLs), reconstituted at 5 mol % in supported planar lipid bilayers composed of 95 mol % POPC, is ca. 10(6) M(-1) for dissolved rgp120 concentrations greater than 25 nM. In contrast, at concentrations of rgp120 between 0.2 and 15 nM, rgp120 does not bind significantly to LacCer and GlcCer, but has a high affinity for GalCer with a measured K(a) value of 1.6 x 10(9) M(-1). However, protein surface coverage measurements show that this strong binding process accounts for very little of the total protein adsorbed over the entire concentration range studied. At a protein concentration of ca. 20 nM, the surface coverage is only 3% of that achieved at apparent saturation (i.e., when the protein concentration is ca. 220 nM). Thus the "high affinity" binding sites comprise only a small fraction of the total number of binding sites. Several other variables were investigated. Rgp120 binding behavior at membranes doped with alpha-hydroxygalactosylceramide (alpha-GalCer) was very similar to that observed with GalCer, showing that the presence/absence of an alpha-hydroxy moiety does not significantly affect galactosylceramide recognition. Phase segregation of GalCer, which occurs when the mole fraction of this GSL in a POPC bilayer exceeds ca. 0.1, was also investigated and showed no effect on binding affinity at low rgp120 concentrations. To investigate the influence of fatty acid chain length, GSLs with monodisperse C(18) and C(24) chain lengths, both with and without an alpha-hydroxy moiety, were synthesized, and their binding affinity to rgp120 was examined. Relative to the natural isolates (which contain a mixture of chain lengths), minimal differences were observed; thus among the compounds tested, fatty acid chain length does not affect GSL recognition. The results of this work should aid efforts to design anti-HIV-1 agents based on membrane-tethered, carbohydrate-based receptors for rgp120.

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