Integration of ganglioside GT1b receptor into DPPE and DPPC phospholipid monolayers: an X-ray reflectivity and grazing-incidence diffraction study

Abstract

Using synchrotron grazing-incidence x-ray diffraction (GIXD) and reflectivity, the in-plane and out-of-plane structures of mixed-ganglioside GT(1b)-phospholipid monolayers were investigated at the air-liquid interface and compared with monolayers of the pure components. The receptor GT(1b) is involved in the binding of lectins and toxins, including botulinum neurotoxin, to cell membranes. Monolayers composed of 20 mol % ganglioside GT(1b), the phospholipid dipalmitoyl phosphatidylethanolamine (DPPE), and the phospholipid dipalmitoyl phosphatidylcholine (DPPC) were studied in the gel phase at 23 degrees C and at surface pressures of 20 and 40 mN/m, and at pH 7.4 and 5. Under these conditions, the two components did not phase-separate, and no evidence of domain formation was observed. The x-ray scattering measurements revealed that GT(1b) was intercalated within the host DPPE/DPPC monolayers, and slightly expanded DPPE but condensed the DPPC matrix. The oligosaccharide headgroups extended normally from the monolayer surfaces into the subphase. This study demonstrated that these monolayers can serve as platforms for investigating toxin membrane binding and penetration.

FIGURE 1

Chemical structure of GT_1b, DPPE, and DPPC. A bar of length 10 Å is shown for reference. The saccharide region of GT_1b is not drawn to scale, for better visibility of its chemical structure.

FIGURE 2

Pressure-area isotherms for GT_1b, DPPE, DPPC, 1:4 mol % GT_1b/DPPE, and 1:4 mol % GT_1b/DPPC. Isotherm of GT_1b/DPPE lipid monolayer could almost be superimposed on isotherm of pure DPPE. This indicated that GT_1b molecules, up to a mole fraction of 20%, are incorporated into DPPE matrix and do not significantly disturb the packing of DPPE. However, the 1:4 mol % GT_1b/DPPC monolayer showed a condensing effect, designated as a lower surface pressure, in the liquid-solid phase transition. Above surface pressures of 10 mN/m, isotherms of DPPC and 1:4 mol % GT_1b/DPPC are almost identical.

FIGURE 3

X-ray reflectivity results for monolayers of pure GT_1b, DPPE, and 1:4 mol % GT_1b/DPPE at pH 7.4 and surface pressure of 20 mN/m. (a) Measured reflectivity plotted as R/R_Fresnel vs. q_z. Error bars for reflectivity data represent statistical errors in these measurements. Measured data are represented as symbols, and solid lines represent fits with lowest χ². Curves were vertically offset by factors of 10 for clarity. (b) Electron-density profiles for pure GT_1b, DPPE, and 1:4 mol % GT_1b/DPPE monolayers at 20 mN/m on water/buffer subphase. The thickness of electron-density profiles, corresponding to reflectivity fits with χ² values at no more than 5% of the minimal value, represents uncertainty in real-space structure. Electron densities ρ(z) are normalized to the electron density of water with buffer, ρ_subphase = 0.339 e⁻/Å⁻³. In the electron-density profile of the GT_1b/DPPE monolayer, the saccharide group of GT_1b is clearly evident as a large electron-density increase extending ∼20 Å into the subphase from the DPPE headgroup region (at ∼22 Å; dashed line). (b) Illustration of one DPPE molecule and one GT_1b molecule in their approximate orientation at the liquid surface. Dashed line at depth equal to 0 Å represents average position of alkyl tails/air interface.

FIGURE 4

X-ray reflectivity results for monolayers of pure GT_1b, DPPC, and 1:4 mol % GT_1b/DPPC at pH = 7.4 and surface pressure of 20 mN/m. (a) Measured reflectivity, plotted as R/R_Fresnel vs. q_z. Error bars for reflectivity data represent statistical errors in these measurements. Measured data are represented as symbols, and solid lines represent fits with the lowest χ². Curves were vertically offset by factors of 10 for clarity. (b) Electron-density profiles for pure GT_1b, DPPC, and 1:4 mol % GT_1b/DPPC monolayers at 20 mN/m on a water/buffer subphase. Electron densities ρ(z) are normalized to electron density of water with buffer, ρ_subphase = 0.339 e⁻/Å⁻³. (b) Illustration of one DPPC molecule and one GT_1b molecule in their approximate orientation at liquid surface. Two dashed lines represent average positions of alkyl tails/air interface (z = 0 Å) and center of headgroup region (z ∼20 Å).

FIGURE 5

Grazing-incidence x-ray diffraction from ordered alkyl tail regions of DPPE, GT_1b/DPPE, DPPC, and GT_1b/DPPC monolayers. (a) Bragg peaks. (b) Bragg rods. The DPPE and GT_1b/DPPE data are offset from DPPC and GT_1b/DPPC data in each case for clarity. The three GIXD Bragg peaks indicate packing of the lipid tails in an oblique 2D unit cell. Miller indices of each peak are provided. (a) Gray arrows highlight unit-cell expanding for GT_1b/DPPE monolayer, and condenseing for GT_1b/DPPC monolayer. Bragg peaks were fit using Voight functions (solid gray lines). (b) Bragg rods were fitted (solid line) by approximating the coherently scattering part of the alkyl tail by a cylinder with length L_c and constant electron density. The sharp peak at q_z = 0.01 Å⁻¹ is so-called Yoneda-Vineyard peak (66), which arises from interference between x-rays diffracted up into a monolayer and x-rays diffracted down and then reflected up by interface. (c) Top view of arrangement of hydrocarbon tails of DPPE and DPPC molecules within unit cells at 20 mN/m. Their azimuthal tilt direction is approximately along the a + b direction, and molecules are tilted from surface normal by angles indicated in the text.