Monosialoganglioside-GM1 triggers binding of the amyloid-protein salmon calcitonin to a Langmuir membrane model mimicking the occurrence of lipid-rafts

Abstract

GM1 ganglioside is known to be involved in the amyloid-associated diseases and it is a crucial factor for the assembly of amyloid proteins on lipid-rafts, which are lipid structures located on the synaptic plasma membranes. Due to its slow aggregation rate, we employed salmon calcitonin (sCT) as a suitable probe representative of amyloid proteins, to study the interaction between this class of proteins and a membrane model. Here, we prepared a neuronal membrane model by depositing onto mica two Langmuir-Blodgett films in liquid-condensed phase: the outer monolayer was characterized by high content of GM1 (50%) and minority parts of cholesterol and POPC (25-25%), while the inner one by plain POPC. To deeply investigate the interaction of sCT with this model and the role-played by GM1, we prepared the outer leaflet adding sCT at a concentration such that the number of proteins equals that of GM1. Atomic Force Microscopy revealed the occurrence of two distinct kinds of flat surfaces, with globular aggregates localized exclusively on top of the highest one. To unravel the nature of the interaction, we studied by ζ-potential technique liposomes composed as the outer leaflet of the model. Results demonstrated that an electrostatic interaction sCT-GM1 occurred. Finally, to investigate the interaction thermodynamics between sCT and the outer leaflet, Langmuir films as the outer monolayer and containing increasing content of sCT were studied by compression isotherms and Brewster Angle Microscopy experiments. Based on the all body of results we propose an interaction model where GM1 plays a pivotal role.

Keywords: AFM; Amyloid; BAM; GM1; Langmuir Films; Lipid-rafts; Membrane Models; sCT; ζ-potential.

Fig. 1

AFM study of the model made of a Chol/POPC (0.5/0.5) layer deposited onto mica covered by POPC. Fig. 1a shows the line-profile obtained after the scratching performed in contact mode (white line in Fig. 1b). Fig. 1c shows the same sample before the scratching, imaged in TM-AFM. A flat surface can be observed.

Fig. 2

AFM images relative to the raft model made of GM1/Chol/POPC (0.50/0.25/0.25) and sCT, deposited onto mica covered by plain POPC. sCT was added before the deposition to the outer monolayer at X=0.34 molar fraction. Fig. 2a shows a wide field, with many repeated features magnified in Fig. 2b. The inset represents the line-profile of the zone indicated in Fig. 2b. Fig. 2c and d are relative to an independent sample.

Fig. 3

Compression isotherms and BAM images (frame of 1×1 mm) relative to the model of the outer membrane leaflet: GM1/Chol/POPC and sCT. Fig. 3a shows isotherms relative to GM1/Chol/POPC (black solid) and sCT (dashed) at different molar ratios: 0.20 (red), 0.34 (green), 0.50 (blue), 0.60 (pink), 0.70 (light-blue), 0.80 (orange). Fig. 3b shows the “area-analysis” of this system at five surface pressures: ■ π=5 mN/m, • π=10 mN/m, ▲ π=15 mN/m, ▼ π=20 mN/m, ★ π=25 mN/m. Errors are within the symbols. The inset of Fig. 3a shows the “ΔG-analysis” at three surface pressures: ■ π=5 mN/m, • π=15 mN/m, ★ π=25 mN/m. BAM images show the monolayer morphology (in the L_c phase at π=22 mN/m) observed without sCT (Fig. 3c), with sCT at 0.34 (Fig. 3d) and at 0.60 (Fig. 3e) molar fraction.

Fig. 4

Partial molecular area results relative to the to the model of the outer membrane leaflet made of GM1/Chol/POPC and sCT. Symbols • indicate data relative to sCT while ■ to the GM1/Chol/POPC matrix. The analysis has been performed at π=10 mN/m (a) and π=25 mN/m (b), which is above and below the sCT knee (π=18 mN/m).

Fig. 5

Compression isotherms relative to the GM1-free control model made of Chol/POPC and sCT. Fig. 5a shows the isotherms of plain lipids (black solid) and sCT (dashed) at different molar ratios: 0.20 (green), 0.40 (blue), 0.60 (light-blue) and 0.80 (pink) for Chol/POPC. Fig. 5b shows the “area-analysis” of this system at five surface pressures: ■ π=5 mN/m, • π=10 mN/m, ▲ π=15 mN/m, ▼ π=20 mN/m, ★ π=25 mN/m. Errors are within the symbols. The inset of Fig. 5a shows the “ΔG-analysis” at three surface pressures: ■ π=5 mN/m, • π=15 mN/m, ★ π=25 mN/m.

Fig. 6

DLS size distributions of the liposomal samples reported in Table 1, obtained from intensity-weighted NNLS analysis. For GM1/Chol/POPC liposomes (panel A, ◯) the presence of sCT in the bilayer (▼, ▲) promotes the increase of mean hydrodynamic radius and the formation of smaller sCT aggregates, independently of the addition protocol. In Chol/POPC liposomes without GM1 (panel B, ⎕), no difference in radius and size distribution is found after sCT addition (panel B, ■).

Fig. 7

Simplified graphical model of the possible interaction of the amyloid protein sCT molecules with the Langmuir monolayer simulating the raft containing outer neuronal membrane leaflet (0.50/0.25/0.25 molar fraction of GM1/Chol/POPC). At X=0.34 molar fraction, the number of sCT molecules equals that of GM1.