A natural lipid membrane exists in a liquid state, but it forms a well ordered structure. The biological membrane contains solid, highly ordered aggregates, which are composed of cholesterol and glycolipids possessing large head groups composed of sugar and/or sialic acid residues.^[1] Thereby glycosphingolipids, glycolipids containing at least one sialic acid residue, are proposed to be a binding partner for siglec proteins (sialic-acid binding immunoglobulin-like lectins), which are an important part of the immune system. In this study Siglec-4, the myelin-associated glycoprotein (MAG) is relevant.^[2]

Unsymmetric three component lipid bilayers containing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 30 mol % of cholesterol and 20 mol % of GD1a ganglioside were prepared on the gold surface. To produce the mixed lipid bilayer vertical Langmuir-Blodgett and horizontal touch Langmuir-Schaeffer methods were used. The produced lipid layer contains the ganglioside only in the outer layer, mimicking a natural cell membrane. Under physiological conditions the lipid membrane is constantly exposed to high electric fields. The stability, potential window of the adsorption of the three component lipid bilayer and the phase transition in the film were tested in the electrochemical cell. Next, the lipid bilayer was exposed to the electrolyte solution containing up to 2 μg/ml MAG siglec protein. The interaction was studied measuring the differential capacitance over a period of several hours. Without the protein present in the solution the capacitance increased over time and the phase transition is found at -0.38 V vs. Ag/AgCl reference electrode. This result indicated that a three component lipid bilayer is not very stable when exposed to electric field. A lowering of the capacitance was observed, indicating that the binding of the protein stabilizes the lipid bilayer. Moreover, upon interaction with the siglec protein the potential of the phase transition is shifted to -0.64 V vs. Ag/AgCl. This result also shows a stabilization of the lipid membrane and its higher resistance to the electric field.

The polarization modulation infrared reflection absorption spectroscopy (PM IRRAS) was used to confirm binding of the protein to the lipid bilayer. The IRRA spectra were collected from lipid bilayers in which the outer-layer was exposed to the solution containing the MAG protein. The amide I and amide II bands are clearly seen in the spectra, indicating attachment of the siglec protein to the lipid bilayer.

[1] K. Simons, E. Ikonen, Nature 1997, 387, 569-572.

[2] S. Kelm, Ligands for Siglecs, Springer-Verlag, Berlin, 2001.

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