Interaction of B2-Glycoprotein I with Negatively Charged Phospholipid Membranes

MARIANA PAOLOROSSI Y GUILLERMO G. MONTICH

Montevideo, Uruguay

Conferencia; 6 International Conference of Biological Physics and 5 Southern Cone Biophysics Congress; 2007

B2-Glycoprotein I (B2-GPI) is a soluble glycoprotein present in plasma. It participates in several physiological and pathogenic events. It was proposed, for example, thatB2-GPI binds to the exposed anionic phospholipids of apoptotic cells producing auto-antigenic lipid-protein complexes. The binding to the membrane is mediated by both electrostatic and hydrophobic forces. Even when the electrostatic interaction is absolutely required, the sole presence of a negative charge does not determine the strength of binding and different affinities are observed for different anionic lipids. We addressed the study of membrane interactions of B2-GPI to understand the balance and mutual influence of electrostatic and hydrophobic driving forces and whether these interactions are coupled to conformational changes of the protein in the interface. B2-GPI was purified from human plasma by heparin affinity chromatography. The interaction of B2-GPI with phospholipid membranes composed of palmitoyl oleoyl phosphatidylglycerol (POPG), palmitoyl oleoyl phosphatidylserine (POPS) and palmitoyl oleoyl phosphatidylcholine (POPC) was studied by differential scanning calorimetry (DSC), fluorescence spectroscopy and infrared spectroscopy (FT-IR). In solution, B2-GPI exhibited a reversible two-state transition with a Tm of 64 ºC. In the presence of large unilamellar vesicles (LUVs) composed of POPC, the thermal unfolding was similar to that observed for pure B2-GPI. The thermal transition of the protein in the presence of negative charged phospholipid LUVs presented remarkable differences when compared with that of the pure protein solution. The addition of POPG/POPC or POPS/POPC LUVs induced an increase in turbidity of the solution of pure B2-GPI immediately after liposomes addition. DSC thermograms show a sharp peak at 60 ºC in the presence of POPG vesicles, while the addition of CL liposomes abolished the thermotropic transition of B2-GPI. The secondary structure of B2-GPI was studied by FT-IR. The presence of POPG or CL multilamellar vesicles did not induce remarkable changes in the spectrum shape of the native conformation at 25 ºC.