Kouhei Hayashi

Thermotropic phase behavior and stability of monosialoganglioside micelles in aqueous solution

The thermotropic phase behavior of monosialoganglioside in a dilute aqueous dispersion at pH 6.8 was measured by using synchrotron radiation small-angle x-ray scattering and was analyzed by a shell-modeling method. Previous calorimetric studies on ganglioside systems have shown quite different thermotropic behaviors from other biological lipid systems, however, the details have still been ambiguous. Because of high statistical data and a shell-modeling analysis, we could elucidate the internal structural change of monosialoganglioside micelle induced by the elevation of temperature from 6 to 60 degrees C, that is, the shrinkage of the hydrophilic region and the slight expansion of the hydrophobic region occurring simultaneously, accompanying the elongation of the axial ratios of the ellipsoidal micelles. The model structures obtained explain the changes in the experimental scattering curves, the distance distribution functions, and the gyration radii. In addition we have also found an evident thermal hysteresis in the scattering curves and in the structural parameters. The present result suggests that the thickness of the hydrophilic region, namely, the conformation of oligosaccharide chains, is sensitive to a change of temperature.

Interaction of gangliosides with proteins depending on oligosaccharide chain and protein surface modification.

By using neutron and synchrotron x-ray small-angle scattering techniques, we investigated the process of the complexation of gangliosides with proteins. We treated monosialoganglioside (G(M1)), disialoganglioside (G(D1a)), and a mixture of G(M1)/G(D1a). Proteins used were bovine serum albumins whose surfaces were modified with different sugars (deoxy-D-galactose, deoxy-L-fucose, deoxymaltitol, and deoxycellobiitol), which were used as model glycoproteins in a membrane. We found that the complexation of gangliosides with albumins greatly depends on the combination of ganglioside species and protein surface modification. With a varying protein/ganglioside ratio in a buffer solution at pH 7, the complexation of G(M1) or G(D1a) with albumins modified by monosaccharides appears to be less destructive for ganglioside aggregate structures in forming large complexes; the complexation of G(D1a) with the albumins modified by disaccharides induces the formation of complexes with a dimeric structure; and the complexation of G(M1) with albumins modified by disaccharides, to form small complexes, is very destructive. The present results show a strong dependence of the interaction between ganglioside and protein on the characteristics of the ganglioside and protein surface, which would relate to a physiological function of gangliosides, such as a function regulating the receptor activity of glycoproteins in a cell membrane.

INTERMICELLAR INTERACTION OF GANGLIOSIDE AGGREGATES AND STRUCTURAL STABILITY ON PH VARIATION

We have studied the effect of concentration and pH variations on micellar structures of sialoglycosphingolipids (gangliosides) by using synchrotron radiation small-angle X-ray scattering. We have treated four different aqueous dispersions of gangliosides containing monosialogangliosides (GM1), disialogangliosides (GD1a) and two different types of the ganglioside mixtures. To discuss the change in the scattering curve with concentration in the range of 0.1–10% w/v, we employed a rescaled mean spherical approximation method (RMSA) for charged particle dispersions combined with a shell-modelling method. The present modelling method well describes the characteristics of the whole experimental scattering curve, namely an intermicellar correlation peak at a very small angle and a rounded peak at a medium angle reflecting an intramicellar scattering density fluctuation. We determined simultaneously the inter- and intra-micellar structures of the ganglioside aggregates under the repulsive screened Coulomb potential between them. The estimated micellar surface charge is ca. 57 e in 0.01 M citrate buffer at pH 6.7, 25 °C. For every sample, the pH elevation from 3.6 to 8.0 at 25 °C caused similar changes in the experimental scattering curve, gyration radius and distance distribution function, suggesting a shrinkage of the micellar dimension and an intramicellar structural change. The modelling analyses can explain the above changes mostly resulting from the change of the oligosaccharide chain portions in the micelles. The structural differences depending on the specimens, containing different ganglioside components are also clarified.

Salt-dependent phase behaviour of the phosphatidylinositol 4,5-diphosphate–water system

We have studied the effect of mono- and di-valent cations on the structure of the phosphatidylinositol 4,5-diphosphate (PIP2)–biological buffer system at pH 7.2. We found that the PIP2 aggregates from prolate ellipsoidal micelles at pH 7.2 and that the addition of a small amount of Ca2+ ions even below 5 mmol dm–3([Ca2+] : [PIP2]= 5/7) induces a drastic structural phase transition from prolate micelles to disordered lamellae. Mg2+ ions induced a similar phase transition around 10 mmol dm–3, which, however, occurred less sensitively. In contrast to the case of divalent cations, the presence of Na+ ions did not affect the PIP2 micellar structures up to 0.1 mol dm–3([Na+] : [PIP2]= 100/7), indicating that the above structural change of the PIP2 aggregates is not attributable to the elevation of the electrostatic shielding effect between the polar heads. Modelling analysis suggests that the observed phase transition results from an increase of the critical packing parameter of the PIP2 molecule accompanying the dehydration of the PIP2 polar-head region.

Complexes of gangliosides with proteins in solution

The interaction of gangliosides with methylated bovine albumins was studied using neutron and synchrotron-radiation X-ray solution scattering methods. The present result suggests that the binding of gangliosides with protein occurs heterogeneously.

Ganglioside structure in solution

Solution scattering methods using neutron and X-ray synchrotron radiation were used complementarily to determine a ganglioside micellar structure. Based on the simple double-shell model, the spread of the sugar head and the structural anisotropy were determined.

Structural Changes in Phosphatidylserine Salts by Hydration and Addition of Calcium Ion

Hydrations and phase transitions of ammonium, sodium and potassium salts of phosphatidylserines (PS) were investigated by small-angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC). The increase in long spacings for NH 4 + -PS by addition of water was slightly smaller than those for the other PS salts, implying that a small amount of water penetrated into the hydrocarbon regions. The long spacings for PS salts and water system were reduced by addition of CaCl 2 . A mixture of phosphatidylcholine (PC) and PS in KCl aqueous solution gave a long spacing of 59 A, while it separated into two long spacings at 65 and 55 A in 1 mol CaCl 2 solution. This SAXS result shows that Ca 2+ ion induces not only two dimensional, but three dimensional phase separation of the mixture of PC and PS. The phase transition temperature of the anhydrous NH 4 + -PS was lowest among those of the three PS salts, but fully hydrated NH 4 + -PS had the highest transition temperature among them.