~2H-NMR study of two probe-labelled glycosphingolipid-derived signalling modulators in bilayer membranes

摘要

We describe here the first report of sphingoid bases bearing non-perturbing 2H probe nuclei. These were produced, by two different routes of partial synthesis, to permit direct assessment of their arrangement and behaviour as minor components in membrane systems. Wideline 2H-NMR spectra of N,N-dimethylsphingosine with deuterated amino-methyl groups ([2H6]dimethylsphingosine), and of lyso-dihydrogalactosylceramide (lyso-GalCer) with deuterium nuclei at C4,C5 of the sphingosine backbone and at C3,C4 of the galactose ring ([2H4]lyso-GalCer), were recorded in unsonicated, cholesterol-containing fluid bilayer membranes. The sphingolipid metabolites behaved as single populations of lipid amphiphiles dispersed uniformly in the membrane and undergoing rapid symmetric motion about their long molecular axes. This was the case throughout the pH ranges examined, which included values generally considered for the cell cytoplasm. Spectra of [2H6]dimethyl sphingosine indicated that the methyl groups are equivalent on the NMR timescale, and that the molecule's orientation and behaviour are largely unaffected by pH over the range, 6 to 10.5. There was no spectral evidence of deprotonation of the tertiary amine function in this range. Similarly, variation of pH between 6.4 and 8.9 had virtually no effect on the average conformation and orientational order of lyso-GalCer at the level of C4,C5 in the sphingosine backbone. pH did, however, exert significant control over the orientation of the galactose residue — the effect being most marked in the region of the sphingoid base pKa. The lyso-glycolipid showed some evidence of being less motionally ordered than the corresponding parent species, presumably as a result of removal of constraints imposed by the fatty acid.