ACYL-CHAIN LENGTH EFFECTS RELATED TO GLYCOSPHINGOLIPID CRYPTICITY IN PHOSPHOLIPID-MEMBRANES - PROBED BY H-2-NMR

Wideline H-2-NMR was used to consider the relationships amongst glycos phingolipid and phospholipid fatty acid chain length and glycosphingol ipid receptor function, in a system classically associated with crypti city. Galactosyl ceramide (GalCer), having 18-or 24-carbon fatty acid, was deuterium labelled at the conformationally-restricted fatty acid alpha-carbon (C-2). H-2-NMR spectra of N-[2,2-H-2(2)]stearoyl and N-[2 ,2-H-2(2)]lignoceroyl GalCer (GalCer with 18-vs. 24-carbon selectively deuterated fatty acid) were then compared over a range of temperature s in phosphatidylcholine/cholesterol membranes in which the host phosp holipid had dimyristoyl, dipalmitoyl, or distearoyl fatty acid composi tion. Findings were evaluated in the light of known sensitivity of ant ibody interaction with GalCer to temperature and to both glycolipid fa tty acid chain length and host matrix fatty acid chain length. Under t he conditions of experimentation, spectra were not obtainable for glyc olipids having rigid body motions that were slow on the NMR timescale (10(-4)-10(-5) s) - i.e.. motions typical of non-fluid (gel phase) mem branes. The systems, DPPC/cholesterol and DSPC/cholesterol, in which t he original observation was made of increased antibody binding to GalC er with long fatty acid, proved to be characterised by receptor motion s that were in this slow timescale for both 18:0 and 24:0 GalCer at 22 -24-degrees-C. Under conditions for which spectra could be obtained, t hose for GalCer with [2,2-H-2(2)]lignoceroyl (24-carbon alpha-deuterat ed) fatty acid were qualitatively similar to those of its 18-carbon an alogue in all (fluid) membranes examined. However, spectral splittings differed quantitatively between deuterated 18:0 and 24:0 GalCer at a given temperature, dependent upon host matrix. These differences were most marked at lower temperatures and in the longer chain (more ordere d) matrices, DPPC/cholesterol and DSPC/cholesterol. This suggests that maximum effects of glycolipid chain length on glycolipid receptor fun ction may be expected to occur in spatially and motionally constrained lipid environments. There was little effect of temperature on spectra l splittings seen for a given sample containing deuterated 18:0 GalCer . The small differences seen could be adequately accounted for by rela tively minor alterations in glycolipid order and backbone conformation . In contrast, 24:0 GalCer in DPPC/cholesterol and DSPC/cholesterol di splayed significant variation in its spectral splittings as the temper ature was reduced; and these proved to be the source of the quantitati ve differences between 18:0 and 24:0 GalCer referred to above. For 18: 0 GalCer, the only spectral feature seen to be notably sensitive to te mperature and to choice of host membrane phospholipid in the range, 22 -65-degrees-C, was whole-body motion; which could be abruptly 'frozen out' with decreasing membrane fluidity. The 24:0 GalCer analogue was s een to undergo the same loss of motional freedom, in the same temperat ure range, as 18:0 GalCer. We suggest therefore that several previous observations of GalCer crypticity in phospholipid/cholesterol systems having longer vs. shorter chain phospholipids, may derive most importa ntly from reduced motional freedom of the glycolipid recognition sites .