Ks. Hamilton et al., ACYL-CHAIN LENGTH EFFECTS RELATED TO GLYCOSPHINGOLIPID CRYPTICITY IN PHOSPHOLIPID-MEMBRANES - PROBED BY H-2-NMR, Biochimica et biophysica acta. Biomembranes, 1190(2), 1994, pp. 367-375
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
.