BEHAVIOR OF COMPLEX OLIGOSACCHARIDES AT A BILAYER-MEMBRANE SURFACE - PROBED BY H-2-NMR

Deuterium wideline NMR was used in an attempt to directly assess oligo saccharide arrangement and motional characteristics of complex glycosp hingolipids dispersed as minor components in phospholipid membranes. A convenient, general synthetic approach was developed which involved r eplacement of the acetate group of amido sugars with deuteroacetate (- COCD3). This provided excellent signal-to-noise when applied to the te rminal GalNAc residue of globoside, and the terminal NANA residue of G M1. Simultaneously, globoside and GM1 fatty acids were replaced with s tearic acid deuterated at C-2 - a probe location sensitive to glycolip id hydrophobic backbone orientation and rigid body motion. Deuterated GM1 and globoside were studied by H-2-NMR in bilayers of 1-palmitoyl-2 -oleoyl phosphatidylcholine, in the presence and absence of physiologi cal quantities of cholesterol. The monoglycosyl glycosphingolipid, glu cosyl ceramide, which is the common skeleton of many complex glycosphi n-golipids including those studied here, was also deuterated at fatty acid C-2 for comparative study in the same matrices. Correlation with spectra of the complex glycolipids demonstrated that, for a given temp erature and membrane composition, ceramide backbone conformation was v ery similar amongst the species studied. Spectral features of GM1 deut erated on terminal NANA and assembled at a membrane surface, were foun d to be highly consistent with the oligosaccharide conformation determ ined in studies of GM1 in solution. In contrast, globoside deuterated in the terminal GalNAc residue gave spectra very different from those predicted on the basis of the conformation considered to exist in solu tion. It seems likely that this result reflects a combination of great er oligosaccharide chain flexibility relative to GM1, and the presence of the membrane environment. Interestingly, although there was highly significant spatial geometry associated with the complex oligosacchar ide chains, and although temperature and the presence of cholesterol e xert measurable effects on the membrane-inserted portion, these factor s had very little impact on the measured spectral parameters associate d with the NANA residue of GM1 or the terminal GalNAc residue of globo side. This seems to indicate lack of sensitivity of the complex oligos accharide chains to conformation and internal motions of the hydrophob ic chain segments in these fluid and semi-fluid membranes; and has imp ortant implications for mechanisms of crypticity.