Headgroup behaviour of an uncharged complex glycolipid

A globoside spin labelled on the terminal sugar residue has been synthesized, and employed in model membranes to study headgroup behaviour of complex uncharged glycolipids. The labelled headgroup demonstrated a high degree of motional freedom limited to the aqueous region of the interface between lipid bilayer and surrounding medium. This observation was unaltered by the presence of a dense, tightly-bound surface layer of peripheral proteins or polysaccharide—which might be expected to reproduce conditions present at a cell surface. Headgroup dynamics were only very modestly correlated with the physical state (i.e., fluidity) of the membrane itself. In spite of the absence of charged sugar residues in globoside, the aspects of its headgroup behaviour monitored here we found to be similar to those of oligosaccharide chains on gangliosides and several sialic acid-rich glycoproteins.     

Headgroup dynamics of an integral membrane glycoprotein.

Glycophorin, an integral membrane glycoprotein known to be a receptor for several lectins, has been spin labelled specifically on headgroup terminal sugars. The labelled derivative has been studied in solution and also in various model membranes in an attempt to determine the factors which control headgroup dynamics. Under conditions which mimic those in a living cell the oligosaccharide chains show a uniform, relatively high freedom of motion, with individual sugar correlation times on the order of 6 × 10^?10 sec to 8 × 10^?10 sec depending upon the extent of glycoprotein headgroup involvement with other glycocalyx components. They exhibit no detectable occupancy of lipid or protein hydrophobic domains. Oligosaccharide dynamics are insensitive to factors which act upon that portion of the polypeptide backbone which inserts into the membrane, however a specific recognition event markedly reduces terminal sugar mobility.     

Dynamics and orientation of glycolipid headgroups by 2H-NMR: gentiobiose

Deuterium nuclear magnetic resonance has been used to investigate the dynamics and determine the orientation of the headgroup of the glycolipid 1,2-di-O-tetradecyl-3-O-(6-O-beta-D-glucopyranosyl-beta-D-glucopyranosyl )-sn- glycerol (beta-DTDGL), in aqueous multilamellar dispersions. In addition, its anomeric analog, having an alpha glucose-glycerol linkage, was prepared and examined. The lipids were labelled with deuterium at specific positions in the disaccharide moiety. Analysis of the deuterium quadrupolar splittings for the first glucose ring (glycerol-linked) gave segmental order parameters of 0.43 and 0.35 for the beta and alpha isomers, respectively. Both isomers had similar orientations of the sugar ring relative to the bilayer surface, as determined for lipid in the liquid-crystalline phase. 2H-NMR results for the lipid labelled at C-6' are consistent with a single conformation about the C-5'-C-6' bond of the first glucose residue, with a dihedral angle (O-5'-C-5'-C-6'-O-6') of -17 degrees. The results obtained for the second sugar ring suggest that two conformers may be present, which are in slow exchange on the 2H-NMR timescale. Measurements of longitudinal relaxation times, T1z, gave similar values for both sugar moieties in the headgroup, suggesting that the disaccharide does not exhibit the flexibility expected about the 1----6 linkage. Since T1z for 2H in these compounds decreases with increasing temperature and increases with magnetic field strength, the motion(s) dominating relaxation is in the long-correlation-time regime [omega 0 tau c)2 greater than 1). Thus, the gentiobiosyl headgroup undergoes the slowest motion of the glycolipid headgroups studied to date.     

Glycosphingolipids: 2H NMR study of the influence of carbohydrate headgroup structure on ceramide acyl chain behavior in glycolipid-phospholipid bilayers

Galactosyl- and glucosylceramide, globoside, and dihydrolactosylceramide, bearing [2,2-2H2]stearic acid, have been studied at a concentration of 10 mol% in bilayers of dimyristoylphosphatidylcholine by 2H NMR. The quadrupolar splitting delta vQ of the C2 deuterons were measured at several temperatures in the range of 30-60 degrees C. Spin-lattice relaxation times T1 of C2 deuterons were determined in the same temperature range for all lipids but globoside. T1 values at 30 and 50 degrees C were unexpectedly short (6-8 ms), indicating reduced mobility of the ceramide acyl chains compared to that of the host phospholipid. At all temperatures, both delta vQ and T1 were essentially identical for the monoglycosylated species, GalCer and GlcCer, indicating that the order and dynamics of the upper portion of the fatty acyl chain are insensitive to this small change in the headgroup structure. In the case of globoside, where the glycolipid headgroup is equivalent to that of GlcCer extended by three sugar residues, values for the quadrupolar splittings associated with the acyl chain C2-position were very close to those obtained for Gal- and GlcCer. In contrast, the delta vQ values obtained for the diglycosyl species, LacCer, were significantly different at all temperatures. This different behavior of LacCer relative to that of the other glycolipids most likely originates from an orientational change of the acyl chain at the C2-position due to the absence of a 4,5 double bond in dihydrosphingosine. T1 values for the GlcCer and GalCer systems increased with temperature, indicating that the motions responsible for relaxation were in the short correlation time regime.(ABSTRACT TRUNCATED AT 250 WORDS)     

Headgroup oligosaccharide dynamics of a transmembrane glycoprotein

Glycophorin, a major integral membrane glycoprotein of the human erythrocyte, has been spin labelled on oligosaccharide chains. Electron paramagnetic resonance studies of this glycoprotein in systems of controlled complexity have provided a degree of insight into its headgroup behaviour. (i) When glycophorin is free in solution its oligosaccharide chains exhibit uniformly high freedom of motion. This motional freedom is not attributable to the presence of N-acetyl-neuraminic acid residues. (ii) No evidence has been found of a finite tendency for headgroup sugars to associate with hydrophobic regions of phospholipid or glycoprotein. (iii) Headgroup oligosaccharide dynamics are essentially independent of the state of and interactions of the polypeptide hydrophobic portion (that portion which traverses the membrane). (iv) Nonspecific interaction with proteins and polysaccharides can readily reduce oligosaccharide chain mobility by some 25%, but does not alter their basic behaviour. (v) Binding of wheat germ agglutinin, dramatically immobilizes (terminal) N-acetylneuraminic acid residues. (vi) The above observations hold over the temperature range 0-40 degrees C. (vii) Headgroup carbohydrate mobility is at a minimum in the region of headgroup neutrality (pH 2.6-3.5) and is pH invariant over several pH units in the physiological range.     

Plasma membrane and intracellular expression of globotetraosylceramide (globoside) in mouse bone marrow-derived mast cells

The cellular localization of globotetraosylceramide (globoside), one of the predominant neutral glycosphingolipids of mouse interleukin 3-dependent, bone marrow culture-derived mast cells (BMMC), has been determined by immunologic and chemical methods. Although less than 10% of BMMC expressed globoside on their surface, as assessed by cytofluorographic analysis of the binding of a mouse monoclonal IgM anti-globoside antibody, treatment of BMMC with nonactivating doses of pronase, trypsin, or neuraminidase increased the percentage of BMMC binding anti-globoside antibody by an average of six, three, or sixfold respectively. That most BMMC had globoside on their plasma membrane was confirmed by the surface radiolabeling of globoside with galactose oxidase and sodium borotritide, as detected by autoradiography of thin layer chromatograms of the extracted neutral glycosphingolipids. Thus, BMMC expressed globoside on their plasma membrane, but accessibility of a large probe such as IgM antibody to the glycosphingolipid was impeded by surrounding surface molecules. All BMMC bound anti-globoside antibody intracellularly, as assessed by indirect immunofluorescence staining and fluorescence microscopy on acetone-permeabilized cells, and the pattern of staining suggested that globoside was associated with the secretory granules of BMMC. Immunologic activation of BMMC resulted in a fivefold increase in the surface expression of globoside, as detected by cytofluorographic analysis of the binding of monoclonal anti-globoside antibody. The findings suggest that activation of BMMC causes a reorganization of the plasma membrane such that globoside is more exposed or that activation is accompanied by movement of globoside from internal membranes to the plasma membrane. The increased expression of globoside is a novel marker of the activated mouse BMMC.     

Glycosphingolipid biosynthesis in early chick embryos

Early chick embryos at the age of 22 h (neurula stage) and 48 h (20-25 somite stage) were explanted from eggs and cultured in vitro in the presence of radioactive sugar precursors. Metabolically labelled glycosphingolipids were isolated. Amongst these, neutral and acidic components, the latter including sulfatide and gangliosides, were identified. Cleavage by exoglycosidases, as well as immunostaining with antibodies on thin-layer plates, showed that at both embryonic stages glycosphingolipids were synthesized that belong to the globo series (globoside, Forssman glycolipid), the lacto series (lactoneotetraosylceramide, nLc4Cer, and two nLc4Cer-based gangliosides, a monosialo and a disialo species), and the ganglio series (ganglioside Gtet1a and higher sialylated derivatives).     

Glycolipid membrane surface structure: orientation, conformation, and motion of a disaccharide headgroup

The orientation of the disaccharide headgroup of a lactose-containing lipid, 3-O-(4-O-beta-D-galactopyranosyl-beta-D-glucopyranosyl)-1,2-di-O-tetrade cyl-sn- glycerol (DTLL), relative to the surface of bilayer membranes has been determined via 2H NMR. The lactosyl headgroup is extended away from the membrane surface into the aqueous phase. The headgroup motion has axial symmetry as evidenced by the spectral line shape and order parameter tensor. 2H NMR of oriented multibilayers of DTLL confirms that the director of motional averaging is the bilayer normal. The two sugar residues have segmental order parameters S (glucose, 0.53; galactose, 0.51) which indicate that the headgroup fluctuates about the bilayer normal as a rigid unit. 2H spin-lattice relaxation times T1z for deuterons on each of the two sugar rings are similar, indicating further that there is no substantial motion about the disaccharide linkage within the headgroup. The magnitude of the relaxation times (4 ms) suggests that the rigid body motions of the headgroup are approaching the Larmor frequency; however, they increase with increasing temperature, indicating that the motions are rapid enough to be in the fast motional regime (omega o2 tau c2 less than 1). The conformation about the galactose-glucose intersaccharide linkage, calculated from the 2H NMR data, is shown to differ substantially from those found in X-ray diffraction studies of crystalline lactose and high-resolution NMR studies of methyl lactoside in nonviscous solution. The orientations of the hydroxymethyl groups in the headgroup have been calculated from the 2H NMR data. For the galactosyl residue the data are consistent with the presence of more than one rotamer about the C5"-C6" bond which are in fast exchange on the 2H NMR time scale. The hydroxymethyl group of the glucose residue exists in two rotameric forms about the C5'-C6' bond which have relative populations of ca. 2:1 and which are in slow exchange on the 2H NMR time scale (10(-5) s). The two rotamers differ from those deduced from X-ray crystallography of crystalline lactose and 13C NMR studies of methyl lactoside in solution.     

A model for ganglioside behaviour in cell membranes.

Gangliosides from beef brain have been spin-labeled using two different attaching groups and employed to investigate the physical nature of ganglioside behaviour in membranes. Results obtained using EPR spectroscopy indicate that, in phosphatidylcholine bilayers at physiological pH, ganglioside oligosaccharide chains are quite mobile and show a measurable tendency towards cooperative interaction amongst themselves. We suggest that the source of this interaction is the formation of H-bonds between sugar residues in adjacent ganglioside molecules. We present evidence that physiological (extracellular fluid) levels of Ca2+ and Mg2+ lead to cross-linking and condensing of ganglioside headgroups by complexing sialic acid carboxyl residues. Ganglioside headgroup interactions are not very sensitive to changes in the buffer ionic strength, suggesting that ionic interactions are of minor importance. We have found no measurable tendency for headgroup carbohydrate to penetrate hydrophobic regions of lipid bilayers. EPR spectroscopy was also used to follow the interaction of spin-labeled gangliosides with the glycoprotein, glycophorin, and with intact BHK cells. We suggest that these carbohydrate-based interactions should contribute significantly to the properties of the eucaryotic cell glycocalyx. We predict that laterally mobile carbohydrate-bearing components of cell surface will show a tendency to cluster about complex glycoprotein arrays, especially if the species involved bear accessible carboxylic acid functions.     

Monoclonal antibodies detecting different epitopes on the Forssman glycolipid hapten

Two hybridomas, derived by fusing mouse myeloma cells with spleen cells from a rat immunized with mouse mammary tumors, have been shown to produce antibodies that recognize cell surface antigens on mesenchymal cells in a variety of tissues. Evidence presented in this report suggests that these antibodies detect overlapping epitopes on the Forssman glycolipid hapten (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer). One antibody (33B12) reacts with the terminal sugar sequence GalNAc alpha 1-3GalNAc and is specific for Forssman. The other antibody (117C9) recognizes the internal sugar sequence GalNAc beta 1-3Gal. The terminal sugar sequence GalNAc beta 1-3Gal in globoside, as well as the internal sugar sequence GalNAc beta 1-4Gal in asialo-GM1, is not recognized as an antigenic determinant by 117C9. Nevertheless, the 117C9 antibody does not react exclusively with the Forssman antigen. In a lipid extract fractionated by Folch partition of mouse mammary tumors, the antibody also detects other glycolipids.     

Association of glycosphingolipids with intermediate filaments of human umbilical vein endothelial cells

Our previous studies of glycosphingolipids (GSLs) of human umbilical vein endothelial cells (HUVECs) established that globoside and ganglioside GM3 are the most abundant GSLs of HUVECs. Both compounds are located intracellularly, as well as on the cell surface. In this study, we demonstrate that the intracellular globoside and GM3 antigens are associated with the vimentin intermediate filaments of the HUVEC cytoskeleton. Immunofluorescence staining of fixed, permeabilized HUVECs showed colocalization of globoside and GM3 with vimentin but not with tubulin or actin. Both GSLs remained associated with intermediate filaments after perinuclear collapse of the filaments induced by colcemid. Indirect evidence that the globoside epitope is present on a GSL is the loss of staining by anti-globoside after methanol fixation and the absence of anti-globoside reactivity with HUVEC proteins on immunoblots. Colocalization of anti-globoside and anti-vimentin was also demonstrated in cryosections of endothelial cells, which indicates that the observed association was not an artifact induced by exposure of cells to detergent or organic solvent. Association of globoside with intermediate filaments was confirmed by immunoelectron microscopy, which demonstrated the presence of antigen along intermediate filaments, as well as on the cell surface and on lipid vesicles. Interferon-gamma decreased the ratio of surface to filamentous globoside staining, but had the opposite effect on GM3 distribution. Less abundant HUVEC GSLs, including Gb3, nLc4, IV2FucnLc4, and IV3NeuAcnLc4, were not detected along filaments. This is the first report of the association of GSLs with intermediate filaments. We suggest that intermediate filaments may play a role in the transport of GSLs.     

Analysis of NMR spectra of sugar chains of glycolipids by multiple relayed COSY and 2D homonuclear Hartman-Hahn spectroscopy

We applied multiple relayed COSY and 2D homonuclear Hartman-Hahn spectroscopy to globoside, a glycolipid purified from human red blood cells. The subspectra corresponding to individual sugar components were extracted even from overlapping proton resonances by taking the cross sections of 2D spectra parallel to the F2 axis at anomeric proton resonances, so that unambiguous assignments of sugar proton resonances were accomplished.     

The conformational behaviour of phosphatidylinositol in model membranes: 2H-NMR studies.

Dimyristoylphosphatidylinositol (DMPI) has been synthesized with the appropriate natural stereochemistry and labelled with deuterium at specific sites in the D-myo-inositol headgroup. 2H-NMR spectroscopy of DMPI in its lamellar phase at a molar ratio of water-to-lipid RW/L of 129 and at 70 degrees C reveals quadrupolar splittings delta v of 3.83 and 2.17 kHz, respectively, for the five axially oriented C-D bonds and the single equatorially oriented C-D bond of the D-myo-inositol headgroup. Between RW/L ratios of 129 and 210 and between 30 degrees C and 80 degrees C the value of the ratio of these splittings delta nu ax/delta nu eq varies significantly (between 1.17 and 4.38). If it is assumed that, at a particular temperature, there is a single preferred orientation of the inositol headgroup, and that motion of the DPMI molecule establishes axial symmetry with respect to the bilayer normal then the ratio of these quadrupolar splittings can be used to impose constraints on that orientation. For example, the data are inconsistent with a situation in which the inositol ring lies parallel to the membrane surface and are difficult to reconcile with an arrangement where the inositol ring lies perpendicular to the surface. Computational modelling identifies four possible 'tilted' orientations, all of which are consistent with the data, and two of these allow good intramolecular hydrogen bonds to be formed. In one there is hydrogen bonding between the inositol C2-OH and the phosphate pro-R oxygen. This is close to the conformation previously identified as being dominant in DMSO solution (Bushby, R.J., Byard, S.J., Hansbro, P.M. and Reid, D.G. (1990) Biochim. Biophys. Acta 1044, 231-236).     

Oligosaccharide order in a membrane-incorporated complex glycosphingolipid.

Galactosylceramide (GalCer) and the ganglioside, GM1, were 2H-labelled at C-6 (the hydroxymethyl moiety) of their single terminal galactosyl residues. Each deuterated glycosphingolipid was incorporated at a biologically relevant low concentration into multibilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). 2H-NMR spectra of aqueous dispersions of GalCer-POPC in the liquid crystal phase were characteristic of restricted headgroup motion (ordering) with effective axial symmetry. The degree of headgroup ordering was analogous to that of GalCer in pure aqueous multibilayers (Skarjune, R. and Oldfield, E. (1979) Biochim. Biophys. Acta 556, 208-218). In the case of GM1, 2H-labelled in the terminal galactose residue of the pentasaccharide headgroup, the 2H-NMR spectra were remarkably like those of the simple glycolipid, GalCer. This suggests substantial restriction of motion about the glycosidic and sugar-ceramide bonds of the complex GM1 headgroup, and that both lipids have comparable degrees of orientational averaging (fluctuation) about the bilayer normal. The result is the first direct demonstration that headgroup orientational order can exist for a complex glycolipid incorporated into 'fluid' bilayer membranes. Such behaviour argues for the possibility of modulation of membrane receptor properties through surface effects on average headgroup orientation and conformation.     

Influence of glycolipids on immune reactions of phospholipid antigens in liposomes

Complement-dependent immune damage to liposomes mediated by a murine monoclonal antibody to the liposomal bilayer was completely inhibited by ceramide tetrasaccharide (globoside) at an 8% concentration in the liposomes. Lower concentrations of globoside, or higher concentrations of ceramide tri-, di-, or monohexoside, were not inhibitory. At a globoside concentration of 5.8%, inhibition of the monoclonal antibody activity was reduced and inhibition was related to antibody concentration; but at 2% globoside, suppression of antibody activity was not observed at all. Analysis of space-filling models revealed that at 8% globoside the distance between adjacent tetrasaccharides of globoside approached the dimensions of the antigen-binding end of a 7S immunoglobulin molecule. We therefore propose that globoside, and perhaps other glycolipids, can exert steric hindrance to the binding of extracellular proteins to nonglycolipid constituents of the lipid bilayer. We conclude that microheterogeneity among polar groups of glycolipids may be a novel mechanism for allowing selective access of proteins to phospholipids on the lipid bilayer.     

Expression cloning of human globoside synthase cDNAs. Identification of beta 3Gal-T3 as UDP-N-acetylgalactosamine:globotriaosylceramide beta 1,3-N-acetylgalactosaminyltransferase

By using a eukaryocytic cell expression cloning system, we have isolated cDNAs of the globoside synthase (beta1, 3-N-acetylgalactosaminyltransferase) gene. Mouse fibroblast L cells transfected with SV40 large T antigen and previously cloned Gb3/CD77 synthase cDNAs were co-transfected with a cDNA library prepared from mRNA from human kidney together with Forssman synthase cDNA, and Forssman antigen-positive cells were panned using an anti-Forssman monoclonal antibody. The isolated cDNAs contained a single open reading frame predicting a type II membrane protein with 351 amino acids. Surprisingly, the cDNA clones turned out to be identical with previously reported beta3Gal-T3, which had been cloned by sequence homology with other galactosyltransferases. Substrate specificity analysis with extracts from cDNA-transfected L cells confirmed that the gene product was actually beta1, 3-N-acetylgalactosaminyltransferase that specifically catalyzes the transfer of N-acetylgalactosamine onto globotriaosylceramide. Results of TLC immunostaining of neutral glycolipids from the cDNA-transfected cells also supported the identity of the newly synthesized component as globoside. The results show that glycosyltransferases apparently belonging to a single glycosyltransferase family do not necessarily catalyze reactions utilizing the same acceptor or even the same sugar donor. The globoside synthase gene was expressed in many tissues, such as heart, brain, testis, etc. We propose the designation beta3GalNAc-T1 for the cloned globoside synthase gene.     

Perturbation of lecithin bilayer structure by globoside.

The ultrastructure of aggregates formed by mixtures of pig erythrocyte lecithin, cholesterol and globoside in aqueous systems was studied by electron microscopy and X-ray diffraction. Globoside and lecithin in up to equimolar amounts formed a lamellar mesophase, although the structure of the lamellae was perturbed. Mixtures containing excess globoside formed complex tubular or reticular aggregates. Cholesterol appeared to promote mixing of lecithin and globoside. The flexibility gradient of the hydrocarbon (hc) region of the lipid bilayers was studied using electron spin resonance (esr) spectroscopy of various nitroxide-labelled stearic acid probes. Globoside in equimolar amounts greatly perturbed the order parameters of lecithin bilayers, reducing the fluidity of the hc region and flattening the flexibility gradient near the polar (p) surface. The effect of globoside on lecithin-cholesterol bilayers was not so pronounced, since the latter was already more ordered than lecithin bilayers. A phase transition of pure globoside at 55 degrees C, involving 'melting' of the hc chains was also detected using X-ray and esr spectroscopic techniques. The interbilayer spacing, dw, of equimolar lecithin-globoside lamellar phase increased by 42% from that of lecithin bilayers, indicating that the glycolipid p group may increase the net repulsive force between bilayers, as was previously predicted theoretically.     

Influence of glycolipids on immune reactions of phospholipid antigens in liposomes

Complement-dependent immune damage to liposomes mediated by a murine monoclonal antibody to the liposomal bilayer was completely inhibited by ceramide tetrasaccharide (globoside) at an 8% concentration in the liposomes. Lower concentrations of globoside, or higher concentrations of ceramide tri-, di-, or monohexoside, were not inhibitory. At a globoside concentration of 5.8%, inhibition of the monoclonal antibody activity was reduced and inhibition was related to antibody concentration; but at 2% globoside, suppression of antibody activity was not observed at all. Analysis of space-filling models revealed that at 8% globoside the distance between adjacent tetrasaccharides of globoside approached the dimensions of the antigen-binding end of a 7S immunoglobulin molecule. We therefore propose that globoside, and perhaps other glycolipids, can exert steric hindrance to the binding of extracellular proteins to nonglycolipid constituents of the lipid bilayer. We conclude that microheterogeneity among polar groups of glycolipids may be a novel mechanism for allowing selective access of proteins to phospholipids on the lipid bilayer.     

Hydrostatic pressure-induced conformational changes in phosphatidylcholine headgroups: a 2H NMR study.

The effects of pressure and temperature on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine headgroup conformations were examined using deuterium nuclear magnetic resonance. Isothermal compression was found to produce a decrease in the choline alpha deuteron quadrupole splitting and increases in the choline beta and gamma deuteron quadrupole splittings. A similar counterdirectional change, seen in the presence of positive surface charge, has been attributed to tilting of the headgroup away from the bilayer surface in response to the torque exerted on the phosphocholine dipole by positive surface charges. The direction of the change in headgroup deuteron quadrupole splitting is consistent with the pressure-induced reduction in area per lipid in the liquid crystalline phase, which can be inferred from the ordering of phospholipid acyl chains under comparable conditions. The temperature dependences of the headgroup deuteron quadrupole splittings were also examined. It was found that at elevated pressure, the alpha splitting was insensitive to temperature, whereas the beta and gamma splittings decreased. The response of the beta deuteron splitting to temperature was found to be weaker at elevated pressure than at ambient pressure.     

Characterization of the L lambda phase in trehalose-stabilized dry membranes by solid-state NMR and X-ray diffraction

Solid-state nuclear magnetic resonance (NMR) spectroscopy and X-ray powder diffraction were used to investigate the mechanism of trehalose (TRE) stabilization of lipid bilayers. Calorimetric investigation of dry TRE-stabilized bilayers reveals a first-order phase transition (L kappa----L lambda) at temperatures similar to the L beta'----(P beta')----L alpha transition of hydrated lipid bilayers. X-ray diffraction studies show that dry mixtures of TRE and 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) have a lamellar structure with excess crystalline TRE being present. The L kappa phase shows typical gel-phase X-ray diffraction patterns. In contrast, the L lambda-phase diffraction patterns indicate disordered hydrocarbon chains. 2H NMR of specifically 2H chain-labeled DPPC confirmed that the acyl chains are disordered in the L lambda phase over their entire lengths. 2H spectra of the choline headgroup show hindered molecular motions as compared to dry DPPC alone, and 13C spectra of the sn-2-carbonyl show rigid lattice powder patterns indicating very little motion at the headgroup and interfacial regions. Thus, the sugar interacts extensively with the hydrophilic regions of the lipid, from the choline and the phosphate moieties in the headgroup to the glycerol and carbonyls in the interfacial region. We postulate that the sugar and the lipid form an extensive hydrogen-bonded network with the sugar acting as a spacer to expand the distance between lipids in the bilayer. The fluidity of the hydrophobic region in the L lambda phase together with the bilayer stabilization at the headgroup contributes to membrane viability in anhydrobiotic organisms.