Tomokazu Yasuda

Detailed Comparison of Deuterium Quadrupole Profiles between Sphingomyelin and Phosphatidylcholine Bilayers

Lipid rafts are microdomains rich in sphingomyelin (SM) and cholesterol (Chol). The essential question is why natural lipid rafts prefer SM rather than saturated diacyl glycerophosphocholine, although both form ordered membranes with Chol in model systems. Hence in this study, we synthesized site-specifically deuterated 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholines that match the acyl chain length of stearoyl-SM (SSM), and compared their deuterium quadrupole coupling profiles in detail. The results suggest a deeper distribution of Chol in the SSM membranes, a lower entropic penalty upon accommodation of Chol in SSM membranes, and a higher thermal stability of acyl-chain orders in the SSM-Chol bilayers than in the 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine-Chol system at various Chol concentrations. The entropy effect and thermal stability should render SM a more preferred raft constituent than saturated diacyl glycerophosphocholine. Our data also demonstrate that the selective and comprehensive deuteration strategy is indispensable for accurate comparison of order profiles.

NMR-based conformational analysis of sphingomyelin in bicelles

Sphingomyelin (SM) is a common sphingolipid in mammalian membranes and is known to be substantially involved in cellular events such as the formation of lipid rafts. Despite its biological significance, conformation of SM in a membrane environment remains unclear because the noncrystalline property and anisotropic environment of lipid bilayers hampers the application of X-ray crystallography and NMR measurements. In this study, to elucidate the conformation of SM in membranes, we utilized bicelles as a substitute for a lipid bilayer membrane. First, we demonstrated through (31)P NMR, (2)H NMR, and dynamic light scattering experiments that SM forms both oriented and isotropic bicelles by changing the ratio of SM/dihexanoyl phosphatidylcholine. Then, we determined the conformation of SM in isotropic bicelles on the basis of coupling constants and NOE correlations in (1)H NMR and found that the C2-C6 and amide groups of SM take a relatively rigid conformation in bicelles.

Deuterium NMR of Raft Model Membranes Reveals Domain-Specific Order Profiles and Compositional Distribution

In this report, we applied site-specifically deuterated N-stearoylsphingomyelins (SSMs) to raft-exhibiting ternary mixtures containing SSM, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol (Chol) and successfully acquired deuterium quadrupole coupling profiles of SSM from liquid-ordered (Lo) and liquid-disordered (Ld) domains. To our knowledge, this is the first report that shows detailed lipid chain dynamics separately and simultaneously obtained from coexisting Lo and Ld domains. We also found that the quadrupole profile of the Lo phase in the ternary system was almost identical to that in the SSM-Chol binary mixture, suggesting that the order profile of the binary system is essentially applicable to more complicated membrane systems in terms of the acyl chain order. We also demonstrated that (2)H NMR spectroscopy, in combination with organic synthesis of deuterated components, could be used to reveal the accurate mole fractions of each component distributed in the Lo and Ld domains. As compared with the reported tie-line analysis of phase diagrams, the merit of our (2)H NMR analysis is that the domain-specific compositional fractions are directly attainable without experimental complexity and ambiguity. The accurate compositional distributions as well as lipid order profiles in ternary mixtures are relevant to understanding the molecular mechanism of lipid raft formation.

Lipid Interactions and Organization in Complex Bilayer Membranes.

Bilayer lipids influence the lateral structure of the membranes, but the relationship between lipid properties and the lateral structure formed is not always understood. Model membrane studies on bilayers containing cholesterol and various phospholipids (PLs) suggest that high and low temperature melting PLs may segregate, especially in the presence of cholesterol. The effect of different PL headgroups on lateral structure of bilayers is also not clear. Here, we have examined the formation of lateral heterogeneity in increasingly complex (up to five-component) multilamellar bilayers. We have used time-resolved fluorescence spectroscopy with domain-selective fluorescent probes (PL-conjugated trans-parinaric acid), and (2)H NMR spectroscopy with site or perdeuterated PLs. We have measured changes in bilayer order using such domain-selective probes both as a function of temperature and composition. Our results from time-resolved fluorescence and (2)H NMR showed that in ternary bilayers, acyl chain order and thermostability in sphingomyelin-rich domains were not affected to any greater extent by the headgroup structure of the monounsaturated PLs (phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine) in the bilayer. In the complex five-component bilayers, we could not detect major differences between the different monounsaturated PLs regarding cholesterol-induced ordering. However, cholesterol clearly influenced deuterated N-palmitoyl sphingomyelin differently than the other deuterated PLs, suggesting that cholesterol favored N-palmitoyl sphingomyelin over the other PLs. Taken together, both the fluorescence spectroscopy and (2)H NMR data suggest that the complex five-component membranes displayed lateral heterogeneity, at least in the lower temperature regimen examined.

Toxin-induced pore formation is hindered by intermolecular hydrogen bonding in sphingomyelin bilayers

Sticholysin I and II (StnI and StnII) are pore-forming toxins that use sphingomyelin (SM) for membrane binding. We examined how hydrogen bonding among membrane SMs affected the StnI- and StnII-induced pore formation process, resulting in bilayer permeabilization. We compared toxin-induced permeabilization in bilayers containing either SM or dihydro-SM (lacking the trans Δ(4) double bond of the long-chain base), since their hydrogen-bonding properties are known to differ greatly. We observed that whereas both StnI and StnII formed pores in unilamellar vesicles containing palmitoyl-SM or oleoyl-SM, the toxins failed to similarly form pores in vesicles prepared from dihydro-PSM or dihydro-OSM. In supported bilayers containing OSM, StnII bound efficiently, as determined by surface plasmon resonance. However, StnII binding to supported bilayers prepared from dihydro-OSM was very low under similar experimental conditions. The association of the positively charged StnII (at pH7.0) with unilamellar vesicles prepared from OSM led to a concentration-dependent increase in vesicle charge, as determined from zeta-potential measurements. With dihydro-OSM vesicles, a similar response was not observed. Benzyl alcohol, which is a small hydrogen-bonding compound with affinity to lipid bilayer interfaces, strongly facilitated StnII-induced pore formation in dihydro-OSM bilayers, suggesting that hydrogen bonding in the interfacial region originally prevented StnII from membrane binding and pore formation. We conclude that interfacial hydrogen bonding was able to affect the membrane association of StnI- and StnII, and hence their pore forming capacity. Our results suggest that other types of protein interactions in bilayers may also be affected by hydrogen-bonding origination from SMs.

Formation of Gel-like Nanodomains in Cholesterol-Containing Sphingomyelin or Phosphatidylcholine Binary Membrane As Examined by Fluorescence Lifetimes and 2H NMR Spectra

In this study, we measured the time-resolved fluorescence of trans-parinaric acid (tPA), steady-state fluorescence anisotropy of diphenylhexatriene (DPH), and (2)H NMR of 10,10-d2-stearoyl lipids in stearoyl sphingomyelin with cholesterol (SSM/Chol) and l-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine with Chol (PSPC/Chol) binary membranes. The results suggest that the membrane order obtained from the fluorescence experiments shows a similar temperature dependency as those of the (2)H NMR data. More importantly, the time-resolved fluorescence data implied the presence of at least two types of domains, cholesterol-poor gel-like domains (CPGLD) and cholesterol-enriched liquid-ordered (Lo) domains. These domains appear on a nano-to-micro second time scale for both SSM-Chol and PSPC-Chol membranes. The relative size of the gel-like domain was also estimated from the temperature-dependent lifetime measurements and (2)H NMR spectral changes. The results imply that the size of the gel-like domains is very small, probably on the nanometer scale, and smaller in SSM-Chol membrane than those in PSPC-Chol bilayers, which could account for the higher thermal stability of SM-Chol membranes. The present study demonstrates that gel-like nanodomains occur in SM-Chol binary membrane even with Chol content of over 33 mol %, which has been thought to consist exclusively of Lo phase, implying that not only Lo domains but also gel-like nanodomains are important for formation of lipid-ordered phase in SM-Chol and PC-Chol membranes.

Phosphatidylcholine bearing 6,6-dideuterated oleic acid: A useful solid-state 2H NMR probe for investigating membrane properties

Lipid organization has been at the center of research on lipid rafts. Dioleoylphosphatidylcholine (DOPC) is a typical unsaturated lipid. Very few studies have reported its thermodynamics in raft-like membranes. Herein, we have developed a highly efficient synthetic method for [C6-(2)H2] oleic acid, and newly synthesized [C6-(2)H2] DOPC. In raft-like oriented bilayers, [C6-(2)H2] DOPC shows clear phase separation and characteristic phase behavior at various temperature. It has been successfully utilized for the comparison of membrane properties between sphingomyelin (SM) and dihydrosphingomyelin (DHSM) membranes.

Nanosized Phase Segregation of Sphingomyelin and Dihydrosphingomyelin in Unsaturated Phosphatidylcholine Binary Membranes without Cholesterol

In this study, we applied fluorescence spectroscopy, differential scanning calorimetry (DSC), and 2H NMR to elucidate the properties of nanoscopic segregated domains in stearoylsphingomyelin (SSM)/dioleoylphosphatidylcholine (DOPC) and dihydrostearoylsphingomyelin (dhSSM)/DOPC binary membranes. The results obtained from fluorescence measurements suggest the existence of gel-like domains with high fluidity in both SSM and dhSSM macroscopic gel phases. The DSC thermograms showed that DOPC destabilizes SM-rich gel-like domains to a much lesser extent compared to the same amount of cholesterol. It was also found that a stable lateral segregation occurs without cholesterol, indicating that SSM itself undergoes homophilic interactions to form small gel-like domains. 2H NMR experiments disclosed differences in the temperature-dependent ordering of SSM/DOPC and dhSSM/DOPC bilayers; the dhSSM membrane showed less miscibility with the DOPC fluid phase, higher thermal stability, and tighter packing. In addition, the NMR results suggest the formation of mid-sized gel-like aggregates consisting of dhSSM. These differences could be accounted for by homophilic interactions, as previously reported ( Yasuda Biophys. J. 2016 , 110 , 431 - 440 ). In the absence of cholesterol, the moderately strong sphingomyelin (SM)/SM affinity results in the formation of small gel-like domains, whereas a stronger dhSSM/dhSSM affinity leads to larger gel-like domains. Considering the similar physicochemical features of SSM and dhSSM, the present results suggest that the formation of nanosized domains of SM is better characterized by homophilic interactions than by SM-cholesterol interplay. These effects are considered important to the ordered domain formation of SMs in biological membranes.

Sphingomyelin Stereoisomers Reveal That Homophilic Interactions Cause Nanodomain Formation

Sphingomyelin is an abundant lipid in some cellular membrane domains, such as lipid rafts. Hydrogen bonding and hydrophobic interactions of the lipid with surrounding components such as neighboring sphingomyelin and cholesterol (Cho) are widely considered to stabilize the raft-like liquid-ordered (Lo) domains in membrane bilayers. However, details of their interactions responsible for the formation of Lo domains remain largely unknown. In this study, the enantiomer of stearoyl sphingomyelin (ent-SSM) was prepared, and its physicochemical properties were compared with the natural SSM and the diastereomer of SSM to examine possible stereoselective lipid-lipid interactions. Interestingly, differential scanning calorimetry experiments demonstrated that palmitoyl sphingomyelin, with natural stereochemistry, exhibited higher miscibility with SSM bilayers than with ent-SSM bilayers, indicating that the homophilic sphingomyelin interactions occurred in a stereoselective manner. Solid-state 2H NMR revealed that Cho elicited its ordering effect very similarly on SSM and ent-SSM (and even on the diastereomer of SSM), suggesting that SSM-Cho interactions are not significantly affected by stereospecific hydrogen bonding. SSM and ent-SSM formed gel-like domains with very similar lateral packing in SSM/Cho/palmitoyloleoyl phosphatidylcholine membranes, as shown by fluorescence lifetime experiments. This observation can be explained by a homophilic hydrogen-bond network, which was largely responsible for the formation of gel-like nanodomains of SSMs (or ent-SSM). Our previous study revealed that Cho-poor gel-like domains contributed significantly to the formation of an Lo phase in sphingomyelin/Cho membranes. The results of the study presented here further show that SSM-SSM interactions occur near the headgroup region, whereas hydrophobic SSM-Cho interactions appeared important in the bilayer interior for Lo domain formation. The homophilic interactions of sphingomyelins could be mainly responsible for the formation of the domains of nanometer size, which may correspond to the small sphingomyelin/Cho-based rafts that temporally occur in biological membranes.