Effect of anti-tumor ether lipids on ordered domains in model membranes

1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (OMPC, edelfosine) and 1-hexadecylphosphocholine (HePC, miltefosine) represent two groups of synthetic ether lipid analogues with anti-tumor activity. Because of their hydrophobic nature, they may become incorporated into plasma membranes of cells, and it has been argued that they may act via association with lipid rafts. With the quenching of steady-state fluorescence of probes preferentially partitioning into sterol-rich ordered domains (cholestatrienol and trans-parinaric acid), we showed that OMPC and HePC by themselves did not form sterol-rich domains in fluid model membranes, in contrast to the two chain ether lipid 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine. Nevertheless, all three ether lipids significantly stabilized palmitoyl-sphingomyelin/cholesterol-rich domains against temperature induced melting. In conclusion, this study shows that anti-tumor ether lipids are likely to affect the properties of cholesterol-sphingomyelin domains (i.e., lipid rafts) when incorporated into cell membranes.     

Phosphatidylglycerol lipids enhance folding of an alpha helical membrane protein

Membrane lipids are increasingly being recognised as active participants in biological events. The precise roles that individual lipids or global properties of the lipid bilayer play in the folding of membrane proteins remain to be elucidated, Here, we find a significant effect of phosphatidylglycerol (PG) on the folding of a trimeric α helical membrane protein from Escherichia coli diacylglycerol kinase. Both the rate and the yield of folding are increased by increasing the amount of PG in lipid vesicles. Moreover, there is a direct correlation between the increase in yield and the increase in rate; thus, folding becomes more efficient in terms of speed and productivity. This effect of PG seems to be a specific requirement for this lipid, rather than a charge effect. We also find an effect of single-chain lyso lipids in decreasing the rate and yield of folding. We compare this to our previous work in which lyso lipids increased the rate and yield of another membrane protein, bacteriorhodopsin. The contrasting effect of lyso lipids on the two proteins can be explained by the different folding reaction mechanisms and key folding steps involved. Our findings provide information on the lipid determinants of membrane protein folding.     

Direct visualization of saposin remodelling of lipid bilayers

Saposins A, B, C and D are soluble, non-enzymatic proteins that interact with lysosomal membranes to activate the breakdown and transfer of glycosphingolipids. The mechanisms of hydrolase activation and lipid transfer by saposins remain unknown. We have used in situ atomic force microscopy (AFM) with simultaneous confocal fluorescence microscopy to investigate the interactions of saposins with lipid membranes. AFM images of the effect of saposins A, B and C on supported lipid bilayers showed a time and concentration-dependent nucleated spread of membrane transformation. Saposin B produced deep gaps that ultimately filled with granular material, while saposins A and C lead to localized areas of membrane that were reduced in height by approximately 1.5 nm. Fluorescence-labeled saposin C co-localized with the transformed areas of the bilayer, indicating stable binding to the membrane. Fluorescence resonance energy transfer confirmed a direct interaction between saposin C and lipid. Under certain conditions of membrane lipid composition and saposin concentration, extensive bilayer lipid removal was observed. We propose a multi-step mechanism that integrates the structural features and amphipathic properties of the saposin proteins.     

NMR studies of molybdate complexes of D-erythro-L-manno-octose and D-erythro-L-gluco-octose and their alditols

Different amounts and various types of bis-dinuclear tetradentate molybdate complexes of D-erythro-L-manno-octose, D-erythro-L-gluco-octose, D-erythro-L-manno-octitol and D-erythro-L-gluco-octitol were characterized by 1H and 13C NMR spectroscopy in aqueous solutions. Detailed analysis of 1H-(1)H coupling constants and NOEs, together with chemical shifts, allowed characterization of the different isomers of these complexes.     

Structure and fluctuations of charged phosphatidylserine bilayers in the absence of salt

Using x-ray diffraction and NMR spectroscopy, we present structural and material properties of phosphatidylserine (PS) bilayers that may account for the well documented implications of PS headgroups in cell activity. At 30 degrees C, the 18-carbon monounsaturated DOPS in the fluid state has a cross-sectional area of 65.3 A(2) which is remarkably smaller than the area 72.5 A(2) of the DOPC analog, despite the extra electrostatic repulsion expected for charged PS headgroups. Similarly, at 20 degrees C, the 14-carbon disaturated DMPS in the gel phase has an area of 40.8 A(2) vs. 48.1 A(2) for DMPC. This condensation of area suggests an extra attractive interaction, perhaps hydrogen bonding, between PS headgroups. Unlike zwitterionic lipids, stacks of PS bilayers swell indefinitely as water is added. Data obtained for osmotic pressure versus interbilayer water spacing for fluid phase DOPS are well fit by electrostatic interactions calculated for the Gouy-Chapman regime. It is shown that the electrostatic interactions completely dominate the fluctuational pressure. Nevertheless, the x-ray data definitively exhibit the effects of fluctuations in fluid phase DOPS. From our measurements of fluctuations, we obtain the product of the bilayer bending modulus K(C) and the smectic compression modulus B. At the same interbilayer separation, the interbilayer fluctuations are smaller in DOPS than for DOPC, showing that B and/or K(C) are larger. Complementing the x-ray data, (31)P-chemical shift anisotropy measured by NMR suggest that the DOPS headgroups are less sensitive to osmotic pressure than DOPC headgroups, which is consistent with a larger K(C) in DOPS. Quadrupolar splittings for D(2)O decay less rapidly with increasing water content for DOPS than for DOPC, indicating greater perturbation of interlamellar water and suggesting a greater interlamellar hydration force in DOPS. Our comparisons between bilayers of PS and PC lipids with the same chains and the same temperature enable us to focus on the effects of these headgroups on bilayer properties.     

N-palmitoyl-sulfatide participates in lateral domain formation in complex lipid bilayers

Sulfatides (galactosylceramidesulfates) are negatively charged glycosphingolipids that are important constituents of brain myelin membranes. These membranes are also highly enriched in galactosylceramide and cholesterol. It has been implicated that sulfatides, together with other sphingolipids, take part in lateral domain formation in biological membranes. This study was conducted to characterize the lateral phase behavior of N-palmitoyl-sulfatide in mixed bilayer membranes. Going from simple lipid mixtures with sulfatide as the only sphingolipid in a fluid matrix of POPC, to more complex membranes including other sphingolipids, we have examined 1) ordered domain formation with sulfatide, 2) sterol enrichment in such domains and 3) stabilization of the domains against temperature by the addition of calcium. Using two distinct phase selective fluorescent probes, trans-parinaric acid and cholestatrienol, together with a quencher in the fluid phase, we were able to distinguish between ordered domains in general and ordered domains enriched in sterol. We found that N-palmitoyl-sulfatide formed ordered domains when present as the only sphingolipid in a fluid phospholipid bilayer, but these domains did not contain sterol and their stability was unaffected by calcium. However, at low, physiologically relevant concentrations, sulfatide partitioned favorably into domains enriched in other sphingolipids and cholesterol. These domains were stabilized against temperature in the presence of divalent cations. We conclude that sulfatides are likely to affect the lateral organization of biomembranes.     

Base catalysed isomerisation of aldoses of the arabino and lyxo series in the presence of aluminate

Base-catalysed isomerisation of aldoses of the arabino and lyxo series in aluminate solution has been investigated. L-Arabinose and D-galactose give L-erythro-2-pentulose (L-ribulose) and D-lyxo-2-hexulose (D-tagatose), respectively, in good yields, whereas lower reactivity is observed for 6-deoxy-D-galactose (D-fucose). From D-lyxose, D-mannose and 6-deoxy-L-mannose (L-rhamnose) are obtained mixtures of ketoses and C-2 epimeric aldoses. Small amounts of the 3-epimers of the ketoses were also formed. 6-Deoxy-L-arabino-2-hexulose (6-deoxy-L-fructose) and 6-deoxy-L-glucose (L-quinovose) were formed in low yields from 6-deoxy-L-mannose and isolated as their O-isopropylidene derivatives. Explanations of the differences in reactivity and course of the reaction have been suggested on the basis of steric effects.     

Facile synthesis of benzyl beta-D-galactofuranoside. A convenient intermediate for the synthesis of D-galactofuranose-containing molecules

Benzyl beta-D-galactofuranoside was efficiently obtained from 1,2,3,5,6-penta-O-benzoyl-alpha,beta-D-galactofuranose, via benzyl 2,3,5,6-tetra-O-benzoyl-beta-D-galactofuranoside. Conditions for the O-debenzylation were investigated in order to evaluate the synthetic application of the benzyl group as an anomeric protector of a galactofuranose moiety in synthetic strategies involving galactofuranose.     

Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 Å, respectively. A subunit of P. cichoriid-TE adopts a (β/α)₈ barrel structure, and a metal ion (Mn²⁺) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the β-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn²⁺, and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.     

Using micellar mole fractions to assess membrane protein stability in mixed micelles

The increased focus on the structural and physical properties of membrane proteins has made it critical to develop methods that provide a reliable estimate of membrane protein stability. A simple approach is to monitor the protein's conformational changes in mixed detergent systems, typically consisting of an anionic (denaturing) and non-ionic (non-denaturing) component. Linear correlations between, e.g., the melting temperature and the bulk mole fraction of the anionic component have been observed. However, a potential complication is that the bulk mole fraction is not identical to the mole fraction in the mixed micelle, which is the local environment experienced by the membrane protein. Here, we present an extensive analysis of the thermal stability of the membrane-integrated domain of the outer membrane protein AIDA in the presence of different mixed micelles. In the micelle system SDS-octyl-polyoxyethylene, the melting temperature in the absence of SDS extrapolates to 113 °C using bulk mole fractions. However, for mixed micelles involving short-chain detergents or phospholipids, the melting temperature calculated using bulk mole fractions reaches values up to several hundred degrees higher than 113 °C and can only be obtained by extrapolation over a narrow mole fraction interval. Furthermore, there is a non-linear relationship between the melting temperature and bulk mole fractions for mixed micelle systems involving cationic detergents (also denaturing). We show that if we instead use the micellar mole fraction as a parameter for denaturing detergent strength, we obtain linear correlations which extrapolate to more or less the same value of the melting temperature. There remains some scatter in the extrapolated values of the melting temperature in different binary systems, which suggest that additional micellar interactions may play a role. Nevertheless, in general terms, the mixed micellar composition is a good parameter to describe the membrane protein's microenvironment. Note, however, that for the mixed micelle system involving SDS and dodecyl maltoside, which has been used by several research groups to determine membrane protein stability, the estimate provided by bulk mole fraction leads to similar values as that of micellar mole fractions.     

Synthesis and intramolecular reactions of Tyr-Gly and Tyr-Gly-Gly related 6-O-glucopyranose esters

6-O-(L-Tyrosylglycyl)- and 6-O-(L-tyrosylglycylglycyl)-D-glucopyranose were synthesized by condensation of the pentachlorophenyl esters of the respective di- and tripeptide with fully unprotected D-glucose. The intramolecular reactivity of the sugar conjugates was studied in pyridine-acetic acid and in dry methanol, at various temperatures and for various incubation times. The composition of the incubation mixtures was monitored by a reversed-phase HPLC method that permits simultaneous analysis of the disappearance of the starting material and the appearance of rearrangement and degradation products. To determine the influence of esterification of the peptide carboxy group on its amino group reactivity, parallel experiments were done in which free peptides were, under identical reaction conditions, incubated with D-glucose (molar ratios 1:1 and 1:5). Depending on the starting compound, different types of Amadori products (cyclic and bicyclic form), methyl ester of peptides, and Tyr-Gly-diketopiperazine were obtained.     

Recognition of selected monosaccharides by Pseudomonas aeruginosa Lectin II analyzed by molecular dynamics and free energy calculations

In this study, interactions of selected monosaccharides with the Pseudomonas aeruginosa Lectin II (PA-IIL) are analyzed in detail. An interesting feature of the PA-IIL binding is that the monosaccharide is interacting via two calcium ions and the binding is unusually strong for protein-saccharide interaction. We have used Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) and normal mode analysis to calculate the free energy of binding. The impact of intramolecular hydrogen bond network for the lectin/monosaccharide interaction is also analyzed.     

Cold temperature-induced modifications to the composition and structure of the lipopolysaccharide of Yersinia pestis

Following a report of variations in the lipopolysaccharide (LPS) structure of Yersinia pestis at mammalian (37 degrees C) and flea (25 degrees C) temperatures, a number of changes to the LPS structure were observed when the bacterium was cultivated at a temperature of winter-hibernating rodents (6 degrees C). In addition to one of the known Y. pestis LPS types, LPS of a new type was isolated from Y. pestis KM218 grown at 6 degrees C. The core of the latter differs in: (i) replacement of terminal galactose with terminal d-glycero-d-manno-heptose; (ii) phosphorylation of terminal oct-2-ulosonic acid with phosphoethanolamine; (iii) a lower content of GlcNAc, and; (iv) the absence of glycine; lipid A differs in the lack of any 4-amino-4-deoxyarabinose and presumably partial (di)oxygenation of a fatty acid(s). The data obtained suggest that cold temperature switches on an alternative mechanism of control of the synthesis of Y. pestis LPS.     

Molecular features of phospholipids that affect glycolipid transfer protein-mediated galactosylceramide transfer between vesicles

The glycolipid transfer protein (GLTP)-mediated movement of galactosylceramide from model membrane donor vesicles to acceptor vesicles is sensitive to the membrane environment surrounding the glycolipid. GLTP can catalyze the transfer of a fluorescently labeled GSL, anthrylvinyl-galactosylceramide (AV-GalCer), from vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and dipalmitoylphosphatidylcholine matrices, but not from vesicles prepared from N-palmitoylsphingomyelin, regardless of the cholesterol content of the vesicles. In this study, we have examined the structural features of sphingomyelin (SM) that are responsible for its inhibition of the rate of GLTP-catalyzed transfer of AV-GalCer. The rate of glycolipid transfer was enhanced when the N-palmitoyl chain of SM was replaced with an N-oleoyl chain. Analogs of N-palmitoyl-SM in which the 4,5-double bond of the long-chain base is reduced or the 3-hydroxy group is removed did not inhibit GLTP-catalyzed transfer of AV-GalCer. When the donor vesicles were prepared with phosphatidylcholines or ether-linked phosphatidylcholine analogs, the transfer rates of AV-GalCer increased with increasing degree of unsaturation. The rate of AV-GalCer transfer was strongly dependent on the unsaturation degree of the acyl and/or alkyl chains. For ester-linked PCs, the transfer rate increased in the order DPPC < POPC < DOPC, which have 0, 1, and 2 cis double bonds, respectively.     

Efficient synthesis of 5-thio-D-arabinopyranose and 5-thio-D-xylopyranose from the corresponding D-pentono-1,4-lactones

5-Thio-D-arabinopyranose (5) and 5-thio-D-xylopyranose (10) were synthesized from the corresponding D-pentono-1,4-lactones. After regioselective bromination at C-5, transformation into 5-S-acetyl-5-thio derivatives, reduction into lactols and deprotection afforded the title compounds in 49 and 42% overall yield, respectively.     

Crystal structure of YihS in complex with D-mannose: structural annotation of Escherichia coli and Salmonella enterica yihS-encoded proteins to an aldose-ketose isomerase

The three-dimensional structure of a Salmonella enterica hypothetical protein YihS is significantly similar to that of N-acyl-d-glucosamine 2-epimerase (AGE) with respect to a common scaffold, an α₆/α₆-barrel, although the function of YihS remains to be clarified. To identify the function of YihS, Escherichia coli and S. enterica YihS proteins were overexpressed in E. coli, purified, and characterized. Both proteins were found to show no AGE activity but showed cofactor-independent aldose-ketose isomerase activity involved in the interconversion of monosaccharides, mannose, fructose, and glucose, or lyxose and xylulose. In order to clarify the structure/function relationship of YihS, we determined the crystal structure of S. enterica YihS mutant (H248A) in complex with a substrate (d-mannose) at 1.6 Å resolution. This enzyme-substrate complex structure is the first demonstration in the AGE structural family, and it enables us to identify active-site residues and postulate a reaction mechanism for YihS. The substrate, β-d-mannose, fits well in the active site and is specifically recognized by the enzyme. The substrate-binding site of YihS for the mannose C1 and O5 atoms is architecturally similar to those of mutarotases, suggesting that YihS adopts the pyranose ring-opening process by His383 and acidifies the C2 position, forming an aldehyde at the C1 position. In the isomerization step, His248 functions as a base catalyst responsible for transferring the proton from the C2 to C1 positions through a cis-enediol intermediate. On the other hand, in AGE, His248 is thought to abstract and re-adduct the proton at the C2 position of the substrate. These findings provide not only molecular insights into the YihS reaction mechanism but also useful information for the molecular design of novel carbohydrate-active enzymes with the common scaffold, α₆/α₆-barrel.     

NAD+-specific D-arabinose dehydrogenase and its contribution to erythroascorbic acid production in Saccharomyces cerevisiae

Erythroascorbic acid (eAsA) is a five-carbon analog of ascorbic acid, and it is synthesized from D-arabinose by D-arabinose dehydrogenase (ARA) and D-arabinono-gamma-lactone oxidase. We found an NAD+-specific ARA activity which is operative under submillimolar level of d-arabinose in the extracts of Saccharomyces cerevisiae. The hypothetical protein encoded by YMR041c showed a significant homology to a l-galactose dehydrogenase which plays in plant ascorbic acid biosynthesis, and we named it as Ara2p. Recombinant Ara2p showed NAD+-specific ARA activity with Km=0.78 mM to d-arabinose, which is 200-fold lower than that for the conventional NADP+-specific ARA, Ara1p. Gene disruptant of ARA2 lost entire NAD+-specific ARA activity and the conspicuous increase in intracellular eAsA by exogenous d-arabinose feeding, while the double knockout mutant of ARA1 and ARA2 still retained measurable amount of eAsA. It demonstrates that Ara2p, not Ara1p, mainly contributes to the production of eAsA from d-arabinose in S. cerevisiae.     

Melting behaviour of D-sucrose, D-glucose and D-fructose

The melting behaviour of d-sucrose, d-glucose and d-fructose was studied. The melting peaks were determined with DSC and the start of decomposition was studied with TG at different rates of heating. In addition, melting points were determined with a melting point apparatus. The samples were identified as d-sucrose, alpha-d-glucopyranose and beta-d-fructopyranose by powder diffraction measurements. There were differences in melting between the different samples of the same sugar and the rate of heating had a remarkable effect on the melting behaviour. For example, T(o), DeltaH(f) and T(i) (initial temperature of decomposition) at a 1 degrees Cmin(-1) rate of heating were 184.5 degrees C, 126.6Jg(-1) and 171.3 degrees C for d-sucrose, 146.5 degrees C, 185.4Jg(-1) and 152.0 degrees C for d-glucose and 112.7 degrees C, 154.1Jg(-1) and 113.9 degrees C for d-fructose. The same parameters at 10 degrees Cmin(-1) rate of heating were 188.9 degrees C, 134.4Jg(-1) and 189.2 degrees C for d-sucrose, 155.2 degrees C, 194.3Jg(-1) and 170.3 degrees C for d-glucose and 125.7 degrees C, 176.7Jg(-1) and 136.8 degrees C d-fructose. At slow rates of heating, there were substantial differences between the different samples of the same sugar. The melting point determination is a sensitive method for the characterization of crystal quality but it cannot be used alone for the identification of sugar samples in all cases. Therefore, the melting point method should be validated for different sugars.     

A short helix in the C-terminal region of LolA is important for the specific membrane localization of lipoproteins

The structures of a lipoprotein carrier, LolA, and a lipoprotein receptor, LolB, are similar except for an extra C-terminal loop containing a 3(10) helix and beta-strand 12 in LolA. Lipoprotein release was significantly reduced when beta-12 was deleted. Deletion of the 3(10) helix also inhibited the lipoprotein release. Furthermore, lipoproteins were non-specifically localized to membranes when LolA lacked the 3(10) helix. Thus, the membrane localization of lipoproteins with the LolA derivative lacking the 3(10) helix was independent of LolB whereas LolB was essential for the outer membrane localization of lipoproteins with the wild-type LolA.     

Two novel oligosaccharides isolated from a beverage produced by fermentation of a plant extract

An extract from 50 kinds of fruits and vegetables was fermented to produce a new beverage. Natural fermentation of the extract was carried out mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Two new saccharides were found in this fermented beverage. The saccharides were isolated using carbon-Celite column chromatography and preparative high performance liquid chromatography. Gas liquid chromatography analysis of methylated derivatives as well as MALDI-TOF MS and NMR measurements were used for structural confirmation. The (1)H and (13)C NMR signals of each saccharide were assigned using 2D-NMR including COSY, HSQC, HSQC-TOCSY, CH(2)-HSQC-TOCSY, and CT-HMBC experiments. The saccharides were identified as beta-D-fructopyranosyl-(2-->6)-beta-D-glucopyranosyl-(1-->3)-D-glucopyranose and beta-D-fructopyranosyl-(2-->6)-[beta-D-glucopyranosyl-(1-->3)]-D-glucopyranose.