Identical Acyl Chains Research Articles

Physicochemical characterization of biological and synthetic forms of two lipid A-based TLR4 agonists

Toll-like receptor (TLR) agonists are recognized as potential immune-enhancing adjuvants and are included in several licensed vaccines. Monophosphoryl lipid A (MPL®, GlaxoSmithKline) is one such TLR4 agonist that has been approved for use in human vaccines, such as Cervarix and Shingrix. Due to the heterogeneous nature of biologically derived MPL and the need for safer and more potent adjuvants, our groups have developed the novel TLR4 agonist candidates, BECC438 and BECC470 using the Bacterial Enzymatic Combinatorial Chemistry (BECC) platform. BECC438 and BECC470 have been included in studies to test their adjuvant potential and found to be effective in vaccines against both viral and bacterial disease agents. Here, we report detailed biophysical characterization of BECC438 and BECC470 purified from a biological source (BECC438b and BECC470b, respectively) and synthesized chemically (BECC438s and BECC470s, respectively). Both BECC438s and BECC470s have identical acyl chain configurations, BECC438s is bis-phosphorylated and BECC470s is mono-phosphorylated with the removal of the 4′ phosphate moiety. We determined the phase transition temperatures for the acyl chains of BECC438b and BECC470b and found them to be different from those exhibited by their synthetic counterparts. Furthermore, the phosphate groups of BECC438b and BECC470b are more highly hydrated than are those of BECC438s and BECC470s. In addition to exploring the BECC molecules’ biophysical features in aqueous solution, we explored potential formulation of BECC438 and BECC470 with the aluminum-based adjuvant Alhydrogel and as part of an oil-in-water emulsion (Medimmune Emulsion or ME). All of the lipid A analogues could be fully absorbed to Alhydrogel or incorporated onto ME. Surprisingly, the BECC470s molecule, unlike the others, displayed a nearly baseline signal when monitored using a Limulus amebocyte lysate (LAL) endotoxin detection system. Despite this, it was shown to behave as an agonist for human and mouse TLR4 when tested using multiple cell-based systems. This work paves the way for further formulation optimization of two chemically defined TLR4 agonists that are showing great promise as vaccine adjuvants.

Zwitterionic and Charged Lipids Form Remarkably Different Structures on Nanoscale Oil Droplets in Aqueous Solution

The molecular structure of zwitterionic and charged monolayers on small oil droplets in aqueous solutions is determined using a combined second harmonic and sum frequency study. From the interfacial vibrational signature of the acyl chains and phosphate headgroups as well as the response of the hydrating water, we find that zwitterionic and charged lipids with identical acyl chains form remarkably different monolayers. Zwitterionic phospholipids form a closely packed monolayer with highly ordered acyl tails. In contrast, the charged phospholipids form a monolayer with a low number density and disordered acyl tails. The charged headgroups are oriented perpendicular to the monolayer rather than parallel, as is the case for zwitterionic lipids. These significant differences between the two types of phospholipids indicate important roles of phospholipid headgroups in the determination of properties of cellular membranes and lipid droplets. The observed behavior of charged phospholipids is different from expectations based on studies performed on extended planar interfaces, at which condensed monolayers are readily formed. The difference can be explained by nanoscale related changes in charge condensation behavior that has its origin in a different balance of interfacial intermolecular interactions.

Associations of systemic sphingolipids with measures of hepatic function in liver cirrhosis are related to cholesterol

Lipoprotein particles are composed of various lipid classes including cholesterol and sphingolipids, and are low in serum of patients with liver cirrhosis. Hepatic decompensation is associated with a further decline of lipoproteins. Aim of the present work was to evaluate whether ceramide and sphingomyelin species are similarly changed in patients with liver cirrhosis and whether these variations are related to systemic cholesterol levels. In a cohort of 45 patients suffering from liver cirrhosis, cholesteryl ester species and subsequently total cholesterol were identified to be negatively associated with model of end stage liver disease (MELD) score. Indeed, the negative correlations of ceramide (Cer) and sphingomyelin (SM) species with MELD score, bilirubin and anti-thrombin 3 were non-significant after adjustment for cholesterol. Cer/SM ratios of species with identical acyl chains were not related to Child-Pugh or MELD score indicating that both lipids are comparably changed. Further, cholesterol levels and concentrations of all sphingolipids measured were similar in systemic, hepatic vein and portal vein blood. Cholesterol and distinct sphingolipids were similar before and 3 months after insertion of a transjugular intrahepatic portosystemic shunt while hexosylceramide 24:1 was significantly induced. It is concluded that analysis of distinct systemic sphingolipid species is not superior to measurement of cholesterol as non-invasive marker of hepatic injury in patients with liver cirrhosis.

Differential Influence of DOPG and DOPA Anionic Phospholipids on Single Channel Conductance of KcsA

Binding of anionic phospholipids to non-annular binding site of KcsA has been shown with crystallographic and biochemical methods. To gain insight into the functional importance of these specific protein-lipid interactions we studied the single channel properties of KcsA in planar lipid bilayer by varying the anionic phospholipid content of the membrane. Phospholipids with identical acyl chains and equivalent headgroup charges have been chosen for this purpose. Single channel properties of KcsA were unchanged, when the content of anionic phospholipids was kept below 30% in planar lipid bilayers formed from DOPC neutral phospholipid as remainder component. Upon gradual increase of membrane DOPG content up to 100%, a significant increase in single channel conductance was observed only at positive potentials (from 82,2±1,4 pS to 116,3±0,7 pS at +100mV in symmetrical 150 mM KCl solution). Surprisingly, equimolar increase of DOPA content of the membrane had no influence on single channel conductance of KscA. Differential influence of DOPG and DOPA phospholipids was preserved at varying K+ concentrations of recording solutions ranging from 50 to 250 mM. Using KcsA-Kv1.3 chimera we could show that the stretch of amino acids between E51 and A65 in KcsA was responsible for differential sensitivity of KcsA for DOPA and DOPG. Taken together, our data show that the structure of the phospholipid headgroup is important for phospholipid-specific modulation of single channel conductance in KcsA.

Phosphatidyl alcohols: Effect of head group size on domain forming properties and interactions with sterols

In this study, we have examined the membrane properties and sterol interactions of phosphatidyl alcohols varying in the size of the alcohol head group coupled to the sn-3-linked phosphate. Phosphatidyl alcohols of interest were dipalmitoyl derivatives with methanol (DPPMe), ethanol (DPPEt), propanol (DPPPr), or butanol (DPPBu) head groups. The Phosphatidyl alcohols are biologically relevant, because they can be formed in membranes by the phospholipase D reaction in the presence of alcohol. The melting behavior of pure phosphatidyl alcohols and mixtures with 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) or cholesterol was assessed using high sensitivity differential scanning calorimetry (DSC). DPPMe had the highest melting temperature (∼ 49 °C), whereas the other phosphatidyl alcohols had similar melting temperatures as DPPC (∼ 40–41 °C). All phosphatidyl alcohols, except DPPMe, also showed good miscibility with DPPC. The effects of cholesterol on the melting behavior and membrane order in multilamellar bilayer vesicles were assessed using steady-state anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DSC. The ordering effect of cholesterol in the fluid phase was lower for all phosphatidyl alcohols as compared to DPPC and decreased with increasing head group size. The formation of ordered domains containing the phosphatidyl alcohols in complex bilayer membranes was determined using fluorescence quenching of DPH or the sterol analogue cholesta-5,7,(11)-trien-3-beta-ol (CTL). The phosphatidyl alcohols did not appear to form sterol-enriched ordered domains, whereas DPPMe, DPPEt appeared to form ordered domains in the temperature window examined (10–50 °C). The partitioning of CTL into bilayer membranes containing phosphatidyl alcohols was to a small extent increased for DPPMe and DPPEt, but in general, sterol interactions were weak or unfavorable for the phosphatidyl alcohols. Our results show that the biophysical and sterol interacting properties of phosphatidyl alcohols, having identical acyl chain structures, are markedly dependent on the size of the head group.

Thematic Review Series: Glycerolipids. Cardiolipin synthesis for the assembly of bacterial and mitochondrial membranes

In this article, the formation of prokaryotic and eukaryotic cardiolipin is reviewed in light of its biological function. I begin with a detailed account of the structure of cardiolipin, its stereochemistry, and the resulting physical properties, and I present structural analogs of cardiolipin that occur in some organisms. Then I continue to discuss i) the de novo formation of cardiolipin, ii) its acyl remodeling, iii) the assembly of cardiolipin into biological membranes, and iv) the degradation of cardiolipin, which may be involved in apoptosis and mitochondrial fusion. Thus, this article covers the entire metabolic cycle of this unique phospholipid. It is shown that mitochondria produce cardiolipin species with a high degree of structural uniformity and molecular symmetry, among which there is often a dominant form with four identical acyl chains. The subsequent assembly of cardiolipin into functional membranes is largely unknown, but the analysis of crystal structures of membrane proteins has revealed a first glimpse into the underlying principles of cardiolipin-protein interactions. Disturbances of cardiolipin metabolism are crucial in the pathophysiology of human Barth syndrome and perhaps also play a role in diabetes and ischemic heart disease.

Detection of charge movements in ion pumps by a family of styryl dyes.

A family of fluorescent styryl dyes was synthesized to apply them as probes that monitor the ion-translocating activity of the Na,K-ATPase and the SR Ca-ATPase, similar to the widely used dye RH421. All dyes had the same chromophore but they differed in the length of the spacer between chromophore and polar head, an isothiocyanate group, and in the lengths of the two identical acyl chains, which form the tail of the dye molecules. A number of substrate-dependent partial reactions of both P-type ATPases affected the fluorescence intensity, and the magnitude of the fluorescence changes was used to characterize the usefulness of the dyes for further application. The experimental results indicate that electrochromy is the major mechanism of these dyes. While in the case of the Na,K-ATPase a single dye, 5QITC, showed larger fluorescence changes than all others, in the case of the SR Ca-ATPase all dyes tested were almost equal in their fluorescence responses. This prominent difference is interpreted as a hint that the position of the ion binding sites in both ion pumps may differ significantly despite their otherwise closely related structural features. Quench experiments with spin-labeled lipids in various positions of their fatty acids were used to gain information on the depth of the chromophore of the different dyes within the membrane dielectric, however, the spatial resolution was so poor that only qualitative information on the position of the chromophore in the lipid phase could be obtained.

Cholesterol-induced interfacial area condensations of galactosylceramides and sphingomyelins with identical acyl chains.

The interfacial interactions occurring between cholesterol and either galactosylceramides (GalCers) or sphingomyelins (SMs) with identical acyl chains have been investigated using Langmuir film balance techniques. Included among the synthesized GalCers and SMs were species containing palmitoyl (16:0), stearoyl (18:0), oleoyl [18:1 delta 9(c)], nervonoyl [24:1 delta 15(c)], or linoleoyl [18:2 delta 9,12(c)] acyl residues. The cholesterol-induced condensations in the average molecular areas of the monolayers were determined by classic mean molecular area vs composition plots as well as by expressing the changes in terms of sphingolipid cross-sectional area reduction over the surface pressure range from 1 to 40 mN/m (at 1 mN/m intervals). The results show that, at surface pressures approximating bilayer conditions (30 mN/m), acyl heterogeneity in naturally occurring SMs (bovine of egg SM) enhanced the area condensation induced by cholesterol compared with their predominant molecular species (e.g. 18:0 SM in bovine SM; 16:0 SM in egg SM). Nonetheless, cholesterol always had a greater condensing effect on SM compared to GalCer when these sphingolipids were acyl chain matched and in similar phase states (prior to mixing with cholesterol). Also, the cholesterol-induced area changes for a given sphingolipid type (e.g. SM or GalCer) were similar whether the acyl chains were saturated, cis-delta 9-monounsaturated, or cis-delta 9,12-diunsaturated if the sphingolipids were in similar phase states (prior to mixing with cholesterol) and compared at equivalent surface pressures. These results indicate that, under conditions were hydrocarbon structure is matched, the sphingolipid head group plays a dominant role in determining the extent to which cholesterol reduces sphingolipid cross-sectional area. Despite the larger cholesterol-induced area condensations observed in SMs compared to those in GalCers, the molecular-packing densities showed that equimolar GalCer-cholesterol films were generally packed as tight as or slightly tighter than those of the SM-cholesterol films. The results are discussed in terms of a molecular model for sphingolipid-cholesterol interactions. Our findings also do only raise questions as to whether cholesterol-induced condensation data provide a reliable measure of the affinity, i.e. interaction strength, between cholesterol and different lipids but also provide insight regarding the stability of sterol/sphingolipid 1-1 rich microdomains thought to exist in caveolae and other cell membrane regions.

Gramicidin A/short-chain phospholipid dispersions: chain length dependence of gramicidin conformation and lipid organization.

Gramicidin-lipid interactions were investigated using diacylphosphatidylcholines that contained two identical acyl chains of varying length, between 6 and 14 carbons. The gramicidin A (gA) conformation was monitored by circular dichroism (CD) spectroscopy and high-performance size-exclusion chromatography, and the lipid organization was investigated using 31P and 1H NMR spectroscopy and negative-stain electron microscopy. Diacylphosphatidylcholine (PC) lipids with chain lengths between 4 and 8 carbons have been previously shown to have a micellar organization in aqueous solution [Lin, T.-L., et al. (1986) J. Am. Chem. Soc. 108, 3499-3507]. CD spectra of aqueous gA/lipid dispersions, at a ratio of 1:28, demonstrated that the channel conformation of gA can be readily obtained when the acyl chain length is > or = 10, but not when the chain length is < or = 7. Size-exclusion chromatography revealed that the fraction of gA that could easily be dissociated into monomers in the dispersions increased with increasing acyl chain length, in agreement with the CD results. For a chain length of 8, the results were intermediate. The formation of the channel structure was found to depend on the "solvent-history", the temperature, the gA and lipid concentrations, the gA:lipid ratio, and consequently on the method of sample preparation. 1H and 31P NMR results suggest that codispersed gA increases the size of dioctanoyl-PC aggregates, but not of dihexanoyl-PC micelles. Negative-stain electron microscopy directly supports these findings. Dihexanoyl-PC (28 mM) was able to solubilize 1 mM gA in H2O, but the gA was not in the "channel" conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of phosphatidylethanolamine and phosphatidylglycerol headgroups on bilayer dynamics: a slow-motion ESR study

A study of the effect of phosphatidylethanolamine and phosphatidylglycerol headgroups on the behaviour of cholestane spin-label (CSL) molecules in three lipid bilayer configurations: planar multibilayers, multilamellar liposomes and small unilamellar vesicles (SUV) is presented. The results are compared with those obtained from the corresponding configurations of phosphatidylcholines with identical acyl chain composition. A comparison of the parameters describing the orientational order and dynamics of CSL in palmitoyloleoylphosphatidyl-ethanolamine (POPE) and palmitoyloleoylphosphatidylcholine (POPC) bilayer configurations indicates that the hydration of the lipid headgroups is the most important factor determining the rates of rotational motion. The orientational order of CSL is found to be lower in fully hydrated multilamellar liposomes and SUVs of negatively charged dioleoylphosphatidylglycerol (DOPG), than in the same configurations of the zwitterionic dioleoylphosphatidylcholine (DOPC). However, the rotational diffusion is not affected by the difference in the lipid headgroup. Apparently, the electrostatic repulsion in DOPG multilamellar liposomes and SUVs causes the orientational order to decrease, but without affecting the rates of rotational diffusion.

Competitive inhibition of lipolytic enzymes. III. Some acylamino analogues of phospholipids are potent competitive inhibitors of porcine pancreatic phospholipase A 2

Competitive inhibition of porcine pancreatic phospholipase A 2 was studied in mixed-micellar systems composed of long-and medium-chain substrates, potential inhibitors and detergents. A number of positional and stereoisomeric monoacylamino, acyloxyglycerophospholipids were investigated for their inhibitory properties, using as substrates the corresponding diacyl- sn-glycero-3-phospholipids possessing the same polar headgroup and identical acyl chain lengths. Based on a kinetic model applicable to water-insoluble inhibitors (see accompanying paper I), which allows a quantitative comparison of the inhibitory power ( Z) of the various phospholipid analogues, the following results were obtained: Substitution of a single acylester bond in a diacylglycerophospholipid by an acylamino group can transform the substrate molecule in a potent competitive inhibitor. This property is acquired only when this substitution occurs on the phospholipase-susceptible ester bond of the substrate. If the acylamino group replaces an ester bond which cannot be attacked by the highly positional and stereospecific phospholipase, the resulting molecule binds with similar affinity to the active site of the enzyme as the parent substrate molecule. Because of its positional and stereospecificity, this so-called inhibitory ‘amide effect’ suggests that these inhibitors behave as substrate-derived analogues. The inhibitory ‘amide-effect’ observed with several medium- and long-chain monoacyloxy-, monoacylamino-deoxyglycerophosphatides is completely lost upon specific alkaline hydrolysis of the single acylester bond. Reesterification of the free glycerol OH group in these lysoacylaminophosphoglycerides, even with an acetyl residue, restores the inhibitory properties. These observations indicate that specific binding of phospholipids to the active site of pancreatic phospholipase A 2, requires the presence of two chains in substrate or inhibitor structure and suggest that those results obtained with lysophospholipids and single-chain analogues may be questionable.

The influence of fatty acyl chain length and head group on the size of multilamellar vesicles

The aggregation properties of diacyl phosphatidyl cholines (PC) with symmetric chains of 8–18 carbons in length and diacyl phospholipids with differing head groups (PC versus phosphatidyl ethanolamine (PE) versus phosphatidyl glycerol (PG) were examined using the techniques of quasi-elastic light scattering (QELS) and freeze fracture microscopy. All of these lipids except for the short chain C8PC (where C8 denotes 8 carbon chains) and CPC form multilamellar vesicles. Within this series we observed a positive correlation between most probable vesicle size and acyl chain length. For lipids with identical acyl chains there was an inverse correlation between vesicle size and effective head group cross-sectional area. We envisage a scheme in which the size of liposomes is determined by the area and thickness of multilamellar sheets which occur upon hydration of the dried lipid.

Scanning calorimetry reveals a new phase transition in L-alpha-dipalmitoylphosphatidylcholine

We report a new phase transition in fully hydrated dispersions of dipalmitoylphosphatidylcholine (DPPC). This new transition, called the sub-subtransition, exhibits a transition enthalpy of 0.25 kcal/mol with a Tm at 6.8 degrees C. Unlike the subtransition, no extended low temperature incubation is required to observe the sub-subtransition. This new sub-subgel (SGII) phase may be a precursor to the subgel (SGI) phase, and this discovery is discussed in relation to the current knowledge regarding the polymorphic gel phases of both ester- and ether-linked lipids with identical acyl chains.

Studies of structural modifications induced by gamma-irradiation in distearoylphosphatidylcholine liposomes.

An investigation of the structural and thermodynamical modifications induced by gamma-irradiation in model membranes is reported. Differential scanning calorimetry and X-ray diffraction were used to study the different phases and associated transitions of distearoylphosphatidylcholine multilamellar liposomes after 60Co gamma-irradiation. Changes were observed in the shape of the calorimetric peaks and in the corresponding phase transition temperatures. In particular a shoulder was observed at about 20 kGy. The three phases characteristic of lecithins with identical acyl chains were detected also for the highest radiation dose. The formation of lysolecithin and stearic acid upon phospholipid degradation was observed. The lysolecithin concentration increases as a function of irradiation dose, until a saturation value is reached at 40 kGy. These results correlate quite well with those obtained for interlayer and interchain distances and for the width of the main phase transition calorimetric peak. At the highest dose (approximately equal to 80 kGy) cross-linked adjacent radicals and other molecular species are also formed. Appreciable differences, and some similarities, in the behaviour of DSPC and DPPC liposomes under gamma-irradiation were observed.

The Stereoselective Deacylation of Long-chain Amino Acid Esters by Comicelles of N-Acyl-l-histidine and Various Cationic Surfactants

Abstract The stereoselective deacylation of H–(CH2)\undersetn−1—CONHCH(R)CO2–C6H4NO2-p (R=CH3, CH(CH3)2, CH2CH(CH3)2, and C6H5CH2; n=2–16) with N-acyl-l-histidine (acyl=octanoyl, dodecanoyl, and hexadecanoyl) and cationic surfactants (CmH2m+1\overset+N(CH3)2R X; (R,X,m)=(CH3,Br,16), (C2H5,Br,16), (C6H5CH2,Cl,16), and (\overset*CH(CH3)C6H5,Br, 14–18) was examined with particular reference to the structural effects of surfactants, nucleophiles, and substrates on both the deacylation rates and the stereoselectivity. The cationic surfactant, R-(+)-C16H33\overset+N(CH3)2\overset*CH(CH3)C6H5 Br, which possesses the hydrophobic and chiral groups near the polar head, formed the stereoselectively most effective comicelles with N-dodecanoyl-l-histidine. The order of stereoselectivity observed in the deacylation of the amino acid esters including the identical acyl chain length (n), phenylalanine&amp;gt;leucine&amp;gt;alanine∼valine. suggested steric effects of the substituent bound to the asymmetric carbon atom of the substrate on the stereoselectivity. The highest stereoselectivity (enantiomer rate ratio=4.4 at 25 °C under pH 7.61) was observed in the deacylation of the N-decanoylphenylalanine p-nitrophenyl ester, involving an appropriately long acyl chain.