Highly active and stable nanobiocatalyst based on in-situ cross-linking of phospholipase D for the synthesis of phosphatidylserine.

Phospholipase D (PLD) was effectively immobilized on a ZnO nanowires/macroporous SiO2 composite support through an in-situ cross-linking method. An anionic and long-chained bi-epoxy cross-linker was used by adsorbing on the surface of ZnO nanowires through static interaction before cross-linking. Under the fine control of in-situ cross-linking the immobilized PLD has loading amount as high as 113.7 mg/gsupport, possessing high specific activity from 13,987 to 16,142 U/gprotein in all the range of loading amount. The immobilized PLD showed high activity and stability in catalyzing the conversion of phosphatidylcholine (PC) to phosphatidylserine (PS). The reaction conditions such as loading amount of PLD, substrate molar ratio, temperature, solution pH, and reaction time were optimized for the finding of best synthetic process. Under optimized conditions and the PS yield reached 94.8% within 40 min at 50 °C. The immobilized PLD exhibited not only better thermostability and resistance to pH inactivation than free PLD but also the greatly improved storage stability and reusability. It was found that 81.5% of initial activity retained after incubation at 4 °C for 60 days and that 80.4% of PS yield retained after 13 cycling reuses.

Investigation of the phosphatidylserine binding properties of the lipid biosensor, Lactadherin C2 (LactC2), in different membrane environments.

Lipid biosensors are robust tools used in both in vitro and in vivo applications of lipid imaging and lipid detection. Lactadherin C2 (LactC2) was described in 2000 as being a potent and specific sensor for phosphatidylserine (PS) (Andersen et al. Biochemistry 39:6200-6206, 2000). PS is an anionic phospholipid enriched in the inner leaflet of the plasma membrane and has paramount roles in apoptosis, cells signaling, and autophagy. The myriad roles PS plays in membrane dynamics make monitoring PS levels and function an important endeavor. LactC2 has functioned as a tantamount PS biosensor namely in the field of cellular imaging. While PS specificity and high affinity of LactC2 for PS containing membranes has been well established, much less is known regarding LactC2 selectivity for subcellular pools of PS or PS within different membrane environments (e.g., in the presence of cholesterol). Thus, there has been a lack of studies that have compared LactC2 PS sensitivity based upon the acyl chain length and saturation or the presence of other host lipids such as cholesterol. Here, we use surface plasmon resonance as a label-free method to quantitatively assess the apparent binding affinity of LactC2 for membranes containing PS with different acyl chains, different fluidity, as well as representative lipid vesicle mimetics of cellular membranes. Results demonstrate that LactC2 is an unbiased sensor for PS, and can sensitively interact with membranes containing PS with different acyl chain saturation and interact with PS species in a cholesterol-independent manner.

Optical and nuclear imaging of glioblastoma with phosphatidylserine-targeted nanovesicles.

Multimodal tumor imaging with targeted nanoparticles potentially offers both enhanced specificity and sensitivity, leading to more precise cancer diagnosis and monitoring. We describe the synthesis and characterization of phenol-substituted, lipophilic orange and far-red fluorescent dyes and a simple radioiodination procedure to generate a dual (optical and nuclear) imaging probe. MALDI-ToF analyses revealed high iodination efficiency of the lipophilic reporters, achieved by electrophilic aromatic substitution using the chloramide 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodogen) as the oxidizing agent in an organic/aqueous co-solvent mixture. Upon conjugation of iodine-127 or iodine-124-labeled reporters to tumor-targeting SapC-DOPS nanovesicles, optical (fluorescent) and PET imaging was performed in mice bearing intracranial glioblastomas. In addition, tumor vs non-tumor (normal brain) uptake was compared using iodine-125. These data provide proof-of-principle for the potential value of SapC-DOPS for multimodal imaging of glioblastoma, the most aggressive primary brain tumor.

Introducing biobased ionic liquids as the nonaqueous media for enzymatic synthesis of phosphatidylserine.

Biobased ionic liquids with cholinium as the cation and amino acids as the anions, which could be prepared from renewable biomaterials by simple neutralization reactions, have recently been described as promising and green solvents. Herein, they were successfully used as the reaction media for enzyme-mediated transphosphatidylation of phosphatidylcholine with l-serine for phosphatidylserine synthesis for the first time. Our results indicated that the highest phosphatidylserine yield of 86.5% was achieved. Moreover, 75% original activity of the enzyme was maintained after being used for 10 batches. The present work could be considered an alternative enzymatic strategy for preparing phosphatidylserine. Additionally, the excellent results make the biobased ionic liquids more promising candidates for use as environmentally friendly solvents in biocatalysis fields.

Improved synthesis of phosphatidylserine using bio-based solvents, limonene and p-cymene.

PURPOSE OF WORK: The bio-based solvents limonene and p-cymene obtained from citrus waste were innovatively employed as the reaction media for enzymatic synthesis of phosphatidylserine. (R)-(+)-Limonene, which is available in large quantities from citrus waste, and its close derivative p-cymene, are promising green solvents. Herein, they were successfully employed as reaction media for enzyme-mediated transphosphatidylation of phosphatidylcholine with L-serine for phosphatidylserine synthesis for the first time. A 95 % yield of phosphatidylserine was achieved after 12 h and the side-reactions (which are the undesirable hydrolysis of phosphatidylcholine and phosphatidylserine) did not happen. This work presents an alternative strategy for preparing phosphatidylserine that possesses obvious advantages over the traditional processes in terms of high efficiency combined with environmental friendliness.

Enzymatic synthesis of phosphatidylserine using bile salt mixed micelles.

Phosphatidylserine (PS) rich in polyunsaturated fatty acids of the n-3 series was obtained by enzymatic synthesis with phospholipase D (PLD) and a marine lipid extract as substrate. Synthesis was performed using mixed micelles composed of either sodium deoxycholate (SDC) or sodium cholate (SC). To limit the use of surfactant and to monitor the performance of PLD, the mixed micelles were characterized both in terms of bile salt/lipid molar ratio in the aggregates and of mean diameter. A fractional factorial experiment was selected to study the effect of pH, temperature, enzyme, L-serine concentrations, bile salt/lipid molar ratio and Ca(2+) content (in the case of SC only) on PS synthesis. The amount of L-serine was the main factor governing the equilibrium between transphosphatidylation and hydrolysis reaction. Increasing the bile salt/lipid molar ratio decreased PS synthesis yield. In contrast, pH (6.5-8) and temperature (35-45°C) did not affect PLD activity in the tested conditions. This statistical approach allowed determining a combination of parameters (pH, temperature, bile salt/lipid molar ratio, enzyme and alcohol acceptor concentrations) for PS synthesis. After 24 h, the transphosphatidylation reaction led to 57±2% and 56±3% of PS in the phospholipid mixtures with SDC and SC, respectively. In both cases, about 10% of phosphatidic acid was present as a side-product. On the whole, this work provided fundamental basis for a possible development of enzymatic PLD technology using food-grade emulsifiers to produce PS complying with industrial constraints for nutritional applications.

New cardiolipin analogs synthesized by phospholipase D-catalyzed transphosphatidylation.

Cardiolipin (CL) and related diphosphatidyl lipids are hardly accessible because of the complexity of their chemical synthesis. In the present paper, the transphosphatidylation reaction catalyzed by phospholipase D (PLD) from Streptomyces sp. has been proven as an alternative enzyme-assisted strategy for the synthesis of new CL analogs. The formation of this type of compounds from phosphatidylcholine was compared for a series of N- and C2-substituted ethanolamine derivatives as well as non-charged alcohols such as glycerol and ethylene glycol. The rapid exchange of the choline head group by ethanolamine derivatives having a low molecular volume (diethanolamine and serinol) gave rise to an efficient production of the corresponding CL analogs. In contrast, the yields were comparably low in the reaction with bulky nitrogenous acceptor alcohols (triethanolamine, tris(hydroxymethyl)aminomethane, tetrakis(hydroxyethyl)ammonium) or the non-charged alcohols. Therefore, a strong dependence of the conversion of the monophosphatidyl to the diphosphatidyl compound on steric parameters and the head group charge was concluded. The enzyme-assisted strategy was used for the preparation of purified diphosphatidyldiethanolamine and diphosphatidylserinol.

Optimized synthesis of phosphatidylserine.

The synthesis of phosphatidyl serine containing saturated fatty acids was thoroughly studied and optimized in order to establish a protocol amenable to large-scale synthesis. The key step was a one-pot multicomponent reaction involving an O-benzyl phosphorodiamidite, protected serine and diacylglycerol, followed by in situ oxidation of the resulting phosphite. In order to replace expensive and poorly stable tetrazole, a screening of substitutes was carried out and imidazolium chloride was selected as the best suited one.

Cisplatin interaction with phosphatidylserine bilayer studied by solid-state NMR spectroscopy.

Cisplatin [cis-diamminedichloridoplatinum(II)] is used in chemotherapy where platinum-DNA adducts initiate tumor cell death. It is possible that side effects such as neurotoxicity and cellular cisplatin resistance can be due to interaction of cisplatin with lipids and the phospholipid bilayer. In this study, (13)C, (31)P, and (15)N solid-state NMR spectra of 1-palmitoyl-2-oleoyl phosphatidylserine (POPS) bilayers, POPS bilayers with 10 mol% cisplatin, and POPS bilayers with 30 mol% cisplatin were acquired. In addition, (15)N{(31)P} rotational echo double resonance spectra of POPS bilayers with 30 mol% cisplatin were acquired. The data demonstrate that the serine head group of phosphatidylserine binds to the aquated form of cisplatin and that cisplatin release of ammine takes place. It appears that the cisplatin release of ammine is followed by another cisplatin-POPS complex formation, possibly with cisplatin binding to one of the oxygen atoms of the POPS phosphate moiety.

Bio-orthogonal phosphatidylserine conjugates for delivery and imaging applications.

The syntheses of phosphatidylserine (PS) conjugates are described, including fluorescent derivatives for potential cellular delivery and bioimaging applications. Installation of terminal functional groups (amine, thiol, or alkyne) onto the sn-2 chain provides reactive sites for bio-orthogonal conjugation of cargo with suitably protected PS derivatives. An amine-containing PS forms amide bonds with peptidic cargo, a thiol derivative is designed for conjugation to cargo that contain alpha-halo carbonyls or Michael acceptors, and the terminal alkyne PS analogue permits "click" conjugation with any azide-tagged molecule. This latter conjugation method is quite versatile as it can be performed without PS headgroup protection, in aqueous media, and with acid-labile cargo.

Uranium(VI) complexes with phospholipid model compounds--a laser spectroscopic study.

We present the complex formation of the uranyl ion (UO(2)(2+)) in the aqueous system with phosphocholine, O-phosphoethanolamine and O-phosphoserine. These phosphonates (R-O-PO(3)(2-)) represent the hydrophilic head groups of phospholipids. The complexation was investigated by time-resolved laser-induced fluorescence spectroscopy (TRLFS) at pH=2-6. An increase of the fluorescence intensity, connected with a strong red-shift of about 8 nm compared to the free uranyl ion, indicates a complex formation between UO(2)(2+) and the phosphonates already at pH=2. Even at pH=6 these complexes prevail over the uranyl hydroxide and carbonate species, which are generated naturally at this pH. At pH=4 and higher a 1:2 complex between uranyl and O-phosphoserine was found. Complexes with a metal-to-ligand ratio of 1:1 were observed for all other ligands. Fluorescence lifetimes, emission maxima and complex stability constants at T=22+/-1 degrees C are reported. The TRLFS spectra of uranyl complexes with two phosphatidic acids (1,2-dimyristoyl-sn-glycero-3-phosphate and 1,2-dipalmitoyl-sn-glycero-3-phosphate), which represent the apolaric site of phospholipids, show in each case two different species.

Enzymatic synthesis of structured lipids.

Structured lipids (SLs) are defined as lipids that are modified chemically or enzymatically in order to change their structure. This review deals with structured triacylglycerols (STGs) and structured phospholipids (SPLs). The most typical STGs are MLM-type STGs, having medium chain fatty acids (FAs) at the 1- and 3-positions and a long chain fatty acid at the 2- position. MLM-type STGs are synthesized by: 1) 1,3-position-specific lipase-catalyzed acyl exchange of TG with FA or with FA ethylester (FAEt); 2) 1,3-position-specific lipase-catalyzed acylation of glycerol with FA, giving symmetric 1,3-diacyl-sn-glycerol, followed by chemical acylation at the sn-2 position, and; 3) 1,3-position-specific lipase-catalyzed deacylation of TG, giving 2-monoacylglycerol, followed by reacylation at the 1- and 3-positions with FA or with (FAEt). Enzymatic preparation of SPLs requires: 1) acyl group modification, and 2) head group modification of phospholipids. Acyl group modification is performed using lipases or phospholipase A2-mediated transesterification or ester synthesis to introduce arbitrary fatty acid to phospholipids. Head group modification is carried out by phospholipase D-catalyzed transphosphatidylation. A wide range of compounds can be introduced into the polar head of phospholipids, making it possible to prepare various SPLs.

N-stearoyl-phosphatidylserine: synthesis and role in divalent-cation-induced aggregation and fusion.

N-Acylphosphatidylserines have been isolated from intact and injured tissues, but the participation of such acidic phospholipids in membrane aggregation and fusion has not been demonstrated. We have synthesized N-stearoylphosphatidylserine (NSPS) and examined divalent-cation-induced aggregation of NSPS-liposomes, which leads to membrane destabilization and fusion. The purified lipid was characterized by its chromatographic and spectroscopic (infrared and 1H nuclear magnetic resonance) properties and by its chemical degradation pattern. Aggregation of unilamellar NSPS-liposomes was studied as a function of calcium and magnesium concentration. The ability of calcium and magnesium to induce vesicle aggregation is higher for phosphatidylserine (PS)-liposomes (threshold concentration 1.5 mM for calcium and 4.6 mM for magnesium) than for NSPS-liposomes (threshold concentration 2.8 mM for calcium and 6.6 mM for magnesium). The irreversibility of the aggregation reactions after adding EDTA suggests that vesicle fusion might occur in the presence of calcium and magnesium. Preliminary studies, based on mixing of both lipid and internal aqueous contents, show that fusion rather than aggregation of NSPS-liposomes occurs in the presence of calcium ions. The tendency of NSPS-liposomes to aggregate at higher cation concentrations than PS-liposomes suggests that N-acylation of phosphatidylserine protects the membrane against degenerative damage caused by aggregation and fusion.

Mutational and crystallographic analyses of interfacial residues in annexin V suggest direct interactions with phospholipid membrane components.

Annexin V belongs to a family of eukaryotic calcium-dependent membrane-binding proteins. The calcium-binding sites at the annexin-membrane interface have been investigated in some detail; however, little is known about the functional roles of highly conserved interfacial residues that do not coordinate calcium themselves. In the present study, the importance of tryptophan 185, and threonine or serine at positions 72, 144, 228, and 303, in rat annexin V is investigated by site-directed mutagenesis, X-ray crystallography, and functional assays. The high-resolution crystal structures of the mutants show that the mutations do not cause structural perturbations of the annexin molecule itself or disappearance of bound calcium ions from calcium-binding sites. The assays indicate that relative to wild-type annexin V, loss of the methyl substituent at position 72 (Thr72-->Ser) has no effect while loss of the hydroxyl group (Thr72-->Ala or Thr72-->Lys) causes reduction of membrane binding. Multiple lysine substitutions (e.g., Thr72,Ser144,Ser228,Ser303-->Lys) have a greater adverse effect than the single lysine mutation, suggesting that in annexin V the introduction of potentially favorable electrostatic interactions between the lysine side chains and the net negatively charged membrane surface is not sufficient to overcome the loss of the hydroxyl side chains. Replacement of the unique tryptophan, Trp185, by alanine similarly decreases membrane binding affinity. Taken together, the data suggest that the side chains mutated in this study contribute to phospholipid binding and participate directly in intermolecular contacts with phospholipid membrane components.

Sphingolipid-derived signalling modulators: interaction with phosphatidylserine.

We previously described the synthesis of two deuterium-labelled sphingoid bases, which made it possible to perform NMR spectroscopy on this family of signalling modulators in membranes (Rigby, A.C, Barber, K.R and Grant, C.W.M. (1995) Biochim. Biophys. Acta 1240, 75-82). In the present work we sought to test the concept that such mediators may display altered physical behaviour through association with anionic lipids - as a possible mechanism of involvement in signal transduction. Lyso-dihydrogalactosylceramide with deuterium nuclei at C4 and C5 of the sphingosine backbone and at C'3 and C'4 of the galactose ring ([2H4]lyso-GalCer), and N,N-dimethylsphingosine with deuterated amino-methyl groups ([2H6]dimethylsphingosine), were assembled as minor components into unsonicated fluid bilayer membranes of 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol. The effect of (anionic) phosphatidylserine was considered in this zwitterionic host matrix. The results present a picture of rapidly reversible interaction. The (-) charged phosphatidylserine exerted readily-measurable control over the orientation of the carbohydrate residue of [2H4]lyso-GalCer. In contrast, surrounding (-) charges exerted little spectral influence at the level of C4 and C5 of the lyso-GalCer, membrane-inserted, backbone; or at the level of the amino group of dimethylsphingosine. It would appear that packing alterations induced by the phosphatidylserine/sphingoid base association can translate into sizeable spatial constraints in the neighbouring aqueous domain.

Synthesis of 1,2-diacyl-sn-glycerophosphatidylserine from egg phosphatidylcholine by phosphoramidite methodology.

A simple chemical method for the synthesis of 1,2-diacyl-sn-glycerophosphatidylserine (PS), with the same fatty acid composition in the sn-1 and sn-2 glycerol positions as egg phosphatidylcholine (PC), is described. PS synthesis was carried out by a phosphite-triester approach, using 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (phosphoramiditate) as the phosphorylating agent, for the formation of phosphate linkage between serine and diacylglycerol. 1,2-Diacylglycerol, obtained from PC hydrolysis by phospholipase C, was coupled with N-t-BOC-L-serinebenzhydryl ester phosphoramidite with tetrazole as catalyst. Phosphite-triester was oxidized to the corresponding phosphate-triester with 30% H2O2 in CH2Cl2. The cyanoethyl group was removed by addition of an Et3N/CH3 CN/pyridine mixture, and trifluoroacetic acid was used to eliminate the protecting groups of O-(1,2-diacylglycero-3-phospho)-N-t-BOC-serinebenzhydryl ester. Purified PS was identified by thin-layer chromatography, infrared, and 1H nuclear magnetic resonance.

Antitumor effects of liposomal IL1 alpha and TNF alpha against the pulmonary metastases of the B16F10 murine melanoma in syngeneic mice.

Interleukin 1 alpha (IL1 alpha) and tumor necrosis factor alpha (TNF alpha) have been successfully incorporated into specific phosphatidylcholine (PC) and phosphatidylserine (PS) multilamellar vesicle (MLV) liposomes by modifying the concentration of calcium ion and pH of the encapsulation buffer. Under these conditions, some of the cytokines may attach to the exterior surface of the MLV and therefore be readily accessible to target cells for receptor binding and signal transduction. These cytokine-associated liposomes are stable for up to 2 weeks in serum-free buffer, and leakage of cytokines into medium containing 10% fetal bovine serum was about 50% at the end of a 3-day incubation period at 37 degrees C. The biological activities mediated by liposomal IL1 alpha and TNF alpha were specific: the stimulation of thymidine uptake in T-helper D10 lymphocytes and the cytolysis of TNF alpha-sensitive L929 target cells could be blocked by specific neutralizing antibodies in a dose-dependent fashion. When administered intravenously into C57BL/6 mice bearing the syngeneic B16F10 murine melanoma cells, dual entrapment of liposomal IL1 alpha and TNF alpha significantly reduced the number of metastatic tumor nodules in the lungs and prolonged the life span of the animals. Thus, liposomal IL1 alpha and TNF alpha displayed significant in vivo antitumor activity against the IL1 alpha- and TNF alpha-resistant B16F10 metastatic murine melanoma.

Synthesis and characterization of N-acylated, pH-sensitive 'caged' aminophospholipids.

A series of acid-labile 'caged' phosphatidylserine (PS) and phosphatidylethanolamine (PE) molecules have been synthesized by N-acylation of the aminophospholipid with maleic, citraconic, dimethylmaleic, phthalic, or 3,4,5,6-tetrahydrophthalic anhydride. N-citraconyl-dioleoylphosphatidylethanolamine (C-DOPE) and N-citraconyl-dioleoyl-phosphatidylserine (C-DOPS) exhibited the highest degree of sensitivity to acidic pH; incubation at pH 5.5 and 6.5, respectively, resulted in 50% cleavage to the native aminophospholipid within 60 min. Significant cleavage of the phthalyl- and maleyl-PE derivatives did not occur at physiologically relevant pH values (pH 5.5-8), while tetrahydrophthalyl-PE and dimethylmaleyl-PE could not be isolated, reflecting their inherent instability. At pH 5.5, the half time for cleavage of C-DOPE and C-DOPS was 110 min and 85 min, respectively. The utility of these 'caged' lipids was demonstrated in human erythrocytes. When mixed with cells, C-DOPS, or the short chain analog, N-citraconyl-dilauroylphosphatidylserine (C-DLPS), transferred from liposome membranes into erythrocytes and remained in the cell outer monolayer. Low pH treatment released the citraconyl group and the free PS was transported to the inner monolayer by the aminophospholipid transporter. These novel 'caged' phospholipids may be useful for the study of transmembrane aminophospholipid transport, protein-lipid interactions and membrane fusion.

Synthesis and characterization of NBD-PS: a fluorescent analog of cerebroside arylsulfatase A deficiency disorders.

N-[7-Nitrobenz-2-oxa-1,3-diazol-4-yl]psychosine sulfate (NBD-PS), a fluorescent analog of cerebroside sulfate (CS), was synthesized and tested as an alternative to the radiolabeled forms of CS used for assaying arylsulfatase A (ASA) in its physiological role as a cerebroside sulfate sulfohydrolase. NBD-PS simulates the natural substrate for ASA. Protocols have been developed for its use in differentiating low enzyme activities in diagnostic samples. Hydrolysis of NBD-PS is specific for ASA and optimal assay parameters were identical to those determined for CS. Differentiations between each of the major phenotypes for ASA activity were possible in the set of samples tested. One particular advantage was the ability to discriminate between individuals exhibiting arylsulfatase A pseudodeficiency and the truly deficient individuals with metachromatic leukodystrophy. Differential diagnosis was possible with fibroblast extracts by an assay that is more sensitive than procedures employing radioisotopes. Reaction products may be analyzed quantitatively by HPLC, or semiquantitatively with TLC. NBD-PS provides a simpler, safer, and more cost-effective means of performing natural substrate enzyme assays for ASA. Phenotyping with the fluorescence assay is an effective alternative to the laborious radioactive CS preparations and tissue culture loading studies that have previously been necessary.

Halo lipids. 10. Synthesis and cytostatic activity of O-alkylglycerophospho-L-serine analogs.

The synthesis of O-alkylglycerophospho-L-serine analogs is described, which represent a new class of cytostatically active agents based on phospholipids. The new compounds were obtained by conversion of O-alkylglycerophosphoric ester analogs by means of phospholipase D and by condensing O-alkylglycerophosphoric acid analogs with protected L-serine followed by the removal of protective groups of the resulting intermediates. The structure of the O-alkylglycerophospho-L-serines was confirmed by fast atom bombardment mass spectrometry. The compounds were found to inhibit the growth of Ehrlich ascites tumor cells in vitro. Half maximum inhibition was observed at concentrations between 7 and 15 microM. For the 1-O-alkyl-2-methoxy glycerophosphoserine only a higher value (30 microM) was found. With most of the substances tested growth was completely inhibited at a concentration of 40 microM.