Phosphatidyl Residue Research Articles

Synthesis of liponucleotides using bacterial phospholipase D

A series of conjugates of pharmacologically promising modified nucleosides with phospholipid was synthesized. Some liponucleotides were originally produced. Soybean lecithin served as the donor of phosphatidyl residue. Phospholipase D from strain Streptomyces netropsis BIM B-428D was engaged as biocatalyst in transphosphatidylation reaction. The yield of liponucleotide synthesis reaction varied in the range 44–95 mol% depending on the acceptor of phosphatidyl residue. 65 mg (about 68 µmoles) of pure phosphatidylclofarabine was recovered by chromatography on silica gel column, resulting in 68 mol% yield calculated from the amount of modified nucleoside supplied into the reaction mixture.

Barth Syndrome: From Mitochondrial Dysfunctions Associated with Aberrant Production of Reactive Oxygen Species to Pluripotent Stem Cell Studies.

Mutations in the gene encoding the enzyme tafazzin, TAZ, cause Barth syndrome (BTHS). Individuals with this X-linked multisystem disorder present cardiomyopathy (CM) (often dilated), skeletal muscle weakness, neutropenia, growth retardation, and 3-methylglutaconic aciduria. Biopsies of the heart, liver and skeletal muscle of patients have revealed mitochondrial malformations and dysfunctions. It is the purpose of this review to summarize recent results of studies on various animal or cell models of Barth syndrome, which have characterized biochemically the strong cellular defects associated with TAZ mutations. Tafazzin is a mitochondrial phospholipidlysophospholipid transacylase that shuttles acyl groups between phospholipids and regulates the remodeling of cardiolipin (CL), a unique inner mitochondrial membrane phospholipid dimer consisting of two phosphatidyl residues linked by a glycerol bridge. After their biosynthesis, the acyl chains of CLs may be modified in remodeling processes involving up to three different enzymes. Their characteristic acyl chain composition depends on the function of tafazzin, although the enzyme itself surprisingly lacks acyl specificity. CLs are crucial for correct mitochondrial structure and function. In addition to their function in the basic mitochondrial function of ATP production, CLs play essential roles in cardiac function, apoptosis, autophagy, cell cycle regulation and Fe-S cluster biosynthesis. Recent developments in tafazzin research have provided strong insights into the link between mitochondrial dysfunction and the production of reactive oxygen species (ROS). An important tool has been the generation of BTHS-specific induced pluripotent stem cells (iPSCs) from BTHS patients. In a complementary approach, disease-specific mutations have been introduced into wild-type iPSC lines enabling direct comparison with isogenic controls. iPSC-derived cardiomyocytes were then characterized using biochemical and classical bioenergetic approaches. The cells are tested in a “heart-on-chip” assay to model the pathophysiology in vitro, to characterize the underlying mechanism of BTHS deriving from TAZ mutations, mitochondrial deficiencies and ROS production and leading to tissue defects, and to evaluate potential therapies with the use of mitochondrially targeted antioxidants.

Ionization constants pKa of cardiolipin.

Cardiolipin is a phospholipid found in the inner mitochondrial membrane and in bacteria, and it is associated with many physiological functions. Cardiolipin has a dimeric structure consisting of two phosphatidyl residues connected by a glycerol bridge and four acyl chains, and therefore it can carry two negative charges. The pKa values of the phosphate groups have previously been reported to differ widely with pKa1 = 2.8 and pKa2 = 7.5–9.5. Still, there are several examples of experimental observations from cardiolipin-containing systems that do not fit with this dissociation behavior. Therefore, we have carried out pH-titration and titration calorimetric experiments on two synthetic cardiolipins, 1,1′,2,2′-tetradecanoyl cardiolipin, CL (C14∶0), and 1,1′,2,2′-tetraoctadecenoyl cardiolipin, CL (C18∶1). Our results show that both behave as strong dibasic acids with pKa1 about the same as the first pKa of phosphoric acid, 2.15, and pKa2 about one unit larger. The characterization of the acidic properties of cardiolipin is crucial for the understanding of the molecular organization in self-assembled systems that contain cardiolipin, and for their biological function.

Mycobacterial Phosphatidylinositol Mannosides Negatively Regulate Host Toll-like Receptor 4, MyD88-dependent Proinflammatory Cytokines, and TRIF-dependent Co-stimulatory Molecule Expression

Mycobacterium tuberculosis modulates host immune responses through proteins and complex glycolipids. Here, we report that the glycosylphosphatidylinositol anchor phosphatidyl-myo-inositol hexamannosides PIM(6) or PIM(2) exert potent anti-inflammatory activities. PIM strongly inhibited the Toll-like receptor (TLR4) and myeloid differentiation protein 88 (MyD88)-mediated release of NO, cytokines, and chemokines, including tumor necrosis factor (TNF), interleukin 12 (IL-12) p40, IL-6, keratinocyte-derived chemokine, and also IL-10 by lipopolysaccharide (LPS)-activated macrophages. This effect was independent of the presence of TLR2. PIM also reduced the LPS-induced MyD88-independent, TIR domain-containing adaptor protein inducing interferon beta (TRIF)-mediated expression of co-stimulatory receptors. PIM inhibited LPS/TLR4-induced NFkappaB translocation. Synthetic PIM(1) and a PIM(2) mimetic recapitulated these in vitro activities and inhibited endotoxin-induced airway inflammation, TNF and keratinocyte-derived chemokine secretion, and neutrophil recruitment in vivo. Mannosyl, two acyl chains, and phosphatidyl residues are essential for PIM anti-inflammatory activity, whereas the inosityl moiety is dispensable. Therefore, PIM exert potent antiinflammatory effects both in vitro and in vivo that may contribute to the strategy developed by mycobacteria for repressing the host innate immunity, and synthetic PIM analogs represent powerful anti-inflammatory leads.

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.

Monovalent Gb3-/Gb2-Derivatives Conjugated with a Phosphatidyl Residue: A Novel Class of Shiga Toxin-Neutralizing Agent

Shiga toxin (Stx) exerts toxic activity by binding to glycosphingolipids, mainly globotriaosyl (Gb(3)) ceramide, on the surface of target cells. The inhibition of toxin-receptor binding is a promising therapeutic approach to prevent Stx-mediated diseases. In this study, we synthesized monovalent Stx-ligands of phosphatidylethanolamine dipalmitoyl-Gb(3) (Gb(3)-PEDP) and galabiosyl (Gb(2))-PEDP and we examined their neutralizing activity against Stx-1 and Stx-2 in vitro. Both Gb(3)-PEDP and Gb(2)-PEDP strongly neutralized the cytotoxicity of Stx-1 and Stx-2. It is likely that the mechanism of neutralization involved formation of liposomes and consequently clustering of sugar units. We propose monovalent Gb(3)-/Gb(2)-derivatives conjugated with phosphatidyl residue as a novel class of Stx-neutralizing agent.

Identification and functional characterization of hCLS1, a human cardiolipin synthase localized in mitochondria

In eukaryotic cells, CLS (cardiolipin synthase) is involved in the final step of cardiolipin synthesis by catalysing the transfer of a phosphatidyl residue from CDP-DAG (diacylglycerol) to PG (phosphatidylglycerol). Despite an important role of cardiolipin in regulating mitochondrial function, a gene encoding the mammalian CLS has not been identified so far. We report in the present study the identification and characterization of a human cDNA encoding the first mammalian CLS [hCLS1 (human CLS1)]. The predicted hCLS1 peptide sequence shares significant homology with the yeast and plant CLS proteins. The recombinant hCLS1 enzyme expressed in COS-7 cells catalysed efficiently the synthesis of cardiolipin in vitro using CDP-DAG and PG as substrates. Furthermore, overexpression of hCLS1 cDNA in COS-7 cells resulted in a significant increase in cardiolipin synthesis in intact COS-7 cells without any significant effects on the activity of the endogenous phosphatidylglycerophosphate synthase of the transfected COS-7 cells. Immunohistochemical analysis demonstrated that the recombinant hCLS1 protein was localized to the mitochondria when transiently expressed in COS-7 cells, which was further corroborated by results from subcellular fractionation analyses of the recombinant hCLS1 protein. Northern-blot analysis showed that the hCLS1 gene was predominantly expressed in tissues that require high levels of mitochondrial activities for energy metabolism, with the highest expression in skeletal and cardiac muscles. High levels of hCLS1 expression were also detected in liver, pancreas, kidney and small intestine, implying a functional role of hCLS1 in these tissues.

Synthesis of Serine‐Linked Phosphatidylinositol Mannosides (PIMs).

The 6-O-allyl group of inositol mannoside (D)-6 was used for the generation of O-linked serine residues via a sequence of dihydroxylation, selective 3′′-O-protection, introduction of the 2′′-amino group, deprotection, and oxidation of the 3′′-position. Attachment of the phosphatidyl residue to 1-O of the inositol moiety and complete deprotection furnished target molecules (D)-1 and (D)-2. Alternatively, instead of introduction of a 6-O-allyl group, 2′′,3′′-dihydroxy propanylation of the 6-O-position of the inositol moiety using a silyl-protected cyclic sulfate of glycerol was possible. Thus, target molecule (L)-3 was obtained. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

Phospholipase D‐catalyzed synthesis of novel phospholipid‐phytosterol conjugates

In the present study, plant sterols were modified to form amphiphiles by synthesizing phospholipid derivatives from them so they could be formulated in different functional foods and possibly improve their effects as therapeutic agents. The new phosphatidyl derivatives, phosphatidyl-sitosterols, were synthesized by the transfer reaction of a phosphatidyl residue from PC to beta-sitosterol by phospholipase D (PLD EC 3.1.4.4) from Streptomyces sp. in a biphasic medium.

Synthesis of Serine‐Linked Phosphatidylinositol Mannosides (PIMs)

AbstractThe 6‐O‐allyl group of inositol mannoside (D)‐6 was used for the generation of O‐linked serine residues via a sequence of dihydroxylation, selective 3′′‐O‐protection, introduction of the 2′′‐amino group, deprotection, and oxidation of the 3′′‐position. Attachment of the phosphatidyl residue to 1‐O of the inositol moiety and complete deprotection furnished target molecules (D)‐1 and (D)‐2. Alternatively, instead of introduction of a 6‐O‐allyl group, 2′′,3′′‐dihydroxy propanylation of the 6‐O‐position of the inositol moiety using a silyl‐protected cyclic sulfate of glycerol was possible. Thus, target molecule (L)‐3 was obtained. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

On the evolutionary conservation of the cell death pathway: mitochondrial release of an apoptosis-inducing factor during Dictyostelium discoideum cell death.

Mitochondria play a pivotal role in apoptosis in multicellular organisms by releasing apoptogenic factors such as cytochrome c that activate the caspases effector pathway, and apoptosis-inducing factor (AIF) that is involved in a caspase-independent cell death pathway. Here we report that cell death in the single-celled organism Dictyostelium discoideum involves early disruption of mitochondrial transmembrane potential (DeltaPsim) that precedes the induction of several apoptosis-like features, including exposure of the phosphatidyl residues at the external surface of the plasma membrane, an intense vacuolization, a fragmentation of DNA into large fragments, an autophagy, and the release of apoptotic corpses that are engulfed by neighboring cells. We have cloned a Dictyostelium homolog of mammalian AIF that is localized into mitochondria and is translocated from the mitochondria to the cytoplasm and the nucleus after the onset of cell death. Cytoplasmic extracts from dying Dictyostelium cells trigger the breakdown of isolated mammalian and Dictyostelium nuclei in a cell-free system, and this process is inhibited by a polyclonal antibody specific for Dictyostelium discoideum apoptosis-inducing factor (DdAIF), suggesting that DdAIF is involved in DNA degradation during Dictyostelium cell death. Our findings indicate that the cell death pathway in Dictyostelium involves mitochondria and an AIF homolog, suggesting the evolutionary conservation of at least part of the cell death pathway in unicellular and multicellular organisms.

Synthesis Of Labeled Glycosyl Phosphatidyl Inositol (GPI) Anchors

The exploration of the molecular and structural basis for the sorting of GPI-anchored proteins is based on labeled partial structures of GPI′s which can be incorporated into the GPI anchor biosynthesis and cellular transport systems. To this end, from mannosyl donor 6 and the D-glucosaminyl-(1→6)-D-myo-inositol derivative 7 as acceptor, the pseudotrisaccharide 8 was prepared. Compound 8 was transformed into the GPI partial structures 5a,b which contain the pseudotrisaccharide ligated to two different phosphatidyl residues. Compounds 5a,b have Boc protection at the 2-amino group of the glucosamine residue (2b-position) and a free amino group at the 6b-position. The 6b-amino group was used for the ligation of the 3-(7-nitrobenzofurazan-4-yl)-aminopropanoyl group as a fluorescent label, the 5-azido-2-nitrobenzoyl and 4-azidophenylaminothiocarbonyl groups as photolabels, and the 4-azido-2-hydroxybenzoyl group as a radiolabel after the introduction of radioactive iodine by an electrophilic aromatic substitution. Thus, after acid-catalyzed removal of the protective groups, the unprotected target molecules 1–4 were obtained.

ホスホリパーゼＤの触媒するホスファチジル基転移反応による機能性リン脂質誘導体の合成

We found that phospholipase D from Streptomyces sp. (PLDP) effectively catalyzed transphosphatidylation, i.e., the regiospecific transfer of the phosphatidyl residue from phosphatidylcholines to alkanols. The scope of this enzymatic reaction was investigated in detail to develop an efficient method for preparing various phospholipid derivatives. Using this enzymatic method, 5'-Ο-phosphatidyl derivatives of antitumor nucleosides were successfully synthesized to identify potent antitumor drugs. 5'-Ο-Phosphatidyl derivatives of DMDC (2'-deoxy-2'-methylenecytidine) and CNDAC [1- (2-C -cyano-2-deoxy-β-arabino-pentofuranosyl) cytosine], antitumor nucleosides developed by us, showed significant antitumor effects in mice which clearly surpassed those of parent compounds. We have also demonstrated that 5'-Ο-phosphatidylnucleosides can be used as a drug delivery system for lymph : 5'-Ο-phosphatidyl-5-fluorouridine given orally is absorbed via the deacylation-reacylation cycle, a specific pathway for natural phospholipids, to be transported selectively to the lymph.

Conductimetric Assays for the Hydrolase and Transferase Activities of Phospholipase D Enzymes

Measurement of solution electrical conductance (conductimetry) is a simple direct assay method for the protogenic, hydrolytic reactions catalyzed by all phospholipase enzymes. The technique is especially suitable for assay of phospholipase D (PLD) enzymes where cleavage of zwitterionic substrates reinforces the pH dependent conductance change and allows the method to be used over a much wider pH range than the equivalent titrimetric assay. The ability to detect zwitterion cleavage enables the method to assay reactions in which phospholipase D transfers neutral, or anionic, alcohol species to the zwitterionic substrates phosphatidyl choline and phosphatidyl ethanolamine. The method can follow the sequential attack by different phospholipases and provides a simple technique for investigating the effect of substrate structure on susceptibility to various phospholipase enzymes. The results confirm that PLD from Streptomyces chromofuscus can attack lysophospholipids, but cannot transfer primary alcohols to the phosphatidyl residue, while the PLD from savoy cabbage is an efficient transferase, but cannot attack lysophospholipids. The data suggest that the bacterial PLD fails to act as a transferase because it hydrolyzes the transphosphatidylation products. Some phosphatidyl alcohols are more highly susceptible to PLA2 attack than the parent phosphatidyl choline derivatives.

Enzymatic Synthesis of Novel Phosphatidylgenipin, and Its Enhanced Cytotoxity

A new phosphatidyl derivative, l,2-dipalmitoyl-3-sn-phosphati-dylgenipin (DPP-genipin), was found to be synthesized by the transfer reaction of a dipalmitoylphosphatidyl residue from l,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) to genipin by phospholipase D (PLD) from Streptomyces sp. in a biphasic system. DPP-genipin also transferred its phosphatidyl residue to choline by PLD, to reproduce DPPC and liberate genipin. DPP-genipin showed 6 to 52 times stronger cytotoxity than genipin to Hela cells, human embryonic lung fibroblasts (HEL), and MT-4 cells.

Phospholipase D-Catalyzed Synthesis of Phosphatidyl Aromatic Compounds

Phospholipase D (PLD) from Streptomyces sp. was found to catalyze the transfer reaction of the dipalmitoylphosphatidyl residue from 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) to an aromatic hydroxy group in a biphasic system of an organic solvent with low water solubility and acetate buffer. The following phosphatidyl aromatic compounds were synthesized: 1,2-dipalmitoyl-3-sn-phosphatidylphenol (1), 1,2-dipalmitoyl-3-sn-phosphatidyl-4-methoxyphenol (2, DPP-PMP), 1,2-dipalmitoyl-3-sn-phosphatidyl-4-methylphenol (3), 1,2-dipalmitoyl-3-sn-phosphatidylhydroquinone (4), 1,2-dipalmitoyl-3-sn-phosphatidyl-4-chlorophenol (5), 1,2-dipalmitoyl-3-sn-phosphatidylcatechol (6), l,2-dipalmitoyl-3-sn-phosphatidyl-3-methoxyphenol (7), 1,2-dipalmitoyl-3-sn-phosphatidyl-2-naphthol (8), and 1,2-dipalmitoyl-3-sn-phosphati-dyl-5-hydroxyindole (9). The transphosphatidylation reaction of DPPC by PLD was accompanied by hydrolysis of DPPC, and the ratios were significantly affected by both organic solvents and water contents in a biphasic system. PLD also catalyzed the transphosphatidylation reaction of DPP-PMP (2) to choline, to reproduce DPPC and liberate 4-methoxyphenol. This is the first report on the enzymatic transfer of phosphatidyl residue to aromatic hydroxy compounds.

On the basic structure of poly (glycerophosphate) lipoteichoic acids

Poly(glycerophosphate) lipoteichoic acids from 24 Gram-positive bacteria of the genera Bacillus, Enterococcus, Lactobacillus, Lactococcus, Listeria, Staphylococcus, and the streptococcal pyogenic and oral group were analyzed. The 1,3-linked poly(glycerophosphate) structure was proved by analysis of glycerol and glycerophosphates after acid and alkaline hydrolysis. Using the molar ratios of glycolipid to phosphorus (A) and phosphomonoester to phosphorus after periodate oxidation followed by hydrazinolysis (B) or beta-elimination (C), we show that all lipoteichoic acids contain a single unbranched poly(glycerophosphate) chain and that the chain is uniformly phosphodiester-linked to C-6 of the nonreducing hexopyranosyl residue of the glycolipid moiety. On some chains minor phosphate-containing substituents were detected whose structure remains to be clarified. The lipoteichoic acids of enterococci and listeria strains were separated by hydrophobic interaction chromatography into glycolipid- and phosphatidylglycolipid-containing molecular species. The phosphatidylglycolipid moieties were structurally characterized after liberation from lipoteichoic acids with moist acetic acid. After periodate oxidation of lipoteichoic acids beta-elimination released both phosphatidic acid and the poly(glycerophosphate) chain. This indicates together with the sequence analysis of the released phosphatidylglycolipid that the phosphatidyl residue is located at C-6 of the reducing hexosyl residue of the glycolipid moiety and the poly(glycerophosphate) chain at C-6 of the nonreducing one. Together with earlier observations these results complete the evidence for the structural and possibly biosynthetic relationship between lipoteichoic acids and glycerophosphoglycolipids.

A facile enzymatic synthesis of 5'-(3-sn-phosphatidyl)nucleosides and their antileukemic activities.

Phospholipase D from Streptomyces effectively catalyzed the transfer reaction of the phosphatidyl residue from 3-sn-phosphatidylcholine to the 5'-hydroxyl group of nuclesides in a two-phase system. Thus, a variety of 5'-(3-sn-phosphatidyl)nucleosides could be readily prepared in high yields by means of this reaction. Among them, phosphatidyl-FUR (3b), phosphatidyl-Ara FC (8b), and phoshatidyl-neplanocin A (12b) each produced a significant increase in the life span mice bearing i.p.-implanted P388 leukemia, being more effective than the parent nucleosides.

ChemInform Abstract: A Facile One‐Step Synthesis of Phosphatidylhomoserines by Phospholipase D‐Catalyzed Transphosphatidylation.

Phospholipase D from Streptomyces effectively catalyzed the transfer reaction of the phosphatidyl residue from phosphatidylcholines to the primary hydroxyl group of homoserine in a two-phase system. Various phosphatidylhomoserines were easily prepared in good yields by this reaction.

A facile one-step synthesis of phosphatidylhomoserines by phospholipase D-catalyzed transphosphatidylation.

Phospholipase D from Streptomyces effectively catalyzed the transfer reaction of the phosphatidyl residue from phosphatidylcholines to the primary hydroxyl group of homoserine in a two-phase system. Various phosphatidylhomoserines were easily prepared in good yields by this reaction.