Translational Approaches Targeting Ceramide Generation From Sphingomyelin in T Cells to Modulate Immunity in Humans.

In T cells, as in all other cells of the body, sphingolipids form important structural components of membranes. Due to metabolic modifications, sphingolipids additionally play an active part in the signaling of cell surface receptors of T cells like the T cell receptor or the co-stimulatory molecule CD28. Moreover, the sphingolipid composition of their membranes crucially affects the integrity and function of subcellular compartments such as the lysosome. Previously, studying sphingolipid metabolism has been severely hampered by the limited number of analytical methods/model systems available. Besides well-established high resolution mass spectrometry new tools are now available like novel minimally modified sphingolipid subspecies for click chemistry as well as recently generated mouse mutants with deficiencies/overexpression of sphingolipid-modifying enzymes. Making use of these tools we and others discovered that the sphingolipid sphingomyelin is metabolized to ceramide to different degrees in distinct T cell subpopulations of mice and humans. This knowledge has already been translated into novel immunomodulatory approaches in mice and will in the future hopefully also be applicable to humans. In this paper we are, thus, summarizing the most recent findings on the impact of sphingolipid metabolism on T cell activation, differentiation, and effector functions. Moreover, we are discussing the therapeutic concepts arising from these insights and drugs or drug candidates which are already in clinical use or could be developed for clinical use in patients with diseases as distant as major depression and chronic viral infection.

Preparation and Membrane Distribution of Fluorescent Derivatives of Ceramide.

Ceramide is a bioactive lipid with significant roles in several biological processes including cell proliferation, apoptosis, and raft formation. Although fluorescent derivatives of ceramide are required to probe the behavior of ceramide in cells and cell membranes, commercial fluorescent ceramide derivatives do not reproduce the membrane behavior of native ceramide because of the introduction of bulky fluorophores in the acyl chain. Recently, we developed novel fluorescent analogs of sphingomyelin in which the hydrophilic fluorophores, ATTO488 and ATTO594, are attached to the polar head of sphingomyelin via a nonaethylene glycol linker and demonstrated that their partition and dynamic behaviors in bilayer membranes are similar to native sphingomyelin. In this report, by extending the concept used for the development of fluorescent analogs of sphingomyelin, we prepared novel fluorescent ceramides that exhibit membrane behaviors similar to native ceramide and succeeded in visualizing ceramide-rich membrane domains segregated from ceramide-poor domains.

Biological functions of sphingomyelins.

Sphingomyelin (SM) is a dominant sphingolipid in membranes of mammalian cells and this lipid class is specifically enriched in the plasma membrane, the endocytic recycling compartment, and the trans Golgi network. The distribution of SM and cholesterol among cellular compartments correlate. Sphingolipids have extensive hydrogen-bonding capabilities which together with their saturated nature facilitate the formation of sphingolipid and SM-enriched lateral domains in membranes. Cholesterol prefers to interact with SMs and this interaction has many important functional consequences. In this review, the synthesis, regulation, and intracellular distribution of SMs are discussed. The many direct roles played by membrane SM in various cellular functions and processes will also be discussed. These include involvement in the regulation of endocytosis and receptor-mediated ligand uptake, in ion channel and G-protein coupled receptor function, in protein sorting, and functioning as receptor molecules for various bacterial toxins, and for non-bacterial pore-forming toxins. SM is also an important constituent of the eye lens membrane, and is believed to participate in the regulation of various nuclear functions. SM is an independent risk factor in the development of cardiovascular disease, and new studies have shed light on possible mechanism behind its role in atherogenesis.

[An expedient synthesis of fluorescent labeled ceramide-1-phosphate analogues].

A synthesis for fluorescent analogs of ceramide-1-phosphate bearing 9-anthrylvinyl or 4,4-difluoro-3a,4a- diaza-s-indacene-8-yl (Me4-BODIPY) fluorophore at o-position of fatty acid residue was carried out. The key stage of the synthesis is hydrolysis of corresponding sphingomyelins catalyzed by phospholipase D from Streptomyces chromofuscus; the enzymatic yield has been raised to 50-70% by appliance of organic solvent in the incubation medium.

Design and synthesis of sphingomyelin-cholesterol conjugates and their formation of ordered membranes.

A lipid raft is a cholesterol (Chol)-rich microdomain floating in a sea of lipid bilayers. Although Chol is thought to interact preferentially with sphingolipids such as sphingomyelin (SM), rather than with glycerophospholipids, the origin of the specific interaction has remained unresolved, primarily because of the high mobility of lipid molecules and weak intermolecular interactions. In this study, we synthesized SM-Chol conjugates with functionally designed linker portions to restrain Chol mobility and examined their formation of ordered membranes by a detergent insolubility assay, fluorescence anisotropy experiments, and fluorescence-quenching assay. In all of the tests, membranes prepared from the conjugates showed properties of ordered domains comparable to a SM-Chol (1:1) membrane. To gain insight into the structure of bilayers composed from the conjugates, we performed molecular dynamics simulations with 64 molecules of the conjugates, which suggested that the conjugates form a stable bilayer structure by bending at the linker portion and, mostly, reproduce the hydrogen bonds between the SM and Chol portions. These results imply that the molecular recognition between SM and Chol in an ordered domain is essentially reproduced by the conjugated molecules and, thus, demonstrates that these conjugate molecules could potentially serve as molecular probes for understanding molecular recognition in lipid rafts.

Selective deuterium labeling of the sphingoid backbone: facile syntheses of 3,4,5-trideuterio-d-erythro-sphingosine and 3-deuterio-d-erythro-sphingomyelin.

Deuteration at C-4 and C-5 of sphingosine was achieved via a hydrogen-deuterium exchange reaction of a ß-ketophosphonate intermediate catalyzed by ND4Cl in D2O/tetrahydrofuran. To install deuterium at C-3 of sphingosine and sphingomyelin, sodium borodeuteride reduction/cerium(III) chloride reduction of an α,ß-enone in perdeuteromethanol was used.

Structural role of mismatched C-C bonds in a series of d-erythro-sphingomyelins as studied by DSC and electron microscopy.

A series of d-erythro (2S, 3R) sphingomyelins (SMs) whose acyl chain was 16, 18, 20, 22, and 24 carbons long, respectively, was synthesized by the acylation of d-erythro-sphingosylphosphorylcholine. For all the SM dispersions, reversible and reproducible thermal behavior was observed to show the gel-to-gel and the main gel-to-liquid crystal phase transition in heating scan. The main transition enthalpy (DeltaH(M)) decreased linearly with increasing acyl chain length. The vesicular structures were observed for all the gel phases at temperatures just below the main transition, but the mean diameter of these vesicles changed markedly from approximately 1.5 to 100nm with increasing acyl chain length. On this basis, the decrease in DeltaH(M) with increasing acyl chain length was discussed from the viewpoint of the effect of the mismatched C-C bonds in the acyl chain on the van der Waals attractive force between the matched acyl chain segment and the sphingoshine chain of the gel phase at temperatures just below the main transition.

A modular synthesis of alkynyl-phosphocholine headgroups for labeling sphingomyelin and phosphatidylcholine.

A general route to phospho- and sphingolipids that incorporate an alkyne in the phosphocholine headgroup is described. The strategy preserves the ammonium functionality of the phosphocholine and can be easily modified to introduce desired functional groups at the N-acyl chain. The targets accessible with this strategy provide a bioorthogonal handle for postsynthetic introduction of fluorophores or other labeling agents with aqueous phase chemistry. We report the synthesis of sphingomyelin derivatives that incorporate a fluorophore and an alkyne. The modified sphingolipids retain activity as substrates for sphingomyelinase, making these compounds viable probes of enzymatic activity. Importantly, the strategy allows modification of the lipid across the phosphodiester, making the alkyne a potential probe of sphingomyelinase activity.

Synthesis of sphingomyelin sulfur analogue and its behavior toward sphingomyelinase.

The sulfur analogue of sphingomyelin was designed and stereoselectively synthesized from S-benzyl-N-Boc-cysteine. The introduction of the phosphoryl choline moiety was successfully achieved by our own method using 2-bromoethyl dimethyl phosphite and carbon tetrabromide followed by a trimethylamine treatment. The synthesized compound proved to be a useful substrate for monitoring the enzyme activity of sphingomyelinase by detecting the liberated thiol group with a thiol-sensitive reagent.

[Synthesis of phosphonic acid and phosphinic acid derivatives for development of biologically active compounds].

This paper covers recent publications from our laboratory on the synthesis of a variety of phosphonate and phosphinate derivatives. New methods for the enantioselective synthesis of alpha-hydroxyphosphonates were established by Lewis acid-mediated cleavage of homochiral 1,3-dioxaneacetals with P(OEt)(3) and chiral metal ligand-mediated hydrophosphonylation of aldehydes. Two diastereomers of HPmp derivatives were prepared by an application of these methods. The HPmp derivatives were convered to FPmp derivatives but with low diastereoselectivity. Hydrophosphonylation of alpha-aminoaldehydes afforded threo- and erythro-beta-amino-alpha-hydroxyphosphonates under chelation and nonchelation controlled conditions, respectively. The asymmetric dihydroxylation of alpha, beta-, and beta, gamma-unsaturated phosphonates with AD-mix-alpha and AD-mix-beta reagents gave alpha, beta- and beta, gamma-dihydroxyphosphonates with high enantioselectivity. The method was applied to the kinetic resolution of racemic alpha-oxygetated beta, gamma-unsaturated phosphonates. Treatment of allyloxymethylphosphonates with the base afforded alpha-hydroxyphosphonates via the [2,3]-Wittig reaction. Threo- and erythro-beta-amino-alpha-hydroxyphosphinates were obtained with high diastereoselectivity by phosphinylation of alpha-aminoaldehydes in the presence of (R)- and (S)-ALB, respectively. The phosphinylation of alpha-oxygenated aldehydes afforded the corresponding alpha, beta-dioxygenated phosphinates, but with low diastereoselectivity. Sphingomyelin analogues containing CF(2)PO(OH)(2) were synthesized starting from (S)- and (R)-Garner aldehyde for the purpose of obtaining potent sphyngomyelinase inhibitors. A useful method for the synthesis of alpha, alpha-difluorobenzylphosphonates was established based on the cross coupling reaction of an iodobenzene derivative with ZnCuBr(2)CF(2)PO(OEt)(2). The synthetic utility of ZnCuBr(2)CF(2)PO(OEt)(2) was examined to obtain alpha, alpha-difluoromethylenenphosphonates. The method was applied to a synthesis of PNP-inhibitory active compounds by combination of the purine base and alcohols containing difluoromethylenephosphonate. The methodology for the beta-selective N-glycosylation of 2,3-dideoxy glucoside was established by introducing phosphonothioates at the 3-position of glycosyl doners instead of phosphonate. Synthesis of new acylic nucleotide analogues designed based on the structural modification of ARS2267 is also described. Finally, kiral synthesis of some phosphonates was achieved using lipase through kinetic resolution.

Overexpression of phosphatidylinositol transfer protein beta in NIH3T3 cells has a stimulatory effect on sphingomyelin synthesis and apoptosis.

Phosphatidylinositol transfer proteins (PI-TPs) consist of two isoforms (PI-TPalpha and PI-TPbeta), which differ in phospholipid transfer properties and intracellular localization. Both PI-TP isoforms are substrates for protein kinase C and contain a minor phosphorylation site (Ser166 in PI-TPalpha; Ser165 in PI-TPbeta). Only PI-TPbeta contains a major phosphorylation site at Ser262, which must be phosphorylated for PI-TPbeta to be associated with the Golgi. The PI-TP isoforms are completely conserved between mammals. Although their function is still not clear, their importance follows from knock-out studies, showing that mice lacking PI-TPalpha die soon after birth and that embryonic stems cells lacking PI-TPbeta cannot be generated [Mol. Biol. Cell 13 (2002) 739]. We determined the levels of the PI-TP isoforms in various mouse tissues by immunoblotting. PI-TPalpha is present in all tissues investigated, with highest levels in brain (167 ng/100 microg total protein). The levels of PI-TPbeta are 50-100 times lower than those of PI-TPalpha, with relatively high levels found in liver and brain (1.2 and 1.8 ng/100 microg of total protein, respectively). In contrast to NIH3T3 cells overexpressing PI-TPalpha, cells overexpressing PI-TPbeta (SPIbeta cells) were able to maintain steady-state levels of sphingomyelin in plasma membrane under conditions where this lipid is degraded by exogenous sphingomyelinase. This process of rapid sphingomyelin replenishment is dependent on PI-TPbeta being associated with the Golgi as cells overexpressing a mutant PI-TPbeta in which the major phosphorylation site is replaced (PI-TPbeta(S262A) behave as wild-type NIH3T3 cells. Since the SPIbeta cells display a decreased growth rate (35 h as compared to 21 h for wtNIH3T3 cells), we have investigated the sensitivity of these cells towards UV-induced apoptosis. We have found that the SPIbeta cells, but not the cells overexpressing PI-TPbeta(S262A), are very sensitive. We are currently investigating whether a relationship exists between PI-TPbeta being involved in maintaining plasma membrane sphingomyelin levels and the enhanced sensitivity towards apoptosis.

Synthesis of a nitrogen analogue of sphingomyelin as a sphingomyelinase inhibitor.

[structure: see text] Sphingomyelin nitrogen analogue 1 was designed and synthesized as a sphingomyelinase inhibitor. The synthesis was established by continuous Hofmann rearrangement and Crutius rearrangement as key steps in constructing the 3-hydroxy-1,2-diamine structure in the backbone of 1. This analogue showed moderate inhibitory activity toward SMase isolated from B. cereus.

Sphingomyelin analogues as inhibitors of sphingomyelinase.

To search for neutral sphingomyelinase inhibitors we designed and synthesized hydrolytically stable analogues of sphingomyelin. The novel compounds 8 and 9 which were replaced the phosphodiester moiety of sphingomyelin with the carbamate moiety showed inhibitory activity with an IC(50) value of micro M on neutral sphingomyelinase in rat brain microsomes. Compound 8i showed a selective neutral sphingomyelinase inhibitory activity.

[Synthesis of sphingomyelin analogues as a sphingomyelinase inhibitor and the application as a blocking agent of extracellular stress signaling].

Sphingomyelin (SM) pathway, where sphingomyelinase (SMase) hydrolyzes SM to produce ceramide, has recently been suggested to link to the signaling pathway that determines cell death. Therefore, elucidation of the mechanism by which SMase is activated and the regulation of SMase activity will be an important therapeutic strategy for various cytokine-related and ischemic diseases. We have synthesized nine difluoromethylene analogues of SM as SMase inhibitors and evaluated their inhibitory potencies. In this study, we show that the inhibitor suppresses ceramide production and cell death of PC-12 neurons that have been induced by deprivation of serum from the culture medium. The SMase inhibitor could also suppress neuronal cell death in a mouse model of brain ischemia. These results suggest a possibility that the prevention of various extracellular stress-induced cell death signalings is accomplished by inhibiting the cell membrane SMase.

Synthesis of sphingomyelin carbon analogues as sphingomyelinase inhibitors.

The highly efficient and stereocontrolled syntheses of sphingomyelin carbon analogues 1 and 2 were achieved by effectively utilizing Hofmann rearrangement of enantiomerically pure beta-hydroxyamide 7, which was prepared by an asymmetric hydrogenation of alpha-acyl-gamma-butyrolactone 9 and ring opening with NH(3). Intermediary isocyanate 6 was selectively trapped with the vicinal hydroxy group in an intramolecular fashion to produce an oxazolidinone derivative, 5. In the synthesis of a quite polar compound such as 1, a convenient one-pot procedure of the introduction of a benzyloxycarbonyl group into the hydroxy group resulting from the oxazolidinone ring opening is another key point, because, in addition to the efficiency, this protecting group was easily removable by a simple procedure and workup at the final step. Both synthesized compounds 1 and 2 showed moderate inhibitory activity toward sphingomyelinase from B. cereus.

Synthesis and evaluation of a difluoromethylene analogue of sphingomyelin as an inhibitor of sphingomyelinase.

A sphingomyelin analogue 2, in which the long alkenyl chain and the phosphodiester moiety of sphingomyelin were replaced by a phenyl and an isosteric difluoromethylenephosphonic acid, was prepared to evaluate its inhibitory potency to sphingomyelinase. The analogue non-competitively inhibited the neutral sphingomyelinase in bovine brain microsomes with an IC50 of 400 microM. The compound had the ability to suppress tumor necrosis factor alpha-induced apoptosis of PC-12 neurons at a low concentration of 0.1 microM.

Stereocontrolled synthesis of a sphingomyelin methylene analogue as a sphingomyelinase inhibitor.

[reaction: see text]Efficient synthesis of a sphingomyelin methylene analogue, which was designed as a sphingomyelinase inhibitor, was stereoselectively achieved. The Hofmann rearrangement of the alpha-hydroxyethyl-beta-hydroxy amide 4 followed by the intramolecular oxazolidinone ring formation was one of the key steps.

Analysis of natural and synthetic sphingomyelins using high-performance thin-layer chromatography.

The chromatographic behaviour of molecular species of sphingomyelin on HPTLC was investigated. Sphingomyelin gave a double band pattern on HPTLC plates developed using chloroform/methanol/acetic acid/water (25 : 15 : 4 : 2, v/v) or chloroform/methanol/water (25 : 10 : 1.1, v/v). HPTLC analysis of acyl chain-defined sphingomyelins showed that the Rf values increased linearly with the length of the N-linked acyl chain. A double-banded pattern was therefore seen for natural sphingomyelins with a bimodal fatty acid composition. Racemic sphingomyelins also gave a double band pattern on HPTLC, where the lower band represented the Derythro and the upper band the Lthreo isomer. We also showed that Derythro-N-16:0-dihydrosphingomyelin migrated faster on HPTLC than Derythro-N-16:0-sphingomyelin. The upper and lower band sphingomyelins from two different cell lines (human skin fibroblasts and baby hamster kidney cells) were separately scraped off the HPTLC plates and the fatty acid and long-chain base profiles were studied using GC-MS. The lower bands contained short-chain fatty acids and most of the fatty acids in the upper bands were long. The predominant long-chain base was sphingosine, which was found in both upper and lower bands, but sphinganine was found only in the upper bands. To conclude, there are at least three possible reasons for the sphingomyelin double bands on HPTLC; acyl chain length, long-chain base composition and stereochemistry. These reasons might sometimes overlap and, therefore, HPTLC alone is insufficient for complete analysis of the molecular species of sphingomyelin.