Masato Oikawa

Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide

Lipopolysaccharide (LPS) is the main inducer of shock and death in Gram-negative sepsis. Recent evidence suggests that LPS-induced signal transduction begins with CD14-mediated activation of 1 or more Toll-like receptors (TLRs). The lipid A analogues lipid IVa and Rhodobacter sphaeroides lipid A (RSLA) exhibit an uncommon species-specific pharmacology. Both compounds inhibit the effects of LPS in human cells but display LPS-mimetic activity in hamster cells. We transfected human TLR4 or human TLR2 into hamster fibroblasts to determine if either of these LPS signal transducers is responsible for the species-specific pharmacology. RSLA and lipid IVa strongly induced NF-kappaB activity and IL-6 release in Chinese hamster ovary fibroblasts expressing CD14 (CHO/CD14), but these compounds antagonized LPS antagonists in CHO/CD14 fibroblasts that overexpressed human TLR4. No such antagonism occurred in cells overexpressing human TLR2. We cloned TLR4 from hamster macrophages and found that human THP-1 cells expressing the hamster TLR4 responded to lipid IVa as an LPS mimetic, as if they were hamster in origin. Hence, cells heterologously overexpressing TLR4 from different species acquired a pharmacological phenotype with respect to recognition of lipid A substructures that corresponded to the species from which the TLR4 transgene originated. These data suggest that TLR4 is the central lipid A-recognition protein in the LPS receptor complex.

Divergent synthesis and biological activities of lipid A analogues of shorter acyl chains

Abstract Novel lipid A analogues which possess four ( R )-3-hydroxyacyl moieties of shorter chain length were synthesized via a new divergent synthetic route in order to clarify the effect of the chain length of acyl groups to the biological activity: a disaccharide 4′-phosphate was first constructed as a common synthetic intermediate and all acyl moieties were then introduced step by step to the respective positions. The hydroxy group in acyl moieties was protected with a benzyl group by novel 1-pot reductive alkylation using benzaldehyde, TMS 2 O, TMSOTf, and Et 3 SiH. Both the glycosyl donor and acceptor were synthesized by using the new method recently reported by ourselves for the regioselective reductive opening of 4,6- O -benzylidene glucosamine derivatives with BH 3 ·Me 2 NH and BF 3 ·OEt 2 . In this reaction, a 3- O -allyloxycarbonylated 4,6- O -benzylidene compound in CH 3 CN afforded the 6- O -benzylated product selectively, which was then converted to a glycosyl trichloroacetimidate used as the donor. The 4- O -benzylated acceptor was synthesized by the same reductive opening of a 3- O -p-methoxybenzylated compound in CH 2 Cl 2 . A disaccharide 4′-phosphate was synthesized by coupling of the imidate donor and the acceptor using TMSOTf as a catalyst. ( R )-3-Benzyloxyacyl groups were then introduced to the 3,3′, 2 and 2′ positions followed by 1- O -phosphorylation and subsequent deprotection by Pd H 2 afforded the desired lipid A analogues. The present divergent route opens an efficient way toward the synthesis of lipid A libraries. Biological tests (inhibition of IL-6 induction) clearly showed the critical importance of the chain length of the acyl moieties in lipid A to the activity.

Endotoxic and immunobiological activities of a chemically synthesized lipid A of Helicobacter pylori strain 206-1

A synthetic lipid A of Helicobacter pylori strain 206-1 (compound HP206-1), which is similar to its natural lipid A, exhibited no or very low endotoxic activities as compared to Escherichia coli-type synthetic lipid A (compound 506). Furthermore, compound HP206-1 as well as its natural lipid A demonstrated no or very low mitogenic responses in murine spleen cell. On the other hand, compound HP206-1 showed a weaker but significant production of interleukin-8 in a gastric cancer cell line, MKN-1, in comparison with compound 506. Furthermore, compound HP206-1 exhibited induction of tumor necrosis factor-alpha production in human peripheral blood mononuclear cells and the cytokine production was clearly inhibited by mouse anti-human Toll-like receptor (TLR) 4 monoclonal antibody HTA125. Our findings indicate that the chemically synthesized lipid A, mimicking the natural lipid A portion of lipopolysaccharide from H. pylori strain 206-1, has a low endotoxic potency and immunobiological activities, and is recognized by TLR4.

Lipopolysaccharide-binding protein-mediated interaction of lipid A from different origin with phospholipid membranes

Investigations are reported into the interaction of lipid A, the ‘endotoxic principle’ of bacterial lipopolysaccharide (LPS), with phospholipid membranes in the absence and presence of an acute-phase lipid transport protein, lipopolysaccharide-binding protein (LBP) applying Fourier-transform infrared (FTIR) and fluorescence resonance energy transfer (FRET) spectroscopy. In the absence of LBP, intermixing of phospholipids with lipid A takes place on the time-scale of hours, while in the presence of LBP this process takes place in the order of minutes. A comparison of chemically different lipid A shows that a prerequisite for the intercalation of lipid A into the phospholipid membrane is a sufficiently high negative charge density of lipid A. Variations in the lipid A acyl chain fluidity may modulate the intercalation, whereas the type of lipid A aggregate structure has no influence on the intercalation.The intercalation is a necessary, but not sufficient prerequisite for cell activation. Only lipid A with a conical molecular shape and a tilt angle of more than 40° of the backbone with respect to the direction of the acyl chains induces cytokine induction in human mononuclear cells, while lipid A with a cylindrical shape and a small tilt angle does not exhibit this biological activity but may act antagonistically. This antagonistic effect may be explained by blocking of the binding-sites of the putative signal-transducing protein, possibly an ion channel, by the antagonist.

Structural basis for endotoxic and antagonistic activities: investigation with novel synthetic lipid A analogs.

Our early work using homogeneous synthetic preparations demonstrated the presence of a lipid A analog which antagonizes endotoxic activities of LPS and lipid A. The first example was a tetraacylated biosynthetic precursor, now known as precursor Ia or lipid IVa, that contains four 3-hydroxytetradecanoyl moieties linked to the bisphosphorylated disaccharide backbone common to the endotoxic hexa-acyl Escherichia coli lipid A. Various compounds with both endotoxic and antagonistic activities have subsequently been reported from either natural or synthetic sources, but little is known about the factors determining the type of the activities of the respective compounds. To approach this issue, we have synthesized a series of lipid A analogs with various numbers and chain lengths of acyl groups on the backbone. Some were prepared by the aid of a novel affinity separation procedure. The phosphate moieties were also synthetically replaced. Biological tests showed that at least three acyl groups are required for antagonistic activity but one or even both of the phosphates can be replaced with other acidic moieties without losing the activity. The effect of Kdo residues linked to lipid A is also briefly discussed. Molecular dynamics calculations reasonably explain possible conformations required for the biological activity.

The spiroketals containing a benzyloxymethyl moiety at C8 position showed the most potent apoptosis-inducing activity

The spiroketals containing a benzyloxymethyl moiety at the C8 position showed the most potent apoptosis-inducing activity, whereas its analogous compounds lacking any substituent at C8 or possessing ones other than the benzyloxymethyl moiety at C8 were all much less active. These results strongly suggest an important role of the benzyloxymethyl moiety linked to the C8 oxygen atom.

Synthesis of Helicobacter pylori lipid A and its analogue using p-(trifluoromethyl)benzyl protecting group

Abstract Synthesis of lipid A 1 isolated from Helicobacter pylori strain 206-1 has been achieved in 2.2% total yield through 14 steps from d -glucosamine by employing a p -(trifluoromethyl)benzyl group for protection of the hydroxy group on the 3-hydroxy fatty acid residue. The synthetic specimen was identical with the natural counterpart in chromatographic, spectroscopic, and biological aspects. A structural analogue 2 which lacks the ethanolamine residue of 1 was also synthesized, and 2 was found to exhibit less potent IL-1β-inducing activity than 1 .

Interference terahertz label-free imaging for protein detection on a membrane

We demonstrate a highly sensitive imaging method combined a terahertz time-domain spectroscopy and an interference effect for label-free protein detection on a polyvinylidene difluoride membrane. The method is based on terahertz time-domain spectroscopy and uses an interference effect. Biotin is linked to the membrane using poly ethylene glycol or poly ethylene glycol methyl ether to prevent it from being washed off. Binding of the biotin with streptavidin is then observed by measuring the terahertz signal change due to the variation of the membrane refractive index. We demonstrate the detection of the binding streptavidin protein in gradually decreasing concentrations, down to 27 ng mm-2, using the image recorded at 1.5 THz.

On a practical synthesis of β-hydroxy fatty acid derivatives

Abstract An efficient synthesis of three homochiral β-hydroxy fatty acid derivatives, which have been utilized in our total synthesis of lipid A, is reported. The synthesis features Sharpless asymmetric dihydroxylation of an unsaturated ester, regioselective conversion of a diol into acyloxy chlorides, and a reductive removal of the chloro group.

Cytochalasin D acts as an inhibitor of the actin–cofilin interaction

Cofilin, a key regulator of actin filament dynamics, binds to G- and F-actin and promotes actin filament turnover by stimulating depolymerization and severance of actin filaments. In this study, cytochalasin D (CytoD), a widely used inhibitor of actin dynamics, was found to act as an inhibitor of the G-actin-cofilin interaction by binding to G-actin. CytoD also inhibited the binding of cofilin to F-actin and decreased the rate of both actin polymerization and depolymerization in living cells. CytoD altered cellular F-actin organization but did not induce net actin polymerization or depolymerization. These results suggest that CytoD inhibits actin filament dynamics in cells via multiple mechanisms, including the well-known barbed-end capping mechanism and as shown in this study, the inhibition of G- and F-actin binding to cofilin.