Atsushi Shimoyama

Chemical synthesis of Helicobacter pylori lipopolysaccharide partial structures and their selective proinflammatory responses.

Helicobacter pylori is a common cause of gastroduodenal inflammatory diseases such as chronic gastritis and peptic ulcers and also an important factor in gastric carcinogenesis. Recent reports have demonstrated that bacterial inflammatory processes, such as stimulation with H. pylori lipopolysaccharide (LPS), initiate atherosclerosis. To establish the structures responsible for the inflammatory response of H. pylori LPS, we synthesized various kinds of lipid A structures (i.e., triacylated lipid A and Kdo-lipid A compounds), with or without the ethanolamine group at the 1-phosphate moiety, by a new divergent synthetic route. Stereoselective α-glycosylation of Kdo N-phenyltrifluoroacetimidate was achieved by use of microfluidic methods. None of the lipid A and Kdo-lipid A compounds were a strong inducer of IL-1β, IL-6, or IL-8, suggesting that H. pylori LPS is unable to induce acute inflammation. In fact, the lipid A and Kdo-lipid A compounds showed antagonistic activity against cytokine induction by E. coli LPS, except for the lipid A compound with the ethanolamine group, which showed very weak agonistic activity. On the other hand, these H. pylori LPS partial structures showed potent IL-18- and IL-12-inducing activities. IL-18 has been shown to correlate with chronic inflammation, so H. pylori LPS might be implicated in the chronic inflammatory responses induced by H. pylori. These results also indicated that H. pylori LPS can modulate the immune response: NF-κB activation through hTLR4/MD-2 was suppressed, whereas production of IL-18 and IL-12 was promoted.

Phylogeny of Galactolipid Synthase Homologs Together with their Enzymatic Analyses Revealed a Possible Origin and Divergence Time for Photosynthetic Membrane Biogenesis

The photosynthetic membranes of cyanobacteria and chloroplasts of higher plants have remarkably similar lipid compositions. In particular, thylakoid membranes of both cyanobacteria and chloroplasts are composed of galactolipids, of which monogalactosyldiacylglycerol (MGDG) is the most abundant, although MGDG biosynthetic pathways are different in these organisms. Comprehensive phylogenetic analysis revealed that MGDG synthase (MGD) homologs of filamentous anoxygenic phototrophs Chloroflexi have a close relationship with MGDs of Viridiplantae (green algae and land plants). Furthermore, analyses for the sugar specificity and anomeric configuration of the sugar head groups revealed that one of the MGD homologs exhibited a true MGDG synthetic activity. We therefore presumed that higher plant MGDs are derived from this ancestral type of MGD genes, and genes involved in membrane biogenesis and photosystems have been already functionally associated at least at the time of Chloroflexi divergence. As MGD gene duplication is an important event during plastid evolution, we also estimated the divergence time of type A and B MGDs. Our analysis indicated that these genes diverged ∼323 million years ago, when Spermatophyta (seed plants) were appearing. Galactolipid synthesis is required to produce photosynthetic membranes; based on MGD gene sequences and activities, we have proposed a novel evolutionary model that has increased our understanding of photosynthesis evolution.

Synthesis and immunomodulatory activities of Helicobacter pylori lipophilic terminus of lipopolysaccharide including lipid A

Helicobacter pylori, a Gram-negative bacterium, causes gastroduodenal inflammatory diseases such as chronic gastritis and peptic ulcers, and is also a risk factor for gastric carcinogenesis. In this article, we review recent developments and findings in the chemical synthesis and immunomodulatory activities of H. pylori lipid A and 3-deoxy-D-manno-2-octulosonic acid (Kdo)-lipid A, to clarify the structural basis for the inflammatory response to H. pylori LPS. The synthetic methods include a new divergent synthetic approach with a widely applicable key intermediate for other types of lipid A structures, as well as a selective α-glycosylation reaction between Kdo and lipid A. Cytokine induction assays of the chemically synthesized lipid A structures showed selective cytokine induction depending on the patterns of acyl groups and phosphate groups. The results of cytokine induction assay suggested that H. pylori LPS can modulate the immune response during infection, and also plays a role in chronic inflammatory responses.

Access to a novel near-infrared photodynamic therapy through the combined use of 5-aminolevulinic acid and lanthanide nanoparticles

BACKGROUND There have been considerable efforts to develop photodynamic therapy (PDT) for cancer, in which photoirradiation of a sensitizer delivered near cancer cells results in the conversion of oxygen into active species, causing cell destruction. Aiming at the best cancer selectivity, one PDT method employed protoporphyrin IX (PPIX), which selectively accumulated in cancer cells after oral administration of 5-aminolevulinic acid (ALA). The drawback, however, is that blue incident lights are required to excite PPIX, resulting in low tissue penetrability, and therefore limiting its application to surface cancers. METHODS To overcome the low penetrability of the incident light, we employed a light energy upconverter, lanthanide nanoparticle (LNP), which, upon irradiation with highly penetrative near-infrared (NIR) radiation, emits visible light within the Q-band region of PPIX absorbance allowing its sensitization. To discover the optimum conditions for the LNP-assisted PDT, the cytotoxicity and PPIX-sensitizability of LNPs were first studied. Then, the LNP-assisted PDT was validated using the MKN45 cell line: cells were pretreated with ALA and LNP, irradiated with a 975-nm diode laser, and subjected to MTT assay to measure cell viability. RESULTS The singlet oxygen generation on NIR-irradiation of the PPIX-LNP mixture was proved, indicating that the emission from LNP could excite the PPIX sensitizer. An intermittent NIR-irradiation for 32 min of MKN45, pretreated with LNP (1mg/mL) and ALA (2mM), caused 87% cell destruction. CONCLUSIONS The potential applicability of the NIR-irradiation PDT with ALA- and LNP-pretreated cancer cells was demonstrated.

Chemical synthesis of peptidoglycan fragments for elucidation of the immunostimulating mechanism.

Partial structures of peptidoglycan were chemically synthesized for elucidation of their precise biological activities. By using an efficient synthetic strategy, mono-, di-, tetra- and octasaccharide fragments of peptidoglycan were synthesized in good yields. The biological activity of synthetic fragments of peptidoglycan was evaluated by induction of TNF-α from human monocytes, and TLR2 and NOD2 dependencies by using transfected HEK293 cells, respectively. We revealed that TLR2 was not stimulated by the series of synthetic peptidoglycan partial structures, whereas NOD2 recognizes the partial structures containing the MDP moiety. We also synthesized potent NOD1 agonists, which showed several hundred-fold stronger activity than γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP). Interaction of PGRPs with synthetic peptidoglycan fragments is also described.

Innate immunomodulation by lipophilic termini of lipopolysaccharide; synthesis of lipid As from Porphyromonas gingivalis and other bacteria and their immunomodulative responses

Synthetic studies of lipid A and LPS partial structures have been performed to investigate the relationship between structures and functions of LPS. Recent studies have suggested several pathological implications of LPS from parasitic bacteria due to its influence on the host immune responses. To address this issue, we established an efficient synthetic strategy that is widely applicable to the synthesis of various lipid As by using a key disaccharide intermediate with selectively cleavable protecting groups. Porphyromonas gingivalis and Helicobacter pylori lipid As were synthesized and their biological activities were evaluated. All synthetic lipid As did not induce strong inflammatory responses: some are very weak cytokine inducers and others are antagonistic in IL-6 and IL-8 induction with E. coli LPS. On the other hand, P. gingivalis lipid As showed potent IL-18 inducing activity. Since IL-18 has been shown to correlate with chronic inflammation, P. gingivalis LPS may be implicated in the chronic inflammatory responses.

Sugar-attached upconversion lanthanide nanoparticles: a novel tool for high-throughput lectin assay.

To create a novel high-throughput lectin assay (HTPLA) method based on the emission of a luminophore by highly penetrable near-infrared excitation, sugar-attached upconversion lanthanide nanoparticles (LNPs) were synthesized as a tool to highlight the aggregates caused by the sugar-mediated specific bridging between LNP and lectin. The emissions from a mannose-coated LNP in the aggregates with a mannose-binding lectin were much stronger than those from the non-aggregated samples, being sensitive enough for HTPLA. A galactose-coated LNP was also applicable to a macrophage aggregation assay for the sugar specificity of its surface lectin.

Self and nonself recognition with bacterial and animal glycans, surveys by synthetic chemistry

In this chapter, we describe synthetic studies on partial structures of lipopolysaccharide (LPS) and peptidoglycan (PGN), which work as tags for nonself recognition in innate immune system. Our previous studies demonstrated that lipid A is the endotoxic principle of LPS. The synthetic homogeneous preparations have enabled not only precise structure-activity relationships, but also recognition mechanisms of LPS with innate immune receptor complexes, including the TLR4/MD-2 complex, to be studied. Synthetic studies of lipid A and Kdo-lipid A from parasitic Helicobacter pylori revealed their low inflammatory activities, suggesting the molecular evolution to escape from the host immune system. A synthetic study of the partial structures of PGN has also contributed to the understanding of the innate immune mechanism. The biological activities of the synthetic fragments have revealed that the intracellular receptor Nod2 recognizes partial structures containing the muramyl dipeptide (MDP) moiety. The PGN of Gram-negative bacteria and some Gram-positive bacteria contain meso-diaminopimelic acid (meso-DAP), and recent studies have revealed that the intracellular receptor Nod1 recognizes DAP-containing peptides. We have synthesized DAP-containing PGN fragments, including the first chemical synthesis of tracheal cytotoxin (TCT). The ability of these fragments to stimulate human Nod1 as well as differences in Nod1 recognition for various synthesized ligand structures was elucidated. Cell-surface glycans such as N-glycans and O-glycans on glycoproteins and glycoconjugates work as signaling molecules for self-recognition and control immune system. Our new strategy using glycan-imaging in whole-body system is expected to unveil the dynamics of glycans in the body. Positron emission tomography (PET) is a noninvasive method that visualizes the locations and levels of radiotracer accumulation. We developed the facile labeling of peptides and proteins for PET imaging. The labeled glycoproteins and glycoclusters were then subjected to PET imaging in order to examine their in vivo dynamics, visualizing the differences in the circulatory residence of glycoproteins and glycoclusters in the presence or absence of sialic acid residues.

Lymphoid tissue-resident Alcaligenes LPS induces IgA production without excessive inflammatory responses via weak TLR4 agonist activity

Alcaligenes are opportunistic commensal bacteria that reside in gut-associated lymphoid tissues such as Peyer’s patches (PPs); however, how they create and maintain their homeostatic environment, without inducing an excessive inflammatory response remained unclear. We show here that Alcaligenes-derived lipopolysaccharide (Alcaligenes LPS) acts as a weak agonist of toll-like receptor 4 and promotes IL-6 production from dendritic cells, which consequently enhances IgA production. The inflammatory activity of Alcaligenes LPS was weaker than that of Escherichia coli-derived LPS and therefore no excessive inflammation was induced by Alcaligenes LPS in vitro or in vivo. Alcaligenes LPS also showed adjuvanticity, inducing antigen-specific immune responses without excessive inflammation. These findings reveal the presence of commensal bacteria-mediated homeostatic inflammatory conditions within PPs that produce optimal IgA induction without causing pathogenic inflammation and suggest that Alcaligenes LPS could be a safe and potent adjuvant.

Bradyrhizobium Lipid A: Immunological Properties and Molecular Basis of Its Binding to the Myeloid Differentiation Protein-2/Toll-Like Receptor 4 Complex

Lipopolysaccharides (LPS) are potent activator of the innate immune response through the binding to the myeloid differentiation protein-2 (MD-2)/toll-like receptor 4 (TLR4) receptor complexes. Although a variety of LPSs have been characterized so far, a detailed molecular description of the structure–activity relationship of the lipid A part has yet to be clarified. Photosynthetic Bradyrhizobium strains, symbiont of Aeschynomene legumes, express distinctive LPSs bearing very long-chain fatty acids with a hopanoid moiety covalently linked to the lipid A region. Here, we investigated the immunological properties of LPSs isolated from Bradyrhizobium strains on both murine and human immune systems. We found that they exhibit a weak agonistic activity and, more interestingly, a potent inhibitory effect on MD-2/TLR4 activation exerted by toxic enterobacterial LPSs. By applying computational modeling techniques, we also furnished a plausible explanation for the Bradyrhizobium LPS inhibitory activity at atomic level, revealing that its uncommon lipid A chemical features could impair the proper formation of the receptorial complex, and/or has a destabilizing effect on the pre-assembled complex itself.