Synthesis and biological activity of phosphoglycolipids from Thermus thermophilus.

An extreme thermophile, Thermus thermophilus, has very unique glycolipids on the cell surface. The acidic immunostimulatory phosphoglycolipid of T. thermophilus was synthesized for the first time, with newly developed glycosylation methods using 3-nitropyridyl (3NPy) and 4,6-dimethoxy-1,3,5-triazin-2-yl (DMT) glycosides as glycosyl donors. The analogues of the phosphoglycolipid, which include a diastereomer possessing the opposite configuration at the diacyl glycerol moiety, were also synthesized. The biological activities of the synthesized compounds were elucidated with cytokine inductions (IL-6 and TNF-α). A synthetic phosphoglycolipid with a natural-type diacyl glycerol configuration showed apparent immunostimulatory activity, whereas its diastereomer did not. The present study revealed that the configuration at the diacyl glycerol moiety of the phosphoglycolipids is important for immunostimulation, suggesting the existence of the particular receptor/recognizing protein that can recognize the stereochemistry of the glycerol part.

MPIase is a glycolipozyme essential for membrane protein integration.

Protein integration into biological membranes is a vital cellular event for all organisms. We previously reported an integration factor in the inner membrane of Escherichia coli, named MPIase (membrane protein integrase). Here we show that in contrast to previously identified integration factors that are proteins, MPIase is a glycolipid composed of diacylglycerol and a glycan chain of three acetylated aminosugars linked through pyrophosphate. Hydrolytic removal of the lipid moiety gives a soluble product with higher integration activity than that of the original MPIase. This soluble form of MPIase directly interacts with a newborn membrane protein, maintaining its integration-competent structure and allowing its post-translational integration. MPIase actively drives protein integration following chaperoning membrane proteins. We further demonstrate with anti-MPIase antibodies that MPIase is likely involved in integration in vivo. Collectively, our results suggest that MPIase, essential for membrane protein integration, is to our knowledge the first glycolipid with an enzyme-like activity.

Structural characterization of neutral and acidic glycolipids from Thermus thermophilus HB8.

The structural characterization of glycolipids from Thermus thermophilus HB8 was performed in this study. Two neutral and one acidic glycolipids were extracted and purified by the modified TLC-blotting method, after which their chemical structures were determined by chemical composition analysis, mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. The structure of one of the neutral glycolipids, NGL-A, was Galp(α1-6)GlcpNacyl(ß1-2)Glcp(α1-)acyl(2)Gro, and the other, NGL-C, was Galf(ß1-2)Galp(α1-6)GlcpNacyl(ß1-2)Glcp(α1-)acyl(2)Gro. The structure of NGL-C was identical to that reported previously [Oshima, M. and Ariga, T. (1976) FEBS Lett. 64, 440]. Both neutral glycolipids shared a common structural unit found in the Thermus species. The acyl groups found in NGL-A and NGL-C, iso-type pentadecanoxy and heptadecanoxy fatty acid, were also the same as those found in this species. In contrast, the acidic glycolipid, AGL-B, possessed the structure of N-(((GlcpNAc(α1-)acyl(2)Gro)P-2)GroA)alkylamine. The alkyl group in AGL-B was an iso-type heptadecanyl, suggesting that the iso-type structure of the long alkyl chain is responsible for the thermal stability of the bacteria.

Lipopolysaccharide induces raft domain expansion in membrane composed of a phospholipid-cholesterol-sphingomyelin ternary system.

The molecular behavior and interaction of Re-type lipopolysaccharide (ReLPS) and phospholipids were investigated in two different types of model membrane systems, a pure phospholipid membrane consisting of 1,2-dielaidoyl-snglycero-3-phosphoethanolamine (DEPE) and a raft-forming membrane composed of equimolar DEPE, sphingomyelin (SM), and cholesterol (Chol) by solid-state NMR spectroscopy. A remarkable influence of ReLPS on the property of lipid bilayer was found by analyzing the (13)C-NMR spectra. Namely, while both liquid-ordered (L(o)) and liquid-disordered (L(d)) phases co-exist in DEPE/SM/Chol, only the L(o) phase is present in DEPE/SM/Chol/ReLPS. This clearly indicates that ReLPS induces expansion of the raft area in the raft-forming membrane. The (1)H spin-lattice relaxation times in the rotating frame T( 1ρ) (H) in the two different membranes, DEPE/ReLPS and DEPE/SM/Chol/ReLPS, indicate that the motion of DEPE is affected by the presence of ReLPS, Chol, and SM, and much faster than that of ReLPS in both membranes. The ReLPS in the raft-forming membrane, in particular, accelerated the movement of DEPE. Thus, this study shows the possibility that LPS induces the expansion of raft region and the rapid motion of the raft-forming membranes to favor molecular interactions in the animal cell membrane during innate immune recognition.

Key structures of bacterial peptidoglycan and lipopolysaccharide triggering the innate immune system of higher animals: Chemical synthesis and functional studies".

Chemistry-based investigation is reviewed which led to identification of the active entities responsible for the immunostimulating potencies of peptidoglycan and lipopolysaccharide. Though these glycoconjugates which ubiquitously occur in wide range of bacteria as the essential components of their cell envelopes have long been known to enhance the immunological responses of higher animals, neither the precise chemical structures required nor the mechanism of their action had been elucidated until early 1970s. Chemical synthesis of partial structures of peptidoglycan proved N-acetylmuramyl-L-alanyl-D-isoglutamine to be the minimum structure responsible for the activity and led to later identification of its receptor protein Nod2 present in animal cells. Another active partial structure of peptidoglycan, g-D-glutamylmeso-diaminopimelic acid, and its receptor Nod1 were also identified as well. With regard to lipopolysaccharide, its glycolipid part named lipid A was purified and the structure studied. Chemically synthesized lipid A according to the newly elucidated structure exhibited full activity described for lipopolysaccharide known as endotoxin. Synthetic homogeneous lipid A and its structural analogues and labeled derivatives enabled precise studies of their interaction with receptor proteins and the mechanism of their action. Chemical synthesis of homogeneous partial structures of peptidoglycan and lipopolysaccharide gave unequivocal evidences for the concept that definite small molecular parts of these complex macromolecular bacterial glycoconjugates are specifically recognized by their respective receptors and trigger our defense system now widely recognized as innate immunity.

Key structures of bacterial peptidoglycan and lipopolysaccharide triggering the innate immune system of higher animals: chemical synthesis and functional studies.

Chemistry-based investigation is reviewed which led to identification of the active entities responsible for the immunostimulating potencies of peptidoglycan and lipopolysaccharide. Though these glycoconjugates which ubiquitously occur in wide range of bacteria as the essential components of their cell envelopes have long been known to enhance the immunological responses of higher animals, neither the precise chemical structures required nor the mechanism of their action had been [corrected] elucidated until early 1970s. Chemical synthesis of partial structures of peptidoglycan proved N-acetylmuramyl-L-alanyl-D-isoglutamine to be the minimum structure responsible for the activity and led to later identification of its receptor protein Nod2 present in animal cells. Another active partial structure of peptidoglycan, gamma-D-glutamyl-meso-diaminopimelic acid, and its receptor Nod1 were also identified as well. With regard to lipopolysaccharide, its glycolipid part named lipid A was purified and the structure studied. Chemically synthesized lipid A according to the newly elucidated structure exhibited full activity described for lipopolysaccharide known as endotoxin. Synthetic homogeneous lipid A and its structural analogues and labeled derivatives enabled precise studies of their interaction with receptor proteins and the mechanism of their action. Chemical synthesis of homogeneous partial structures of peptidoglycan and lipopolysaccharide gave unequivocal evidences for the concept that definite small molecular parts of these complex macromolecular bacterial glycoconjugates are specifically recognized by their respective receptors and trigger our defense system now widely recognized as innate immunity.

A novel complete reconstitution system for membrane integration of the simplest membrane protein.

A complete reconstitution system for membrane integration of the simplest protein was developed by means of defined factors. A mutant version of Pf3 coat protein, 3L-Pf3 coat, requires neither signal recognition particle/Sec factors nor a membrane potential for its integration into the cytoplasmic membrane of Escherichia coli. Although 3L-Pf3 coat is spontaneously integrated into liposomes composed of phospholipids, diacylglycerol completely blocks such spontaneous integrations at a physiological level. Under the conditions where spontaneous integration does not occur, 3L-Pf3 coat integration was absolutely dependent on a novel integration-stimulating factor. Combination of the PURE system, an in vitro translation system composed of the purified factors involved in translation in E. coli, with liposomes containing the highly purified integration-stimulating factor revealed multiple cycles of 3L-Pf3 coat integration, achieving the complete reconstitution of membrane integration. Based on the function of the factor, we propose that the factor is named MPIase (Membrane Protein Integrase).

Toll-like receptors of the ascidian Ciona intestinalis: prototypes with hybrid functionalities of vertebrate Toll-like receptors.

Key transmembrane proteins in the innate immune system, Toll-like receptors (TLRs), have been suggested to occur in the genome of non-mammalian organisms including invertebrates. However, authentic invertebrate TLRs have been neither structurally nor functionally investigated. In this paper, we originally present the structures, localization, ligand recognition, activities, and inflammatory cytokine production of all TLRs of the ascidian Ciona intestinalis, designated as Ci-TLR1 and Ci-TLR2. The amino acid sequence of Ci-TLR1 and Ci-TLR2 were found to possess unique structural organization with moderate sequence similarity to functionally characterized vertebrate TLRs. ci-tlr1 and ci-tlr2 genes were expressed predominantly in the stomach and intestine as well as in hemocytes. Ci-TLR1 and Ci-TLR2 expressed in HEK293 cells, unlike vertebrate TLRs, were localized to both the plasma membrane and endosomes. Intriguingly, both Ci-TLR1 and Ci-TLR2 stimulate NF-kappaB induction in response to multiple pathogenic ligands such as double-stranded RNA, and bacterial cell wall components that are differentially recognized by respective vertebrate TLRs, revealing that Ci-TLRs recognize broader pathogen-associated molecular patterns than vertebrate TLRs. The Ci-TLR-stimulating pathogenic ligands also induced the expression of Ci-TNFalpha in the intestine and stomach where Ci-TLRs are expressed. These results provide evidence that the TLR-triggered innate immune systems are essentially conserved in ascidians, and that Ci-TLRs possess "hybrid" biological and immunological functions, compared with vertebrate TLRs. Moreover, it is presumed that chordate TLR ancestors also acquired the Ci-TLR-like multiple cellular localization and pathogen-associated molecular pattern recognition.

Interaction of lipopolysaccharide and phospholipid in mixed membranes: solid-state 31P-NMR spectroscopic and microscopic investigations.

Lipopolysaccharide (LPS), which constitutes the outermost layer of gram-negative bacterial cells as a typical component essential for their life, induces the first line defense system of innate immunity of higher animals. To understand the basic mode of interaction between bacterial LPS and phospholipid cell membranes, distribution patterns were studied by various physical methods of deep rough mutant LPS (ReLPS) of Escherichia coli incorporated in phospholipid bilayers as simple models of cell membranes. Solid-state (31)P-NMR spectroscopic analysis suggested that a substantial part of ReLPS is incorporated into 1,2-dimyristoyl-sn-glycero-3-phosphocholine lipid bilayers when multilamellar vesicles were prepared from mixtures of these. In egg L-alpha-phosphatidylcholine (egg-PC)-rich membranes, ReLPS undergoes micellization. In phosphatidylethanolamine-rich membranes, however, micellization was not observed. We studied by microscopic techniques the location of ReLPS in membranes of ReLPS/egg-PC (1:10 M/M) and ReLPS/egg-PC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (1:9:1 M/M/M). The influence of ReLPS on the physicochemical properties of the membranes was studied as well. Microscopic images of both giant unilamellar vesicles and supported planar lipid bilayers showed that LPS was uniformly incorporated in the egg-PC lipid bilayers. In the egg-PC/POPG (9:1 M/M) lipid bilayers, however, ReLPS is only partially incorporated and becomes a part of the membrane in a form of aggregates (or as mixed aggregates with the lipids) on the bilayer surface. The lipid lateral diffusion coefficient measurements at various molar ratios of ReLPS/egg-PC/POPG indicated that the incorporated ReLPS reduces the diffusion coefficients of the phospholipids in the membrane. The retardation of diffusion became more significant with increasing POPG concentrations in the membrane at high ReLPS/phospholipid ratios. This work demonstrated that the phospholipid composition has critical influence on the distribution of added ReLPS in the respective lipid membranes and also on the morphology and physicochemical property of the resulting membranes. A putative major factor causing these phenomena is reasoned to be the miscibility between ReLPS and individual phospholipid compositions.

Differential activation of human TLR4 by Escherichia coli and Shigella flexneri 2a lipopolysaccharide: combined effects of lipid A acylation state and TLR4 polymorphisms on signaling.

The lipid A of LPS activates TLR4 through an interaction with myeloid differentiation protein-2 (MD-2) and the degree of lipid A acylation affects TLR4 responsiveness. Two TLR4 single nucleotide polymorphisms (Asp299Gly and Thr399Ile) have been associated with LPS hyporesponsiveness. We hypothesized that the combination of hypoacylation and these single nucleotide polymorphisms would exhibit a compounded effect on TLR4 signaling. HEK293T transfectants expressing wild-type or polymorphic TLR4 were stimulated with Escherichia coli (predominantly hexaacylated lipid A) or Shigella flexneri 2a (a mixture of hexaacylated, pentaacylated, and predominantly tetraacylated lipid A) LPS, or hexaacylated vs pentaacylated synthetic lipid As. NF-kappaB-reporter activity was significantly lower in response to S. flexneri 2a than E. coli LPS and further decreased in polymorphic transfectants. Neither hexaacylated nor pentaacylated synthetic lipid A induced NF-kappaB activity in wild-type transfectants under the identical transfection conditions used for LPS; however, increasing human MD-2 expression rescued responsiveness to hexaacylated lipid A only, while murine MD-2 was required to elicit a response to pentaacylated lipid A. Adherent PBMC of healthy volunteers were also compared for LPS-induced TNF-alpha, IL-6, IL-1beta, and IL-10 production. Cytokine levels were significantly lower (approximately 20-90%) in response to S. flexneri than to E. coli LPS/lipid A and PBMC from polymorphic individuals secreted decreased cytokine levels in response to both LPS types and failed to respond to pentaacylated lipid A. Thus, the combination of acylation state and host genetics may significantly impact vaccine immunogenicity and/or efficacy, whether LPS is an integral component of a whole organism vaccine or included as an adjuvant.

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-alpha 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 gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP). Interaction of PGRPs with synthetic peptidoglycan fragments is also described.

Evidence of immunostimulating lipoprotein existing in the natural lipoteichoic acid fraction.

Lipoteichoic acid (LTA) is a cell surface glycoconjugate of gram-positive bacteria and is reported to activate the innate immune system. We previously reported that purified LTA obtained from Enterococcus hirae has no immunostimulating activity, but a subfraction (Eh-AF) in an LTA fraction possesses activity. In this study, we established a mouse monoclonal antibody neutralizing the activity of Eh-AF and investigated its inhibitory effects. Monoclonal antibody (MAbEh1) was established by the immunization of BALB/c mice with Eh-AF, followed by hybridoma screening based on its inhibitory effect for the production of interleukin-6 (IL-6) induced by Eh-AF. MAbEh1 neutralized the production of IL-6 by LTA fraction from not only E. hirae but also Staphylococcus aureus, while it failed to block that of lipopolysaccharide, suggesting that the antibody recognized a common active structure(s) in LTA fractions. Synthetic glycolipids in these LTAs did not induce cytokine production, at least in our system. Interestingly, the antibody was found to inhibit the activity of immunostimulating synthetic lipopeptides, Pam(3)CSK(4) and FSL-1. These results suggest that MAbEh1 neutralizes the activity of lipoprotein-like compounds which is responsible for the activity of the LTA fraction of E. hirae and S. aureus.

Virulence factors of Yersinia pestis are overcome by a strong lipopolysaccharide response.

At mammalian body temperature, the plague bacillus Yersinia pestis synthesizes lipopolysaccharide (LPS)-lipid A with poor Toll-like receptor 4 (TLR4)-stimulating activity. To address the effect of weak TLR4 stimulation on virulence, we modified Y. pestis to produce a potent TLR4-stimulating LPS. Modified Y. pestis was completely avirulent after subcutaneous infection even at high challenge doses. Resistance to disease required TLR4, the adaptor protein MyD88 and coreceptor MD-2 and was considerably enhanced by CD14 and the adaptor Mal. Both innate and adaptive responses were required for sterilizing immunity against the modified strain, and convalescent mice were protected from both subcutaneous and respiratory challenge with wild-type Y. pestis. Despite the presence of other established immune evasion mechanisms, the modified Y. pestis was unable to cause systemic disease, demonstrating that the ability to evade the LPS-induced inflammatory response is critical for Y. pestis virulence. Evading TLR4 activation by lipid A alteration may contribute to the virulence of various Gram-negative bacteria.

Immobilization and clustering of structurally defined oligosaccharides for sugar chips: an improved method for surface plasmon resonance analysis of protein-carbohydrate interactions.

Oligosaccharides are increasingly being recognized as important partners in receptor-ligand binding and cellular signaling. Surface plasmon resonance (SPR) is a very powerful tool for the real-time study of the specific interactions between biological molecules. We report here an advanced method for the immobilization of oligosaccharides in clustered structures for SPR and their application to the analysis of heparin-protein interactions. Reductive amination reactions and linker molecules were designed and optimized. Using mono-, tri-, or tetravalent linker compounds, we incorporated synthetic structurally defined disaccharide units of heparin and immobilized them as ligands for SPR. Their binding to an important hemostatic protein, von Willebrand factor (vWf), and its known heparin-binding domain was quantitatively analyzed. These multivalent ligand conjugates exhibited reproducible binding behavior, with consistency of the surface conditions of the SPR chip. This novel technique for oligosaccharide immobilization in SPR studies is accurate, specific, and easily applicable to both synthetic and naturally derived oligosaccharides.

Meso-diaminopimelic acid and meso-lanthionine, amino acids specific to bacterial peptidoglycans, activate human epithelial cells through NOD1.

Peptidoglycans (PGNs) are ubiquitous constituents of bacterial cell walls and exhibit various immunobiological activities. Two types of minimum essential PGN structures for immunobiological activities were chemically synthesized and designated as muramyldipeptide; N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) and gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP), which are common constituents of both Gram-positive and Gram-negative bacteria, as well as most Gram-negative and some Gram-positive bacteria, respectively. Recently, intracellular receptors for MDP and iE-DAP have been demonstrated to be nucleotide-binding oligomerization domain (NOD)1 and NOD2, respectively. In this study, we demonstrated that chemically synthesized meso-DAP itself activated human epithelial cells from various tissues, through NOD1 to generate antibacterial factors, PGN recognition proteins and beta-defensin 2, and cytokines in specified cases, although the activities of meso-DAP were generally weaker than those of known NOD agonists. However, stereoisomers of meso-DAP, LL-DAP, and DD-DAP were only slightly activated or remained inactive, respectively. Synthetic meso-lanthionine, which is another diamino-type amino acid specific to PGN of the specified Gram-negative bacteria, was also recognized by NOD1. In human monocytic cells, in the presence of cytochalasin D meso-DAP induced slightly but significantly increased production of cytokines, although the cells did not respond to meso-DAP in the absent of cytochalasin D. Our findings suggest that NOD1 is a special sentinel molecule, especially in the epithelial barrier, allowing the intracellular detection of bacteria through recognizing meso-DAP or comparable moiety of PGN from specified bacteria in cooperation with NOD2, thereby playing a key role in innate immunity.

Synthesis of lipid A monosaccharide analogues containing acidic amino acid: exploring the structural basis for the endotoxic and antagonistic activities.

For elucidation of the structural and conformational requirements on the endotoxic and antagonistic activity of lipid A derivatives, we designed and synthesized lipid A analogues containing acidic amino acid residues in place of the non-reducing end phosphorylated glucosamine. Definite switching of the endotoxic or antagonistic activity was observed depending on the difference of the acidic groups (phosphoric acid or carboxylic acid) in the lipid A analogues.

Synthesis of peptidoglycan fragments and evaluation of their biological activity.

The peptidoglycan (PG) bacterial cell wall glycoconjugate has been well known as a strong immunopotentiator. Partial structures of PG were chemically synthesized for elucidation of precise biological activities. Effective construction of distinct repeating glycans of PG was accomplished by the coupling of a key disaccharide glucosaminyl-beta(1-4)-muramic acid unit. Stereoselective glycosylation of disaccharide units was achieved by neighboring group participation of the N-Troc (Troc = 2,2,2-trichloroethoxycarbonyl) group and appropriate reactivity of N-Troc-glucosaminyl trichloroacetimidate. By using an efficient synthetic strategy, mono-, di-, tetra- and octasaccharide fragments of PG were synthesized in high yields. The biological activity of synthetic fragments of PG was evaluated by induction of tumor necrosis factor-alpha (TNF-alpha) from human monocytes, and toll-like receptor 2 (TLR2) and Nod2 dependencies by using transfected HEK293 cells, respectively. Here we reveal that TLR2 was not stimulated by the series of synthetic PG partial structures, whereas Nod2 recognizes the partial structures containing the MDP moiety.