Phagocytes, such as neutrophils, macrophages and dendritic cells, play crucial roles in the innate immune system. Innate immune responses are initiated by the binding of pathogens expressing highly conserved pathogen-associated molecular patterns (PAMPs) to a diverse array of pattern-recognition receptors (PRRs) expressed by phagocytes. On phagocytes, glycosphingolipids (GSLs) cluster together with cholesterol, glycophosphatidylinositol-anchored proteins, and various signaling molecules to form GSL-enriched lipid rafts. These GSL-enriched lipid rafts bind to several types of pathogens and their PAMPs, suggesting that these lipid rafts are essential for host-pathogen interactions in innate immunity. The neutral GSL lactosylceramide (LacCer, CDw17) binds to various microorganisms, including Candida and Mycobacteria. LacCer is expressed on plasma and granular membranes of mature human neutrophils and forms lipid rafts together with members of the Src family tyrosine kinases. These LacCer-enriched lipid rafts can mediate migration, phagocytosis of non-opsonized microorganisms and superoxide generation. On plasma membranes, LacCer-enriched lipid rafts serve as signal transduction platform for CD11b/CD18 integrin (Mac-1, CR3, or α_Mβ₂-integrin), indicating the importance of LacCer in a wide range of human innate immune responses. Especially, the interactions between LacCer-enriched lipid rafts and mycobacterial lipoarabinomannan (LAM) are significant for neutrophil phagocytosis of non-opsonized mycobacteria in human. We describe here the functional role of LacCer-enriched lipid rafts in human phagocytes.

Membrane proteins are present in the cell membranes of all organisms and are deeply involved in basic biological phenomena such as signal transduction and metabolite transport between the inside and the outside of the cell. Recently, we discovered a novel essential factor for membrane protein integration, a glycolipid named MPIase (Membrane Protein Integrase), in the inner membrane of Escherichia coli. Structural analysis revealed that MPIase has approximately ten repeating trisaccharide units, featureing 4-acetamido-4-deoxyfucose (Fuc4NAc), 2-acetamido-2-deoxymannuronic acid (ManNAcA), and 2-acetamido-2-deoxyglucose (GlcNAc), and diacylglycerol at the reducing end of the glycan through a pyrophosphate linkage. About 30% of the 6-hydroxy groups of GlcNAc are acetylated. In order to elucidate the mechanism of membrane protein integration, we chemically synthesized trisaccharyl pyrophospholipid (mini-MPIase-3), which is the minimum unit of MPIase, and its derivatives. Structure–activity relationship studies demonstrated that mini-MPIase-3 showed significant activity, indicating that it contains the minimum active structure. The phosphorylated glycan part of MPIase has chaperone-like activity to suppress the aggregation of proteins, and anchoring of MPIase by the lipid moiety in the membrane is essential for the integration activity. Since chemically synthesized mini-MPIase-3 is active, properly designed synthetic molecules will enable the determination of the detailed structure–activity relationship. Clarifying the molecular basis of preprotein translocation and membrane protein integration in E. coli would bring new insights not only for uncovering the biological functions of glycolipids, but also for developing antibacterial agents, protein aggregation inhibitors, and membrane protein reconstruction techniques.

Gram-negative bacteria are to a large extent covered by lipopolysaccharide (LPS) anchored in the outer leaflet of their outer membrane. There are presently four described pathways for the O-antigen assembly of LPS, viz., synthase-, Wzk-, ABC-transporter- and Wzx/Wzy-dependent pathways, where the latter two are used in Escherichia coli, subject to the O-antigen polysaccharide to be made. NDP-sugar monosaccharides are used by glycosyltransferases in the process of linking sugar residues together in the cytoplasm and depending on the biosynthetic pathway polymerization of the O-antigen takes place either in the cytoplasm (ABC-transporter pathway) or in the periplasm, where an oligosaccharide anchored in the inner membrane is flipped to the periplasmic side to this end (Wzx/Wzy-dependent pathway). Additions of sugars to form side-chains on the O-antigen may also occur in the periplasmic space. The degree of polymerization of the O-antigen is regulated to give a modal distribution, i.e., a narrow distribution around the most probable chain-length. The O-antigen is subsequently conjugated to the lipid A-core to form the LPS, which is transported across the periplasmic region by an ATP-driven mechanism as part of an LPS transport (Lpt) system. By using predictions of NDP-monosaccharide pathways and glycosyltransferase function it is shown how O-antigen structure can be elucidated rapidly by the computer program CASPER using bioinformatics data in conjunction with unassigned NMR data of the polysaccharide.

Phagocytes, such as neutrophils, macrophages and dendritic cells, play crucial roles in the innate immune system. Innate immune responses are initiated by the binding of pathogens expressing highly conserved pathogen-associated molecular patterns (PAMPs) to a diverse array of pattern-recognition receptors (PRRs) expressed by phagocytes. On phagocytes, glycosphingolipids (GSLs) cluster together with cholesterol, glycophosphatidylinositol-anchored proteins, and various signaling molecules to form GSL-enriched lipid rafts. These GSL-enriched lipid rafts bind to several types of pathogens and their PAMPs, suggesting that these lipid rafts are essential for host-pathogen interactions in innate immunity. The neutral GSL lactosylceramide (LacCer, CDw17) binds to various microorganisms, including Candida and Mycobacteria. LacCer is expressed on plasma and granular membranes of mature human neutrophils and forms lipid rafts together with members of the Src family tyrosine kinases. These LacCer-enriched lipid rafts can mediate migration, phagocytosis of non-opsonized microorganisms and superoxide generation. On plasma membranes, LacCer-enriched lipid rafts serve as signal transduction platform for CD11b/CD18 integrin (Mac-1, CR3, or α_Mβ₂-integrin), indicating the importance of LacCer in a wide range of human innate immune responses. Especially, the interactions between LacCer-enriched lipid rafts and mycobacterial lipoarabinomannan (LAM) are significant for neutrophil phagocytosis of non-opsonized mycobacteria in human. We describe here the functional role of LacCer-enriched lipid rafts in human phagocytes.

Membrane proteins are present in the cell membranes of all organisms and are deeply involved in basic biological phenomena such as signal transduction and metabolite transport between the inside and the outside of the cell. Recently, we discovered a novel essential factor for membrane protein integration, a glycolipid named MPIase (Membrane Protein Integrase), in the inner membrane of Escherichia coli. Structural analysis revealed that MPIase has approximately ten repeating trisaccharide units, featureing 4-acetamido-4-deoxyfucose (Fuc4NAc), 2-acetamido-2-deoxymannuronic acid (ManNAcA), and 2-acetamido-2-deoxyglucose (GlcNAc), and diacylglycerol at the reducing end of the glycan through a pyrophosphate linkage. About 30% of the 6-hydroxy groups of GlcNAc are acetylated. In order to elucidate the mechanism of membrane protein integration, we chemically synthesized trisaccharyl pyrophospholipid (mini-MPIase-3), which is the minimum unit of MPIase, and its derivatives. Structure–activity relationship studies demonstrated that mini-MPIase-3 showed significant activity, indicating that it contains the minimum active structure. The phosphorylated glycan part of MPIase has chaperone-like activity to suppress the aggregation of proteins, and anchoring of MPIase by the lipid moiety in the membrane is essential for the integration activity. Since chemically synthesized mini-MPIase-3 is active, properly designed synthetic molecules will enable the determination of the detailed structure–activity relationship. Clarifying the molecular basis of preprotein translocation and membrane protein integration in E. coli would bring new insights not only for uncovering the biological functions of glycolipids, but also for developing antibacterial agents, protein aggregation inhibitors, and membrane protein reconstruction techniques.

Gram-negative bacteria are to a large extent covered by lipopolysaccharide (LPS) anchored in the outer leaflet of their outer membrane. There are presently four described pathways for the O-antigen assembly of LPS, viz., synthase-, Wzk-, ABC-transporter- and Wzx/Wzy-dependent pathways, where the latter two are used in Escherichia coli, subject to the O-antigen polysaccharide to be made. NDP-sugar monosaccharides are used by glycosyltransferases in the process of linking sugar residues together in the cytoplasm and depending on the biosynthetic pathway polymerization of the O-antigen takes place either in the cytoplasm (ABC-transporter pathway) or in the periplasm, where an oligosaccharide anchored in the inner membrane is flipped to the periplasmic side to this end (Wzx/Wzy-dependent pathway). Additions of sugars to form side-chains on the O-antigen may also occur in the periplasmic space. The degree of polymerization of the O-antigen is regulated to give a modal distribution, i.e., a narrow distribution around the most probable chain-length. The O-antigen is subsequently conjugated to the lipid A-core to form the LPS, which is transported across the periplasmic region by an ATP-driven mechanism as part of an LPS transport (Lpt) system. By using predictions of NDP-monosaccharide pathways and glycosyltransferase function it is shown how O-antigen structure can be elucidated rapidly by the computer program CASPER using bioinformatics data in conjunction with unassigned NMR data of the polysaccharide.