Yasuko Nagatsuka

Association of FcγRII with Low-Density Detergent-Resistant Membranes Is Important for Cross-Linking-Dependent Initiation of the Tyrosine Phosphorylation Pathway and Superoxide Generation

IgG immune complexes trigger humoral immune responses by cross-linking of FcRs for IgG (FcγRs). In the present study, we investigated role of lipid rafts, glycolipid- and cholesterol-rich membrane microdomains, in the FcγR-mediated responses. In retinoic acid-differentiated HL-60 cells, cross-linking of FcγRs resulted in a marked increase in the tyrosine phosphorylation of FcγRIIa, p58lyn, and p120c-cbl, which was inhibited by a specific inhibitor of Src family protein tyrosine kinases. After cross-linking, FcγRs and tyrosine-phosphorylated proteins including p120c-cbl were found in the low-density detergent-resistant membrane (DRM) fractions isolated by sucrose-density gradient ultracentrifugation. The association of FcγRs as well as p120c-cbl with DRMs did not depend on the tyrosine phosphorylation. When endogenous cholesterol was reduced with methyl-β-cyclodextrin, the cross-linking did not induce the association of FcγRs as well as p120c-cbl with DRMs. In addition, although the physical association between FcγRIIa and p58lyn was not impaired, the cross-linking did not induce the tyrosine phosphorylation. In human neutrophils, superoxide generation induced by opsonized zymosan or chemoattractant fMLP was not affected or increased, respectively, after the methyl-β-cyclodextrin treatment, but the superoxide generation induced by the insoluble immune complex via FcγRII was markedly reduced. Accordingly, we conclude that the cross-linking-dependent association of FcγRII to lipid rafts is important for the activation of FcγRII-associated Src family protein tyrosine kinases to initiate the tyrosine phosphorylation cascade leading to superoxide generation.

Carbohydrate-dependent signaling from the phosphatidylglucoside-based microdomain induces granulocytic differentiation of HL60 cells

Glycosphingolipids form glycosphingolipid signaling microdomains. Here, we report an unrecognized type of phosphatidylglucoside (PhGlc)-based lipid microdomain in HL60 cells. Treatment of cells with rGL-7, which preferentially reacts with PhGlc, induced differentiation of HL60 cells. This was manifested by the appearance of nitroblue tetrazolium-positive cells together with CD38 expression and c-Myc down-regulation. We determined the molecular mechanisms underlying early stages of signal transduction. rGL-7 treatment induced rapid tyrosine phosphorylation of Src family protein kinases Lyn and Hck. Reduction of endogenous cholesterol after application of methyl-β-cyclodextrin suppressed rGL-7-stimulated tyrosine phosphorylation. Phosphorylated proteins and PhGlc colocalized in the Triton X-100 insoluble, light buoyant density fraction after sucrose gradient ultracentrifugation of HL60 cell lysates. This suggests PhGlc-based microdomain is involved in GL-7 signaling. Ligation of known components of microdomains, such as sphingomyelin and ganglioside GM1, with corresponding antibodies failed to induce differentiation and tyrosine phosphorylation. These results show that PhGlc constitutes a previously undescribed lipid signaling domain, and the glucose residue of PhGlc is critical for organization of the carbohydrate-dependent signaling domain involved in cellular differentiation of HL60 cells.

A 35-kDa co-aggregation factor is a hemin binding protein in Porphyromonas gingivalis.

It has been known that Porphyromonas gingivalis has an obligate requirement for hemin or selected heme- or Fe-containing compounds for its growth. In addition, the influence of hemin on the expression of several putative virulence factors produced by this bacterium has also been recently documented; however, the mechanisms involved in hemin uptake are poorly defined. We succeeded in cloning the gene coding for the 35-kDa protein, which was specifically expressed in P. gingivalis and seemed to confer colonizing activities. Recently, we have constructed the P. gingivalis 381 mutant defective in the 35-kDa protein by insertion mutagenesis. The beige mutant exhibited little co-aggregation and the virulence was also decreased. Based on these results and homology search analysis, we focused on assessing the hemin bindings and found the heme regulatory motif (HRM) as a hemin direct binding site. The 35-kDa protein did possess the binding ability of selected protoporphyrins involving the hemin. These results demonstrated that 35-kDa protein is one of the hemin binding proteins in P. gingivalis and suggested that hemin binding ability of 35-kDa protein is important for the expression of virulence in P. gingivalis.

A new phosphoglycerolipid, ‘phosphatidylglucose’, found in human cord red cells by multi-reactive monoclonal anti-i cold agglutinin, mAb GL-1/GL-2

Cord red cell membranes express many differentiation‐related molecules. To study such molecules, we have established human cell lines, termed GL‐1 and GL‐2, by the Epstein–Barr virus transformation method, both of which produce monoclonal anti‐i cold agglutinin [Y. Nagatsuka et al., Immunol. Lett. 46 (1995) 93–100]. Thin layer chromatography immunoblotting analysis revealed that these antibodies had broad specificities reacting with a variety of glycolipid antigens. Of the immunoreactive lipid antigens, a new phosphoglycerolipid containing glucose from human cord red cells was found. The isolated lipid was unstable to alkaline hydrolysis and contained glucose as a sole sugar. Secondary ion mass spectrum–collision‐induced dissociation mass spectrometric analysis of this lipid gave the main molecular ion peak at m/z 885 corresponding to phosphatidylhexose. This antigen was susceptible to phospholipases A2, C and D but resistant to phosphatidylinositol‐specific phospholipase C. Two‐dimensional nuclear magnetic resonance spectroscopy confirmed that glucose is linked to the sn‐glycerol 3‐phosphate residue with a β‐anomeric configuration. Based upon these combined results, we identified this lipid as phosphatidyl‐β‐D‐glucose. This is the first report showing the presence of the glucosylated glycerophospholipid in mammalian sources.

Glycerophospholipid regulation of modality-specific sensory axon guidance in the spinal cord

Axon paths in developing spinal cords Sensory neurons entering the spinal cord take different paths as inputs for pain and proprioception diverge. Working with chick and mouse embryos, Guy et al. found that glycerophospholipids produced by radial glial cells guide these neural fibers, or axons, in the developing spinal cord. A soluble glycerophospholipid released by the cells provided an inhibitory signal to the pain-sensitive axons, keeping them on their own unique pathway. Science, this issue p. 974 Axons follow glycerophospholipids to find their way in the developing spinal cord. Glycerophospholipids, the structural components of cell membranes, have not been considered to be spatial cues for intercellular signaling because of their ubiquitous distribution. We identified lyso-phosphatidyl-β-d-glucoside (LysoPtdGlc), a hydrophilic glycerophospholipid, and demonstrated its role in modality-specific repulsive guidance of spinal cord sensory axons. LysoPtdGlc is locally synthesized and released by radial glia in a patterned spatial distribution to regulate the targeting of nociceptive but not proprioceptive central axon projections. Library screening identified the G protein–coupled receptor GPR55 as a high-affinity receptor for LysoPtdGlc, and GPR55 deletion or LysoPtdGlc loss of function in vivo caused the misallocation of nociceptive axons into proprioceptive zones. These findings show that LysoPtdGlc/GPR55 is a lipid-based signaling system in glia-neuron communication for neural development.

Lipid rafts enriched in phosphatidylglucoside direct astroglial differentiation by regulating tyrosine kinase activity of epidermal growth factor receptors

Membrane lipid rafts provide a specialized microenvironment enriched with sphingolipids and phospholipids containing saturated fatty acids and serve as a platform for various intracellular signalling pathways. PtdGlc (phosphatidylglucoside) is a type of glycophospholipid localized in the outer leaflet of the plasma membrane. Owing to PtdGlcs unique fatty acid composition, exclusively composed of C(18:0) at sn-1 and C(20:0) at sn-2 of the glycerol backbone, it tends to form PGLRs (PtdGlc-enriched lipid rafts). Previously, we demonstrated that PGLRs reside on the cell surface of astroglial cells from fetal rat brain [Nagatsuka, Horibata, Yamazaki, Kinoshita, Shinoda, Hashikawa, Koshino, Nakamura and Hirabayashi (2006) Biochemistry 45, 8742-8750]. In the present study, we observed PGLRs in astroglial lineage cells at mid-embryonic to early-postnatal stages of developing mouse cortex. This suggests that PGLRs are developmentally correlated with astroglial differentiation during fetal cortical development. Our cell culture studies with multipotent neural progenitor cells prepared from fetal mouse telencephalon demonstrated that treatment with EGF (epidermal growth factor) or anti-PtdGlc antibody caused recruitment of EGFRs (EGF receptors) into lipid raft compartments, leading to activation of EGFRs. Moreover, the activation of EGFRs by antibody triggered downstream tyrosine kinase signalling and induced marked GFAP (glial fibrillary acidic protein) expression via the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) signalling pathway. These findings strongly suggest that PGLRs are physiologically coupled to activated EGFRs on neural progenitor cells during fetal cortical development, and thereby play a distinct role in mediating astrogliogenesis.

Phosphatidylglucoside forms specific lipid domains on the outer leaflet of the plasma membrane.

Phosphatidylglucoside (PtdGlc) is a recently discovered unique glycophospholipid involved in granulocytic differentiation of human promyelocytic leukemia cell line HL60 and in astrocytic differentiation in developing rodent brains. Using a PtdGlc-specific monoclonal antibody in immunofluorescence and immunoelectron microscopy, we showed that PtdGlc forms distinct lipid domains on the outer leaflet of the plasma membrane of HL60 cells and the human alveolar epithelial cell line, A549. Similar to glycosphingolipid, glucosylceramide (GlcCer), the natural form of PtdGlc exhibited a high main phase transition temperature in differential scanning calorimetry (DSC). However, unlike GlcCer, PtdGlc did not exhibit a large difference in the main phase transition temperature between the heating and cooling scans. DSC further indicated that GlcCer, but not PtdGlc, was miscible with sphingomyelin. In addition, DSC and small-angle X-ray scattering (SAXS) experiments revealed that PtdGlc was poorly miscible with phosphatidylcholine. Our results suggest that the lack of tight intermolecular interaction excludes PtdGlc from other lipid domains on the plasma membrane.

Bacterial expression of a human recombinant monoclonal antibody Fab fragment against hepatitis B surface antigen

The Fab fragment was cloned from the monoclonal cell line TAPC301‐CL4, which was produced using the Epstein‐Barr virus (EBV) transformation method. This cell line produces a human monoclonal antibody (CL4MAb) against the hepatitis B surface antigen (HBsAg). This MAb was shown to have hepatitis B virus (HBV) neutralizing activity in chimpanzees. The Fab fragment was produced by subjecting the heavy and light chain antibody genes of the TAPC301‐CL4 cell line to reverse transcription‐polymerase chain reaction, cloning the products in the plasmid vector pFab1‐His2 and introducing the plasmid into bacteria. Sequence analyses of the CL4Fab fragment revealed that the light and heavy chains belong to the Vk3a and VH3 groups of the immunoglobulin (Ig) family, respectively. An enzyme‐linked immunosorbent assay confirmed that specificity of the recombinant CL4Fab antibody against HBsAg was the same as that of the parental MAb. Flow cytometric analysis using PLC/PRF/5 (Alexander) cells, which express HBsAg, showed the reactivities of the CL4MAb and CL4Fab antibody were the same. These results suggest that the recombinant CL4Fab antibody produced by Escherichia coli using the new vector‐primer system developed for human IgG Fab fragments has a very high affinity for the HBsAg and may be useful clinically. A source for generation of human MAb for human therapy with very stable and specific expression was thus produced by isolating antibodies from EBV‐transformed cell lines. J. Med. Virol. 58:338–345, 1999.

The Novel Neutrophil Differentiation Marker Phosphatidylglucoside Mediates Neutrophil Apoptosis

A new type of glycolipid, phosphatidylglucoside (PtdGlc), was identified as a component of raft-like membrane domains of the human leukemia cell line HL-60. In this study, we show that PtdGlc forms functional domains that are different from those produced by lactosylceramide (LacCer)-enriched lipid rafts. These rafts initiate neutrophil apoptosis. Neutrophils are the only type of human peripheral blood leukocyte or monocyte-derived dendritic cell to express large amounts of PtdGlc on their cell surfaces. PtdGlc was not colocalized with LacCer. Anti-PtdGlc IgM DIM21 did not induce neutrophil chemotaxis or superoxide generation, whereas anti-LacCer IgM T5A7 induced these activities. DIM21, but not T5A7, significantly induced neutrophil apoptosis. DIM21-induced apoptosis was inhibited by specific inhibitors of cysteine-containing aspartate-specific proteases (caspases)-8, -9, and -3 but not by the Src family kinase inhibitor PP1, PIP3 kinase inhibitor LY294002, NADPH oxidase inhibitor diphenyleneiodonium, superoxide dismutase, or catalase. PtdGlc was colocalized with Fas on the neutrophil plasma membrane. DIM21 and the agonist anti-Fas Ab DX2 induced the formation of large Fas-colocalized clusters of PtdGlc on the plasma membrane. Furthermore, the antagonistic anti-Fas Ab ZB4 significantly inhibited DIM21-induced neutrophil apoptosis. These results suggest that PtdGlc is specifically expressed on neutrophils and mediates apoptosis of these cells, and that the Fas-associated death signal may be involved in PtdGlc-mediated apoptosis.

Fucoganglioside α-fucosyl(α-galactosyl)-GM1: a novel member of lipid membrane microdomain components involved in PC12 cell neuritogenesis

In order to search for novel components of lipid membrane microdomains involved in neural signalling pathways, mAbs (monoclonal antibodies) were raised against the detergent-insoluble membrane fraction of PC12 (pheochromocytoma) cells. Among the 22 hybrid clones, mAb PR#1 specifically detected a fucoganglioside Fuc(Gal)-GM1 [a-fucosyl(a-galactosyl)-GM1], a ganglioside homologous with GM1a (II3NeuAc,GgOse4Cer), as a novel member of microdomain components with biological functions. In the presence of mAb PR#1 in the culture medium, the outgrowth of neurites was induced in PC12 cells in a dose-dependent manner, with no effects on cell proliferation, suggesting that Fuc(Gal)-GM1 is preferentially involved in PC12 cell neuritogenesis. Effects through Fuc(Gal)-GM1 were different from those through GM1a during differentiation, e.g. under PR#1 treatment on Fuc(Gal)-GM1, round cell bodies with thinner cell processes were induced, whereas treatment with CTB (cholera toxin B subunit), a specific probe for GM1a, produced flattened cell bodies with thicker pro-cesses. Molecular analysis demonstrated that the PR#1-Fuc(Gal)-GM1 pathway was associated with Fyn and Yes of the Src family of kinases, although Src itself was not involved. No association was found with TrkA (tropomyosin receptor kinase A) and ERKs (extracellular-signal-regulated kinases), which are responsible for GM1a-induced differentiation. From these findings, it is suggested that a fucoganglioside Fuc(Gal)-GM1 provides a functional platform distinct from that of GM1a for signal transduction in PC12 cell differentiation.