A unique phosphatidylinositol bearing a novel branched-chain fatty acid from Rhodococcus equi binds to influenza virus hemagglutinin and inhibits the infection of cells.

From the aquatic bacterium Rhodococcus equi strain S(420), we isolated a substance that strongly binds to influenza viruses. Structural analyses revealed that it is a unique type of phosphatidylinositol (PtdIns) bearing a branched-chain fatty acid (14-methyloctadecanoic acid). In a TLC/virus-binding immunostaining assay, this PtdIns bound to all subtypes of hemagglutinin (HA) of influenza A viruses tested, isolated from humans, ducks and swine, and also to human influenza B viruses. Furthermore, the PtdIns significantly prevented the infection of MDCK cells by influenza viruses, and also inhibited the virus-mediated hemagglutination and low pH-induced hemolysis of human erythrocytes, which represents the fusogenic activities of the viral HA. We also used purified hemagglutinin instead of virions to examine the interaction between viral HA and PtdIns, showing that the PtdIns binds to hemagglutinin. These findings indicate that the inhibitory mechanism of PtdIns on the influenza virus infection may be through its binding to viral HA spikes and host cell endosomal/lysosomal membranes, which are mediated by the function of viral HA.

Duck and human pandemic influenza A viruses retain sialidase activity under low pH conditions.

The majority of influenza A viruses isolated from wild birds, but not humans, can replicate in the duck intestinal tract. Here we demonstrate that all duck isolates tested universally retain sialidase activities under low pH conditions independent of their neuraminidase (NA) subtypes. In contrast, the sialidase activities of most isolates from humans and pigs practically disappear below pH 4.5, with the exception of four human pandemic viruses isolated in 1957 and 1968. Sequence comparisons among duck, human, and swine N2 NA subtypes indicate that amino acids at positions 153, 253, 307, 329, 344, 347, 356, 368, 390, and 431 may be associated with the low pH stability of duck and human pandemic N2 NAs. This finding suggests that the low pH stability of duck influenza A virus NA may be a critical factor for replication in the intestinal tract through the digestive tract of ducks, and that the properties of NAs are important for understanding the epidemiology of the influenza virus.

Engagement of endogenous ganglioside GM1a induces tyrosine phosphorylation involved in neuron-like differentiation of PC12 cells.

Using the cholera toxin B subunit (CTB) that specifically binds to ganglioside GM1a on the plasma membrane, we investigated intracellular signaling mediated by endogenous GM1a involved in neuronal differentiation of PC12 cells. The treatment with CTB induced morphological alternations of PC12 cells, such as augmentation of the cell body, neurite extension, and branched spikes of tips of neurites. The neurite extension induced with CTB was strongly suppressed by the pretreatment of tyrosine kinase inhibitors in a dose-dependent manner. Western blotting analysis showed that CTB induced tyrosine phosphorylation of several cellular proteins with molecular masses around 120, 70, and 45-40 kDa in PC12 cells. Some of the proteins identified were extracellular-signal regulated kinase (ERKs) (ERK1 and ERK2). The peak activation of ERKs lasted for 60-90 min and gradually decreased thereafter. Immunoprecipitation analysis demonstrated that the intracellular events induced with CTB are not related with the activation of Trk proteins, suggesting that signals evoked by ligation of endogenous GM1a are unique and distinct from those induced with exogenous GM1a. Although the presence of a tyrosine kinase inhibitor, genistein, at a concentration of 10 microM diminished the neurite extension of PC12 cells induced with CTB, ERK activation was still observed. However, pretreatment with a MEK inhibitor, PD98059, abolished the activation of ERKs induced with CTB in a dose-dependent manner and only attenuated the morphological alternations of PC12 cells. Considered together, we concluded that tyrosine phosphorylation induced with CTB was responsible for neuron-like differentiation of PC12 cells and that the MEK-ERK cascade is part of the biological signals mediated by endogenous ganglioside GM1a on PC12 cells.

Inhibition of human parainfluenza virus type 1 sialidase by analogs of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid.

Eleven novel analogs of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en) modified at the C-4 and C-9 positions were designed and tested for their ability to inhibit sialidase of human parainfluenza virus type 1 (hPIV-1). The analogs modified by the cyanomethyl, amidinomethyl, and thiocarbamoylmethyl groups at the C-4 position exhibited potent inhibition against hPIV-1 sialidase compared with Neu5Ac2en. The most effective compound was thiocarbamoylmethyl analog (4-O-thiocarbamoylmethyl-Neu5Ac2en). The activity of 4-O-thiocarbamoylmethyl-Neu5Ac2en causing 50% enzyme inhibition at a concentration of approximately 1.0x10(-5) M was 30-fold larger than Neu5Ac2en. While, the analogs of Neu5Ac2en modified by the azido and N-acetyl groups at the C-9 showed a decrease in inhibition of sialidase compared with the 9-hydroxy analogs. In addition, 4-O-thiocarbamoylmethyl-Neu5Ac2en strongly inhibited hPIV-1 infections of Lewis lung carcinoma-monkey kidney cells in comparison with Neu5Ac2en. The present findings would provide useful information for the development of anti-human parainfluenza virus compounds.

Substitution of amino acid residue in influenza A virus hemagglutinin affects recognition of sialyl-oligosaccharides containing N-glycolylneuraminic acid.

Sialic acids are essential components of cell surface receptors used by influenza viruses. To determine the molecular mechanisms of viral recognition of two major species of sialic acids, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), we tested the binding reactivity of nine human H3 influenza A viruses to sialylglycolipids containing type II sugar chain and different molecular species of terminal sialic acids. All human H3 viruses tested except A/Memphis/1/71 bound both Neu5Ac and Neu5Gc. Nucleotide sequence analysis suggests that amino acids at 143, 155, and 158 are linked to the viral recognition of Neu5Gc.

Ganglioside GM(1a) on the cell surface is involved in the infection by human rotavirus KUN and MO strains.

Rotavirus is the most common cause of severe gastroenteritis in infants and children worldwide. The cell attachment of most animal rotaviruses, which belong to the neuraminidase-sensitive strains, requires sialic acid residues on the host cell membranes. On the other hand, most human rotaviruses are classified as neuraminidase-insensitive strains. The involvement of gangliosides on the host cell surface in human rotavirus infection was investigated by immunostaining analysis of target cells, and by assaying the neutralization of infection by rotavirus and the blocking of target cellular receptors. In host cells (MA104 cells) pretreated with Arthrobacter ureafaciens neuraminidase, which were still infected by human rotaviruses (KUN and MO strains), GM(3) was hydrolyzed markedly by the neuraminidase, while GM(1a) was not hydrolyzed at all. Infection by the rotaviruses was strongly inhibited by exogenous ganglioside GM(1a), but not GA(1). Infection was also inhibited by pretreatment of the MA104 cells with cholera toxin B-subunit, which specifically blocked ganglioside GM(1a) on the plasma membrane. The treatment of MA104 cells with the endoglycoceramidase attenuated human rotavirus infection. From these findings, we concluded that GM(1a) on the plasma membrane of the host cells was involved in the infection by human rotavirus KUN and MO strains.

Engagement of P-selectin glycoprotein ligand-1 enhances tyrosine phosphorylation and activates mitogen-activated protein kinases in human neutrophils.

During inflammation, P-selectin on activated platelets and endothelial cells initiates adhesion of leukocytes through interactions with P-selectin glycoprotein ligand-1 (PSGL-1). We investigated whether ligation of PSGL-1 also transmits signals into leukocytes. Neutrophils incubated with anti-PSGL-1 monoclonal antibodies, but not with Fab fragments of these antibodies, rapidly increased tyrosine phosphorylation of proteins with relative molecular masses of 105-120, 70-84, and 42-44 kDa. PSGL-1-dependent adhesion of neutrophils to P-selectin increased tyrosine phosphorylation of similarly sized proteins. Cytochalasin B did not prevent the tyrosine phosphorylation induced by ligation of PSGL-1, suggesting that an intact cytoskeleton is not required for signaling. Engagement of PSGL-1 activated the GTPase Ras through a mechanism that did not require tyrosine phosphorylation of PSGL-1 or association of the Shc.Grb2.Sos1 complex with PSGL-1. Engagement of PSGL-1 activated the 42-44-kDa extracellular signal-regulated kinase family of mitogen-activated protein (MAP) kinases through a pathway that required activation of the MAP kinase kinase. Ligation of PSGL-1 also stimulated secretion of interleukin-8. The tyrosine kinase inhibitor, genistein, blocked tyrosine phosphorylation and secretion of interleukin-8, whereas the MAP kinase kinase inhibitor PD98059 partially inhibited secretion of interleukin-8. Tyrosine phosphorylation stimulated through PSGL-1 on selectin-tethered leukocytes may propagate a signaling cascade that is integrated with signals generated by other mediators.

Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis.

We have isolated a cDNA encoding human ceramide glucosyltransferase (glucosylceramide synthase, UDP-glucose:N-acylsphingosine D-glucosyltransferase, EC 2.4.1.80) by expression cloning using as a recipient GM-95 cells lacking the enzyme. The enzyme catalyzes the first glycosylation step of glycosphingolipid synthesis and the product, glucosylceramide, serves as the core of more than 300 glycosphingolipids. The cDNA has a G+C-rich 5' untranslated region of 290 nucleotides and the open reading frame encodes 394 amino acids (44.9 kDa). A hydrophobic segment was found near the N terminus that is the potential signal-anchor sequence. In addition, considerable hydrophobicity was detected in the regions close to the C terminus, which may interact with the membrane. A catalytically active enzyme was produced from Escherichia coli transfected with the cDNA. Northern blot analysis revealed a single transcript of 3.5 kb, and the mRNA was widely expressed in organs. The amino acid sequence of ceramide glucosyltransferase shows no significant homology to ceramide galactosyltransferase, which indicates different evolutionary origins of these enzymes.

Synthesis and characterization of a carbene-generating biotinylated lactosylceramide analog as a novel chromogenic photoprobe for GM3 synthase.

A new biotinylated lactose derivative bearing a nitro-substituted chromogenic diazirine was synthesized. The biotinyl group within the structure enabled the performance of a convenient assay of GM3 synthase based on avidin-biotin technology, and the Km values of this biotinylated photoprobe were determined as 40 and 47 microM using bovine brain and rat liver Golgi as enzyme sources, respectively. Furthermore, the sialylation of lactosylceramide, a natural acceptor substrate for GM3 synthase, was competitively inhibited by this synthetic analog. The reagent could be a useful chromogenic photoprobe for GM3 synthase.

Occurrence of a novel fucose-containing pentaglycosylceramide with blood-group-B active determinant in Xenopus blastula cells: its possible involvement in cell-cell adhesion.

For understanding of the biological function of glycoconjugates during embryogenesis and morphogenesis, Xenopus laevis is considered a very useful animal model. We have found that blood-group-active molecules characteristically were distributed in the cell-cell contact region of Xenopus blastula cells. The chemical nature of blood-group-active glycoconjugates, including glycosphingolipids, is little known. T.l.c.-immunostaining using anti-blood-group-antigen antibodies showed that many species of blood group-B-active glycosphingolipids existed in the neutral glycosphingolipid fraction extracted from Xenopus laevis eggs. Among the B-active glycosphingolipids detected, two major components with the fastest mobility on a t.l.c. plate, tentatively termed XN-1 and XN-2, were isolated, and their chemical structures were characterized by gas chromatography-mass spectrometry, immunological anlaysis, fast-atom-bombardment mass spectrometry and 1H-n.m.r. spectroscopy. Both XN-1 and XN-2 had an identical pentaoligosaccharide structure, but differed in their ceramide moiety. The chemical structure is: [table: see text]. This is a novel type of pentaglycosylceramide with blood-group B activity, in that it lacks N-acetylhexosamine in its core carbohydrate structure. In this paper, a possible involvement of the blood-group antigen in the cell-adhesion process of Xenopus embryonic cells is discussed.

A unique biosynthetic pathway for gangliosides exists in Xenopus laevis oocytes.

It was previously reported that monosialosylgangliopentaosyl ceramide (GalNAc-GM1b) was a major ganglioside in Xenopus laevis oocytes. Here we determined biosynthetic pathways for the ganglioside by detailed measurements of glycosyltransferase activities. CMP-NeuAc:asialo-GM1 alpha 2-3 sialyltransferase (alpha 2-3 ST) and UDP-GalNAc:GM1b beta 1-4 N-acetylgalactosaminyltransferase (beta 1-4 GalNAcT) exhibited much higher activity than CMP-NeuAc:GalNAc-GA1 alpha 2-3 ST and UDP-GalNAc:asialo-GM1 beta 1-4 GalNAcT, respectively. These observations indicated the existence of a unique biosynthetic pathway in the oocytes as follows; asialo-GM1-->GM1b-->GalNAc-GM1b.

Biosynthetic pathway for a new series of gangliosides, GT1a alpha and GQ1b alpha.

A new class of gangliosides, GT1a alpha and GQ1b alpha, were initially identified as cholinergic neuron-specific antigens in bovine brain. These gangliosides have in common alpha 2-6 NeuAc linked to the GalNAc residue in the gangliotetraose core structure. In this study, we have determined the biosynthetic pathways of GT1a alpha and GQ1b alpha using rat liver Golgi fraction. The results showed that GT1a alpha and GQ1b alpha were synthesized from GD1a and GT1b, respectively, by the action of a GalNAc alpha 2-6 sialyltransferase. It was also demonstrated that these two gangliosides were found to exist as extremely minor components in rat liver.

In vitro synthesis of disialoganglioside (GD1 alpha) from asialo-GM1 using sialyltransferases in rat liver Golgi vesicles.

Two gangliosides were efficiently synthesized from asialo-GM1 (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1 Cer) and cytidine 5'-phosphate-N-acetylneuraminic acid (CMP-NeuAc) by using sialyltransferases in rat liver Golgi vesicles in vitro. These gangliosides were rapidly purified by a combination of anion exchange and reverse-phase column chromatographies. The ganglioside structures were determined by TLC analysis, treatment with a sialidase from Salmonella typhimurium LT2, which specifically hydrolyzes alpha 2-3 N-acetylneuraminic acid (NeuAc alpha 2-3) linkages, TLC immunostaining, and 1H-NMR spectroscopy. One of the gangliosides was identified as GD1 alpha [Neu-Ac alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAc beta 1-4Gal beta 1-4Glc beta 1-1 Cer]. The other ganglioside was determined to be GM1b (NeuAc alpha 2-3Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1 Cer), which has been reported in a previous study [Pohlentz, G., Klein, D., Schmitz, D., Schwarzmann, G., Peter-Katalinic, J. & Sandhoff, K. (1988) Biol. Chem. Hoppe-Seyler 369, 55-63]. Finally, GM1b and GD1 alpha were obtained from asialo-GM1 as a starting material in 8.1% and 1.2% overall yields, respectively. This study also suggests that the novel synthetic pathway asialo-GM1-->GM1b-->GD1 alpha may exist in rat liver.

A novel ganglioside with a free amino group in bovine brain.

A novel ganglioside which binds cholera-toxin B-subunit was purified from bovine brain by an h.p.l.c. system using an Aquasil column subsequent to Q-Sepharose column chromatography. T.l.c./immunostaining showed that the isolated ganglioside had about 60% of the binding reactivity of the authentic ganglioside GM1 for cholera-toxin B-subunit. On h.p.l.c., this ganglioside migrated between ganglioside GD1a and GD1b, and was found to give positive reactions with ninhydrin and fluorescamine reagents which specifically react with amino groups. The presence of a free amino group was further confirmed by chemical re-N-acetylation. The N-acetylated product had an identical RF value on h.p.l.c. and similar reactivity with cholera-toxin B-subunit as the authentic GM1. H.p.t.l.c., t.l.c./immunostaining, negative-ion fast-atom-bombardment (f.a.b.)-m.s., and 1H-n.m.r. spectroscopy of the novel ganglioside unequivocally demonstrated that it has the basal structure of GM1 with de-N-acetylated neuraminic acid instead of N-acetylneuraminic acid. In the present study we report for the first time that a ganglioside derivative containing de-N-acetylated neuraminic acid, de-N-acetylated GM1, exists in natural brain tissues.