Dengue virus type 2 recognizes the carbohydrate moiety of neutral glycosphingolipids in mammalian and mosquito cells

Dengue viruses infect cells by attaching to a surface receptor which remains unknown. The putative receptor molecules of dengue virus type 2 on the surface of mosquito (AP-61) and mammalian (LLC-MK2) cell lines were investigated. The immunochemical detection and structural analysis of carbohydrates demonstrated that the neutral glycosphingolipids, L-3 (GlcNAcβ1-3Manβ1-4Glcβ1-1'Cer) in AP-61 cells, and nLc(4) Cer (Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1'Cer) in LLC-MK2 cells were recognized by the virus. These findings strongly suggest that neutral glycosphingolipids share the key determinant for virus binding and that the β-GlcNAc residue may play an important role in dengue virus binding to the host cell surface.     

Structure and anti-dengue virus activity of sulfated polysaccharide from a marine alga

A sulfated polysaccharide, named fucoidan, from the marine alga Cladosiphon okamuranus is comprised of carbohydrate units containing glucuronic acid and sulfated fucose residues. Here we found this compound potently inhibits dengue virus type 2 (DEN2) infection. Viral infection was inhibited when DEN2, but not other serotypes, was pretreated with fucoidan. A carboxy-reduced fucoidan derivative in which glucuronic acid was converted to glucose did not inhibit viral infection. Elimination of the sulfated function group from fucoidan significantly attenuated the inhibitory activity on DEN2 infection with <1% fucoidan. DEN2 particles bound exclusively to fucoidan, indicating that fucoidan interacts directly with envelope glycoprotein (EGP) on DEN2. Structure-based analysis suggested that Arg323 of DEN2 EGP, which is conformationally proximal to one of the putative heparin binding residues, Lys310, is critical for the interaction with fucoidan. In conclusion, both the sulfated group and glucuronic acid of fucoidan account for the inhibition of DEN2 infection.     

Human gastric glycosphingolipids recognized by Helicobacter pylori vacuolating cytotoxin VacA

Many bacterial toxins utilize cell surface glycoconjugate receptors for attachment to target cells. In the present study the potential carbohydrate binding of Helicobacter pylori vacuolating cytotoxin VacA was investigated by binding to human gastric glycosphingolipids on thin-layer chromatograms. Thereby a distinct binding of the toxin to two compounds in the non-acid glycosphingolipid fraction was detected. The VacA-binding glycosphingolipids were isolated and characterized by mass spectrometry and proton NMR as galactosylceramide (Galbeta1Cer) and galabiosylceramide (Galalpha4Galbeta1Cer). Comparison of the binding preferences of the protein to reference glycosphingolipids from other sources showed an additional recognition of glucosylceramide (Glcbeta1Cer), lactosylceramide (Galbeta4Glcbeta1Cer) and globotriaosylceramide (Galalpha4Galbeta4Glcbeta1Cer). No binding to the glycosphingolipids recognized by the VacA holotoxin was obtained with a mutant toxin with deletion of the 37 kDa fragment of VacA (P58 molecule). Collectively our data show that the VacA cytotoxin is a glycosphingolipid binding protein, where the 37 kDa moiety is required for carbohydrate recognition. The ability to bind to short chain glycosphingolipids will position the toxin close to the cell membrane, which may facilitate toxin internalization.     

Studies on Galalpha3-binding proteins: comparison of the glycosphingolipid binding specificities of Marasmius oreades lectin and Euonymus europaeus lectin

The carbohydrate binding preferences of the Galalpha3Galbeta4 GlcNAc-binding lectins from Marasmius oreades and Euonymus europaeus were examined by binding to glycosphingolipids on thin-layer chromatograms and in microtiter wells. The M. oreades lectin bound to Galalpha3-terminated glycosphingolipids with a preference for type 2 chains. The B6 type 2 glycosphingolipid (Galalpha3[Fucalpha2]Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) was preferred over the B5 glycosphingolipid (Galalpha3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer), suggesting that the alpha2-linked Fuc is accommodated in the carbohydrate binding site, providing additional interactions. The lectin from E. europaeus had broader binding specificity. The B6 type 2 glycosphingolipid was the best ligand also for this lectin, but binding to the B6 type 1 glycosphingolipid (Galalpha3[Fucalpha2]Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer) was also obtained. Furthermore, the H5 type 2 glycosphingolipid (Fucalpha2Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer), devoid of a terminal alpha3-linked Gal, was preferred over the the B5 glycosphingolipid, demonstrating a significant contribution to the binding affinity by the alpha2-linked Fuc. The more tolerant nature of the lectin from E. europaeus was also demonstrated by the binding of this lectin, but not the M. oreades lectin, to the x2 glycosphingolipid (GalNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer. The A6 type 2 glycosphingolipid (GalNAcalpha3[Fucalpha2]Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and GalNAcalpha3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1-Cer were not recognized by the lectins despite the interaction with B6 type 2 glycosphingolipid and the B5 glycosphingolipid. These observations are explained by the absolute requirement of a free hydroxyl in the 2-position of Galalpha3 and that the E. europaea lectin can accommodate a GlcNAc acetamido moiety close to this position by reorienting the terminal sugar, whereas the M. oreades lectin cannot.     

The human H3N2 influenza viruses A/Victoria/3/75 and A/Hiroshima/52/2005 preferentially bind to α2-3-sialylated monosialogangliosides with fucosylated poly-N-acetyllactosaminyl chains

Among influenza A viruses, subtype H3N2 is the major cause of human influenza morbidity and is associated with seasonal epidemics causing annually half million deaths worldwide. Influenza A virus infection is initiated via hemagglutinin that binds to terminally sialylated glycoconjugates exposed on the surface of target cells. Gangliosides from human granulocytes were probed using thin-layer chromatography overlay assays for their binding potential to H3N2 virus strains A/Victoria/3/75 and A/Hiroshima/52/2005. Highly polar gangliosides with poly-N-acetyllactosaminyl chains showing low chromatographic mobility exhibited strong virus adhesion which was entirely abolished by sialidase treatment. Auxiliary overlay assays using anti-sialyl Lewis(x) (sLe(x)) monoclonal antibodies showed identical binding patterns compared with those performed with the viruses. A comprehensive structural analysis of fractionated gangliosides by electrospray ionization quadrupole time-of-flight mass spectrometry revealed sLe(x) gangliosides with terminal Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc epitope and extended neolacto (nLc)-series core structures as the preferential virus binding gangliosides. More precisely, sLe(x) gangliosides with nLc8, nLc10 and nLc12Cer cores, carrying sphingosine (d18:1) and a fatty acid with variable chain length (mostly C24:0, C24:1 or C16:0) in the ceramide moiety and one or two additional internal fucose residues in the oligosaccharide portion, were identified as the preferred receptors recognized by H3N2 virus strains A/Victoria/3/75 and A/Hiroshima/52/2005. This study describes glycan-binding requirements of hemagglutinin beyond binding to glycans with a specific sialic acid linkage of as yet undefined neutrophil receptors acting as ligands for H3N2 viruses. In addition, our results pose new questions on the biological and clinical relevance of this unexpected specificity of a subtype of influenza A viruses.     

Galactose-rich glycoproteins are on the cell surface of herpes virus-infected cells. 1. Surface labeling and serial lectin binding studies of Asn-linked oligosaccharides of glycoprotein gC

Cell-surface glycoproteins of mock-infected and herpes simplex virus type 1 (HSV-1)-infected BHK-21 and HEp-2 cells were radiolabeled by incubation with galactose oxidase followed by reduction with NaB3H4. The incorporation of radiolabel into glycoconjugates in both BHK-21 and HEp-2 cells was increased several fold following infection with HSV, showing an increase in surface-exposed Gal residues in the infected cells. This was further confirmed by an increase in binding of cell-surface-labeled glycoproteins gC and gB from HSV-infected BHK-21 cells to Ricinus communis agglutinin I, which is specific for beta-D-Gal residues. Prior treatment of cells with Clostridium perfringens neuraminidase enhanced the surface radiolabeling by the galactose oxidase/NaB3H4 method: HEp-2 cells exhibited over sixfold enhancement in labeling, while BHK-21 cells showed only a slight increase. HSV glycoprotein gC was the predominant cell-surface glycoprotein radiolabeled by the galactose oxidase/NaB3H4 method in virus-infected BHK-21 cells. The glycoprotein gC was purified by immunoaffinity column chromatography on monoclonal anti-gC-antibody-Sepharose. The radiolabel in the glycopeptides of gC was resistant to beta elimination, showing that it was associated only with Asn-linked oligosaccharides. A serial lectin affinity chromatography of glycopeptides on columns of concanavalin A-Sepharose, lentil (Lens culinaris) lectin-Sepharose, and Ricin I-agarose allowed the assignment of minimal oligosaccharide structures bearing terminal Gal residues in gC.     

Soluble fibronectin interaction with cell surface and extracellular matrix is mediated by carbohydrate-to-carbohydrate interaction

Cell adhesion and spreading on solid phase fibronectin (FN), coated on plate or presented in extracellular matrix, are mediated by integrin receptors alpha5beta1, alpha4beta1, etc., although binding of "soluble-form FN" to cell surface varies extensively depending on glycosylation status of FN per se. Deposition or incorporation at the cell surface or pericellular matrix of soluble-form FN from body fluids or synthesized de novo takes place through a yet-unknown (perhaps integrin-independent) mechanism. Here we present evidence that the mechanism involves carbohydrate-to-carbohydrate interaction. Binding or incorporation of soluble-form placental or hepatoma FN to cell surface or pericellular matrix is highly dependent on the specific glycosylation status of FN per se and combination with glycosylation status of the cell surface, and is greatly promoted by a certain type of coexisting (shedded) glycosphingolipid. A few lines of study indicate that the process is mediated by interaction of FN carbohydrate with cell surface carbohydrate. The great enhancement of the binding process by glycosphingolipid is based on dual interaction of glycosphingolipid carbohydrate with FN carbohydrate and with cell surface carbohydrate. Here we present an example of promotion of binding of soluble-form FN from placenta or from hepatoma cells, having a specific carbohydrate epitope termed "disialyl-I," to K562 or VA13 cell surface in the presence of glycosphingolipid Gg3, which interacts specifically with disialyl-I.     

A new monoclonal antibody directed to sialyl alpha 2-3lactoneotetraosylceramide and its application for detection of human gastrointestinal neoplasms

A new monoclonal antibody (NS24) directed to the N-acetylneuraminyl alpha 2-3Gal beta 1-4GlcNAc residue in type II sugar chain of N-acetylneuraminyllactoneotetraosylceramide [sialylparagloboside, IV3(NeuAc)nLc4Cer] was prepared by hybridoma technique. Liposomes composed of dipalmitoylphosphatidylcholine, cholesterol, IV3(NeuAc)nLc4Cer, and lipopolysaccharides from Salmonella minnesota R595 were used for immunization with IV3(NeuAc)nLc4Cer isolated from human erythrocytes. This method allowed the fusion of spleen cells of immunized mouse with myeloma cells only three days after immunization. NS24 reacted specifically to both naturally occurring and chemically synthesized IV3-(NeuAc)nLc4Cer, whereas it has no reactivity to structurally related gangliosides, such as IV6(NeuAc)nLc4Cer, N-glycolylneuraminyl alpha 2-3lactoneotetraosylceramide [IV3(NeuGc)-nLc4Cer], i-active ganglioside [VI3(NeuAc)nLc6Cer], I-active ganglioside [VIII3(NeuAc)-VI3(NeuAc)IV6kladoLc8Cer], GM4(NeuAc), GM3(NeuAc), GM3(NeuGc), GM1b(NeuAc), GD3-(NeuAc), other ganglio-series gangliosides, sulfatide, and paragloboside (nLc4Cer). Synthetic N-acetylneuraminyl alpha 2-3lactotetraosylceramide [IV3(NeuAc)Lc4Cer] and its asialo-derivative (Lc4Cer) carrying type I sugar chain also showed no reaction with NS24. One to 100 pmol of IV3(NeuAc)nLc4Cer was detected dose-dependently by a thin-layer chromatography/enzyme immunostaining procedure. Human gastric carcinomas showed positive reactions with NS24 immunochemically and histochemically. NS24 reacted preferentially with poorly differentiated adenocarcinomas rather than well differentiated ones.     

Immunological analysis of glycolipids on cell surfaces of cultured human tumor cell lines: expression of lactoneotetraosylceramide on tumor cell surfaces

Glycolipids of human cell lines of colonic adenocarcinoma (Colo 205 and BM 314), gastric tumor (AZ 521 and KATO-III), and lung tumor (A 549) were studied by the immunohistochemical fluorescence technique, flow cytometric analysis and immunostaining on thin layer chromatoplates with antibodies against gangliotriaosylceramide (Gg3Cer), gangliotetraosylceramide (Gg4Cer), fucogangliotetraosylceramide (Fuc-Gg4Cer), blood group B active lipid, globopentaosylceramide (Gb5Cer) and lactoneotetraosylceramide (nLc4Cer). Anti-nLc4Cer antibody was the only antibody which reacted with all the tumor cell lines used. The glycolipid fractions of each cell line separated by Iatrobeads column chromatography were immunostained with the six antibodies mentioned above on thin layer plates. The presence of nLc4Cer was detected in all cell lines. On the other hand, Gg4Cer was detected in gastric tumor cell lines, and Gg3Cer was detected in AZ 521. Based on these results, the tumor cell lines were analyzed by flow cytometry using anti-nLc4Cer antibody. About 70% of total cells in each cell line were separated as nLc4Cer-expressing cells. The present findings, together with the occurrence of nLc4Cer in ascitic fluids of cancer patients (Taki, T., Kojima, S., Seto, H., Yamada, H., & Matsumoto, M. (1984) J. Biochem. 96, 1257-1265), suggest that nLc4Cer may be a tumor-associated lipid.     

Neuroblastoma Cell Membranes: Specificity for Cell Fusion Mediated by a Temperature-Sensitive Mutant of Vesicular Stomatitis Virus

Abstract: Temperature-sensitive mutant G3 1 of vesicular stomatitis virus induces mouse neuroblastoma N-18 cells to fuse during infections that are nonpermissive for virus replication, but BHK-21 cells do not undergo the viral glycoprotein-mediated cell fusion. The viral glycoprotein was expressed at the cell surface of both N-18 and BHK-21 cells; therefore, the host cell specificity did not stem from an absence of the viral glycoprotein at the surface of BHK-21 cells. Cell fusion readily occurred between infected and uninfected N-18 cells in mixed cultures, demonstrating that the viral glycoprotein was interacting with an uninfected cell for the initial cell-cell interaction of the cell fusion. Mixing infected BHK-21 cells with uninfected N-18 cells resulted in cell fusion initiated by BHK-21 cell-synthesized viral glycoprotein, but 88% of the nucleiin polykaryocytes were N-18 nuclei. The N-18 cell fusion specificity was readily apparent when infected N-18 cells were mixed with uninfected BHK-21 cells; 98% of the nuclei in polykaryocytes were N-18 nuclei. Similar results also were obtained with mixed cultures of N-18 cells and primary astroglial cells. Thus, the viral glycoprotein synthesized in any of the cell types could initiate cell fusion, but the properties of plasma membranes of neuroblastoma cells appeared to be much more suitable for cell-cell fusion.     

Differential exposure of the major gangliosides in rabbit thymocytes to Vibrio cholerae neuraminidase

Neuraminidase treatment of lymphocytes is known to cause changes of cellular responses in several biological phenomena, but the molecules modified on the cell surface by neuraminidase are not known in detail. Rabbit thymocytes, which contain tissue-characteristic gangliosides, were treated with Vibrio cholerae neuraminidase, and the susceptibility of the cell surface sialic acid residues was examined. The amount of sialic acid released from the thymocytes at the highest level was 42.4 nmol per 1 X 10(9) cells, among which 26.5% was from gangliosides. Ninety-three percent of the VI3NeuGc-nLc6Cer, 84% of the IV3NeuGc-nLc4Cer, and 50% of the II3NA2-LacCer in the thymocytes was hydrolyzed to nLc6Cer, nLc4Cer, and LacCer, respectively, but II3NA-LacCer was completely cryptic. Also, among the molecular species of II3NA2-LacCer, C20:0- to C24:0-containing, but not C16:0- to C18:0-containing molecules, were susceptible to neuraminidase. After neuraminidase treatment, nLc4Cer and nLc6Cer became the major glycosphingolipids, and a 15-fold increase of radioactivity incorporated into the glycosphingolipids was observed by the galactose oxidase-sodium borotritide procedure, suggesting that the beta-galactose of the glycosphingolipids produced by neuraminidase treatment is accessibly to the several ligands which are functionally associated with lymphocytes.     

Carbohydrate recognition by enterohemorrhagic Escherichia coli: characterization of a novel glycosphingolipid from cat small intestine

A key virulence trait of pathogenic bacteria is the ability to bind to receptors on mucosal cells. Here the potential glycosphingolipid receptors of enterohemorrhagic Escherichia coli were examined by binding of 35S-labeled bacteria to glycosphingolipids on thin-layer chromatograms. Thereby a selective interaction with two nonacid glycosphingolipids of cat small intestinal epithelium was found. The binding-active glycosphingolipids were isolated and, on the basis of mass spectrometry, proton NMR spectroscopy, and degradation studies, identified as Galalpha3Galbeta4Glcbeta1Cer (isoglobotriaosylceramide) and Galalpha3Galalpha3Galbeta4Glcbeta1Cer. The latter glycosphingolipid has not been described before. The interaction was not based on terminal Galalpha3 because the bacteria did not recognize the structurally related glycosphingolipids Galalpha3Galalpha4Galbeta4Glcbeta1Cer and Galalpha3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer (B5 glycosphingolipid). However, further binding assays using reference glycosphingolipids showed that the enterohemorrhagic E. coli also bound to lactosylceramide with phytosphingosine and/or hydroxy fatty acids, suggesting that the minimal structural element recognized is a correctly presented lactosyl unit. Further binding of neolactotetraosylceramide, lactotetraosylceramide, the Le(a)-5 glycosphingolipid, as well as a weak binding to gangliotriaosylceramide and gangliotetraosylceramide, was found in analogy with binding patterns that previously have been described for other bacteria classified as lactosylceramide-binding.     

3-O-sulfated glucuronide derivative as a potential anti-dengue virus agent

A series of 12 carbohydrate compounds were synthesized by introduction of a sulfated group at specific positions and evaluated for their activities against dengue virus (DENV) infection as well as binding to BHK-21 cells. 3-O-sulfated GlcA was active against DENV infection, whereas 2-O-sulfated GlcA and 3,6-di-O-sulfated Glc showed negligible activity. Persulfated compounds did not inhibit DENV infection. These results provided a rationale for designing sulfated carbohydrate compounds with low molecular mass as anti-DENV agents targeting E protein functions. 3-O-Sulfated GlcA showed no significant cytotoxicity at 1mM. The EC(50) value (120μM) was lower than that of sucrose octasulfate (SOS), a small molecular weight inhibitor of DENV infection. Two negatively charged groups, 3-O-sulfate and 6-C-carboxylic acid, appear to be essential for anti-DENV activity. We performed docking study to investigate the binding potential of 3-O-sulfated GlcA with respect to DENV E protein. The docking study showed that distance and conformation of these negative charges on the carbohydrate may be suitable for association with three amino acid residues of E protein critically involved in virus adsorption (Lys295, Ser145, and Gly159). This interaction may competitively prevent functional DENV binding to receptor(s) on host cells. In conclusion, 3-O-sulfated GlcA is a chemical probe that may facilitate exploration of the molecular mechanisms underlying manifestations of dengue diseases.     

Characteristic binding of human plasma apolipoprotein B to gangliotetraosylceramide and gangliotriaosylceramide.

The binding of human plasma low-density lipoproteins (LDL), freshly prepared by discontinuous ultracentrifugation, to several neutral and acidic glycosphingolipids was examined by TLC immunostaining with the anti [apolipoprotein B (apoB)] antibody. ApoB was found to bind characteristically to the asialogangliosides, gangliotetraosylceramide (Gg4Cer) and gangliotriaosylceramide (Gg3Cer), the former being a more potent receptor than the latter, indicating that the sequences Gal beta 1-3GalNAc beta 1-4Gal and GalNAc beta 1-4Gal are involved in the binding of apoB. A weak positive reaction with fucosylgangliotetraosylceramide (IV2Fuc-Gg4Cer), which has the same internal recognition sequences, was also observed (the binding ability was only 1/7 of that in the case of Gg4Cer). No binding to other neutral glycosphingolipids, or glycosphingolipid sulfates (I3-SO3-GalCer) and gangliosides, was detected, and therefore substitution of the receptor glycolipid with sialic acid was thought to inhibit the binding. The results indicate that, along with the binding of apoB to the LDL-binding domain of the receptor glycoprotein, interaction with some carbohydrate chains in the receptor, or with glycolipids coexisting on the plasma membrane, may be important for the binding of apoB to cells.     

Secretory IgA specific for a conserved epitope on gp41 envelope glycoprotein inhibits epithelial transcytosis of HIV-1

As one of the initial mucosal transmission pathways of HIV (HIV-1), epithelial cells translocate HIV-1 from apical to basolateral surface by nondegradative transcytosis. Transcytosis is initiated when HIV-1 envelope glycoproteins bind to the epithelial cell membrane. Here we show that the transmembrane gp41 subunit of the viral envelope binds to the epithelial glycosphingolipid galactosyl ceramide (Gal Cer), an alternative receptor for HIV-1, at a site involving the conserved ELDKWA epitope. Disrupting the raft organization of the Gal Cer-containing microdomains at the apical surface inhibited HIV-1 transcytosis. Immunological studies confirmed the critical role of the conserved ELDKWA hexapeptide in HIV-1 transcytosis. Mucosal IgA, but not IgG, from seropositive subjects targeted the conserved peptide, neutralized gp41 binding to Gal Cer, and blocked HIV-1 transcytosis. These results underscore the important role of secretory IgA in designing strategies for mucosal protection against HIV-1 infection.     

Immobilization of fluorous oligosaccharide recognized by influenza virus on polytetrafluoroethylene filter

The lactoside with PEG-fluorous tag was introduced to BHK-21(C-13) cells to generate a GM3-type oligosaccharide (Siaα2-3Galβ1-4Glc). The GM3-type oligosaccharide obtained was easily immobilized by spotting onto commercially available polytetrafluoroethylene (PTFE) filter through non-covalent fluorous affinity and simply assessed by dot blot method using the interaction of carbohydrate- with proteins which recognize sialic acid such as virus membrane proteins.     

Variation in herpes simplex virus type 1 glycoproteins produced in kidney cells from different species

Viral glycoproteins from herpes simplex virus, type 1 (HSV-1) infected NBL-1, Vero, and BHK-21 cells were labelled with 14C-glucosamine and studied by SDS-PAGE and Con-A chromatography. SDS-PAGE analysis demonstrated differences in the number and molecular weight of glycoproteins from these cells. Con-A chromatography resulted in similar binding of glycoproteins from NBL-1 and Vero cells of 10.5 and 18.6%, respectively, whereas BHK-21 cells showed binding of 65%. These studies indicate that HSV-1 glycoprotein oligosaccharide processing is variable in kidney cells of different species.     

Galectin-4 binds to sulfated glycosphingolipids and carcinoembryonic antigen in patches on the cell surface of human colon adenocarcinoma cells

Galectin-4, a member of the galectin family, is expressed in the epithelium of the alimentary tract. It has two tandemly repeated carbohydrate recognition domains and specifically binds to an SO3- -->3Galbeta1-->3GalNAc pyranoside with high affinity (Ideo, H., Seko, A., Ohkura, T., Matta, K. L., and Yamashita, K. (2002) Glycobiology 12, 199-208). In this study, we found that galectin-4 binds to glycosphingolipids carrying 3-O-sulfated Gal residues, such as SB1a, SM3, SM4s, SB2, SM2a, and GM1, but not to glycosphingolipids with 3-O-sialylated Gal, such as sLc4Cer, snLc4Cer, GM3, GM2, and GM4, using both an enzyme-linked immunosorbent assay and a surface plasmon resonance assay. A confocal immunocytochemical assay showed that galectin-4 was colocalized with SB1a, GM1, and carcinoembryonic antigen (CEA) in the patches on the cell surface of human colon adenocarcinoma CCK-81 and LS174T cells. This localization was distinct from caveolin/VIP21 localization. Furthermore, immobilized galectin-4 promoted adhesion of CCK-81 cells through the sulfated glycosphingolipid, SB1a. CEA also bound to galectin-4 with KD value of 2 x 10(-8) m by surface plasmon resonance and coimmunoprecipitated with galectin-4 in LS174T cell lysates. These findings suggest that SB1a and CEA in the patches on the cell surface of human colon adenocarcinoma cells could be biologically important ligands for galectin-4.     

Interaction of Theiler’s Virus with Intermediate Filaments of Infected Cells

Theiler’s murine encephalomyelitis virus is a neurotropic murine picornavirus which replicates permissively and causes a cytopathic effect in the BHK-21 cell line. We examined the interactions between the GDVII and DA strains of Theiler’s virus and BHK-21 host cell proteins in a virus overlay assay. We observed binding of the virions to two proteins of approximately 60 kDa. These proteins were microsequenced and identified as desmin and vimentin, two main components of the intermediate filament network. The association between desmin or vimentin and virions was demonstrated by immunoprecipitation. Anti-desmin and anti-vimentin monoclonal antibodies precipitated GDVII or DA virions from extracts of infected BHK-21 cells. The intracellular distributions of virions and of the desmin and vimentin intermediate filaments of BHK-21 cells were investigated by two-color immunofluorescence confocal microscopy. Following infection, the intermediate filament network was rearranged into a shell-like structure which surrounded a viral inclusion. Finally, close contact between GDVII virus particles and 10-nm intermediate filaments was observed by electron microscopy.