A strain of human influenza A virus binds to extended but not short gangliosides as assayed by thin-layer chromatography overlay

Halina Miller-Podraza1, Lena Johansson, Petra Johansson, Thomas Larsson, Mikhail Matrosovich2,3 and Karl-Anders Karlsson

Institute of Medical Biochemistry, Göteborg University, P.O. Box 440, SE 405 30 Göteborg, Sweden and 3M. P. Chumakov Institute of Poliomyelitis and Viral Encephalitides, Russian Academy of Medical Sciences, 142 782 Moscow, Russia

Received on January 18, 2000; revised on April 4, 2000; accepted on April 12, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
A human strain of influenza virus (A, H1N1) was shown to bind in an unexpected way to leukocyte and other gangliosides when compared with avian virus (A, H4N6) as assayed on TLC plates. The human strain bound only to species with about 10 or more sugars, while the avian strain bound to a wide range of gangliosides including the 5-sugar gangliosides. By use of specific lectins, antibodies, and FAB and MALDI-TOF mass spectrometry an attempt was done to preliminary identify the sequences of leukocyte gangliosides recognized by the human strain. The virus binding pattern did not follow binding by VIM-2 monoclonal antibody and was not identical with binding by anti-sialyl Lewis x antibody. There was no binding by the virus of linear NeuAc{alpha}3- or NeuAc{alpha}6-containing gangliosides with up to seven monosaccharides per mol of ceramide. Active species were minor NeuAc{alpha}6-containing molecules with probably repeated HexHexNAc units and fucose branches. This investigation demonstrates marked distinctions in the recognition of gangliosides between avian and human influenza viruses. Our data emphasize the importance of structural factors associated with more distant parts of the binding epitope and the complexity of carbohydrate recognition by human influenza viruses.

Key words: influenza virus/ganglioside/sialic acid/human leukocyte/receptor


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Influenza virus attachment to host cells is mediated by specific interactions of the viral envelope protein hemagglutinin (HA) with sialylated carbohydrate chains of cell surface glycoproteins and glycolipids (for reviews, see Paulson, 1985; Wiley and Skehel, 1987Go; Suzuki, 1994Go; Herrler et al., 1995Go). Natural sialylglycoproteins and gangliosides exhibit significant structural diversity, and different receptors are probably utilized by the viruses in different host tissues. It has been shown that influenza A viruses isolated from avian species preferentially bind to NeuAc{alpha}3Gal-terminated sugar chains, while closely related human viruses reveal a higher binding affinity towards the NeuAc{alpha}6Gal-terminated structures (Paulson, 1985; Connor et al., 1994Go; Suzuki, 1994Go; Gambaryan et al., 1997Go; Matrosovich et al., 1997Go). Additional influential features for binding are inner parts of saccharide chains (Rogers and Paulson, 1983Go; Suzuki et al., 1987Go; Suzuki et al., 1992Go; Gambaryan et al., 1995Go; Eisen et al., 1997Go; Matrosovich et al., 1997Go), polyvalency of receptor saccharides (Matrosovich, 1989Go; Pritchett and Paulson, 1989Go; Mammen et al., 1995Go), spatial arrangement of sialyloligosaccharides in receptor glycoproteins (Pritchett and Paulson, 1989Go), or glycosylation of the viral hemagglutinins (Ohuchi et al., 1997Go; Gambaryan et al., 1998Go). Detailed molecular mechanisms of these effects and the importance of a variation in fine structure of sialylated receptors for the virulence and pathogenicity of individual viral strains are not known. For example, the virulence of the 1918 influenza pandemic still remains unexplained (Laver et al., 1999Go), and the actual strains have not been assayed for receptor specificity.

Studies on the structural characterization of biological receptors for human influenza viruses are hampered by the limited availability of the human respiratory tract tissues. However, characterization of the binding molecules from other human tissues may permit further specification of the receptor binding epitopes. Human leukocytes represent an attractive experimental model because they contain a series of gangliosides with high binding affinity for the virus. Binding species were detected in human leukocytes among common gangliosides (Müthing et al., 1993Go; Müthing, 1996Go) and among highly complex glycolipid fractions, polyglycosylceramides (Matrosovich et al., 1996Go). Influenza viruses are known to cause neutrophil dysfunction (Abramson and Mills, 1988Go; Cassidy et al., 1989Go; Daigneault et al., 1992Go; Abramson and Hudnor, 1995Go), and therefore their interaction with these as yet undefined neutrophil receptors may be biologically and clinically relevant.

In this study, we analyzed the binding of the avian and human viruses to gangliosides from different human tissues and cells, including leukocytes. Unlike the avian virus, which bound to a variety of common gangliosides, the human virus bound to only minor extended ganglioside species. The structure of these species from leukocyte gangliosides was analyzed using various overlay techniques and mass spectrometry.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Figure 1 shows binding of human and avian influenza viruses to reference gangliosides (lane 9) and to mixtures of gangliosides isolated from different human tissues (lanes 1–8). The human influenza virus did not bind in the assay conditions to shorter gangliosides including abundant 5s and 7s species, but displayed a strong and selective binding to some extended glycolipids of human leukocytes (lane 1 in Figure 1). There was also a weak binding to slow-moving species of other human tissues, in particular small intestine and pancreas (lanes 4 and 8). The avian virus bound to a variety of gangliosides in all lanes including two fractions of reference brain gangliosides, and displayed a preference for NeuAc{alpha}3Gal-terminated species as compared with NeuAc{alpha}6Gal-terminated species and species with sialic acid as internal branches. As shown in the figure, there was a binding to NeuAc{alpha}3-paragloboside (S-3-PG) and gangliosides GD1a and GT1b (see Table I for structures), but not to NeuAc{alpha}6-paragloboside (S-6-PG) or gangliosides GM1 or GD1b. These results agree with earlier reports on avian influenza virus binding specificities (see references in Introduction). The binding to GM3 was however not observed, although some lanes were overloaded with respect to less complex components. Binding to the fastest-moving band by both viruses in lane 6 of Figure 1 was probably unspecific interaction with overloaded and charged sulfatide. To exclude binding to sulfatides in other lanes we used mild periodate oxidation and reduction which shortens specifically the sialic acid glycerol tail in gangliosides by one or two carbon atoms (Veh et al., 1977Go). Binding of influenza viruses to sulfatide was previously reported by Suzuki et al., 1996Go. The mild oxidation eliminated in our studies completely (human influenza) or almost completely (avian virus) binding to leukocyte glycolipids, as shown in Figure 2. The result confirms the specific importance of the sialic acid for the virus attachment, and agrees with previous studies on binding of influenza viruses to chemically modified carbohydrates (Suttajit and Winzler, 1971Go; Matrosovich et al., 1991Go).



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Fig. 1. Binding of HRP conjugates of human (H1N1) and avian (H4N6) influenza A viruses to gangliosides isolated from different human tissues and separated on silica gel thin-layer plates. Anis, plate sprayed with anisaldehyde (4-methoxybenzaldehyde); Human virus and Avian virus, plates overlaid with respective conjugates. Lane 1, upper phase gangliosides (after Folch’s partition) from human leukocytes; lane 2, upper phase gangliosides from human erythrocytes; lane 3, total gangliosides from human small intestine, sample 1; lane 4, total gangliosides from human small intestine, sample 2; lane 5, total gangliosides from human stomach; lane 6, total gangliosides from human meconium; lane 7, total gangliosides from human colon; lane 8, total gangliosides from human pancreas; lane 9, bovine brain gangliosides. The plates were developed in chloroform/methanol/0.25% KCl in H2O, 50:40:10. S-3-PG, sialyl-3-paragloboside; S-6-PG, sialyl-6-paragloboside; 5s, 7s, and 8s, 5-, 7-, and 8-sugar-containing monosialoganglioside fractions (NeuAc1Hex3HexNAc1Cer, NeuAc1Hex4HexNAc2Cer and Fuc1NeuAc1Hex4HexNAc2Cer, respectively). To visualize slowly migrating gangliosides some lanes were overloaded. Dotted lines were drawn to facilitate interpretation.

 

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Table I. Carbohydrate and glycolipid structures discussed in this paper
 


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Fig. 2. Binding of human and avian influenza viruses HRP conjugates to leukocyte gangliosides on silica gel TLC plates before (lane 1) and after (lane 2) mild oxidation and reduction. Lane 3, reference brain gangliosides (from top: GM1, GD1a, GD1b, GT1b). Anis, gangliosides stained with anisaldehyde. Chromatographic conditions were as in Figure 1. x, nonsugar spot acquired during dialysis.

 
The oxidized and reduced gangliosides were tested by FAB MS and by EI MS after permethylation. In FAB MS spectra the pseudomolecular ions [M-H] seen clearly for 3s, 5s, and 7s gangliosides were reduced by 30 or 60 (± 1) mass units, see Figure 3. Thus, the main ions in Figure 3A at m/z 1151.7 (GM3, d18:1–16:0), 1517.2 (SPG, d18:1–16:0), and 1882.7 (7s, d18:1–16:0) were replaced after oxidation and reduction in Figure 3B by ions at 1091.1 and 1121.1, 1456.4 and 1486.4, and 1821.9 and 1851.8, respectively. In EI MS spectra the NeuAc fragment ions at m/z 376 and 344, were replaced after oxidation and reduction by m/z at 332 and 300, and 288 and 256 (not shown). Degradation of the core chains was not observed.



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Fig. 3. Negative ion FAB mass spectra of gangliosides from human leukocytes before (A) and after (B) mild periodate oxidation and reduction.

 
To test if the binding of the human virus was to the monofucosylated sialyl-Lewis x or VIM-2-active saccharides as reported earlier (Suzuki, 1994Go; Müthing, 1996Go), we used overlay of TLC plates with antibodies which react with these structures (Figure 4). The polar solvent (A) allowed clear separation of VIM-2- and virus-positive fractions (Figure 4A, lanes VIM and Virus), at least within the less complex region. There was an overlapping of anti-sialyl-Lewis x and human virus bindings (Figure 4A and 4B, lanes SLX and Virus), but the patterns were not identical, and there was no recognition by the virus of less complex sialyl-Lewis x-positive molecules.



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Fig. 4. Binding of anti-sialyl-Lewis x (SLX) and Vim-2 (VIM) monoclonal antibodies to upper phase gangliosides (after Folch’s partition) of human leukocytes separated on silica gel TLC plates. Anis, gangliosides stained with anisaldehyde; Virus, gangliosides overlaid with human influenza virus HRP conjugate. The plates were developed in (A) chloroform/methanol/0.25% KCl, 50:55:13, and (B) chloroform/methanol/0.25% KCl, 50:40:10.

 
Figure 5 shows binding of MAA and SNA (lectins specific for {alpha}3- and {alpha}6-linked NeuAc, respectively) to leukocyte gangliosides in comparison with binding by human influenza virus. There was a comigration of the more complex virus- and SNA-positive fractions, however, there was no binding of the virus to the less complex SNA-positive bands. The latter were earlier identified as 5s and 7s gangliosides with 6-linked NeuAc (Johansson and Miller-Podraza, 1998Go). MAA, which binds specifically to NeuAc{alpha}3-containing carbohydrate chains of the neolacto series (Knibbs et al., 1991Go; Johansson et al., 1999Go), displayed a completely different pattern of binding than the virus. Binding of the human virus to minor NeuAc{alpha}6-containing species were reproduced after ganglioside separation in a second TLC solvent (not shown). The weak binding of the virus seen in lane 3 of Figure 5 could represent cross-reaction with brain gangliosides. However, this binding was not reproducible (see Figure 1).



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Fig. 5. Binding of NeuAc{alpha}3- and NeuAc{alpha}6-specific lectins from Maackia amurensis (MAA) and Sambucus nigra (SNA) to gangliosides separated by TLC and blotted to PVDF membranes; human virus, binding of human virus HRP conjugate on the corresponding TLC plate; Anis, TLC plate with separated gangliosides and visualized with anisaldehyde. The plates were developed in chloroform/methanol/0.25%KCl, 50:50:13. Lane 1, upper phase gangliosides (after Folch’s partition) from human leukocytes; lane 2, sialyl-3-paragloboside; lane 3, bovine brain gangliosides (GM1, GD1a, GD1b, and GT1b).

 
In an attempt to identify sequences that display efficient binding of human virus, gangliosides from human leukocytes were separated by preparative TLC and tested again for the binding (Figure 6). The most complex fractions were not analyzed in this way because of inadequate amounts and poor separation. Two fractions were shown to contain active components, see Fr. 5 and Fr. 6 in the figure. Tests in different chromatographic systems (TLC not shown) revealed however that the main components in these subfractions were inactive and that binding was to some minor overlapping fractions. This agrees with the fact that the binding was to slowly migrating NeuAc{alpha}6-containing species (Figure 5), which have earlier been detected by SNA lectin (Johansson and Miller-Podraza, 1998Go). They occur in human leukocytes in very small amounts and so far only NeuAc{alpha}3-containing structures were detected by chemical methods among more complex gangliosides isolated from this source (Stroud et al., 1995Go, 1996; Müthing et al., 1996Go;). The detection level on TLC plates by the human virus was 40–80 pmol in relation to the total ganglioside mixture, and apparently much lower for the active species, indicating that the binding was highly efficient (see binding to trace fractions in lane 6 of Figure 6)



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Fig. 6. Binding of HRP conjugate of human (A, H1N1) influenza virus to ganglioside subfractions (Fr. 1–6) obtained from a mixture of human leukocyte gangliosides after preparative TLC; TG, total ganglioside mixture. Anis, plate visualized with anisaldehyde; Human virus, plate overlaid with virus conjugate.

 
The most active isolated fraction (Fr. 6 of Figure 6) was shown by MALDI-TOF mass spectrometry to contain complex gangliosides with 8–11 monosaccharides per mol of ceramide (Figure 7 and Table II). Fragmentation pattern seen in FAB MS analysis confirmed the presence of oligosaccharide chains with repeated HexHexNAc units (spectra not shown). The most abundant molecular ions corresponded to    NeuAc1Fuc1Hex5HexNAc3Cer at m/z 2394.0 and NeuAc1Hex5HexNAc3Cer at 2248.7. As mentioned, the main components were excluded as binding molecules by TLC in different solvent systems. The most probable candidates of the active species were therefore minor disialylated or difucosylated gangliosides with 2 or 3 lactosamine units. Monofucosylated gangliosides NeuAc1Fuc1Hex4HexNAc2Cer, (8s), (m/z at 2030.4 in Figure 7) with two N-acetyllactosamine units are less likely as binding molecules, as judged from chromatographic mobility and binding tests, see Figures 1, 4B, and 6, (8s region). In Fr. 5 of Figure 6 the main (nonactive) component was NeuAc1Hex5HexNAc3Cer.



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Fig. 7. MALDI TOF spectrum of ganglioside fraction 6 of Figure 6. Mass spectrometer was operated in negative-ion reflectron mode.

 

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Table II. Ganglioside composition of fraction 6 (Fr. 6 in Figure 6) based on MALDI-TOF-MS mass spectrometry
 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
In this work we tested binding of human and avian influenza viruses to gangliosides isolated from different human tissues. The important finding was selective binding of the human virus to some complex fractions present in human leukocytes. We could not identify the binding structure because of the small amount of the material. However, we have excluded sialyl-Lewis x and VIM-2-active saccharides (Table I, Figure 4) as the binding sequences. We have also shown using SNA lectin that the binding was to some minor NeuAc{alpha}6-containing species (Figure 5). In addition, we have demonstrated that the binding was dependent on the presence of the unchanged glycerol sialic acid tail (Figures 2 and 3). Our results therefore do not support earlier suggestions on a preferential binding of human influenza A viruses to sialyl-Lewis x (Suzuki, 1994Go) or VIM-2-active structures (Suzuki, 1994Go; Müthing, 1996Go). This discrepancy may be caused by differences in virus strains. The cited authors used influenza viruses A/PR/8/34 (H1N1) and the reassortant virus strain X-31, which bear HA and NA genes of A/Aichi/2/28 (H3N2), while we studied human influenza virus X-113 (HA and NA genes of A/Texas/36/91, H1N1). PR/8/34 and X-31 are known to have a higher affinity to NeuAc{alpha}3-containing receptors than more recently circulated influenza A viruses (Rogers and D'Souza, 1989Go; Matrosovich et al., 1997Go), and this may explain strong interaction of PR/8/34 and X-31 with sialyl-Lewis x and VIM-2 structures and no binding to these structures in our tests. There was an overlapping binding by anti-sialyl-Lewis x antibody and the human virus in our studies (Figure 4). However, the overall patterns were not identical and there was no interaction of the virus with less complex sialyl-Lewis x gangliosides reported to be present in human leukocytes (Müthing et al., 1996Go). We have detected by FAB MS in fractions 1 through 3 (Figure 6) increasing amounts of 8s gangliosides with a potential sialyl-Lewis x composition of Fuc1NeuAc1Hex4HexNAc2Cer with various ceramides (m/z at 2028.7 and 2138.4; not shown). These gangliosides migrated in the 7s region overlapping with more abundant nonfucosylated NeuAc1Hex4HexNAc2Cer fractions. Of importance is a binding in this region of anti-sialyl-Lewis x antibody, but not of either VIM-2 antibody or virus (Figure 4). Structural microheterogeneity associated with ceramide parts and NeuAc {alpha}3/{alpha}6 substitutions may explain the overlapping migration of different gangliosides and the complex multi-band patterns in lanes SLX of Figure 4. The cross-binding to other fucosylated structures (Stroud et al., 1995Go) of the lower TLC regions, may also contribute to these complex patterns.

We have excluded that the main components of fractions 5 and 6 in Figure 6 are binding molecules by TLC analysis in different solvent systems and overlay tests. Table II lists (in boldface type) candidates of active species which we could detect by MALDI-TOF MS. Of importance for the binding could be repeated fucose branches and/or oligosialylation, as judged from the presence of difucosylated and/or disialylated molecules in the mixture (difference between masses of 2Fuc and 1NeuAc is only 1.03 amu). Also length of the sugar should be considered as an important factor, since only complex gangliosides were binding. The extended carbohydrate chains may serve as spacers which reduce steric hindrance to recognition by viral hemagglutinins. It has been shown, that glycosylation of viral HAs in the vicinity of the receptor binding sites may decrease the virus binding to target cells and immobilized receptors (Matrosovich et al., 1997Go; Ohuchi et al., 1997Go; Gambaryan et al., 1998Go). The impaired accessibility of the receptor binding pocket could explain why we did not see binding of the human influenza to less complex gangliosides like GM3 or SPG. In fact, the hemagglutinin of X-113 reassortant human virus, although not yet sequenced, is likely to contain glycans at Asn129 and Asn163 close to the tip of the HA globular head, similar to the HAs of other contemporary H1N1 human viruses, for which sequences are available. In contrast, the avian virus strain A/duck/Czehoslovakia/56 (H4N6) lacks carbohydrates in this portion of the HA (Matrosovich et al., 1999Go). Also Müthing (1996)Go emphasized a stronger binding to longer fucosylated species (sialyl-Lewis x- and VIM-2-active species) compared to 5s and 7s gangliosides using X-31 (H3N2) influenza A strain.

In our studies there was, however, no binding at all to 5s NeuAc{alpha}6-containing SPG nor to its 7s homologue, although the interaction of the human virus with some selected complex species was very strong (for characterization of 5s and 7s gangliosides of human leukocytes see Johansson and Miller-Podraza, 1998Go). Technical assay reasons for this unusual binding are unlikely, since the avian virus bound to S-3-PG and to other well defined common gangliosides under the same experimental conditions (Figure 1). It is reasonable to assume that both length and structural features of the receptor chain contributed to this result. Human leukocytes are known to contain neolacto glycolipids with repeated fucose residues as Fuc{alpha}3GlcNAc units (Stroud et al., 1995Go, 1996; Müthing, 1996Go), seen also in our analyses (see Table II). Fucose may possibly interact with the hydrophobic methyl group with spots outside the NeuAc binding site of the viral HA. In fact, synthetic NeuAc analogues with hydrophobic neighboring groups have been shown to interact with hydrophobic patches adjoining the receptor binding site of influenza virus A hemagglutinin, considerably improving affinity (Watowich et al., 1994Go). Gangliosides with branched N-acetyl-lactosamine chains and NeuAc on more than one arm should also be considered as highly efficient binding molecules. Polyvalency has earlier been shown as an important factor enhancing binding affinity of influenza virus to synthetic sialylated compounds (Mammen et al., 1995Go), and branched polyglycosylceramides were very effective receptors for human influenza A and B viruses (Matrosovich et al., 1996Go).

The complete characterization of the binding epitope will require more ganglioside material. NeuAc{alpha}6-containing glycolipids of human leukocytes with more than two lactosamine units in the core chain have not yet been characterized (Müthing et al., 1993Go, 1996; Müthing, 1996Go; Stroud et al., 1995Go, 1996). They occur in human white cells in very small amounts and their existence has so far been neglected. However, these minor species may be of biological importance for in vivo events during influenza infections and may explain virulence variations between strains (Laver et al., 1999Go). Binding of influenza viruses to sialic acid-containing neutrophil receptor(s) depresses bactericidal activity of neutrophils (Abramson and Mills, 1988Go; Cassidy et al., 1989Go; Daigneault et al., 1992Go; Abramson and Hudnor, 1995Go) and stimulates apoptosis of these cells by a yet undefined mechanism (Colamussi et al., 1999Go). This virus-mediated neutrophil dysfunction is a likely contributor to the development of secondary bacterial infections, which are the main cause of morbidity and mortality during influenza epidemics.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
Horseradish peroxidase (HRP) labeled egg-grown influenza A viruses (human X-113 reassortant vaccine strain bearing hemagglutinin and neuraminidase of A/Texas/36/91, H1N1, and avian virus A/duck/Czehoslovakia/56, H4N6) were prepared as described previously (Matrosovich et al., 1996Go). Total ganglioside fractions were obtained from the Institute of Medical Biochemistry, Göteborg University, Sweden, and prepared according to Karlsson, 1987Go. Some fractions were purified by phase partition (Folch et al., 1957Go) before analysis, as indicated in figure captions. The human virus-binding subfractions of human leukocyte gangliosides (Figure 6) were prepared by preparative thin-layer chromatography (Miller-Podraza et al., 1992Go) followed by further purification. After separation the fractions were suspended in C/M/H2O, 60:30:4.5, by vol, applied to a small (0.25 ml) silica gel column packed in C/M, 2:1, by vol., and the sugar-positive fractions were eluted with C/M/H2O, 60:35:8, by vol. Anti-sialyl-Lewis x and CDw65/clone VIM-2 monoclonal antibodies were from Seikagaku (Japan) and Dianova GmbH (Germany), respectively, and Maackia amurensis (MAA) and Sambucus nigra (SNA) lectins from Boehringer-Mannheim (Germany). Silica gel aluminum plates 60 were purchased from Merck (Germany).

Preparation of leukocytes
Mixtures of human white cells were prepared from venous blood of healthy donors. The buffy coats were lysed in 0.8% NH4Cl (removal of erythrocytes; Fredlund et al., 1988Go) and centrifuged at 400 x g. Fractions used contained from 70% to 85% of polymorphonuclear leukocytes.

Mild periodate oxidation of gangliosides (Veh et al., 1977)
Gangliosides (0.05–0.1 mM) were incubated in 1–2 mM NaIO4 in 0.05 mM acetate buffer, pH 5.5, for 40 min on ice, after which an excess of Na2SO3 was added. The sample was concentrated by freeze-drying (about 5-fold) and reduced with an excess of NaBH4 at room temperature overnight. Finally, the sample was dialyzed against distilled water and freeze-dried.

TLC-overlay binding assays
The general overlay technique was previously described (Karlsson and Strömberg, 1987Go). Specific applications of this technique that we utilized in this study are given below.

Overlay with influenza viruses.
Plates with separated glycolipids were treated with 0.3% polyisobutylmethacrylate (Aldrich Chemical Company, Inc., Milwaukee, WI) in diethyl ether: hexane, 5:1, by vol., for 1 min, dried, and incubated in 2% BSA and 0.1% Tween 20 in PBS for 2 h at room temperature. The plates were then overlaid with HRP-labeled virus suspension in 0.2% BSA, 0.01% Tween 20 in PBS and incubated as above for additional 2 h. After washing four times with PBS, the plates were visualized by incubating at room temperature (in dark) in 0.02% DAB (3,3'-diaminobenzidine tetrahydrochloride; Pierce, Rockford, IL) in PBS containing 0.03% H2O2.

Overlay with antibodies.
Overlay with antibodies was performed as described previously (Miller-Podraza et al., 1997Go).

Overlay with lectins on membrane blots.
Detection of {alpha}3- and {alpha}6-linked sialic acids on membrane blots with lectins from Maackia amurensis (MAA) and Sambucus nigra (SNA), was performed as described previously (Johansson et al., 1999Go).

Mass spectrometry
MALDI-TOF MS was performed on a TofSpec-E (Micromass, UK) mass spectrometer operated in a reflectron mode. The acceleration voltage was 20 kV and sampling frequency 500 MHz. The matrix was 6-aza-2-thiothymine dissolved in CH3CN. FAB MS was performed on a SX102A mass spectrometer (JEOL) operated in a negative ion mode. The spectra were produced by Xe atoms (8 kV) using triethanolamine as matrix. EI MS of permethylated glycolipids was performed as described previously (Breimer et al., 1980Go) using the same JEOL mass spectrometer.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This work was supported by grants from the Swedish Medical Research Council (Nos. 3967, 10435, 13395-01), the Foundations of the National Board of Health and Welfare, the Swedish Research Council for Engineering Sciences, the Swedish Foundation for Strategic Research, the Knut and Alice Wallenberg Foundation, the IngaBritt and Arne Lundberg Foundation, and the Russian Foundation of Fundamental Investigations (95/97–04–11079). During the preparation of the manuscript, M.N.Matrosovich was supported by a Karnofsky fellowship from St. Jude Children’s Research Hospital.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
TLC, thin-layer chromatography; C, chloroform; M, methanol; MAA, Maackia amurensis lectin; SNA, Sambucus nigra lectin; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; FAB MS, fast atom bombardment mass spectrometry; EI MS, electron ionization mass spectrometry. Glycolipid and carbohydrate nomenclature is according to recommendations by the IUPAC-IUB Commission on Biochemical Nomenclature (Lipids (1977) 12:455–68; J. Biol. Chem. (1982) 257:3347–51; J. Biol. Chem. (1987) 262:13–18). We use denotations 3s to 8s to indicate migration regions on TLC plates for three- to eight-sugar-containing monosialogangliosides. S-3-PG, sialyl-3-paragloboside (NeuAc{alpha}3Galß4GlcNAcß3Galß4GlcCer); S-6-PG, sialyl-6-paragloboside (NeuAc{alpha}6Galß4GlcNAcß3Galß4GlcCer).


    Footnotes
 
1 To whom correspondence should be addressed Back

2 Present address: Department of Virology and Molecular Biology, St. Jude Children’s Hospital, Memphis, Tennessee 38105, USA Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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Breimer,M., Hansson,G.C., Karlsson,K.-A., Larsson,G., Leffler,H., Pascher,I., Pimlot,W. and Samuelsson,B.E. (1980) Fingerprinting of lipid-linked oligosaccharides by mass spectrometry. In Quayle,A. (ed.), Advances in Mass Spectrometry, Vol. 8. Heyden & Son, London, pp. 1097–1108.

Cassidy,L.F., Lyles,D.S. and Abramson,J.S. (1989) Depression of polymorphonuclear leukocyte functions by purified influenza virus hemagglutinin and sialic acid-binding lectins. J. Immunol., 142, 4401–4406.[Abstract/Free Full Text]

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