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.
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Abstract |
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Key words: influenza virus/ganglioside/sialic acid/human leukocyte/receptor
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Introduction |
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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., 1993; Müthing, 1996
) and among highly complex glycolipid fractions, polyglycosylceramides (Matrosovich et al., 1996
). Influenza viruses are known to cause neutrophil dysfunction (Abramson and Mills, 1988
; Cassidy et al., 1989
; Daigneault et al., 1992
; Abramson and Hudnor, 1995
), 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.
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Results |
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Discussion |
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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., 1997; Ohuchi et al., 1997
; Gambaryan et al., 1998
). 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., 1999
). Also Müthing (1996)
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 NeuAc6-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, 1998
). 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
3GlcNAc units (Stroud et al., 1995
, 1996; Müthing, 1996
), 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., 1994
). 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., 1995
), and branched polyglycosylceramides were very effective receptors for human influenza A and B viruses (Matrosovich et al., 1996
).
The complete characterization of the binding epitope will require more ganglioside material. NeuAc6-containing glycolipids of human leukocytes with more than two lactosamine units in the core chain have not yet been characterized (Müthing et al., 1993
, 1996; Müthing, 1996
; Stroud et al., 1995
, 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., 1999
). Binding of influenza viruses to sialic acid-containing neutrophil receptor(s) depresses bactericidal activity of neutrophils (Abramson and Mills, 1988
; Cassidy et al., 1989
; Daigneault et al., 1992
; Abramson and Hudnor, 1995
) and stimulates apoptosis of these cells by a yet undefined mechanism (Colamussi et al., 1999
). 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.
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Materials and methods |
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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., 1988) 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 (0.050.1 mM) were incubated in 12 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, 1987). 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., 1997).
Overlay with lectins on membrane blots.
Detection of 3- and
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., 1999
).
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., 1980) using the same JEOL mass spectrometer.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 Present address: Department of Virology and Molecular Biology, St. Jude Childrens Hospital, Memphis, Tennessee 38105, USA
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References |
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