Sialylated glycans as receptor and inhibitor of enterovirus 71 infection to DLD-1 intestinal cells
Many viruses recognize specific sugar residues, particularly sulfated or sialylated glycans, as the infection receptors. A change of sialic acid (2-6)-linked galactose (SA-α2,6Gal) to SA-α2,3Gal determines the receptor for avian flu infection. The receptor for enterovirus 71 (EV71) infection that frequently causes fatal encephalitis in Asian children remains unclear. Currently, there is no effective vaccine or anti-virus agent for EV71 infection. Using DLD-1 intestinal cells, this study investigated whether SA-linked glycan on DLD-1 intestinal cells was a receptor for EV71, and whether natural SA-linked sugars from human milk could block EV71 infection.
EV71 specifically infected DLD-1 intestinal cells but not K562 myeloid cells. Depletion of O-linked glycans or glycolipids, but not N-linked glycans, significantly decreased EV71 infection of DLD-1 cells. Pretreatment of DLD-1 cells with sialidase (10 mU, 2 hours) significantly reduced 20-fold EV71 replication (p < 0.01). Taken together, these results suggest that SA-linked O-glycans and glycolipids, but not N-glycans, on DLD-1 cells were responsible for EV71 infection. Purified SA-α2,3Gal and SA-α2,6Gal from human milk significantly inhibited EV71 infection of DLD-1 cells, indicating terminal SA-linked glycans could be receptors and inhibitors of EV71 infection.
This is the first in the literature to demonstrate that EV71 uses SA-linked glycans as receptors for infection, and natural SA-linked glycans from human milk can protect intestinal cells from EV71 infection. Further studies will test how a SA-containing glycan can prevent EV71 in the future.
KeywordsSialic Acid Human Milk Avian Influenza EV71 Infection Sugar Residue
- Abbreviations used are EV71
3Gal: sialic acid: alpha 2,3 galactose
multiplication of index
- and IFA
Many viruses recognize specific sugar residues, particularly sulfated or sialylated glycans, as the infection receptors. Avian influenza virus and human influenza virus use different sugar residues as their receptors, resulting in different host range of infections . Enterovirus 71 (EV71) which prevails almost every summer season and causes hand-foot-mouth disease is frequently complicated with fatal encephalitis in Asia, and even Europe [2, 3, 4, 5, 6]. Currently, there is neither vaccine available for prevention of EV71, nor antiviral treatment for EV71 infection. Before development of effective antiviral agents or specific vaccine available to control epidemics of EV71, identification of the receptor(s) for EV71 and block of the receptor(s) may be a good regimen for prevention of EV71 infection.
Sialic acid (SA) also known as neuraminic acid is usually linked to galactose or other sugar residues as an antenna of blood group antigens, tumor antigens or viral receptors . Gastrointestinal and respiratory epithelial cells expressed abundant SA-containing glycoproteins and SA-containing glycolipids [1, 8, 9]. It is known that sialic acid-α2,6 galactose (SA-α2,6Gal) epitope is a receptor for human influenza virus  and sialic acid-α2,3galactose (SA-α2,3Gal) is a receptor for coxackievirus A24 . The transmission route of the EV71 is fecal-oral and/or droplet-aerosol route, and the receptor for EV71 is unknown . We, therefore, postulated that EV71 might use the SA-linked glycan on intestinal epithelial cells as a receptor, and natural SA-linked glycans may prevent human intestinal cells from EV71 infection.
This study was conducted to investigate whether depletion of glycolipids, N-linked glycans or O-linked glycans on DLD-1 intestinal cells could avoid EV71 infection, and administration of SA-linked sugars from human milk could block EV71 infections. If natural SA-linked sugars could block EV71 infection, SA-linked glycans may be made to prevent EV71 infections.
Results and Discussion
EV71 infection of DLD-1 intestinal cells
SA-linked O-glycan and glycolipid but not N-glycan responsible for EV71 infection
Inhibition of EV71 infection in DLD-1 cells by sialidase treatment
Blockade of EV71 infection by SA-derived glycans from milk
Materials and methods
Study design. This study initially studied whether EV71 specifically infected DLD-1 intestinal cells, but not K562 myeloid cells. Using DLD-1 intestinal cells which possess abundant sialylated glycans on cell surface, we tested whether depletion of glycolipids or glycoproteins (N-linked glycoprotein or O-linked glycoproteins) blocked EV71 infection, and sialidase depletion of SA residues on DLD-1 cells protected DLD-1 cells from EV71 infection, indicating SA-linked glycan responsible for the entry of EV71 infection. Finally, we used SA-linked glycans purified from human milk to block EV71 infection of DLD-1 cells in order to validate SA-linked galactose residues responsible for EV71 infection.
Culture of human intestine epithelial cell line. We used DLD-1 intestinal epithelial cells as a target of EV71 infection, and K562 myeloid leukemia cells were used as control cells for comparison. The DLD-1 and K562 cell lines were obtained from Food Industrial Research Institute, Hsin-chu, Taiwan. The reason to use intestinal cell line is because it can express different levels of SA-linked glycans resembling neonatal rat intestine . DLD-1 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI1640) medium with 10% fetal bovine serum, and harvested into 2 × 106 cells/ml for testing whether depletion of SA-linked glycans on DLD-1 cells by sialidase treatment reduced EV71 infection. Experiments were also performed to differentiate whether different natural SA-linked glycans such as SA-α2,3Gal or SA-α2,6Gal could protect against EV71 infections.
Determination of EV71 infection by indirect immunofluorescence and RT-PCR analysis. Infection of EV71 was assessed by an indirect immunofluorescent staining with an EV71-specific monoclonal antibody (Chemicon Inc. CA). DLD-1 cells (2 × 105 cells/ml) with and without sialidase pre-treatment for 2 hours were subjected to EV71 infection. The EV71 infected DLD-1 cells were harvested in one day for staining with mouse anti-EV71 monoclonal antibody or nonspecific antibody after cold acetone fixation, followed by FITC-labeled goat anti-mouse immunoglobulin antibody for fluorescent visualization. Cells were also harvested in 3 days for quantification of EV71 replication by a real time quantitative RT-PCR (qRT-PCR) analysis as previously described . The primers used to detect EV71 RNA copies by SYBR Green fluorescent RT-PCR were forward: 5'-CCCCTGAATGCGGCTAATC-3' and reverse: 5'-CCATATAGCTATTGGATTGGCCA-3'. The copies of virus titers were calculated based on a standard curve made by a series of well-known RNA copies of EV71.
Differentiation of the SA-containing glycoproteins and glycolipids responsible for EV71 infection. Sugar residues on cell surface are usually linked to protein, called glycoprotein, or linked to lipid, called glycolipid. Employing inhibitors of protein glycosylation and lipid glycosylation synthesis, we investigated if the SA-based residue responsible for EV71 infection stems from glycoprotein or glycolipid. To test whether N-linked or O-linked sialylglycoprotein on DLD-1 cells was the receptor for EV71, DLD-1 cells (1 × 106 cells/ml) were respectively incubated with 3 mM benzyl N-acetyl-α-D-galactosaminide (Sigma-Aldrich Inc., St. Louis, MO) for 48 hours or with 0.2 mg/ml tunicamycin (Sigma-Aldrich Inc.) for 24 hours before subjected to EV71 binding assay at MOI = 10. To test whether sialylglycolipid on DLD-1 cells was the receptor for EV71, DLD-1 cells (1 × 106 cells/50 μl) were incubated with 50 μl phosphotidylinositol-specific phospholipase (5 U/ml) purchased from Sigma-Aldrich Inc. for 90 minutes before subjected to the test of EV71 binding assay. The EV71 binding assay was performed within one hour after washing out the treatment of specific inhibitor because DLD-1 cells in the inhibitor-free condition could re-express glycoprotein or glycolipid that might interfere the experimental interpretation.
Sources of sialidase and natural SA-linked glycans: The sialidase (α2-3, 6-sialidase, Clostridium perfringens) that can cleave SA from SA-α2,3Gal and/or SA-α2,6Gal compounds was purchased from Calbiochem Inc., Darmstadt, Germany. For experiments, DLD-1 cells were pretreated with 2, 10 or 50 mU/ml sialidase for 2 hours before subjected to EV71 infections for 24 hours at MOI = 10. Human milk SA-α2,3Gal (97% purity) and SA-α2,6Gal (98% purity) were purchased from Sigma-Aldrich Inc. (St. Louis, MO) and tested for inhibition of EV71 infections.
Determination of SA-linked sugar residue as a receptor and inhibitor of EV 71 infection by qRT-PCR. To validate whether SA-linked sugar residues were receptors for EV71 infection, EV71 (2 × 106 copies/ml) were co-incubated with SA-α2,3Gal (0.25 mg/ml) or SA-α2,6Gal (0.25 mg/ml) 15 minutes before added to infect DLD-1 cells (2 × 105 cells/ml) for 3 days. Replication of EV71 in DLD-1 cells was determined by qRT-PCR detection as described above.
Data management and statistics. Specific infection of EV71 to DLD-1 cells was compared to the control cell line, K562, myeloid leukemia cells. Binding of EV71 to SA-linked sugars was validated by depletion of SA by sialidase treatment. Inhibition of EV71 by different SA-linked sugar residues was analyzed by non-parametric analysis of Mann-Whitney U test. Chemical structures of the SA-linked compounds were drawn by the software of Chemwindow 6.0 (Bio-Rad Inc., Hercules, CA).
The authors would like to thank Mr. Jeremy Melton in Kaohsiung American School, Taiwan for his advice on the study progress and review of the manuscript. This study has been partly presented in the 15th European Carbohydrate Symposium held in Vienna, Austria, July 19-24, 2009. This study was in part supported by a grant CMRPG880661 from Chang Gung Memorial Hospital, Taiwan.
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