Entamoeba histolytica - binding of the lipopeptidophosphoglycan - specific monoclonal antibody EH5 to dextrans

The human intestinal parasite Entamoeba histolytica remains the cause of significant morbidity and mortality. Glycans often play an important role in its interactions with the host. A major surface component of E. histolytica, the lipopeptidophosphoglycan (LPPG), is a glycosylphosphatidylinositol- (GPI)- linked glycoprotein with a large amount of O-glycosylation with dextran-like oligoglucosyl side chains. We had previously isolated an LPPG-specific monoclonal antibody named EH5. This antibody protected SCID (severe combined immunodeficient) mice from amoebic liver abscess. To date, the sequence of the core polypeptide of LPPG structure is unknown. Recently, we described an improved method to extract LPPG from whole amoebae with 2-butanol saturated water. The extracted material was rather resistant to proteolysis; however, in the current study, we found that the epitope of EH5 in LPPG was sensitive to treatment with Chaetomium gracile dextranase. Moreover, antibody EH5 bound to commercially available dextrans of higher molecular weights blotted onto nitrocellulose membranes. Preincubation of antibody EH5 with dextrans inhibited its binding to LPPG in immunofluorescence microscopy. The commercially available anti-dextran monoclonal antibody DX1 also bound to the amoebae in a dextran-inhibitable manner. Moreover, anti-Entamoeba antibody EH5 and antibody DX1, tested in confocal immunofluorescence microscopy, bound to dextrans produced by the bacterium Leuconostoc mesenteroides, but not to Escherichia coli cultures. Using a glycan array of dextrans with defined short chain lengths, it was shown that three to five α1,6-linked glucose residues could be sufficient for binding antibody EH5. We conclude that the EH5 antibody binds dextran chains as a carbohydrate epitope.