Localisation to lipid rafts correlates with increased function of the Gal/GalNAc lectin in the human protozoan parasite, Entamoeba histolytica

Entamoeba histolytica is the causative agent of dysentery and liver abscess and is prevalent in developing countries. Adhesion to the host is critical to infection and is mediated by amoebic surface receptors. One such receptor, the Gal/GalNAc lectin, binds to galactose or N-acetylgalactosamine residues on host components and consists of heavy (Hgl), light (Lgl) and intermediate (Igl) subunits. The mechanism by which the lectin assembles into a functional complex is not known. The parasite also relies on cholesterol-rich domains (lipid rafts) for adhesion. Therefore, it is conceivable that rafts regulate the assembly or function of the lectin. To test this, amoebae were loaded with cholesterol and lipid rafts were purified and characterised. Western blotting showed that cholesterol loading resulted in co-compartmentalisation of all three subunits in rafts. This co-compartmentalisation was accompanied by an increase in the ability of the amoebae to bind to host cells in a galactose-specific manner, suggesting that there is a correlation between location and function of the Gal/GalNAc lectin. Cholesterol loading did not increase the surface levels of the lectin subunits. Therefore, the cholesterol-induced increase in adhesion was not the result of externalisation of an internal pool of subunits. A mutant cell line that modestly responded to cholesterol with a slight increase in adhesion exhibited only a slight enrichment of Hgl and Lgl in rafts. This supports the connection between location and function of the Gal/GalNAc lectin. Actin can also influence the interaction of proteins with rafts. Therefore, the sub-membrane distribution of the lectin subunits was also assessed after treatment with an actin depolymerising agent, cytochalasin D. Cytochalasin D-treatment had no effect on the submembrane distribution of the subunits, suggesting that actin does not prevent the association of lectin subunits with rafts in this system. Together, these data provide insight into the molecular mechanisms regulating the location and function of this adhesin