Exposure to Host Ligands Correlates with Colocalization of Gal/GalNAc Lectin Subunits in Lipid Rafts and Phosphatidylinositol (4,5)-Bisphosphate Signaling in Entamoeba histolytica

Abstract

bEntamoeba histolytica is an intestinal parasite that causes dysentery and liver abscess. Parasite cell surface receptors, such as the Gal/GalNAc lectin, facilitate attachment to host cells and extracellular matrix. The Gal/GalNAc lectin binds to galactose or N-acetylgalactosamine residues on host components and is composed of heavy (Hgl), intermediate (Igl), and light (Lgl) subunits. Although Igl is constitutively localized to lipid rafts (cholesterol-rich membrane domains), Hgl and Lgl transiently associate with this compartment in a cholesterol-dependent fashion. In this study, trophozoites were exposed to biologically relevant ligands to determine if ligand binding influences the submembrane distribution of the subunits. Exposure to human red blood cells (hRBCs) or collagen, which are bona fide Gal/GalNAc lectin ligands, was correlated with enrichment of Hgl and Lgl in rafts. This enrichment was abrogated in the presence of galactose, suggesting that direct lectin-ligand interactions are necessary to influence subunit location. Using a cell line that is able to attach to, but not phagocytose, hRBCs, it was shown that physical attachment to ligands was not sufficient to induce the enrichment of lectin subunits in rafts. Additionally, the mutant had lower levels of phosphatidylinositol (4,5)-bisphosphate (PIP 2 ); PIP 2 loading restored the ability of this mutant to respond to ligands with enrichment of subunits in rafts. Finally, intracellular calcium levels increased upon attachment to collagen; this increase was essential for the enrichment of lectin subunits in rafts. Together, these data provide evidence that ligand-induced enrichment of lectin subunits in rafts may be the first step in a signaling pathway that involves both PIP 2 and calcium signaling.

Localization of Phosphatidylinositol 4,5-Bisphosphate to Lipid Rafts and Uroids in the Human Protozoan Parasite Entamoeba histolytica

“…We previously demonstrated that membrane fractions 9 to 14 possess high levels of cholesterol and contain lipid rafts (31). Less buoyant fractions (fractions 17 to 19) and the pellet fraction (fraction 20) are actin rich (26,28,31). Similar to the enrichment seen in mammalian cells, we observed an enrichment of PI(4,5)P 2 in lipid rafts (fractions 9 to 14) ( Fig.…”

“…1A). We previously demonstrated that membrane fractions 9 to 14 possess high levels of cholesterol and contain lipid rafts (31). Less buoyant fractions (fractions 17 to 19) and the pellet fraction (fraction 20) are actin rich (26,28,31).…”

“…To measure the effect of cholesterol treatment on intracellular Ca 2ϩ levels, a Ca 2ϩ assay was employed as described previously (31). Briefly, cells were incubated in serum-free medium supplemented with the fluorescent Ca 2ϩ indicator Fluo-4/AM (Invitrogen) or dimethyl sulfoxide (DMSO; diluent control) for 30 min at 37°C.…”

“…The net densitometry values were corrected to reflect the differences in membrane fraction volume. Whole-cell lipid extraction and lipid dot blot analyses were performed as previously described (31).…”

“…Since PLCD1 also localizes to rafts (42), it is reasonable to hypothesize that the accumulation of PI(4,5)P 2 in rafts after cholesterol treatment might lead to an increase in intracellular Ca 2ϩ . Therefore, we measured intracellular Ca 2ϩ levels after cholesterol treatment using a previously published protocol (31). Several studies have demonstrated that bona fide ligands of E. histolytica, such as collagen and fibronectin themselves, induce signaling leading to in- fluorescein isothiocyanate-ConA (green) for 1 h on ice, followed by incubation at 37°C for 15 min to initiate uroid formation.…”