Revisiting the isolation and characterisation of Entamoeba histolytica lipopeptidophosphoglycan.

The parasite Entamoeba histolytica is the cause of amoebic dysentery and liver abscess in humans. On the protozoan cell surface, a variety of glycosylated molecules are involved in the interaction with the environment, such as attachment to the colonic mucus. One of these molecules is the lipopeptidophosphoglycan (LPPG), a complex surface component with antigenic properties. Its structure is only partly known, it is a glycosylphosphatidylinositol (GPI)-linked glycoprotein with a large amount of O-glycosylation. To date, the sequence of a core protein has not been identified. In this study, we further investigated this complex surface molecule aided by the availability of the monoclonal antibody EH5, which had been raised in our laboratory. We studied the extraction of LPPG in various solvent mixtures and discovered that 2-butanol saturated water was simple and superior to other solvents used in the past. The isolated LPPG was subjected to treatment with several proteases and the Ser/Thr specific cleavage agent scandium (III) trifluoromethanesulfonate (scandium triflate). The products were probed with antibody EH5 and the blots showed that the LPPG preparation was largely resistant to standard proteases, but could be cleaved by the scandium compound. These observations could point to the existence of a Ser- or Thr-rich core protein structure.

Molecular characterisation of Entamoeba histolytica UDP-glucose 4-epimerase, an enzyme able to provide building blocks for cyst wall formation.

In the human host, the protozoan parasite Entamoeba histolytica is adapted to a non-invasive lifestyle in the colon as well as to an invasive lifestyle in the mesenterial blood vessels and the liver. This means to cope with bacteria and human cells as well as various metabolic challenges. Galactose and N-acetylgalactosamine (GalNAc) are sugars of great importance for the amoebae, they attach to the host mucus and enterocytes via their well-studied Gal/GalNAc specific lectin, they carry galactose residues in their surface glycans, and they cleave GalNAc from host mucins. The enzyme UDP-glucose 4-epimerase (GalE) works as a bridge between the galactose and glucose worlds, it can help to generate glucose for glycolysis from phagocytosis products containing galactose as well as providing UDP-galactose necessary for the biosynthesis of galactose-containing surface components. E. histolytica contains a single galE gene. We recombinantly expressed the enzyme in Escherichia coli and used a spectrophotometric assay to determine its temperature and pH dependency (37°C, pH 8.5), its kinetics for UDP-glucose (Km = 31.82 µM, Vmax = 4.31 U/mg) and substrate spectrum. As observed via RP-HPLC, the enzyme acts on UDP-Glc/Gal as well as UDP-GlcNAc/GalNAc. Previously, Trypanosoma brucei GalE and the bloodstream form of the parasite were shown to be susceptible to the three compounds ebselen, a selenoorganic drug with antioxidant properties, diethylstilbestrol, a mimic of oestrogen with anti-inflammatory properties, and ethacrynic acid, a loop diuretic used to treat oedema. In this study, the three compounds had cytotoxic activity against E. histolytica, but only ebselen inhibited the recombinant GalE with an IC50 of 1.79 µM (UDP-Gal) and 1.2 µM (UDP-GalNAc), suggesting that the two other compounds are active against other targets in the parasite. The importance of the ability of GalE to interconvert UDP-GalNAc and UDP-GlcNAc may be that the trophozoites can generate precursors for their own cyst wall from the sugar subunits cleaved from host mucins. This finding advances our understanding of the biochemical interactions of E. histolytica in its colonic environment.