Abstract
Posttranslational modifications provide Entamoeba histolytica proteins the timing and signaling to intervene during different processes, such as phagocytosis. However, SUMOylation has not been studied in E. histolytica yet. Here, we characterized the E. histolytica SUMO gene, its product (EhSUMO), and the relevance of SUMOylation in phagocytosis. Our results indicated that EhSUMO has an extended N-terminus that differentiates SUMO from ubiquitin. It also presents the GG residues at the C-terminus and the ΨKXE/D binding motif, both involved in target protein contact. Additionally, the E. histolytica genome possesses the enzymes belonging to the SUMOylation-deSUMOylation machinery. Confocal microscopy assays disclosed a remarkable EhSUMO membrane activity with convoluted and changing structures in trophozoites during erythrophagocytosis. SUMOylated proteins appeared in pseudopodia, phagocytic channels, and around the adhered and ingested erythrocytes. Docking analysis predicted interaction of EhSUMO with EhADH (an ALIX family protein), and immunoprecipitation and immunofluorescence assays revealed that the association increased during phagocytosis; whereas the EhVps32 (a protein of the ESCRT-III complex)-EhSUMO interaction appeared stronger since basal conditions. In EhSUMO knocked-down trophozoites, the bizarre membranous structures disappeared, and EhSUMO interaction with EhADH and EhVps32 diminished. Our results evidenced the presence of a SUMO gene in E. histolytica and the SUMOylation relevance during phagocytosis. This is supported by bioinformatics screening of many other proteins of E. histolytica involved in phagocytosis, which present putative SUMOylation sites and the ΨKXE/D binding motif.
Highlights
As in other eukaryotes, in Entamoeba histolytica, the protozoan causative of human amoebiasis, cellular activities, including the attack to the target cell, are widely controlled by posttranslational modifications (PTMs) of proteins
To investigate whether the proteins involved in phagocytosis go through SUMOylation, we first performed bioinformatics analysis to search for SUMO genes in the AmoebaDB, using as a template the SUMO sequence from G. lamblia [11,19]
SUMOylation, which consists of the non-covalent binding of SUMO protein with target molecules, is one of the main changes suffered by proteins to enable them to participate in cellular functions
Summary
In Entamoeba histolytica, the protozoan causative of human amoebiasis, cellular activities, including the attack to the target cell, are widely controlled by posttranslational modifications (PTMs) of proteins. Together with the perpetual movement of trophozoites, virulence expression requires intensive vesicular traffic and association to and disassociation from proteins performing the concatenated events that conduct target molecules through several compartments, for recycling or digestion. PTMs range from peptide bond cleavage and addition of phosphate and other small chemical groups, carbohydrates, and lipids to the alteration of proteins by the conjugation of modifiers such as ubiquitin and SUMO. In the N-terminus, SUMO has an extended 10 to 25 amino acid chain, absent in ubiquitin. As this later, SUMO conjugates to its target by an isopeptide bond formed on a lysin present in the consensus sequence ΨKXE/D (ψ: hydrophobic amino acid, K: target lysin, X: any amino acid, E and D: glutamic and aspartic acids, respectively) [2]. DeSUMOylation, the reverse process, occurs by the specific proteases UIp1a and UIp1b [7]
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