Research Article: Bioinformatics and Functional Analysis of an Entamoeba histolytica Mannosyltransferase Necessary for Parasite Complement Resistance and Hepatical Infection

Date Published: February 13, 2008

Publisher: Public Library of Science

Author(s): Christian Weber, Samantha Blazquez, Sabrina Marion, Christophe Ausseur, Divya Vats, Mickael Krzeminski, Marie-Christine Rigothier, Rachid C. Maroun, Alok Bhattacharya, Nancy Guillén, Anuradha Lohia

Abstract: The glycosylphosphatidylinositol (GPI) moiety is one of the ways by which many cell surface proteins, such as Gal/GalNAc lectin and proteophosphoglycans (PPGs) attach to the surface of Entamoeba histolytica, the agent of human amoebiasis. It is believed that these GPI-anchored molecules are involved in parasite adhesion to cells, mucus and the extracellular matrix. We identified an E. histolytica homolog of PIG-M, which is a mannosyltransferase required for synthesis of GPI. The sequence and structural analysis led to the conclusion that EhPIG-M1 is composed of one signal peptide and 11 transmembrane domains with two large intra luminal loops, one of which contains the DXD motif, involved in the enzymatic catalysis and conserved in most glycosyltransferases. Expressing a fragment of the EhPIG-M1 encoding gene in antisense orientation generated parasite lines diminished in EhPIG-M1 levels; these lines displayed reduced GPI production, were highly sensitive to complement and were dramatically inhibited for amoebic abscess formation. The data suggest a role for GPI surface anchored molecules in the survival of E. histolytica during pathogenesis.

Partial Text: Glycosylphosphatidylinositol (GPI) is a glycolipid required for anchoring many cell surface proteins and glycoconjugates to the surface of a wide range of human parasites including Trypanosoma brucei, the agent of sleeping sickness, Leishmania the causative agent of leishmaniasis, Plasmodium falciparum the agent of malaria and Entamoeba histolytica responsible for amoebiasis [1]. A common feature of the surface of these parasites is the presence of a large glycocalyx containing the GPI-anchored compounds that allow them to interact with their external environment. During invasion of human cells or tissues, the glycocalyx contributes to the adhesive mechanisms sustaining interaction of parasites with their target cells. GPI anchors are structurally complex glycophospholipids that are added to carbohydrate chains, as in the case of glycosylinositolphospholipids (GIPLs) and lipophosphoglycan (LPG) or post-translationally to the C-terminal end of many membrane proteins in the ER. Studies on the variant surface glycoproteins of T. brucei led to discovery of the role of GPI in anchoring proteins to the cell surface [2]. During parasitic infections, GPIs of various protozoan parasites, particularly those of P. falciparum and various Trypanosoma and Leishmania species, can activate host macrophages, triggering the production of proinflammatory cytokines and nitric oxide contributing to disease pathogenesis [1]. Recent studies have suggested that GPI and/or many GPI-anchored molecules could be secreted by the parasites during their invasive process. In the context of human infection by P. falciparum, it has been proposed that the secreted GPI of parasite origin functions as the dominant malarial toxin [3],[4]. GPIs of T. cruzi have the same function [5]. These data support the view that GPIs of the parasitic protozoa are dominant proinflammatory agents playing a role in the immunopathology of these parasitic infections. GPI-anchored molecules also play vital roles in amoebic pathogenesis. During dysentery, amoeba trophozoites bind to colonic mucins and to epithelial cells through the Gal/GalNAc lectin, an immunodominant protein complex containing a GPI-anchored subunit [6]. This lectin associates with another GPI-anchored protein, the intermediate IgL sub-unit. E. histolytica also expresses at its surface an abundant second class of GPI-linked molecules referred as GPI-anchored proteophosphoglycan (PPG) [7],[8],[9]. The GPI anchor of PPGs is unusual because it contains a highly acidic polypeptide backbone modified by 1-6 glucan side-chains and this core is also modified by heterogeneous galactose side-chains. Interestingly, the non-virulent E. histolytica strain Rahman synthesizes one class of PPGs containing short disaccharide side-chains [8] and no similar molecule was detected in the non-pathogenic species Entamoeba dispar. PPGs are important virulence factors during hepatic amoebiasis since monoclonal antibodies recognizing these compounds protect animals from abscess development [10]. The E. histolytica PPGs in addition diverge from the conserved sequence because they contain an anchor with the core structure Gal1Man2GlcN-myoinositol, where the terminal Gal residue replaces the 1-2 linked mannose residue of other eukaryotic GPIs. A large number of studies in yeast and mammalian cells allowed to conclude that steps in GPI biosynthesis are conserved in eukaryotes [11]. In general, biosynthesis of GPI begins in the Endoplasmic Reticulum (ER) with the transfer of N-Acetyl Glucosamine from UDP-N-Acetyl Glucosamine to ER membrane residing phosphatidylinositol (PI). This step is catalyzed by N-Acetylglucosamine transferase located in the ER membranes. This intermediate is then deacetylated to form GlcN-PI by another ER enzyme-GPI-deacetylase (PIG-L). It is thought that GlcN-PI is then flipped to the ER lumen by a set of flippases. Next, a set of mannosyltransferases acts on GlcN-PI to add on three mannose moieties successively to form (Man) 3-GlcN-PI [11]. This intermediate is recognized as a substrate by ethanolamine phosphotransferase to add on an ethanolamine phosphate group to the terminal mannose of the extending GPI glycan core, which is conserved in most eukaryotic cells. The mannose groups in the GPI core are all derived from dolichol-phosphate-mannose (Dol-P-Man). Thus, three Dol-P-Man dependent mannosyl transferases are required for independent addition of mannoses to the GPI-core. The first of these mannosyltransferases is PIG-M1 (Phosphatidylinositol glycan mannosyltransferase ) which transfers the first mannose to the growing GPI anchor from the luminal side [12]. The analysis of E. histolytica genome sequence allowed identification of genes involved in the GPI biosynthetic pathway [13]. Among a total of 22 genes in yeast and 23 in humans, 15 genes were identified in E. histolytica; these genes include all catalytic subunits of the enzymatic complexes sustaining GPI biosynthesis. Studies on the GPI biosynthetic pathway in E. histolytica are rather scarce. Nevertheless, it has been found recently that the antisense RNA-mediated inhibition of EhPIG-L, the GPI-deacetylase, has an important effect on cell growth, endocytosis and parasite adhesion to human cells [13].

In this work we have identified an E. histolytica protein homolog of mammalian PIG-M, an endoplasmic reticulum–localized α1,4 mannosyltransferase required for synthesis of the glycosylphosphatidylinositol (GPI) anchor. PIG-M is the enzyme that incorporates the first mannose into the GPI core [11]. The EhPIG-M1encoding gene (XM_644988) is transcribed in growing parasites as assessed by RACE-PCR experiments and by cDNA sequencing. Bioinformatics analysis of EhPIG-M1 secondary structure reveals that it is a transmembrane protein probably residing in the ER. The C-terminus containing the retention motif is intra-cytoplasmic indicating that it can be recognized by COP I, a specific cytoplasmic protein that retrieves proteins from the Golgi after interaction with the ER retention motif [33],[34]. However, the potential ER retention motif -403LRKQKQLKLN412- is atypical. Since the organization of the ER and Golgi of this early branching eukaryote E. histolytica is different from other eukaryotes it is possible that the ER retention signal may be different [33],[34]. The cellular location and trafficking of EhPIG-M1 are matters under investigation.



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