Cloning of the Entamoeba histolytica STT3 Gene, a Subunit of the Oligosaccharyltransferase Complex

Abstract

Entamoeba histolytica , a protozoan parasite and causative agent of human amebiasis, has unusual cellular and biochemical features. It lacks mitochondria and peroxisomes. The presence of a structured tubovesicular network such as endoplasmic reticulum (ER) and Golgi complex has not been clearly established, raising the question of which cellular and molecular mechanisms of protein traffic exist in this organism. Despite this, secretion of several amebic proteins to the extracellular environment is well documented. Furthermore, E. histolytica glycosylates some of its proteins. Glycosylation is a classic activity marker for transit of proteins through the eukaryotic secretory pathway. Two enzymes (GlcNAc-1-P transferase and GlcNAc transferase) involved in early steps leading to N-glycosylation have been characterized at the biochemical level in E. histolytica (1). However, the functional compartments associated to these activities are still to be identified. To provide molecular evidence supporting the existence of an ER-like functional compartment in this protozoan, we searched for evolutionary conserved genes coding for eukaryotic ER resident proteins. A good candidate was the STT3 gene, a subunit of the oligosaccharyltransferase complex. An entamebal STT3-expressed sequence tag (EST) has been reported (2). N-linked glycosylation of proteins is carried out in the lumen of the ER. The first step involves the en bloc transfer of the precursor Glc 3 Man 9 GlcNAc 2 from the dolichol diphosphate-linked core-oligosaccharide assembled at the membrane of the ER to the appropriate Asn residues (AsnX-Ser/Thr) of nascent polypeptide chains. The reaction is catalyzed by the transmembranal ER lumen-oriented enzyme complex oligosaccharyltransferase (OTase). In yeast, the STT3 gene is essential for growth and protein glycosylation. Sc Stt3p regulates the substrate specificity and assembly of the OTase complex (3). Depletion of the Stt3 protein results in loss of transferase activity in vivo and a deficiency in the assembly of the OTase complex. The 78 kDa Sc Stt3 protein has an N-terminal hydrophobic domain with 12 predicted membrane-spanning segments and a carboxy-terminal hydrophilic domain that is located in the lumen of the ER. Homologues of the STT3 yeast gene have been isolated from C. elegans (4), mouse, and human (5), among others. In this report, we describe the cloning of the E. histolytica STT3 gene, a molecular probe for the presence of a functional compartment equivalent to the eukaryotic ER in this parasite.