Ubiquitin of Entamoeba histolytica deviates in six amino acid residues from the consensus of all other known ubiquitins

Abstract

The antino acid tmquencc of ubiquilin from ~tmm~elm histMyticu, as dedu~d from a eDNA nucl¢otid¢ Paluen~. deviated at six positions from the consensus of all other known ubiquitins (run,rig from To '/umosm;u, ¢ru:i to Hmm, x#pienx). The corresponding re,..~idaes were r,e.~ttcre..d over the primary ~quence. but came ¢lorm together on the surfa= of tl~e folded prot~'in structure. We conclude that (i) E. hixtd.vtiea bnumhed olT wry early from the main eakaryoti¢ line, and (ii) thi:¢ or~'mism may yield clues as to the evolutionary development of the ubiquitin sy=t¢m. Against this background we thought it interesting to investigate the amebal ubiquitin, Ubiquitin is a 76. amino acid protein found in all eukaryotic cells where people have looked for it; it has not been found in any prokaryote yet, The amino acid sequence of ubiquitin has been highly conserved throughout evolution, which may be a necessary consequence of its astonishing multifunctionality. The best.characterized biological role of ubiquitin is that of a covalently bound recognition signal for non-lysosomal proteolysis; ubiquitin is also found in linear ubiquitin-prot¢in fusions. Putative functions of ubiquitination include DNA repair, ¢¢11 cycle control, stress response and ribosome biogenesis. In a recent review [3] it has been estimated that over forty Below we present the coding sequence for ubiquitin derived from an amebal eDNA done. The amino acid sequence deduced from this nucleotide sequence devi. ated substantially from that of all other ubiquitins ann. lyzed so far-in particular, six of the variant positions were unique for the E, histolytica ubiquitin. We discuss the implications of this finding for the evolutionary history of' both E, hl#tolytica and the ubiquitin system. EXPERIMENTAL I. Cell~E, hixtMj,rir# HM hIMSS trophozoites were 8town ax=nically at 36*C in TY1-$.33 medium [5] with 15% serum, supplemgnted with penicillin (lO0/Jgml "~) and streptomycin .~ulfate (i00 mg.ml'~), The ameba= were harvested in lat¢-IoiPtrithmi¢ growth by chilling on ice and a 10.rain ccnzrifuBation at 400 x g, and washed twice in phos. phate-buffered laiia=. "E~ridmwnl of ubiqultia io amebal protoitJ extractThe ~lis were suspended in phozphatc-bu•wd saline with iodoacctamid¢ (2 raM) to suppress amebal protear¢ activity [6]. and disrupted with a Branson sonifyer (25 puir,¢,~ of 0.5 s at 40.-50 W), The pro~dur¢ for the enrichment of ubiquilin followed that describ¢d in [7] up to and including the ammonium sulfate fractionations. The ubiquitin.comaining material pr~ipitatcd by 80% (w/v) ammonium sulfate was diraoived in 50 mM Tris.HCl, pH 7,4, and subjected to SOS-PAGE and immunoblot analysis, SDS.PAGE and hmmmoblot analj,aisProteins were separated by SDS-PAG E ( 16% acwlamide. 6 M urea) a~ordin= to ~cMngor and yon Ja~ow [8]. and transferred to nitrootllulose or hnmohilon.P membrane by ¢lcciroblotting. Ubiquitia was detected with polyclonal antibodies ag:tin~t SDS-denatured bovine 54Puh/ished hy Etaevter Science Puhlixher$ B, V,

In silico analysis of ubiquitin/ubiquitin-like modifiers and their conjugating enzymes in Entamoeba species

“…Since a detailed analysis of the ubiquitin C-terminal hydrolases from various protozoa has been reported earlier (Ponder and Bogyo 2007) we have excluded these proteins from our analysis. Based on the differences in Ub sequence of Entamoeba from all other characterized Ubs, it has been suggested earlier that the Ub system of Entamoeba may have stalled and one or more functions of the Ub system may have developed later in the evolutionary process (Wöstmann et al 1996;Wöstmann et al 1992). The present genome scale study suggests that the complexity that is observed in higher eukaryotes due to inter-connections between various Ub-Ubls pathways may be present in this early eukaryote as well.…”

EhVps23: A Component of ESCRT-I That Participates in Vesicular Trafficking and Phagocytosis of Entamoeba histolytica

“…In western blot assays, the a-ubiquitin commercial antibody recognized several bands (Figure 5C) corresponding to EhUbiquitin-conjugated proteins. The 10 and 15 kDa bands detected by the antibody exhibited the molecular weight deduced by the amino acids forming the E. histolytica ubiquitin (Wostmann et al, 1992;Arya et al, 2012).…”

Amebic cysteine proteinase 2 (EhCP2) plays either a minor or no role in tissue damage in acute experimental amebic liver abscess in hamsters

“…Sequencing of fraction II of the purified protein revealed that the final product is a mixture of amebic CP2 and ubiquitin(s), which may be one of the forms in which such molecules normally exist in the parasite. We believe that most of the protein in our purified preparation is EhCP2 and that ubiquitin(s) is present in trace amounts, since in SDS-PAGE ubiquitin migrates as an 8.5-kDa band (Wostmann et al 1992), which we never observed despite conducting many runs and even staining with silver.…”