Unique Asn-linked Oligosaccharides of the Human Pathogen Entamoeba histolytica

Abstract

N-Glycans of Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, are of great interest for multiple reasons. E. histolytica makes an unusual truncated N-glycan precursor (Man 5 GlcNAc 2 ), has few nucleotide sugar transporters, and has a surface that is capped by the lectin concanavalin A. Here, biochemical and mass spectrometric methods were used to examine N-glycan biosynthesis and the final N-glycans of E. histolytica with the following conclusions. Unprocessed Man 5 GlcNAc 2 , which is the most abundant E. histolytica N-glycan, is aggregated into caps on the surface of E. histolytica by the N-glycan-specific, anti-retroviral lectin cyanovirin-N. Glc 1 Man 5 GlcNAc 2 , which is made by a UDP-Glc: glycoprotein glucosyltransferase that is part of a conserved N-glycan-dependent endoplasmic reticulum quality control system for protein folding, is also present in mature N-glycans. A swainsonine-sensitive ␣-mannosidase trims some N-glycans to biantennary Man 3 GlcNAc 2 . Complex N-glycans of E. histolytica are made by the addition of ␣1,2-linked Gal to both arms of small oligomannose glycans, and Gal residues are capped by one or more Glc. In summary, E. histolytica N-glycans include unprocessed Man 5 GlcNAc 2 , which is a target for cyanovirin-N, as well as unique, complex N-glycans containing Gal and Glc.Entamoeba histolytica is the protist (single cell eukaryote) that causes millions of cases of amebic dysentery and liver abscess in regions where its fecal-oral spread cannot be prevented (1, 2). Asn-linked glycans (N-glycans) of E. histolytica are of great interest for seven reasons.First, E. histolytica is missing many of the glycosyltransferases that make lipid-linked precursors to N-glycans and so makes a Man 5 GlcNAc 2 -PP-dolichol rather than Glc 3 Man 9 GlcNAc 2 -PPdolichol, which is present in most animals, plants, and fungi (3, 4). Second, E. histolytica N-glycans contribute to the quality control of protein folding in the endoplasmic reticulum (ER) 2 (4 -6). In particular, E. histolytica has UDP-Glc:glycoprotein glucosyltransferase, calreticulin, glucosidase II, and ERGIC-53, which are the essential components of an N-glycan-dependent quality control system (4 -6).Third, the E. histolytica oligosaccharyltransferase (OST), which transfers N-glycans from the dolichol pyrophosphatelinked precursor to Asn on the nascent peptide in the lumen of the ER is composed of four subunits rather than seven or eight subunits found in metazoan, fungi, and plants (7). As a result, the E. histolytica OST has different kinetics than the OSTs of higher eukaryotes (8). As well, the E. histolytica OST prefers to transfer N-glycans, which resemble its own (Man 5 GlcNAc 2 ), rather than the longer N-glycans of metazoa and fungi (Glc 3 Man 9 GlcNAc 2 ) (8).Fourth, E. histolytica has a limited set of nucleotide sugar transporters (UDP-Gal and UDP-Glc), which transport activated sugars into the lumen of the ER and Golgi (9). UDP-Gal and UDP-Glc are used to make unique O-phosphodiesterlinked glycans ...

Changes in the N -Glycome, Glycoproteins with Asn-Linked Glycans, of Giardia lamblia with Differentiation from Trophozoites to Cysts

“…Fourth, WGA more strongly labeled the intracellular vesicles than the plasma membrane. This result is similar to our experience with antiretroviral lectin cyanovirin-N binding to Entamoeba N-glycans, which is greater on the internal membranes of permeabilized trophozoites than on the surface (29). Fifth, WGA bound to the same acidified vesicles in the body of Giardia trophozoites that accumulate LysoTracker (Fig.…”

“…In contrast, the results here, which focus on N-glycans that are added in the ER lumen and do not appear to be subsequently modified, shed little light on glycosylation in the Golgi apparatus and do not address other functions of the Golgi apparatus (29,42,45,49).…”

cis-Prenyltransferase: New Insights into Protein Glycosylation, Rubber Synthesis, and Human Diseases

“…lack orthologs of cisPTs. Because Entamoeba use dolichol in glycosylation reactions, the lack of a homolog of cis-PT could be explained by an alternative enzyme or co-opting dolichol from the host (34,61).…”

Strategies To Discover the Structural Components of Cyst and Oocyst Walls

“…Cryptococcus is covered by a carbohydrate capsule that is important for pathogenesis (65). While cyst walls of Entamoeba and oocyst walls of Toxoplasma and Cryptosporidium also contain proteins with N-glycans, the mannose chains of these parasites are not nearly as long as those in fungi (47,48,60). The most abundant carbohydrate on Entamoeba cysts is a polymer of ␣-1,6-linked glucose similar to that of bacterial dextran but made by distinct enzymes (51,61).…”

“…Proteins may then be released with trypsin and analyzed with mass spectrometry, using whole-genome sequences of each parasite to predict tryptic peptides (10,51). N-glycans released with peptide:N-glycanaseF (PNGaseF) and O-glycans released by ␤-elimination may be digested with glycohydrolases and identified by gel filtration or mass spectrometry (48,51,60).…”