The Antiretroviral Lectin Cyanovirin-N Targets Well-Known and Novel Targets on the Surface of Entamoeba histolytica Trophozoites

Abstract

Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, has a truncated Asn-linked glycan (N-glycan) precursor composed of seven sugars (Man 5 GlcNAc 2 ). Here, we show that glycoproteins with unmodified N-glycans are aggregated and capped on the surface of E. histolytica trophozoites by the antiretroviral lectin cyanovirin-N and then replenished from large intracellular pools. Cyanovirin-N cocaps the Gal/GalNAc adherence lectin, as well as glycoproteins containing O-phosphodiester-linked glycans recognized by an anti-proteophosphoglycan monoclonal antibody. Cyanovirin-N inhibits phagocytosis by E. histolytica trophozoites of mucin-coated beads, a surrogate assay for amebic virulence. For technical reasons, we used the plant lectin concanavalin A rather than cyanovirin-N to enrich secreted and membrane proteins for mass spectrometric identification. E. histolytica glycoproteins with occupied N-glycan sites include Gal/GalNAc lectins, proteases, and 17 previously hypothetical proteins. The latter glycoproteins, as well as 50 previously hypothetical proteins enriched by concanavalin A, may be vaccine targets as they are abundant and unique. In summary, the antiretroviral lectin cyanovirin-N binds to well-known and novel targets on the surface of E. histolytica that are rapidly replenished from large intracellular pools.Entamoeba histolytica causes amebic dysentery and liver abscess in the developing world (10,20,29). We are interested in E. histolytica glycoproteins containing Asn-linked glycans (Nglycans) for numerous reasons. E. histolytica makes an N-glycan precursor that contains 7 sugars (Man 5 GlcNAc 2 -PP-dolichol) rather than 14 sugars (Glc 3 Man 9 GlcNAc 2 -PP-dolichol) made by most animals, plants, and fungi (21,31,44). E. histolytica N-glycans are used for quality control of glycoprotein folding in the endoplasmic reticulum (ER) lumen, and there is positive selection for sites of N-linked glycosylation in secreted and membrane proteins of E. histolytica (5,11,53).Unprocessed Man 5 GlcNAc 2 , by far the most abundant E. histolytica N-glycan, is present on the plasma membrane and vesicular membranes (31). The antiretroviral lectin cyanovirin-N, which is specific for ␣-1,2-linked mannose present on unprocessed N-glycans, binds E. histolytica N-glycans and forms aggregates or caps on the surface of E. histolytica trophozoites (1,25,31,44,45). E. histolytica glycoproteins are also capped by the plant lectin concanavalin A (ConA), which has a broader carbohydrate specificity (mannose and glucose) than cyanovirin- N (3, 16, 18, 19). Heavy subunits of the Gal/GalNAc lectin, the most important E. histolytica vaccine candidate, have 7 to 10 potential sites for N-linked glycosylation (32,39,43). Inhibition of N-glycan synthesis results in Gal/GalNAc lectins that are unable to bind to sugars on host epithelial cells.Carbohydrates appear to be an important target on the surface of E. histolytica as anti-proteophosphoglycan (PPG) monoclonal antibodies bind to O-phosphodiester-linked glycans...

Proteomic Analysis of the Cyst Stage of Entamoeba histolytica

“…Two LC-MS/MS proteomic datasets were available for the trophozoite stage of E. histolytica comprising a total of 1825 proteins (as of November 30, 2011) [29], [30]. There was also available an EST database for E. histolytica trophozoite comprising a total of 1223 proteins (as of October 1, 2011; from a total of 20,812 public entries at http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summary.html).…”

“…Since the total number of proteins identified in the naturally-occurring cyst samples was small, we asked how unique these proteins are in comparison with the publicly available protein datasets in AmoebaDB ( http://amoebadb.org/amoeba/ ). Two LC-MS/MS proteomic datasets were available for the trophozoite stage of E. histolytica comprising a total of 1825 proteins (as of November 30, 2011) [29] , [30] . There was also available an EST database for E. histolytica trophozoite comprising a total of 1223 proteins (as of October 1, 2011; from a total of 20,812 public entries at http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summary.html ).…”

Asparagine-Linked Glycans of Cryptosporidium parvum Contain a Single Long Arm, Are Barely Processed in the Endoplasmic Reticulum (ER) or Golgi, and Show a Strong Bias for Sites with Threonine

“…However, we observed a large difference in the rate of occupancy of potential N-glycosylation sequons. Despite the 5:3 ratio of Thr (100) and Ser (61) in the second position relative to Asn, the number of occupied N-glycan sites overwhelmingly (9:1) favors Thr (35) over Ser (4), as shown in the WebLogo in Fig. 6 and the data in Table I (46).…”

“…The binding of the anti-retroviral lectin cyanovirin-N to C. parvum strongly suggested that the parasite contains a high mannose N-glycan (23,24). Cyanovirin-N also binds to Entamoeba and Trichomonas, each of which builds its N-glycans from a precursor composed of Man 5 GlcNAc 2 (61)(62)(63). The N-glycan profile of C. parvum differs from those of the other parasites in the relative abundance of GlcMan 5 GlcNAc 2 and the absence of mannosidase products (Man 4 GlcNAc 2 and Man 3 GlcNAc 2 ) and/or hybrid and complex N-glycans, which contain LacNAc arms (Trichomonas) or galactose capped with Glc (Entamoeba) (63,64).…”

Effects of N-glycan precursor length diversity on quality control of protein folding and on protein glycosylation

“…Many N -glycans, however, remain unmodified by ER mannosidases and Golgi glycosyltransferases and so are recognized by lectins that bind to the single 1,3-linked mannose arm of Man 5 GlcNAc 2 (e.g. cyanovirin- N binding to Entamoeba , Trichomonas , Toxoplasma , and Cryptosporidium ) [29, 42, 43]. Similarly, wheat germ agglutinin or Griffonia simplicifolia lectin II binds to unmodified GlcNAc 2 of Giardia and Plasmodium [28, 29].…”