This revised, competitive renewal application is based on the observation that medically important protists make truncated Asn-linked (N-glycan) precursors, which contain 1-2 sugars (Giardia and Plasmodium) or 7 sugars (Entamoeba and Trichomonas) rather than the 14 sugars of most animals and fungi. This remarkable finding, which was made in collaboration with my colleague John Samuelson, who is an expert in parasitic protists, has numerous important and exciting consequences. First, while the truncated N-glycan precursors of Entamoeba and Trichomonas are sufficient for N- glycan-dependent quality control (QC) of protein folding, N-glycan-dependent QC is absent from Giardia and Plasmodium. Second, there is positive selection for N-linked glycosylation in eukaryotes with N- glycan-dependent QC. Third, truncated N-glycans of the protists affect the substrate specificity of the oligosaccharyltransferase (OST) that transfers N-glycans from the lipid precursor to the nascent peptide. Fourth, wheat germ agglutinin and Concanavalin A affinity chromatography dramatically enriches the N-glycomes (glycoproteins with N-glycans composed of GlcNAc2) of Giardia and Entamoeba, respectively. Fifth, Giardia has a single nucleotide sugar transporter (NSTs) for UDP-GlcNAc, such that this protist may be used as an alternative to Saccharomyces for testing NSTs. Sixth, Entamoeba and Trichomonas each have unprocessed N-glycans on their surface, which are the target of anti-retroviral lectins. Seventh, Entamoeba makes unique complex N-glycans, in which 1-1,2-linked Gal and then poly- 1,3-linked Glc are added to both arms of biantennary GlcNAcMan3. Eighth, because the N-glycan precursor of Trichomonas lacks Glc, it is puzzling that its membranes have 200 times the dolichol phosphate glucose (Dol-P-Glc) synthase activity of yeast or mammalian membranes. Preliminary studies suggest Trichomonas uses Dol-P-Glc for O-linked glycosylation and for synthesis of novel glycolipids. In light of these and other results, three Specific Aims attempt to answer the following questions:
Specific Aim 1. What are the functions of the very short (GlcNAc and GlcNAc2) N-glycans of Plasmodium and Giardia (40% of the effort)? How do the short N-glycans of these protists affect their OSTs? What glycoproteins compose the Giardia and Plasmodium N-glycome? What is the effect of inhibiting or deleting N-glycosylation in these organisms? Aim 2. What are the functions of the short N-glycan precursors of Entamoeba and Trichomonas (40% of effort)? What are the final N-glycans of Trichomonas, which has glycosyltransferases absent in other protists? What glycoproteins compose the Entamoeba and Trichomonas N-glycome? Can we use anti-retroviral lectins to target the unprocessed N-glycans of Entamoeba and Trichomonas? Aim 3. What are the novel uses of Dol-P-Glc and Dol-P-Man by Trichomonas (20% of effort)? Are Trichomonas homologs of yeast PMTs and mammalian POMTs involved in synthesis of O-glycans from Dol-P-Glc and Dol-P-Man? What are the glycolipid products from these substrates? Lay description: Addition of sugars to proteins is essential for all organisms, and deficiencies in protein glycosylation are associated with human genetic diseases. The focus here is on sugars added to parasites that cause malaria, dysentery, diarrhea, and vaginitis, with the goals of understanding better how these organisms cause disease and how protein glycosylation works in all cells.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Membrane Biology and Protein Processing (MBPP)
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Marino, Pamela
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Boston University
Schools of Dentistry
United States
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Haserick, John R; Leon, Deborah R; Samuelson, John et al. (2017) 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. Mol Cell Proteomics 16:S42-S53
Bandini, Giulia; Haserick, John R; Motari, Edwin et al. (2016) O-fucosylated glycoproteins form assemblies in close proximity to the nuclear pore complexes of Toxoplasma gondii. Proc Natl Acad Sci U S A 113:11567-11572
Chatterjee, Aparajita; Ratner, Daniel M; Ryan, Christopher M et al. (2015) Anti-Retroviral Lectins Have Modest Effects on Adherence of Trichomonas vaginalis to Epithelial Cells In Vitro and on Recovery of Tritrichomonas foetus in a Mouse Vaginal Model. PLoS One 10:e0135340
Samuelson, John; Robbins, Phillips W (2015) Effects of N-glycan precursor length diversity on quality control of protein folding and on protein glycosylation. Semin Cell Dev Biol 41:121-8
Bushkin, G Guy; Motari, Edwin; Carpentieri, Andrea et al. (2013) Evidence for a structural role for acid-fast lipids in oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria. MBio 4:e00387-13
Samuelson, John; Bushkin, G Guy; Chatterjee, Aparajita et al. (2013) Strategies to discover the structural components of cyst and oocyst walls. Eukaryot Cell 12:1578-87
Bushkin, G Guy; Motari, Edwin; Magnelli, Paula et al. (2012) ýý-1,3-glucan, which can be targeted by drugs, forms a trabecular scaffold in the oocyst walls of Toxoplasma and Eimeria. MBio 3:
Samuelson, John; Robbins, Phillips (2011) A simple fibril and lectin model for cyst walls of Entamoeba and perhaps Giardia. Trends Parasitol 27:17-22
Chatterjee, Anirban; Banerjee, Sulagna; Steffen, Martin et al. (2010) Evidence for mucin-like glycoproteins that tether sporozoites of Cryptosporidium parvum to the inner surface of the oocyst wall. Eukaryot Cell 9:84-96
Mitra, Sanghamitra; Cui, Jike; Robbins, Phillips W et al. (2010) A deeply divergent phosphoglucomutase (PGM) of Giardia lamblia has both PGM and phosphomannomutase activities. Glycobiology 20:1233-40

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