Infection with the protozoan parasite Toxoplasma gondii is a major health risk owing to birth defects, its chronic nature, ability to reactivate to cause blindness and encephalitis, and high prevalence in human populations. Unlike most eukaryotes, Toxoplasma propagates in intracellular parasitophorous vacuoles, but like nearly all other eukaryotes, Toxoplasma glycosylates many cellular proteins and lipids and assembles polysaccharides. Toxoplasma glycans resemble those of other eukaryotes, but species-specific variations have prohibited deeper investigations into their roles in parasite biology and virulence. The Toxoplasma genome encodes a suite of likely glycogenes expected to assemble N-glycans, O-glycans, a C-glycan, GPI-anchors, and polysaccharides, along with their precursors and membrane transporters. To investigate the roles of specific glycans in Toxoplasma, here we coupled genetic and glycomics approaches to map the connections between 67 glycogenes, their enzyme products, the glycans to which they contribute, and cellular functions. We applied a double-CRISPR/Cas9 strategy, in which two guide RNAs promote replacement of a candidate gene with a resistance gene; adapted MS-based glycomics workflows to test for effects on glycan formation; and infected fibroblast monolayers to assess cellular effects. By editing 17 glycogenes, we discovered novel Glc 0-2-Man 6-GlcNAc 2-type N-glycans, a novel HexNAc-GalNAc-mucin-type O-glycan, and Tn-antigen; identified the glycosyltransferases for assembling novel nuclear O-Fuc-type and cell surface Glc-Fuc-type O-glycans; and showed that they are important for in vitro growth. The guide sequences, editing constructs, and mutant strains are freely available to researchers to investigate the roles of glycans in their favorite biological processes. Toxoplasma gondii is a worldwide, obligate intracellular apicomplexan parasite that can infect most nucleated cells of warm-blooded animals (1), with up to 80% of some human populations being seropositive (2). Toxoplasmosis, the disease caused by Toxoplasma, is associated with encephalitis and blindness in individuals whose parasites are reactivated, as can occur in AIDS and other immunosuppressed patients (3). In utero infections can cause mental retardation, blindness, and death (4). Toxoplasma is transmitted by digesting parasites from feline feces (as oocysts) or undercooked meat (as tissue cysts). Once in the host, parasites convert to the tachyzoite form that disseminates to peripheral tissues (e.g. brain, retina, and muscle). The resulting immune response and/or drugs can control tachyzoite replication, but the parasite survives by encysting into slowly growing bradyzoites. Sporadically, burst of cysts allows the parasites to convert to tachyzoites, whose unchecked growth results in cell and tissue damage (5, 6). Currently, no Toxoplasma vaccine exists, anti-toxoplasmosis drugs have severe side effects, and resistance is developing to these drugs (7-11). As individuals remain infected for life, new anti-Toxoplasma drugs a...

O 2 sensing in diverse protozoa depends on the prolyl 4 hydroxylation of Skp1 and modification of the resulting hydroxyproline with a series of five sugars. In yeast, plants, and animals, Skp1 is associated with F-box proteins. The Skp1-F-box protein heterodimer can, for many F-box proteins, dock onto cullin-1 en route to assembly of the Skp1-cullin-1-F-box protein-Rbx1 subcomplex of E3 SCF Ub ligases. E3 SCF Ub ligases conjugate Lys48-polyubiquitin chains onto targets bound to the substrate receptor domains of F-box proteins, preparing them for recognition by the 26S proteasome. In the social amoeba Dictyostelium, we found that O 2 availability was rate-limiting for the hydroxylation of newly synthesized Skp1. To investigate the effect of reduced hydroxylation, we analyzed knockout mutants of the Skp1 prolyl hydroxylase and each of the Skp1 glycosyltransferases. Proteomic analysis of co-immunoprecipitates showed that wild-type cells able to fully glycosylate Skp1 had a greater abundance of an SCF complex containing the cullin-1 homolog CulE and FbxD, a newly described WD40-type F-box protein, than the complexes that predominate in cells defective in Skp1 hydroxylation or glycosylation. Similarly, the previously described FbxA-Skp1CulA complex was also more abundant in glycosylation-competent cells. The CulE interactome also included higher levels of proteasomal regulatory particles when Skp1 was glycosylated, suggesting increased activity consistent with greater association with F-box proteins. Finally, the interactome of FLAG-FbxD was modified when it harbored an F-box mutation that compromised Skp1 binding, consistent with an effect on the abundance of potential substrate proteins. We Timely protein degradation is a cornerstone of cell cycling and the regulation of numerous physiological and developmental processes. Eukaryotes have evolved an extensive array of polyubiquitination enzymes to tag proteins on a proteinby-protein basis as a recognition marker for degradation in the 26S proteasome. The cullin-RING ubiquitin ligases (CRLs) 1 are a prominent subgroup of these enzymes (1) and consist of an E3 architecture that includes a substrate receptor, an adaptor (in most cases), the cullin scaffold, the RING protein, and an exchangeable E2 ubiquitin donor that has been charged with ubiquitin (Ub) by an E1 enzyme. The first discovered and still prototypic example is the CRL1 class (2), also referred to as SCF on account of the names of its founding subunits, Skp1, cullin-1, and F-box proteins (FBPs). The CRL1 (or SCF) complexes utilize FBPs as substrate receptors, Skp1 as the adaptor linking the FBP to the N-terminal region of cullin-1 (Cul1), and Rbx1 as the RING protein that tethers the E2 Ub donor to the Cul1 C-terminal region (see Fig. 2B). CRL1s can be activated by neddylation of Cul1 by a Nedd8-specific E2, which mobilizes Rbx1 to afford rotational flexibility of the E2 and displaces the inhibitor Cand1, permitting docking of the Skp1-FBP heterodimer (3-5). Deneddylation mediated by the eight-subunit COP...

Cell-surface engineering strategies that permit long-lived display of well-defined, functionally active molecules are highly attractive for eliciting desired cellular responses and for understanding biological processes. Current methodologies for the exogenous introduction of synthetic biomolecules often result in short-lived presentations, or require genetic manipulation to facilitate membrane attachment. Herein, we report a cell-surface engineering strategy that is based on the use of a CMP-Neu5Ac derivative that is modified at C-5 by a bifunctional entity composed of a complex synthetic heparan sulfate (HS) oligosaccharide and biotin. It is shown that recombinant ST6GAL1 can readily transfer the modified sialic acid to N-glycans of glycoprotein acceptors of living cells resulting in long-lived display. The HS oligosaccharide is functionally active, can restore protein binding, and allows activation of cell signaling events of HS-deficient cells. The cell-surface engineering methodology can easily be adapted to any cell type and is highly amenable to a wide range of complex biomolecules.

The post-translational glycosylation of select proteins by O-linked mannose (O-mannose or O-man) is a conserved modification from yeast to humans and has been shown to be necessary for proper development and growth. The most well studied O-mannosylated mammalian protein is α-dystroglycan (α-DG). Hypoglycosylation of α-DG results in varying severities of congenital muscular dystrophies, cancer progression and metastasis, and inhibited entry and infection of certain arenaviruses. Defects in the gene products responsible for post-translational modification of α-DG, primarily glycosyltransferases, are the basis for these diseases. The multitude of clinical phenotypes resulting from defective O-mannosylation highlights the biomedical significance of this unique modification. Elucidation of the various O-mannose biosynthetic pathways is imperative to understanding a broad range of human diseases and for the development of novel therapeutics. In this review, we will focus on recent discoveries delineating the various enzymes, structures and functions associated with O-mannose-initiated glycoproteins. Additionally, we discuss current gaps in our knowledge of mammalian O-mannosylation, discuss the evolution of this pathway, and illustrate the utility and limitations of model systems to study functions of O-mannosylation.

Multiple glycosyltransferases are essential for the proper modification of alpha-dystroglycan, as mutations in the encoding genes cause congenital/limb-girdle muscular dystrophies. Here we elucidate further the structure of an O-mannose-initiated glycan on alpha-dystroglycan that is required to generate its extracellular matrix-binding polysaccharide. This functional glycan contains a novel ribitol structure that links a phosphotrisaccharide to xylose. ISPD is a CDP-ribitol (ribose) pyrophosphorylase that generates the reduced sugar nucleotide for the insertion of ribitol in a phosphodiester linkage to the glycoprotein. TMEM5 is a UDP-xylosyl transferase that elaborates the structure. We demonstrate in a zebrafish model as well as in a human patient that defects in TMEM5 result in muscular dystrophy in combination with abnormal brain development. Thus, we propose a novel structure—a ribitol in a phosphodiester linkage—for the moiety on which TMEM5, B4GAT1, and LARGE act to generate the functional receptor for ECM proteins having LG domains.DOI: http://dx.doi.org/10.7554/eLife.14473.001