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Abbreviations: AAL, Aleuria aurantia agglutinin or lectin; Dol-P-sugar, a conjugate of Glc or Man to dolichol phosphate; EIC, extracted ion chromatogram; FBS, fetal bovine serum; GPI, glycophosphatidylinositol; GT, glycosyltransferase; HAc, acetic acid; HdH, a disaccharide consisting of a hexose and a deoxy-hexose residue; HFF, human foreskin fibroblasts; hTERT, HFF immortalized with human telomerase reverse transcriptase; H6N2, an oligosaccharide consisting of 6 hexose (Hex) and 2 Nacetylhexosamine (HexNAc) residues; NHEJ, non-homologous end-joining; nLC-MS/MS, nano-liquid chromatography coupled to multi-dimensional mass spectrometry; mAb, monoclonal antibody; pp-αGalNAcT, UDP-GalNAc-dependent polypeptide α-N-acetyl-D-galactosaminyltransferase; Tn antigen, a structure corresponding to αGalNAc-S/T; TSR, thrombospondin type 1 repeats ABSTRACT Infection by the protozoan parasite Toxoplasma gondii is a major health risk owing to its chronic nature, ability to reactivate to cause blindness and encephalitis, and high prevalence in human populations. Like nearly all eukaryotes, Toxoplasma glycosylates many of its proteins and lipids and assembles polysaccharides. Unlike most eukaryotes, Toxoplasma divides and differentiates in vacuoles within host cells. While their glycans resemble canonical models, they exhibit species-specific variations that have inhibited deeper investigations into their roles in parasite biology and virulence. The genome of Toxoplasma encodes a suite of likely glycogenes expected to assemble a range of N-glycans, O-glycans, a C-glycan, GPI-anchors, and polysaccharides, along with their requisite precursors and membrane transporters. To facilitate genetic approaches to investigate the roles of specific glycans, we mapped probable connections between 59 glycogenes, their enzyme products, and the glycans to which they contribute. We adapted a double-CRISPR/Cas9 strategy and a mass spectrometry-based glycomics workflow to test these relationships, and conducted infection studies in fibroblast monolayers to probe cellular functions. Through the validated disruption of 17 glycogenes, we also discovered novel Glc0-2-Man6-GlcNAc2-type N-glycans, GalNAc2-and Glc-Fuc-type O-glycans, and a nuclear O-Fuc type glycan. We describe the guide sequences, disruption constructs, and mutant strains, which are freely available to practitioners for application in the context of the relational map to investigate the roles of glycans in their favorite biological processes.

Glycan biosynthesis relies on nucleotidesugars (NS), abundant metabolites that serve as monosaccharide donors for glycosyltransferases. In vivo, signal-dependent fluctuations in NS levels are required to maintain normal cell physiology and are dysregulated in disease, but how mammalian cells regulate NS levels and pathway flux remains largely uncharacterized. To address this knowledge gap, we examined uridine diphosphate (UDP)galactose 4'-epimerase (GALE), which interconverts two pairs of essential NSs. GALE deletion in human cells triggered major imbalances in its substrate NSs and consequent dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the Fas death receptor. NS dysregulation also directly impacted cell signaling, as GALE -/cells exhibit Fas hypoglycosylation and hypersensitivity to Fas ligand-induced apoptosis. Our results reveal a new role for GALE-mediated NS regulation in supporting death receptor signaling and may have implications for the molecular etiology of illnesses characterized by NS imbalances, including galactosemia and metabolic syndrome. Control (HeLa)GALE -/-(HeLa)

The O-GlcNActransferase (OGT) is localized to the nucleus and cytoplasm where it regulates nucleocytoplasmic proteins by modifying serine and threonine residues with a non-extended monosaccharide, b-N-Acetyl-Glucosamine (O-GlcNAc). With thousands ofknown O-GlcNAcmodifiedproteinsbut only oneOGTencoded in the mammalian genome, a prevailing question is howOGTselects its substrates. Prior work has indicated that theN-terminaltetratricopeptide repeat (TPR) domain of OGT, rather than itsC-terminalcatalytic domain, is responsible forsubcellular targeting andsubstrate selection.An additional impetus for exploring the OGT TPR domain interactome is the fact that missense mutations inOGTassociated with X-linked intellectual disability (XLID) are primarily localized to the TPR domain without substantial impact on activity or stability of the enzyme.Therefore, we adapted theBioIDlabeling method to identify interactors of a TPR-BirA* fusion protein in HeLa cells. We identified 115high confidenceinteractors representing both known and novel O-GlcNAcmodified proteins and OGT interactors. The TPR interactors are highly enriched in processes in which OGT has a known role (e.g. chromatin remodeling, cellular survival of heat stress, circadian rhythm), as well as processesin which OGT has yet to be implicated (e.g. pre-mRNA processing). Importantly,the identified TPR interactors are involved in several disease states but most notably are highly enriched in pathologies featuring intellectual disability.Theseproteinsrepresent candidateinteractors that may underlie the mechanismby which mutations in OGT lead to XLID. Furthermore, the identified interactors provide additional evidence of the importance of the TPR domain for OGT targeting and/or substrate selection.Thus, this defined interactome for the TPR domain of OGT serves as ajumping off point for future researchexploringthe role of OGT, the TPR domain, and its protein interactorsin multiple cellular processes and disease mechanisms, including intellectual disability.

Skp1, a subunit of E3-Skp1/Cullin-1/F-box protein ubiquitin ligases, is uniquely modified in protists by an O 2 -dependent prolyl hydroxylase, which forms the attachment site for a novel pentasaccharide. Mutational studies demonstrate the importance of the core glycan for growth of the parasite Toxoplasma gondii in fibroblasts, but the significance of the non-reducing terminal sugar is unknown. Here we investigated a homolog of glycogenin, an enzyme that can initiate and prime glycogen synthesis in yeast and animals. Gat1 is required for pentasaccharide assembly in cells and catalyzes the addition of an α galactose in 3-linkage to the subterminal α 3-linked glucose residue in vitro. A strong selectivity of Gat1 for Skp1 in extracts is consistent with evidence that Skp1 is the sole target of the glycosyltransferase pathway. gat1-disruption resulted in slow growth indicating the importance of the complete glycan. Molecular dynamics simulations suggested that the full glycan helps organize Skp1 as previously described in the amoebozoan Dictyostelium where a distinct glycosyltransferase assembles a different terminal disaccharide. The crystal structure of Gat1 from the plant pathogen Pythium ultimum confirmed the striking similarity to glycogenin, with differences in the active sites providing an explanation for its distinct substrate preference and regiospecificity. Gat1 also exhibited low α -glucosyltransferase activity like glycogenin, but autoglycosylation was not detected and gat1-disruption revealed no effect on starch accumulation in Toxoplasma. A phylogenetic analysis suggested that Gat1 was a progenitor of glycogenin, and acquired its role in glycogen formation following the ancestral disappearance of the underlying Skp1 glycosyltransferase Glt1 prior to amoebozoan evolution.

Recent studies demonstrated that mutations in B3GNT1, an enzyme proposed to be involved in poly-N-acetyllactosamine synthesis, were causal for congenital muscular dystrophy with hypoglycosylation of α-dystroglycan (secondary dystroglycanopathies). Since defects in the O-mannosylation protein glycosylation pathway are primarily responsible for dystroglycanopathies and with no established O-mannose initiated structures containing a β3 linked GlcNAc known, we biochemically interrogated this human enzyme. Here we report this enzyme is not a β-1,3-Nacetylglucosaminyltransferase with catalytic activity towards β-galactose but rather a β-1,4glucuronyltransferase, designated B4GAT1, towards both αand β-anomers of xylose. The dual-activity LARGE enzyme is capable of extending products of B4GAT1 and we provide experimental evidence that B4GAT1 is the priming enzyme for LARGE. Our results further define the functional O-mannosylated glycan structure and indicate that B4GAT1 is involved in the initiation of the LARGE-dependent repeating disaccharide that is necessary for extracellular matrix protein binding to O-mannosylated α-dystroglycan that is lacking in secondary dystroglycanopathies.

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