Distinct
            <i>C-</i>
            mannosylation of netrin receptor thrombospondin type 1 repeats by mammalian DPY19L1 and DPY19L3

Shcherbakova, Aleksandra; Tiemann, Birgit; Buettner, Falk F. R.; Bakker, Hans

doi:10.1073/pnas.1613165114

Cited by 73 publications

(100 citation statements)

References 42 publications

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“…5C), but it is unclear whether this affects C-␣ManT activity. The fitness score (Ϫ2.4) indicates importance for growth, potentially related to quality control of secretion of the proteins carrying TSRs, and possibly in concert with Glc-Fuc-modifications (69). Because there is no method known to release C-Man, monitoring the outcome will depend on glycopeptide analyses beyond the scope of this project.…”

Section: Other Glycogenes Associated With the Secretory Pathwaymentioning

confidence: 99%

CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology

Gas-Pascual

Ichikawa

Sheikh

et al. 2019

Journal of Biological Chemistry

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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...

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Section: Other Glycogenes Associated With the Secretory Pathwaymentioning

confidence: 99%

CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology

Gas-Pascual

Ichikawa

Sheikh

et al. 2019

Journal of Biological Chemistry

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“…TSRs glycosylation is carried out in the endoplasmic reticulum (22,26), and in studies performed in mammalian cell and Caenorhabditis elegans, defects in both C-mannosylation and O-fucosylation have been shown to affect protein stabilization and secretion, albeit with protein-to-protein variability (19,23,(27)(28)(29)(30)(31). A knockout of pofut2 is embryonically lethal in mice (31).…”

mentioning

confidence: 99%

O-Fucosylation of thrombospondin-like repeats is required for processing of microneme protein 2 and for efficient host cell invasion by Toxoplasma gondii tachyzoites

Bandini

Leon

Hoppe

et al. 2019

Journal of Biological Chemistry

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Toxoplasma gondii is an intracellular parasite that causes disseminated infections that can produce neurological damage in fetuses and immunocompromised individuals. Microneme protein 2 (MIC2), a member of the thrombospondin-related anonymous protein (TRAP) family, is a secreted protein important for T. gondii motility, host cell attachment, invasion, and egress. MIC2 contains six thrombospondin type I repeats (TSRs) that are modified by C-mannose and O-fucose in Plasmodium spp. and mammals. Here, using MS analysis, we found that the four TSRs in T. gondii MIC2 with protein O-fucosyltransferase 2 (POFUT2) acceptor sites are modified by a dHexHex disaccharide, whereas Trp residues within three TSRs are also modified with C-mannose. Disruption of genes encoding either POFUT2 or the putative GDP-fucose transporter (NST2) resulted in loss of MIC2 O-fucosylation, as detected by an antibody against the GlcFuc disaccharide, and in markedly reduced cellular levels of MIC2. Furthermore, in 10 -15% of the ⌬pofut2 or ⌬nst2 vacuoles, MIC2 accumulated earlier in the secretory pathway rather than localizing to micronemes. Dissemination of tachyzoites in human foreskin fibroblasts was reduced for these knockouts, which both exhibited defects in attachment to and invasion of host cells comparable with the ⌬mic2 phenotype. These results, indicating that O-fucosylation of TSRs is required for efficient processing of MIC2 and for normal parasite invasion, are consistent with the recent demonstration that Plasmodium falciparum ⌬pofut2 strain has decreased virulence and also support a conserved role for this glycosylation pathway in quality control of TSR-containing proteins in eukaryotes.

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“…Moreover, Shcherbakova et al . demonstrated that murine DPY19L1 and DPY19L3 are C ‐mannosyltransferases of the netrin receptor UNC5A .…”

Section: Introductionmentioning

confidence: 99%

“…Recently, we first reported human DPY19L3 to be a C-mannosyltransferase of R-spondin1 (Rspo1), which harbors the C-mannosylation consensus sequence-Trp 156 -Gly-Pro-Cys-in its TSR [13]. Moreover, Shcherbakova et al demonstrated that murine DPY19L1 and DPY19L3 are C-mannosyltransferases of the netrin receptor UNC5A [14].…”

Section: Introductionmentioning

confidence: 99%

Topological analysis of DPY19L3, a human C‐mannosyltransferase

et al. 2018

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C-mannosylation is a rare type of protein glycosylation, the functions and mechanisms of which remain unclear. Recently, we identified DPY19L3 as a C-mannosyltransferase of R-spondin1 in human cells. DPY19L3 is predicted to be a multipass transmembrane protein that localizes to the endoplasmic reticulum (ER); however, its structure is undetermined. In this study, we propose a topological structure of DPY19L3 by in silico analysis and experimental methods such as redox-sensitive luciferase assay and introduction of N-glycosylation sites, suggesting that DPY19L3 comprises 11 transmembrane regions and two re-entrant loops with the N- and C-terminal ends facing the cytoplasm and ER lumen, respectively. Furthermore, DPY19L3 has four predicted N-glycosylation sites, and we have demonstrated that DPY19L3 is N-glycosylated at Asn and Asn but not Asn and Asn , supporting our topological model. By mass spectrometry, we measured the C-mannosyltransferase activity of N-glycosylation-defective mutants of DPY19L3 and isoform2, a splice variant, which lacks the C-terminal luminal region of DPY19L3. Isoform2 does not possess C-mannosyltransferase activity, indicating the importance of the C-terminal region; however, N-glycosylations of DPY19L3 do not have any roles for its enzymatic activity. These novel findings on DPY19L3 provide important insights into the mechanism of C-mannosylation.

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Distinct C- mannosylation of netrin receptor thrombospondin type 1 repeats by mammalian DPY19L1 and DPY19L3

Cited by 73 publications

References 42 publications

CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology

CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology

O-Fucosylation of thrombospondin-like repeats is required for processing of microneme protein 2 and for efficient host cell invasion by Toxoplasma gondii tachyzoites

Topological analysis of DPY19L3, a human C‐mannosyltransferase

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