The Drosophila Neurally Altered Carbohydrate Mutant Has a Defective Golgi GDP-fucose Transporter

Geisler, Christoph; Kotu, Varshika; Sharrow, Mary; Rendić, Dubravko; Pöltl, Gerald; Tiemeyer, Michael; Wilson, Iain B. H.; Jarvis, Donald L.

doi:10.1074/jbc.m112.379313

Cited by 11 publications

(9 citation statements)

References 93 publications

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“…In previous work, knocking out an individual GDP-Fuc transporter only caused mild phenotypes in flies and in mice, likely due to functional redundancy between transporters (Geisler et al, 2012; Hellbusch et al, 2007; Ishikawa et al, 2010; Lu et al, 2010). We reasoned that over-expressing a transporter might provide a more robust means to perturb fucosylation.…”

Section: Resultsmentioning

confidence: 91%

Negative feedback regulation of Wnt signaling via N-linked fucosylation in zebrafish

Feng

Jiang

et al. 2014

Developmental Biology

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Summary L-fucose, a monosaccharide widely distributed in eukaryotes and certain bacteria, is a determinant of many functional glycans that play central roles in numerous biological processes. The molecular mechanism, however, by which fucosylation mediates these processes remains largely elusive. To study how changes in fucosylation impact embryonic development, we up-regulated N-linked fucosylation via over-expression of a key GDP-Fucose transporter, Slc35c1, in zebrafish. We show that Slc35c1 overexpression causes elevated N-linked fucosylation and disrupts embryonic patterning in a transporter activity dependent manner. We demonstrate that patterning defects associated with enhanced N-linked fucosylation are due to diminished canonical Wnt signaling. Chimeric analyses demonstrate that elevated Slc35c1 expression in receiving cells decreases the signaling range of Wnt8a during zebrafish embryogenesis. Moreover, we provide biochemical evidence that this decrease is associated with degradation of Wnt8 ligand and elevated Lrp6 coreceptor, which we show are both substrates for N-linked fucosylation in zebrafish embryos. Strikingly, slc35c1 expression is regulated by canonical Wnt signaling. These results suggest that Wnt limits its own signaling activity in part via up-regulation of a transporter, slc35c1 that promotes terminal fucosylation and thereby limits Wnt activity.

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Section: Resultsmentioning

confidence: 91%

Negative feedback regulation of Wnt signaling via N-linked fucosylation in zebrafish

Feng

Jiang

et al. 2014

Developmental Biology

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“…2010; Geisler et al . 2012). As Gal and GalNAc are often added at or near nonreducing ends of glycans, Csat, a UDP-Gal/UDP-GalNAc transporter (Segawa et al .…”

Section: Resultsmentioning

confidence: 99%

“…6, Table 3). Da6Fut/FucT6 adds a fucose moiety to the core region of N-linked glycans via a1,6linkage (Paschinger et al 2005), whereas Gfr/Nac transports GDP-fucose to the Golgi lumen for fucose addition, including a1,3-fucosylation of the core Nlinked glycans (Ishikawa et al 2010;Geisler et al 2012). As Gal and GalNAc are often added at or near nonreducing ends of glycans, Csat, a UDP-Gal/UDP-GalNAc transporter (Segawa et al 2002), may be involved in terminal glycosylation.…”

Section: Gene Silencing In a Spatiotemporally Regulated Mannermentioning

confidence: 99%

Phenotype‐based clustering of glycosylation‐related genes by RNAi‐mediated gene silencing

et al. 2015

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Glycan structures are synthesized by a series of reactions conducted by glycosylation-related (GR) proteins such as glycosyltransferases, glycan-modifying enzymes, and nucleotide-sugar transporters. For example, the common core region of glycosaminoglycans (GAGs) is sequentially synthesized by peptide-O-xylosyltransferase, β1,4-galactosyltransferase I, β1,3-galactosyltransferase II, and β1,3-glucuronyltransferase. This raises the possibility that functional impairment of GR proteins involved in synthesis of the same glycan might result in the same phenotypic abnormality. To examine this possibility, comprehensive silencing of genes encoding GR and proteoglycan core proteins was conducted in Drosophila. Drosophila GR candidate genes (125) were classified into five functional groups for synthesis of GAGs, N-linked, O-linked, Notch-related, and unknown glycans. Spatiotemporally regulated silencing caused a range of malformed phenotypes that fell into three types: extra veins, thick veins, and depigmentation. The clustered phenotypes reflected the biosynthetic pathways of GAGs, Fringe-dependent glycan on Notch, and glycans placed at or near nonreducing ends (herein termed terminal domains of glycans). Based on the phenotypic clustering, CG33145 was predicted to be involved in formation of terminal domains. Our further analysis showed that CG33145 exhibited galactosyltransferase activity in synthesis of terminal N-linked glycans. Phenotypic clustering, therefore, has potential for the functional prediction of novel GR genes.

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“…Key to this were the definition of a relevant core α1,3-fucosyltransferase (FucTA) in D. melanogaster , RNAi knock-down of FucTA in an insect neural cell line, overexpression of FucTA in D. melanogaster embyros and the proof that the anti-HRP-defective nac mutant was due to a point mutation in the Golgi GDP-Fuc transporter (Fabini et al, 2001, Rendić et al, 2006, Rendic et al, 2010, Geisler et al, 2012). In comparison to the fruit fly, there has been no data to date regarding anti-HRP epitopes in mosquitoes; previous studies were limited primarily to lectin blots and histochemistry with actual mass spectrometric N-glycan analyses performed only on a couple of glycoproteins from mosquitoes or mosquito cell lines (Wilkins and Billingsley, 2001, Rudin and Hecker, 1989, Andrews et al, 1997, Li and Li, 2005, Crispin et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

The fucomic potential of mosquitoes: Fucosylated N-glycan epitopes and their cognate fucosyltransferases

Kurz

King

Dinglasan

et al. 2016

Insect Biochemistry and Molecular Biology

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Fucoconjugates are key mediators of protein-glycan interactions in prokaryotes and eukaryotes. As examples, N-glycans modified with the non-mammalian core α1,3-linked fucose have been detected in various organisms ranging from plants to insects and are immunogenic in mammals. The rabbit polyclonal antibody raised against plant horseradish peroxidase (anti-HRP) is able to recognize the α1,3-linked fucose epitope and is also known to specifically stain neural tissues in the fruit fly Drosophila melanogaster. In this study, we have detected and localized the anti-HRP cross-reactivity in another insect species, the malaria mosquito vector Anopheles gambiae. We were able to identify and structurally elucidate fucosylated N-glycans including core mono- and difucosylated structures (responsible for anti-HRP cross reactivity) as well as a Lewis-type antennal modification on mosquito anionic N-glycans by applying enzymatic and chemical treatments. The three mosquito fucosyltransferase open reading frames (FucT6, FucTA and FucTC) required for the in vivo biosynthesis of the fucosylated N-glycan epitopes were identified in the Anopheles gambiae genome, cloned and recombinantly expressed in Pichia pastoris. Using a robust MALDI-TOF MS approach, we characterised the activity of the three recombinant fucosyltransferases in vitro and demonstrate that they share similar enzymatic properties as compared to their homologues from D. melanogaster and Apis mellifera. Thus, not only do we confirm the neural reactivity of anti-HRP in a mosquito species, but also demonstrate enzymatic activity for all its α1,3- and α1,6-fucosyltransferase homologues, whose specificity matches the results of glycomic analyses.

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The Drosophila Neurally Altered Carbohydrate Mutant Has a Defective Golgi GDP-fucose Transporter

Cited by 11 publications

References 93 publications

Negative feedback regulation of Wnt signaling via N-linked fucosylation in zebrafish

Negative feedback regulation of Wnt signaling via N-linked fucosylation in zebrafish

Phenotype‐based clustering of glycosylation‐related genes by RNAi‐mediated gene silencing

The fucomic potential of mosquitoes: Fucosylated N-glycan epitopes and their cognate fucosyltransferases

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