Identification of a UDP-GlcNAc:Skp1-Hydroxyproline GlcNAc-transferase in the Cytoplasm of Dictyostelium

Teng-umnuay, Patana; Wel, Hanke van der; West, Christopher M.

doi:10.1074/jbc.274.51.36392

Cited by 29 publications

(58 citation statements)

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“…This is ϳ2 orders of magnitude higher than that of pp ␣-GlcNAc-T1 (23), the cytoplasmic GlcNAc-Tase that modifies Skp1, but consistent with values reported for Golgi glycosyltransferases (9). The V max of the enzyme calculated from these data was ϳ20 mol/mg of protein/min, which dramatically exceeds the value for pp ␣-GlcNAc-T1; however, this enzyme has not been assayed with a native acceptor (23). Therefore, cis4c(gntB) encodes a pp GlcNAc-Tase that is able to modify, in vitro, peptides from the same proteins the O-glycosylation of which depends on this gene in vivo.…”

Section: Figmentioning

confidence: 56%

“…Recently, the gene that encodes the enzyme that transfers GlcNAc to a hydroxyproline residue in the cytoplasmic/nuclear protein Skp1 of Dictyostelium was identified (23,24). Although this linkage involves a distinct acceptor hydroxyamino acid and the enzyme resides in the cytoplasm rather than the Golgi apparatus, its sequence exhibits homology with those of the animal pp ␣-GalNAc-Tases (25,26).…”

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confidence: 99%

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Initiation of Mucin-type O-Glycosylation in Dictyostelium Is Homologous to the Corresponding Step in Animals and Is Important for Spore Coat Function

Wang

Metcalf

Wel

et al. 2003

Journal of Biological Chemistry

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Like animal cells, many unicellular eukaryotes modify mucin-like domains of secretory proteins with multiple O-linked glycans. Unlike animal mucin-type glycans, those of some microbial eukaryotes are initiated by ␣-linked GlcNAc rather than ␣-GalNAc. Based on sequence similarity to a recently cloned soluble polypeptide hydroxyproline GlcNAc-transferase that modifies Skp1 in the cytoplasm of the social ameba Dictyostelium, we have identified an enzyme, polypeptide ␣-N-acetylglucosaminyltransferase (pp ␣-GlcNAc-T2), that attaches GlcNAc to numerous secretory proteins in this organism. Unlike the Skp1 GlcNAc-transferase, pp ␣-GlcNAc-T2 is predicted to be a type 2 transmembrane protein. A highly purified, soluble, recombinant fragment of pp ␣-GlcNAc-T2 efficiently transfers GlcNAc from UDP-GlcNAc to synthetic peptides corresponding to mucin-like domains in two proteins that traverse the secretory pathway. pp ␣-GlcNAc-T2 is required for addition of GlcNAc to peptides in cell extracts and to the proteins in vivo. Mass spectrometry and Edman degradation analyses show that pp ␣-GlcNAc-T2 attaches GlcNAc in ␣-linkage to the Thr residues of all the synthetic mucin repeats. pp ␣-GlcNAc-T2 is encoded by the previously described modB locus defined by chemical mutagenesis, based on sequence analysis and complementation studies. This finding establishes that the many phenotypes of modB mutants, including a permeability defect in the spore coat, can now be ascribed to defects in mucin-type O-glycosylation. A comparison of the sequences of pp ␣-GlcNAc-T2 and the animal pp ␣-GalNAc-transferases reveals an ancient common ancestry indicating that, despite the different N-acetylhexosamines involved, the enzymes share a common mechanism of action.

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Section: Figmentioning

confidence: 56%

mentioning

confidence: 99%

Initiation of Mucin-type O-Glycosylation in Dictyostelium Is Homologous to the Corresponding Step in Animals and Is Important for Spore Coat Function

Wang

Metcalf

Wel

et al. 2003

Journal of Biological Chemistry

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“…These sequences are not similar enough to suggest that they are FT85 orthologs. Another enzyme in the Skp1 glycosylation pathway, the Skp1 GlcNAcTase (8), is also related to Family 2 GTases in its NRD2 domain. 3 Role in Cell Physiology-Glycosylation is important for nuclear accumulation of Skp1 based on immunofluorescence studies on mutant Skp1s, and either the native pentasaccharide or the disaccharide produced in GDP-Fuc Ϫ mutant cells is sufficient for localization (4).…”

Section: Discussionmentioning

confidence: 99%

“…Incorporation was measured by liquid scintillation counting of trichloroacetic acid precipitates or counting of the Skp1 band after SDS-PAGE, as described previously (8).…”

Section: Methodsmentioning

confidence: 99%

“…2 Two of the GTases, the GlcNAcTase that adds the first sugar and the ␣1,2-fucosyltransferase (FTase) that adds the third sugar, have been purified to near homogeneity from the cytosolic fraction of Dictyostelium cells and characterized. The GlcNAcTase transfers [ 3 H]GlcNAc from UDP- [ 3 H]GlcNAc to HyPro143 of a mutant Skp1 that is mostly hydroxylated but not glycosylated in vivo (8). This activity exhibits submicromolar K m values for both its donor and acceptor substrates, cannot be detected in the vesicular fraction of the cell, and requires the presence of a reducing agent in vitro.…”

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A Non-Golgi α1,2-Fucosyltransferase That Modifies Skp1 in the Cytoplasm of Dictyostelium

Wel¹,

Morris²,

Panico³

et al. 2001

Journal of Biological Chemistry

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Skp1 is a subunit of the SCF-E3 ubiquitin ligase that targets cell cycle and other regulatory factors for degradation. In Dictyostelium, Skp1 is modified by a pentasaccharide containing the type 1 blood group H trisaccharide at its core. To address how the third sugar, fucose ␣1,2-linked to galactose, is attached, a proteomics strategy was applied to determine the primary structure of FT85, previously shown to copurify with the GDPFuc:Skp1 ␣1,2-fucosyltransferase. Tryptic-generated peptides of FT85 were sequenced de novo using Q-TOF tandem mass spectrometry. Degenerate primers were used to amplify FT85 genomic DNA, which was further extended by a novel linker polymerase chain reaction method to yield an intronless open reading frame of 768 amino acids. Disruption of the FT85 gene by homologous recombination resulted in viable cells, which had altered light scattering properties as revealed by flow cytometry. FT85 was necessary and sufficient for Skp1 fucosylation, based on biochemical analysis of FT85 mutant cells and Escherichia coli that express FT85 recombinantly. FT85 lacks sequence motifs that characterize all other known ␣1,2-fucosyltransferases and lacks the signal-anchor sequence that targets them to the secretory pathway. The C-terminal region of FT85 harbors motifs found in inverting Family 2 glycosyltransferase domains, and its expression in FT85 mutant cells restores fucosyltransferase activity toward a simple disaccharide substrate. Whereas most prokaryote and eukaryote Family 2 glycosyltransferases are membranebound and oriented toward the cytoplasm where they glycosylate lipid-linked or polysaccharide precursors prior to membrane translocation, the soluble, eukaryotic Skp1-fucosyltransferase modifies a protein that resides in the cytoplasm and nucleus.

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[Skp1-Protein]-hydroxyproline N-acetylglucosaminyltransferase

Springer Handbook of Enzymes

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Identification of a UDP-GlcNAc:Skp1-Hydroxyproline GlcNAc-transferase in the Cytoplasm of Dictyostelium

Cited by 29 publications

References 27 publications

Initiation of Mucin-type O-Glycosylation in Dictyostelium Is Homologous to the Corresponding Step in Animals and Is Important for Spore Coat Function

Initiation of Mucin-type O-Glycosylation in Dictyostelium Is Homologous to the Corresponding Step in Animals and Is Important for Spore Coat Function

A Non-Golgi α1,2-Fucosyltransferase That Modifies Skp1 in the Cytoplasm of Dictyostelium

[Skp1-Protein]-hydroxyproline N-acetylglucosaminyltransferase

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