Glycosylation Defect in Lec1 Chinese Hamster Ovary Mutant Is Due to a Point Mutation in N-Acetylglucosaminyltransferase I Gene

Puthalakath, Hamsa; Burke, Jo; Gleeson, Paul A.

doi:10.1074/jbc.271.44.27818

Cited by 59 publications

(40 citation statements)

References 34 publications

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“…Previously, it has been shown that a point mutation in the coding region of N-acetylglucosaminyltransferase I and V leads into the glycosylation defect in Lec1 and Lec4A cells, respectively (39,40). In carbohydrate-deficient glycoprotein syndrome type II, a point mutation was discovered in the nucleotide sequence encoding the catalytic domain of ␤-1,2-Nacetylglucosaminyltransferase II (41).…”

Section: Discussionmentioning

confidence: 99%

Molecular Cloning and Expression of GDP-d-mannose-4,6-dehydratase, a Key Enzyme for Fucose Metabolism Defective in Lec13 Cells

Οhyama¹,

Smith²,

Angata³

et al. 1998

Journal of Biological Chemistry

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Subsets of mammalian cell surface oligosaccharides contain specific fucosylated moieties expressed in lineage-and/or temporal-specific patterns. The functional significance of these fucosylated structures is incompletely defined, although there is evidence that subsets of them, represented by the sialyl Le x determinant, are important participants in leukocyte adhesion and trafficking processes. Genetic deletion of these fucosylated structures in the mouse has been a powerful tool to address functional questions about fucosylated glycans. However, successful use of such approaches can be problematic, given the substantial redundancy in the mammalian ␣-1,3-fucosyltransferase and ␣-1,2-fucosyltransferase gene families. To circumvent this problem, we have chosen to clone the genetic locus encoding a mammalian GDP-D-mannose-4,6-dehydratase (GMD). This enzyme generates GDP-mannose-4-keto-6-D-deoxymannose from GDP-mannose, which is then converted by the FX protein (GDP-4-keto-6-D-deoxymannose epimerase/GDP-4-keto-6-L-galactose reductase) to GDP-L-fucose. GMD is thus imperative for the synthesis of all fucosylated oligosaccharides. An expression cloning approach and the GMD-deficient CHO host cell line Lec13 were used to generate a population of cDNA molecules enriched in GMD cDNAs. This enriched plasmid population was then screened using a human expressed sequence tag (EST AA065072) with sequence similarity to an Arabidopsis thaliana GMD cDNA. This approach, together with 5-rapid amplification of cDNA ends, yielded a human cDNA that complements the fucosylation defect in the Lec13 cell line. Northern blot analyses indicate that the GMD transcript is absent in Lec13 cells, confirming the genetic deficiency of this locus in these cells. By contrast, the transcript encoding the FX protein, which forms GDP-L-fucose from the ketosugar intermediate produced by GMD, is present in increased amounts in the Lec13 cells. These results suggest that metabolites generated in this pathway may participate in the transcriptional regulation of the FX protein and possibly the GMD protein. The results also suggest that the genomic structure encoding GMD in Lec13 cells likely has a defect different from a point mutation in the coding region.

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Section: Discussionmentioning

confidence: 99%

Molecular Cloning and Expression of GDP-d-mannose-4,6-dehydratase, a Key Enzyme for Fucose Metabolism Defective in Lec13 Cells

Οhyama¹,

Smith²,

Angata³

et al. 1998

Journal of Biological Chemistry

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“…To confirm that the inability of influenza virus to infect Lec1 cells was solely due to the presence of terminal N-linked carbohydrate, we transiently expressed the wild-type GnT1 gene (17) in Lec1 cells and examined virus infection. GnT1 expression was confirmed by the ability of transfected cells to bind L-PHA.…”

Section: Influenza Virus Infection Of Lec1 Cells Is Rescued By Expresmentioning

confidence: 99%

“…Lec1 cells were isolated by selection of CHO cells with the cytotoxic lectin Phaseolus vulgaris leukoagglutinin (L-PHA) (15), which binds to complex carbohydrate structures, such as tri-and tetraantennary glycopeptides containing outer galactose residues and an ␣-linked mannose residue substituted at positions C-2 and C-6 (16). Lec1 cells have been characterized as having a defect in the N-acetylglucosaminyltransferase I (GnT1) gene (17,18). As such, they are unable to synthesize mature N-glycans containing terminal branches ending with galactose and sialic acid due to the absence of the glycosyltransferase GnT1, the outcome of which is that they are deficient in receptor sialo-Nglycans (19), although they are not deficient in glycosphingolipids and should not be deficient in O-linked glycans (P. Stanley, personal communication).…”

mentioning

confidence: 99%

Influenza virus entry and infection require host cell N-linked glycoprotein

Chu

Whittaker

2004

Proc. Natl. Acad. Sci. U.S.A.

182

144

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A widely held view of influenza virus infection is that the viral receptor consists of cell surface carbohydrate sialic acid, which can be present as glycoprotein or glycolipid. Here, we examined influenza virus entry and infection in Lec1 cells, a mutant CHO cell line deficient in terminal N-linked glycosylation caused by a mutation in the N-acetylglucosaminyltransferase I (GnT1) gene. We show that influenza virus cannot infect Lec1 cells, despite having full capacity to undergo virus binding and fusion. Lec1 cells also show no virus replication defect, and infection was restored in Lec1 cells expressing wild-type GnT1. Viruses were apparently arrested at the level of internalization from the plasma membrane and were not endocytosed. Lec1 cells were refractory to infection by several strains of influenza virus, including H1 and H3 strains of influenza A, as well as influenza B virus. Finally, cleavage of N-glycans from wild-type CHO cells markedly reduced infection by influenza virus. We suggest that influenza virus specifically requires N-linked glycoprotein for entry into cells, and that sialic acid, although acting as an efficient attachment factor, is not sufficient as an influenza virus receptor in vivo.

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“…The Mgat1 gene encoding GlcNAcT1 adds GlcNAc to high-mannose sites (Schachter, 2010); an essential early step in producing all complex and hybrid N-glycans (Pownall et al, 1992;Ye and Marth, 2004). Thus, MGAT1 generates the entire repertoire of polymeric branched Nglycans destined for secretion or presentation on the cell surface (Puthalakath et al, 1996); and Mgat1 null mutants, containing high mannose in place of complex/hybrid N-glycans, are the earliest Nglycan pathway block available to study N-glycosylation requirements in neural development (Schachter and Boulianne, 2011). Mouse Mgat1 knockouts are lethal at embryonic day (E) 9.5, but conditional mutants show movement defects, tremors, paralysis and early death characteristic of neurodevelopmental impairments (Campbell et al, 1995;Grasa et al, 2012;Shi et al, 2004;Ye and Marth, 2004).…”

Section: Introductionmentioning

confidence: 99%

N-glycosylation requirements in neuromuscular synaptogenesis

et al. 2013

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Neural development requires N-glycosylation regulation of intercellular signaling, but the requirements in synaptogenesis have not been well tested. All complex and hybrid N-glycosylation requires MGAT1 (UDP-GlcNAc:α-3-D-mannoside-β1,2-N-acetylglucosaminyltransferase I) function, and Mgat1 nulls are the most compromised N-glycosylation condition that survive long enough to permit synaptogenesis studies. At the Drosophila neuromuscular junction (NMJ), Mgat1 mutants display selective loss of lectin-defined carbohydrates in the extracellular synaptomatrix, and an accompanying accumulation of the secreted endogenous Mind the gap (MTG) lectin, a key synaptogenesis regulator. Null Mgat1 mutants exhibit strongly overelaborated synaptic structural development, consistent with inhibitory roles for complex/hybrid Nglycans in morphological synaptogenesis, and strengthened functional synapse differentiation, consistent with synaptogenic MTG functions. Synapse molecular composition is surprisingly selectively altered, with decreases in presynaptic active zone Bruchpilot (BRP) and postsynaptic Glutamate receptor subtype B (GLURIIB), but no detectable change in a wide range of other synaptic components. Synaptogenesis is driven by bidirectional trans-synaptic signals that traverse the glycan-rich synaptomatrix, and Mgat1 mutation disrupts both anterograde and retrograde signals, consistent with MTG regulation of trans-synaptic signaling. Downstream of intercellular signaling, pre-and postsynaptic scaffolds are recruited to drive synaptogenesis, and Mgat1 mutants exhibit loss of both classic Discs large 1 (DLG1) and newly defined Lethal (2) giant larvae [L(2)GL] scaffolds. We conclude that MGAT1-dependent N-glycosylation shapes the synaptomatrix carbohydrate environment and endogenous lectin localization within this domain, to modulate retention of trans-synaptic signaling ligands driving synaptic scaffold recruitment during synaptogenesis.

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Glycosylation Defect in Lec1 Chinese Hamster Ovary Mutant Is Due to a Point Mutation in N-Acetylglucosaminyltransferase I Gene

Cited by 59 publications

References 34 publications

Molecular Cloning and Expression of GDP-d-mannose-4,6-dehydratase, a Key Enzyme for Fucose Metabolism Defective in Lec13 Cells

Molecular Cloning and Expression of GDP-d-mannose-4,6-dehydratase, a Key Enzyme for Fucose Metabolism Defective in Lec13 Cells

Influenza virus entry and infection require host cell N-linked glycoprotein

N-glycosylation requirements in neuromuscular synaptogenesis

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