Membrane mutants of animal cells: rapid identification of those with a primary defect in glycosylation.

Stanley, Pamela

doi:10.1128/mcb.5.5.923

Cited by 60 publications

(45 citation statements)

References 39 publications

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“…The Lec1 CHO cell line lacks N-acetylglucosaminyltransferase I, the enzyme responsible for the committed step in the reelaboration and diversification of carbohydrates in the glycosylation pathway (18,26,27). Therefore, Lec1 cells are unable to synthesize complex-type N-linked oligosaccharides.…”

Section: Discussionmentioning

confidence: 99%

Glycosylation of the Major Polar Tube Protein ofEncephalitozoon hellem, a Microsporidian Parasite That Infects Humans

Xu¹,

Takvorian

Cali

et al. 2004

Infect Immun

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The microsporidia are ubiquitous, obligate intracellular eukaryotic spore-forming parasites infecting a wide range of invertebrates and vertebrates, including humans. The defining structure of microsporidia is the polar tube, which forms a hollow tube through which the sporoplasm is transferred to the host cell. Research on the molecular and cellular biology of the polar tube has resulted in the identification of three polar tube proteins: PTP1, PTP2, and PTP3. The major polar tube protein, PTP1, accounts for at least 70% of the mass of the polar tube. In the present study, PTP1 was found to be posttranslationally modified. Concanavalin A (ConA) bound to PTP1 and to the polar tube of several different microsporidia species. Analysis of the glycosylation of Encephalitozoon hellem PTP1 suggested that it is modified by O-linked mannosylation, and ConA binds to these O-linked mannose residues. Mannose pretreatment of RK13 host cells decreased their infection by E. hellem, consistent with an interaction between the mannosylation of PTP1 and some unknown host cell mannosebinding molecule. A CHO cell line (Lec1) that is unable to synthesize complex-type N-linked oligosaccharides had an increased susceptibility to E. hellem infection compared to wild-type CHO cells. These data suggest that the O-mannosylation of PTP1 may have functional significance for the ability of microsporidia to invade their host cells.

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Section: Discussionmentioning

confidence: 99%

Glycosylation of the Major Polar Tube Protein ofEncephalitozoon hellem, a Microsporidian Parasite That Infects Humans

Xu¹,

Takvorian

Cali

et al. 2004

Infect Immun

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“…Transport of UDP-galactose into Golgi vesicles from these cells was 90% defective compared to vesicles from wild-type cells, whereas transport of other nucleotide sugars, including uridine derivatives and CMP-sialic acid, was normal (25). Biochemical studies had previously shown that the resulting phenotype was not due to an impairment in the biosynthesis of nucleotide sugars, glycosyltransferases, or macromolecular and lipid acceptors (37,(77)(78)(79).…”

Section: Phenotype Of Mutants Defective In Transport Of Nucleotide Sumentioning

confidence: 99%

“…These mutants were isolated because of their resistance to plant lectins. CHO Lec8 (77,78), CHO clone 13 (78), and MDCK II-RCA R (37) belong to the same genetic complementation group. They have a 70-90% deficiency of galactose in their glycoproteins, glycosphingolipids, and selective proteoglycans.…”

Section: Phenotype Of Mutants Defective In Transport Of Nucleotide Sumentioning

confidence: 99%

Transporters of Nucleotide Sugars, Atp, and Nucleotide Sulfate in the Endoplasmic Reticulum and Golgi Apparatus

Hirschberg

Robbins²,

Abeijón³

1998

Annu. Rev. Biochem.

346

306

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The lumens of the endoplasmic reticulum and Golgi apparatus are the subcellular sites where glycosylation, sulfation, and phosphorylation of secretory and membrane-bound proteins, proteoglycans, and lipids occur. Nucleotide sugars, nucleotide sulfate, and ATP are substrates for these reactions. ATP is also used as an energy source in the lumen of the endoplasmic reticulum during protein folding and degradation. The above nucleotide derivatives and ATP must first be translocated across the membrane of the endoplasmic reticulum and/or Golgi apparatus before they can serve as substrates in the above lumenal reactions. Translocation of the above solutes is mediated for highly specific transporters, which are antiporters with the corresponding nucleoside monophosphates as shown by biochemical and genetic approaches. Mutants in mammals, yeast, and protozoa showed that a defect in a specific translocator activity results in selective impairments of the above posttranslational modifications, including loss of virulence of pathogenic protozoa. Several of these transporters have been purified and cloned. Experiments with yeast and mammalian cells demonstrate that these transporters play a regulatory role in the above reactions. Future studies will address the structure of the above proteins, how they are targeted to different organelles, 49 0066-4154/98/0701-0049$08.00 Annu. Rev. Biochem. 1998.67:49-69

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“…The ␤ 1 subunit is also heavily glycosylated, with four potential Nglycosylation motifs within its extracellular N-terminal domain (24). To determine whether the observed isoform-specific shifts in Na v gating produced by ␤ 1 are caused by ␤ 1 sialic acids, we co-expressed each of the ␣ subunits with ␤ 1 in two well characterized CHO cell lines that produce proteins with differing amounts of attached sialic acids (41)(42)(43). The Pro5 cell line allows normal CHO cell protein sialylation, whereas Lec2 cells, deficient in the CMP-sialic acid transporter, produce proteins that are essentially nonsialylated.…”

Section: Table Imentioning

confidence: 99%

The Sialic Acid Component of the β1 Subunit Modulates Voltage-gated Sodium Channel Function

Johnson

Montpetit

Stocker

et al. 2004

Journal of Biological Chemistry

106

101

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Voltage-gated sodium channels (Na v ) are responsible for initiation and propagation of nerve, skeletal muscle, and cardiac action potentials. Na v are composed of a pore-forming ␣ subunit and often one to several modulating ␤ subunits. Previous work showed that terminal sialic acid residues attached to ␣ subunits affect channel gating. Here we show that the fully sialylated ␤ 1 subunit induces a uniform, hyperpolarizing shift in steady state and kinetic gating of the cardiac and two neuronal ␣ subunit isoforms. Under conditions of reduced sialylation, the ␤ 1 -induced gating effect was eliminated. Consistent with this, mutation of ␤ 1 N-glycosylation sites abolished all effects of ␤ 1 on channel gating. Data also suggest an interaction between the cis effect of ␣ sialic acids and the trans effect of ␤ 1 sialic acids on channel gating. Thus, ␤ 1 sialic acids had no effect on the gating of the heavily glycosylated skeletal muscle ␣ subunit. However, when glycosylation of the skeletal muscle ␣ subunit was reduced through chimeragenesis such that ␣ sialic acids did not impact gating, ␤ 1 sialic acids caused a significant hyperpolarizing shift in channel gating. Together, the data indicate that ␤ 1 N-linked sialic acids can modulate Na v gating through an apparent saturating electrostatic mechanism. A model is proposed in which a spectrum of differentially sialylated Na v can directly modulate channel gating, thereby impacting cardiac, skeletal muscle, and neuronal excitability.

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Membrane mutants of animal cells: rapid identification of those with a primary defect in glycosylation.

Cited by 60 publications

References 39 publications

Glycosylation of the Major Polar Tube Protein ofEncephalitozoon hellem, a Microsporidian Parasite That Infects Humans

Glycosylation of the Major Polar Tube Protein ofEncephalitozoon hellem, a Microsporidian Parasite That Infects Humans

Transporters of Nucleotide Sugars, Atp, and Nucleotide Sulfate in the Endoplasmic Reticulum and Golgi Apparatus

The Sialic Acid Component of the β1 Subunit Modulates Voltage-gated Sodium Channel Function

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