Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation

Cherepanova, Natalia A.; Gilmore, Reid

doi:10.1038/srep20946

Cited by 87 publications

(119 citation statements)

References 44 publications

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“…N-linked glycosylation of the ERLucT reporter inhibits luciferase activity and inhibition of glycosylation thus increases luciferase activity and measureable bioluminescence. To definitively assign inhibitory activities of small molecule inhibitors of the OST to STT3A, STT3B or both catalytic subunits, HEK293 cells with a CRISPR/Cas9 mediated knockout (ko) of one catalytic subunit (either STT3A or STT3B) (Cherepanova & Gilmore 2016) were stably transfected with the ERLucT reporter (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

Editing N-Glycan Site Occupancy with Small-Molecule Oligosaccharyltransferase Inhibitors

Rinis

Golden

Marceau

et al. 2018

Cell Chemical Biology

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The oligosaccharyltransferase (OST) is a multisubunit enzyme complex that N-glycosylates proteins in the secretory pathway and is considered to be constitutive and unregulated. However, small-molecule OST inhibitors such as NGI-1 provide a pharmacological approach for regulating N-linked glycosylation. Herein we design cell models with knockout of each OST catalytic subunit (STT3A or STT3B) to screen the activity of NGI-1 and its analogs. We show that NGI-1 targets the function of both STT3A and STT3B and use structure-activity relationships to guide synthesis of catalytic subunit-specific inhibitors. Using this approach, pharmacophores that increase STT3B selectivity are characterized and an STT3B-specific inhibitor is identified. This inhibitor has discrete biological effects on endogenous STT3B target proteins such as COX2 but does not activate the cellular unfolded protein response. Together this work demonstrates that subsets of glycoproteins can be regulated through pharmacologic inhibition of N-linked glycosylation.

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Section: Resultsmentioning

confidence: 99%

Editing N-Glycan Site Occupancy with Small-Molecule Oligosaccharyltransferase Inhibitors

Rinis

Golden

Marceau

et al. 2018

Cell Chemical Biology

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“…It is noteworthy that silencing MagT1 did not alter total intracellular Mg (Supplementary S6D). Moreover, since MagT1 is a subunit of the oligosaccharyltransferase complex and, therefore, is implicated in protein glycosylation24, it is possible that its silencing impacts on post-translational modification of proteins involved in controlling cell proliferation.…”

Section: Discussionmentioning

confidence: 99%

The different expression of TRPM7 and MagT1 impacts on the proliferation of colon carcinoma cells sensitive or resistant to doxorubicin

Cazzaniga

Moscheni

Trapani

et al. 2017

Sci Rep

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The processes leading to anticancer drug resistance are not completely unraveled. To get insights into the underlying mechanisms, we compared colon carcinoma cells sensitive to doxorubicin with their resistant counterpart. We found that resistant cells are growth retarded, and show staminal and ultrastructural features profoundly different from sensitive cells. The resistant phenotype is accompanied by the upregulation of the magnesium transporter MagT1 and the downregulation of the ion channel kinase TRPM7. We demonstrate that the different amounts of TRPM7 and MagT1 account for the different proliferation rate of sensitive and resistant colon carcinoma cells. It remains to be verified whether they are also involved in the control of other “staminal” traits.

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“…The cDNA clones for STT3B and MAGT1 were described previously (19). The TUSC3 gene was cloned by PCR from 293T cDNA.…”

Section: Methodsmentioning

confidence: 99%

Dengue Virus Hijacks a Noncanonical Oxidoreductase Function of a Cellular Oligosaccharyltransferase Complex

Lin

Cherepanova

Bozzacco

et al. 2017

mBio

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Dengue virus (DENV) is the most common arboviral infection globally, infecting an estimated 390 million people each year. We employed a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen to identify host dependency factors required for DENV propagation and identified the oligosaccharyltransferase (OST) complex as an essential host factor for DENV infection. Mammalian cells express two OSTs containing either STT3A or STT3B. We found that the canonical catalytic function of the OSTs as oligosaccharyltransferases is not necessary for DENV infection, as cells expressing catalytically inactive STT3A or STT3B are able to support DENV propagation. However, the OST subunit MAGT1, which associates with STT3B, is also required for DENV propagation. MAGT1 expression requires STT3B, and a catalytically inactive STT3B also rescues MAGT1 expression, supporting the hypothesis that STT3B serves to stabilize MAGT1 in the context of DENV infection. We found that the oxidoreductase CXXC active site motif of MAGT1 was necessary for DENV propagation, as cells expressing an AXXA MAGT1 mutant were unable to support DENV infection. Interestingly, cells expressing single-cysteine CXXA or AXXC mutants of MAGT1 were able to support DENV propagation. Utilizing the engineered peroxidase APEX2, we demonstrate the close proximity between MAGT1 and NS1 or NS4B during DENV infection. These results reveal that the oxidoreductase activity of the STT3B-containing OST is necessary for DENV infection, which may guide the development of antiviral agents targeting DENV.

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Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation

Cited by 87 publications

References 44 publications

Editing N-Glycan Site Occupancy with Small-Molecule Oligosaccharyltransferase Inhibitors

Editing N-Glycan Site Occupancy with Small-Molecule Oligosaccharyltransferase Inhibitors

The different expression of TRPM7 and MagT1 impacts on the proliferation of colon carcinoma cells sensitive or resistant to doxorubicin

Dengue Virus Hijacks a Noncanonical Oxidoreductase Function of a Cellular Oligosaccharyltransferase Complex

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