Mammalian STT3A/B oligosaccharyltransferases segregate N-glycosylation at the translocon from lipid-linked oligosaccharide hydrolysis

Lu, Hua; Fermaintt, Charles S.; Cherepanova, Natalia A.; Gilmore, Reid; Yan, Nan; Lehrman, Mark A.

doi:10.1073/pnas.1806034115

Cited by 30 publications

(31 citation statements)

References 51 publications

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“…5, the membrane-bound OST transferase activities of DC2 and MagT1/TUSC3-deficient permeabilized cells resembled their solubilized counterparts, forming G 3 M 9 Gn 2 products with synthetic acceptor peptides. DC2-KO cells had transferase activity levels similar to STT3A-KO cells but approximately double the activity of MagT1/TUSC3-DKO cells which, in turn, were similar to the previously reported activity in STT3B-KO cells (35). LLO hydrolysis in MagT1/TUSC3-DKO cells was greatly reduced and also reminiscent of STT3B-KO cells.…”

Section: Manipulation Of Ost Accessory Subunits Supports a Primary Rosupporting

confidence: 87%

“…This apparent off-target effect may be related to the assay using high concentrations of small peptide acceptors presented in solution rather than cotranslationally, a situation not naturally encountered by STT3A-OST. As expected (35), STT3A-OST had no significant LLO hydrolysis activity, and neither NGI-1 nor C19 affected LLO synthesis.…”

Section: Hsv-1 As An Experimental Model For Ost Functionsupporting

confidence: 79%

“…Assays for OST transferase and LLO hydrolysis activities were performed exactly as previously described (35). ALOpermeabilized cells were incubated 30-60 min in a reaction buffer consisting of transport buffer plus 0 or 400 mM uridine diphosphate-glucose (UDP-Glc), 200 mM uridine diphosphate N-acetylglucosamine, 50 mM guanosine diphosphate mannose, 0.2 mM AMP, 10 mg/ml castanospermine, and 10 mg/ml deoxymannojirimycin, in the absence or presence of 50 mM control (acetyl-Gln-Tyr-Thr-CONH 2 ) or acceptor peptide (acetyl-Asn-Tyr-Thr-CONH 2 ) as indicated.…”

Section: Ost Reactionsmentioning

confidence: 99%

“…In healthy intact cells, NGI-1 inhibits both STT3A-OST and STT3B-OST (22,29), whereas C19 is selective for STT3B-OST (21). Because HSV-1-infected cells may undergo extensive cytopathic effects to determine whether these inhibitors were also generally efficacious with disrupted cells, we employed a system for assaying OST isoforms using cells with permeabilized plasma membranes (35). Consistent with prior results from intact cells, in permeabilized cells, NGI-1 inhibited the transferase activities of both STT3A-OST and STT3B-OST as well as the LLO hydrolysis activity of STT3B-OST, and C19 inhibited the transferase and hydrolase activities of STT3B-OST ( Fig.…”

Section: Hsv-1 As An Experimental Model For Ost Functionmentioning

confidence: 99%

“…Assays were performed in the absence or presence of UDP-Glc to favor synthesis of M 9 Gn 2 -LLO (double red arrowhead) or G 3 M 9 Gn 2 -LLO (single red arrowhead), respectively. Even without UDP-Glc, some G 3 M 9 Gn 2 -LLO is evident (35). A) Representative FACE gels.…”

Section: Manipulation Of Ost Accessory Subunits Supports a Primary Romentioning

confidence: 99%

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Targeting STT3A‐oligosaccharyltransferase with NGI‐1 causes herpes simplex virus 1 dysfunction

Cherepanova

Gilmore

et al. 2019

FASEB j.

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Herpes simplex virus 1 (HSV‐1) is a contagious neurotropic herpesvirus responsible for oral lesions and herpesviral encephalitis. The HSV‐1 envelope contains N‐glycosylated proteins involved in infection and that are candidate drug targets. NGI‐1 is a small‐molecule inhibitor of oligosaccharyltransferase (OST) complexes STT3A‐OST and STT3B‐OST, which catalyze cotranslational and post‐translational N‐glycosylation, respectively. Because host OSTs attach HSV‐1 glycans, NGI‐1 might have anti–HSV‐1 activity. We evaluated HSV‐1 function using NGI‐1 and human embryonic kidney 293 knockout lines for OST isoform‐specific catalytic and accessory subunits. N‐glycosylation of 2 representative envelope proteins (gC and gD) was primarily dependent upon STT3A‐OST, but to a large extent replaceable by STT3B‐OST. Knockouts impairing STT3A‐ or STT3B‐OST activity, by themselves, did not appreciably affect HSV‐1 function (plaque‐forming units, normalized to viral particles measured by unglycosylated capsid protein VP5 content). However, with cells lacking STT3B‐OST activity (missing the catalytic subunit STT3B or the oxidoreductase subunits magnesium transporter 1/tumor suppressor candidate 3) and thus solely dependent upon STT3A‐OST for N‐glycosylation, NGI‐1 treatment resulted in HSV‐1 having cell type–dependent dysfunction (affecting infectivity with Vero cells much more than with the 293 lines). Ablation of post‐translational N‐glycosylation can therefore make HSV‐1 infectivity, and possibly masking of immunogenic peptide epitopes by glycans, highly sensitive to pharmacological inhibition of cotranslational N‐glycosylation.—Lu, H., Cherepanova, N. A., Gilmore, R., Contessa, J. N., Lehrman, M. A. Targeting STT3A‐oligosaccharyltransferase with NGI‐1 causes herpes simplex virus 1 dysfunction. FASEB J. 33, 6801–6812 (2019). http://www.fasebj.org

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Section: Manipulation Of Ost Accessory Subunits Supports a Primary Rosupporting

confidence: 87%

Section: Hsv-1 As An Experimental Model For Ost Functionsupporting

confidence: 79%

Section: Ost Reactionsmentioning

confidence: 99%

Section: Hsv-1 As An Experimental Model For Ost Functionmentioning

confidence: 99%

Section: Manipulation Of Ost Accessory Subunits Supports a Primary Romentioning

confidence: 99%

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Targeting STT3A‐oligosaccharyltransferase with NGI‐1 causes herpes simplex virus 1 dysfunction

Cherepanova

Gilmore

et al. 2019

FASEB j.

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The occurrence of nonglycosylated forms of N‐glycoprotein upon proteasome inhibition does not confirm cytosolic deglycosylation

Huang

Suzuki

2020

FEBS Letters

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N‐Glycosylated substrates that undergo ER‐associated degradation (ERAD) are often deglycosylated by the cytosolic peptide:N‐glycanase (Ngly1) during their proteasomal degradation in the cytosol. Consequently, the presence of non‐ or deglycosylated forms of such proteins after treatment with proteasome inhibitors is widely used as evidence for cytosolic deglycosylation by Ngly1. However, in this study, the accumulation of nonglycosylated RTA∆m, a model ERAD substrate, was still observed in mouse cells lacking cytosolic de‐N‐glycosylating enzymes, when treated with proteasome inhibitors. It was found that RTA∆m is normally partially N‐glycosylated, while the nonglycosylated form is rapidly degraded by proteasomes in the cytosol. Our results suggest that the occurrence of ‘nonglycosylated’ ERAD substrates upon treatment with proteasome inhibitors is not necessarily a clue for cytosolic deglycosylation.

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Protein posttranslational modifications in health and diseases: Functions, regulatory mechanisms, and therapeutic implications

Zhong

Xiao

Xie

et al. 2023

MedComm

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Protein posttranslational modifications (PTMs) refer to the breaking or generation of covalent bonds on the backbones or amino acid side chains of proteins and expand the diversity of proteins, which provides the basis for the emergence of organismal complexity. To date, more than 650 types of protein modifications, such as the most well‐known phosphorylation, ubiquitination, glycosylation, methylation, SUMOylation, short‐chain and long‐chain acylation modifications, redox modifications, and irreversible modifications, have been described, and the inventory is still increasing. By changing the protein conformation, localization, activity, stability, charges, and interactions with other biomolecules, PTMs ultimately alter the phenotypes and biological processes of cells. The homeostasis of protein modifications is important to human health. Abnormal PTMs may cause changes in protein properties and loss of protein functions, which are closely related to the occurrence and development of various diseases. In this review, we systematically introduce the characteristics, regulatory mechanisms, and functions of various PTMs in health and diseases. In addition, the therapeutic prospects in various diseases by targeting PTMs and associated regulatory enzymes are also summarized. This work will deepen the understanding of protein modifications in health and diseases and promote the discovery of diagnostic and prognostic markers and drug targets for diseases.

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Mammalian STT3A/B oligosaccharyltransferases segregate N-glycosylation at the translocon from lipid-linked oligosaccharide hydrolysis

Cited by 30 publications

References 51 publications

Targeting STT3A‐oligosaccharyltransferase with NGI‐1 causes herpes simplex virus 1 dysfunction

Targeting STT3A‐oligosaccharyltransferase with NGI‐1 causes herpes simplex virus 1 dysfunction

The occurrence of nonglycosylated forms of N‐glycoprotein upon proteasome inhibition does not confirm cytosolic deglycosylation

Protein posttranslational modifications in health and diseases: Functions, regulatory mechanisms, and therapeutic implications

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