Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation

Wild, Rebekka; Kowal, Julia; Eyring, Jillianne; Ngwa, Elsy M.; Aebi, Markus; Locher, Kaspar P.

doi:10.1126/science.aar5140

Cited by 175 publications

(214 citation statements)

References 88 publications

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“…However, glyco-peptides containing L28N were not detected by mass spectrometry, suggesting that the site may have remained unmodified. According to the recent cryo-EM structure of oligosaccharyltransferase (OST) [34][35][36], which catalyzes the initial transfer of glycan from the [37][38][39]. IFNβ is a well-known treatment for multiple sclerosis, with a number of different versions available (Rebif -IFNβ 1a, liquid form, Avonex -IFNβ 1a, lyophilized, Cinnovex -IFNβ 1a, biogeneric, Betaseron -IFNβ 1b, Plegridy -PEGylated IFNβ 1a) [40][41][42][43].…”

Section: Discussionmentioning

confidence: 99%

Structure-based glycoengineering of interferon lambda 4 enhances its productivity and anti-viral potency

et al. 2020

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Interferon lambda 4 (IFNλ4) has been recently known and studied for its role in hepatitis C virus (HCV) infection, but its clinical potential is significantly hampered due to its poor expression in vitro. Our study reports the successful production of IFNλ4 from a mammalian cell line through a glycoengineering and structure-based approach. We introduced de novo N-glycosylation of IFNλ4, guided by structural analysis, and produced IFNλ4 variants in Expi293F that displayed improved expression and potency. To preserve the structure and functionality of IFNλ4, the model structure of the IFNλ4 signaling complex was analyzed and the N-glycosylation candidate sites were selected. The receptor binding activity of engineered IFNλ4 variants and their receptormediated signaling pathway were similar to the E. coli version of IFNλ4 (eIFNλ4), while the antiviral activity and induction levels of interferon-stimulated gene (ISG) were all more robust in our variants. Our engineered IFNλ4 variants may be further developed for clinical applications and utilized in basic research to decipher the immunological roles of IFNλ4. Previously, the transient expression of wild-type IFNλ4 in mammalian cells failed to produce significant amounts of recombinant

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Section: Discussionmentioning

confidence: 99%

Structure-based glycoengineering of interferon lambda 4 enhances its productivity and anti-viral potency

et al. 2020

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“…For STT3A, all TMs except TM helix 10 (H10), and for STT3B all except H5 and H9, exhibit at least one positively charged residue ( Figure S4A). Most of these charges either face other complex subunits or are located at the predicted membrane head group region facing either the cytosol or the ER lumen (Braunger et al, 2018;Wild et al, 2018). Interestingly, for STT3A 5 positive charges remain that can be clustered into two groups facing the plane of the membrane on different sides.…”

Section: Two Unique Charges In Stt3a Facilitate Rhbdl4-catalyzed Cleamentioning

confidence: 99%

Intramembrane protease RHBDL4 cleaves oligosaccharyltransferase subunits to target them for ER-associated degradation

Knopf

Landscheidt

Pegg

et al. 2019

Preprint

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The Endoplasmic Reticulum (ER)-resident intramembrane rhomboid protease RHBDL4 generates metastable protein fragments and together with the ER-associated degradation (ERAD) machinery provides a clearance mechanism for aberrant and surplus proteins.However, the endogenous substrate spectrum and with that the role of RHBDL4 in physiological ERAD is mainly unknown. Here, we use quantitative proteomics to identify physiological RHBDL4 substrates. This revealed oligosacharyltransferase (OST) complex subunits such as the catalytic active subunit STT3A as substrates for the RHBDL4dependent ERAD pathway. RHBDL4-catalyzed cleavage inactivates OST subunits by triggering dislocation into the cytoplasm and subsequent proteasomal degradation. RHBDL4catalyzed cleavage is enhanced by positive charged transmembrane residues, which are recognized by a putative negative-charged docking site at the rhomboid active site gate.RHBDL4 controls the abundance and activity of OST, suggesting a novel link between the ERAD machinery and glycosylation tuning in the ER. AbbreviationsERAD, ER-associated degradation; OST, oligosacharyltransferase; TM, transmembrane; UIM, ubiquitin-interacting motif.

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“…[31][32][33] For N-linked glycosylation, [34][35][36] often a critical quality attribute for therapeutic proteins, 37,38 HEK293 and CHO share very similar cellular modification machineries in the compartments of endoplasmic reticulum (ER) and Golgi. A 14-saccharide (Glc 3 Man 9 GlcNAc 2 ) core structure is transferred en bloc to the Asn residues by oligosaccharyltransferase complex 39 and subsequently trimmed by a series of ER and Golgi glycosidases. These glycans are further modified by various Golgi glycosyltransferases to form mature complex glycan structures composed of fucose, galactose, and sialic acids.…”

Section: Introductionmentioning

confidence: 99%

Transient CHO expression platform for robust antibody production and its enhanced N‐glycan sialylation on therapeutic glycoproteins

Zhong

Meade

et al. 2018

Biotechnology Progress

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Large‐scale transient expression in mammalian cells is a rapid protein production technology often used to shorten overall timelines for biotherapeutics drug discovery. In this study we demonstrate transient expression in a Chinese hamster ovary (CHO) host (ExpiCHO‐S™) cell line capable of achieving high recombinant antibody expression titers, comparable to levels obtained using human embryonic kidney (HEK) 293 cells. For some antibodies, ExpiCHO‐S™ cells generated protein materials with better titers and improved protein quality characteristics (i.e., less aggregation) than those from HEK293. Green fluorescent protein imaging data indicated that ExpiCHO‐S™ displayed a delayed but prolonged transient protein expression process compared to HEK293. When therapeutic glycoproteins containing non‐Fc N‐linked glycans were expressed in transient ExpiCHO‐S™, the glycan pattern was unexpectedly found to have few sialylated N‐glycans, in contrast to glycans produced within a stable CHO expression system. To improve N‐glycan sialylation in transient ExpiCHO‐S™, we co‐transfected galactosyltransferase and sialyltransferase genes along with the target genes, as well as supplemented the culture medium with glycan precursors. The authors have demonstrated that co‐transfection of glycosyltransferases combined with medium addition of galactose and uridine led to increased sialylation content of N‐glycans during transient ExpiCHO‐S™ expression. These results have provided a scientific basis for developing a future transient CHO system with N‐glycan compositions that are similar to those profiles obtained from stable CHO protein production systems. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2724, 2019

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Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation

Cited by 175 publications

References 88 publications

Structure-based glycoengineering of interferon lambda 4 enhances its productivity and anti-viral potency

Structure-based glycoengineering of interferon lambda 4 enhances its productivity and anti-viral potency

Intramembrane protease RHBDL4 cleaves oligosaccharyltransferase subunits to target them for ER-associated degradation

Transient CHO expression platform for robust antibody production and its enhanced N‐glycan sialylation on therapeutic glycoproteins

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