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

Significance Specialized sugar polymers (oligosaccharides) are necessary for life at the protein, organelle, cell, and organism levels. Processes for degrading oligosaccharides enhance their repertoire of functions, but a potential problem is short circuiting between degradation and synthesis. With the endoplasmic reticulum of mammalian cells, we show how lipid-linked oligosaccharides (LLOs) meant for attachment to proteins are segregated from an immunogenic process that involves their degradation to free oligosaccharides. Specifically, the STT3A isoform of oligosaccharyltransferase only transfers oligosaccharides, while the STT3B isoform can hydrolyze LLOs when not preoccupied with attachment of oligosaccharides to protein. This allows cells to perform both important processes in concert, without problems due to competition. Oligosaccharyltransferases (OSTs) N-glycosylate proteins by transferring oligosaccharides from lipid-linked oligosaccharides (LLOs) to asparaginyl residues of Asn-Xaa-Ser/Thr acceptor sequons. Mammals have OST isoforms with STT3A or STT3B catalytic subunits for cotranslational or posttranslational N-glycosylation, respectively. OSTs also hydrolyze LLOs, forming free oligosaccharides (fOSs). It has been unclear whether hydrolysis is due to one or both OSTs, segregated from N-glycosylation, and/or regulated. Transfer and hydrolysis were assayed in permeabilized HEK293 kidney and Huh7.5.1 liver cells lacking STT3A or STT3B. Transfer by both STT3A-OST and STT3B-OST with synthetic acceptors was robust. LLO hydrolysis by STT3B-OST was readily detected and surprisingly modulated: Without acceptors, STT3B-OST hydrolyzed Glc3Man9GlcNAc2-LLO but not Man9GlcNAc2-LLO, yet it hydrolyzed both LLOs with acceptors present. In contrast, LLO hydrolysis by STT3A-OST was negligible. STT3A-OST however may be regulatory, because it suppressed STT3B-OST–dependent fOSs. TREX1, a negative innate immunity factor that diminishes immunogenic fOSs derived from LLOs, acted through STT3B-OST as well. In summary, only STT3B-OST hydrolyzes LLOs, depending upon LLO quality and acceptor site occupancy. TREX1 and STT3A suppress STT3B-OST–dependent fOSs. Without strict kinetic limitations during posttranslational N-glycosylation, STT3B-OST can thus moonlight for LLO hydrolysis. In contrast, the STT3A-OST/translocon complex preserves LLOs for temporally fastidious cotranslational N-glycosylation.

• Publication year

  2018

• Venue

  Proceedings of the National Academy of Sciences of the United States of America

• Publication date

  2018-09-04

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1073/pnas.1806034115PMID 30181269PMCID PMC6156661
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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

  2018cited by this paper
• Structural basis for coupling protein transport and N-glycosylation at the mammalian endoplasmic reticulum

  2018cited by this paper
• The atomic structure of a eukaryotic oligosaccharyl transferase complex

  2018cited by this paper
• Structure of the yeast oligosaccharyltransferase complex gives insight into protein N-glycosylation

  2018cited by this paper
• Mitotic Phosphorylation of TREX1 C Terminus Disrupts TREX1 Regulation of the Oligosaccharyltransferase Complex

  2017cited by this paper
• One flexible loop in OST lassos both substrates

  2017influential reference
• Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase

  2017influential reference
• A conserved DGGK motif is essential for the function of the PglB oligosaccharyltransferase from Campylobacter jejuni

  2017cited by this paper
• DC2 and KCP2 mediate the interaction between the oligosaccharyltransferase and the ER translocon

  2017cited by this paper
• N-linked glycosylation and homeostasis of the endoplasmic reticulum.

  2016cited by this paper
• Oligosaccharyltransferase inhibition induces senescence in RTK-driven tumor cells.

  2016influential reference
• Biological roles of glycans

  2016cited by this paper
• Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens

  2016influential reference
• Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation

  2016cited by this paper
• Cytosolic Nuclease TREX1 Regulates Oligosaccharyltransferase Activity Independent of Nuclease Activity to Suppress Immune Activation.

  2015influential reference
• Cytosolic-free oligosaccharides are predominantly generated by the degradation of dolichol-linked oligosaccharides in mammalian cells.

  2015influential reference
• Endo-β-N-acetylglucosaminidase forms N-GlcNAc protein aggregates during ER-associated degradation in Ngly1-defective cells

  2015cited by this paper
• Generation and degradation of free asparagine-linked glycans

  2015cited by this paper
• Oxidoreductase activity is necessary for N-glycosylation of cysteine-proximal acceptor sites in glycoproteins

  2014cited by this paper
• Letter to the Glycoforum: Improved protocols for preparing lipid-linked and related saccharides for Fluorophore-Assisted Carbohydrate Electrophoresis (FACE).

  2013influential reference
• Eukaryotic Oligosaccharyltransferase Generates Free Oligosaccharides during N-Glycosylation*

  2013cited by this paper
• Mutations in STT3A and STT3B cause two congenital disorders of glycosylation.

  2013cited by this paper
• Extreme C-terminal sites are posttranslocationally glycosylated by the STT3B isoform of the OST

  2013cited by this paper
• Flagging and docking: dual roles for N-glycans in protein quality control and cellular proteostasis

  2012influential reference
• A zebrafish model of PMM2-CDG reveals altered neurogenesis and a substrate-accumulation mechanism for N-linked glycosylation deficiency

  2012cited by this paper
• Trex1 regulates lysosomal biogenesis and interferon-independent activation of antiviral genes

  2012cited by this paper
• Mannose-6-phosphate regulates destruction of lipid-linked oligosaccharides

  2011cited by this paper
• X-ray structure of a bacterial oligosaccharyltransferase

  2011cited by this paper
• Study of free oligosaccharides derived from the bacterial N-glycosylation pathway

  2009cited by this paper
• Cotranslational and posttranslational N-glycosylation of polypeptides by distinct mammalian OST isoforms.

  2009cited by this paper
• Defining the glycan destruction signal for endoplasmic reticulum-associated degradation.

  2008cited by this paper
• Free oligosaccharide regulation during mammalian protein N-glycosylation.

  2007cited by this paper
• Unexpected basis for impaired Glc3Man9GlcNAc2-P-P-dolichol biosynthesis by elevated expression of GlcNAc-1-P transferase.

  2007cited by this paper
• Free N-linked oligosaccharide chains: Formation and degradation

  2006cited by this paper
• An evolving view of the eukaryotic oligosaccharyltransferase.

  2006cited by this paper
• Non-radioactive analysis of lipid-linked oligosaccharide compositions by fluorophore-assisted carbohydrate electrophoresis.

  2006influential reference
• z-VAD-fmk inhibits peptide:N-glycanase and may result in ER stress

  2006cited by this paper
• Analysis of Glycosylation in CDG-Ia Fibroblasts by Fluorophore-assisted Carbohydrate Electrophoresis

  2005cited by this paper
• The diversity of dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases.

  2005cited by this paper
• Roles of N-linked glycans in the endoplasmic reticulum.

  2004cited by this paper
• Using a small molecule inhibitor of peptide: N-glycanase to probe its role in glycoprotein turnover.

  2004influential reference
• Q-VD-OPh, a broad spectrum caspase inhibitor with potent antiapoptotic properties

  2003influential reference
• Oligosaccharyltransferase isoforms that contain different catalytic STT3 subunits have distinct enzymatic properties.

  2003influential reference
• Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex

  2003cited by this paper
• Coupling of the Dolichol-P-P-Oligosaccharide Pathway to Translation by Perturbation-sensitive Regulation of the Initiating Enzyme, GlcNAc-1-P Transferase*

  2002cited by this paper
• Endo-β-N-acetylglucosaminidase, an enzyme involved in processing of free oligosaccharides in the cytosol

  2002cited by this paper
• Allosteric regulation provides a molecular mechanism for preferential utilization of the fully assembled dolichol-linked oligosaccharide by the yeast oligosaccharyltransferase.

  2001cited by this paper
• Requirement of the Lec35 gene for all known classes of monosaccharide-P-dolichol-dependent glycosyltransferase reactions in mammals.

  2001cited by this paper
• Oligosaccharide Modification in the Early Secretory Pathway Directs the Selection of a Misfolded Glycoprotein for Degradation by the Proteasome*

  1999cited by this paper
• Site-specific de-N-glycosylation of diglycosylated ovalbumin in hen oviduct by endogenous peptide: N-glycanase as a quality control system for newly synthesized proteins.

  1997cited by this paper
• Studies of Transferrin Recycling Reconstituted in Streptolysin O Permeabilized Chinese Hamster Ovary Cells (*)

  1995cited by this paper
• Release of glucose-containing polymannose oligosaccharides during glycoprotein biosynthesis. Studies with thyroid microsomal enzymes and slices.

  1983cited by this paper

• STT3A-mediated FCN3 N-glycosylation promotes Treg cell activation to drive hepatocellular carcinoma progression via Wnt/β-catenin

  2026cites this paper
• EROS Protein: Decoding Its Pivotal Role in Redox Homeostasis and Disease Pathogenesis

  2025cites this paper
• Systematic profiling reveals hepatic immune and metabolic dysregulation in DNASE1L3-deficient mice

  2025cites this paper
• Integrated analysis of N-glycosylation and Alzheimer’s disease: identifying key biomarkers and mechanisms

  2025cites this paper
• Protein post-translational modifications and tumor immunity: A pan-cancer perspective.

  2025cites this paper
• Quantitative proteomic and glycoproteomic analysis identifies CLCA1, FBN1, and FGB as potential biomarkers for ulcerative colitis

  2025cites this paper
• LLP1 is a pyrophosphatase involved in homeostasis/quality control of dolichol-linked oligosaccharide.

  2025cites this paper
• Unveiling the Role of Oligosaccharyltransferase STT3B in Colorectal Cancer Tissues: Clinical significance and Molecular Mechanisms Driving the Formation of Tertiary Lymphoid Structures.

  2025cites this paper
• Heterozygous pathogenic STT3A variation leads to dominant congenital glycosylation disorders and functional validation in zebrafish

  2025cites this paper
• Free oligosaccharides in serum

  2025cites this paper
• A novel allosteric driver mutation of β‐glucuronidase promotes head and neck squamous cell carcinoma progression through STT3B‐mediated PD‐L1 N‐glycosylation

  2025cites this paper
• Brucella-driven host N-glycome remodeling controls infection.

  2024cites this paper
• Occurrence of free glycans in salmonid serum.

  2024cites this paper
• Processing of N-glycans in the ER and Golgi influences the production of surface sialylated glycoRNA

  2024cites this paper
• Rat hepatocytes secrete free oligosaccharides

  2024cites this paper
• Characterization of PcSTT3B as a Key Oligosaccharyltransferase Subunit Involved in N-glycosylation and Its Role in Development and Pathogenicity of Phytophthora capsici

  2023cites this paper
• Glycomic, Glycoproteomic, and Proteomic Profiling of Philippine Lung Cancer and Peritumoral Tissues: Case Series Study of Patients Stages I–III

  2023cites this paper
• Protein posttranslational modifications in health and diseases: Functions, regulatory mechanisms, and therapeutic implications

  2023cites this paper
• Influence of glycosylation on the immunogenicity and antigenicity of viral immunogens.

  2023cites this paper
• Hydrolytic activity of yeast oligosaccharyltransferase is enhanced when misfolded proteins accumulate in the endoplasmic reticulum

  2023cites this paper
• Advances in Bacterial Oligosaccharyltransferase Structure Elucidation and Potential Application to Glycoconjugate Vaccine Design.

  2023cites this paper
• B7 family protein glycosylation: Promising novel targets in tumor treatment

  2022cites this paper
• TREX1 plays multiple roles in human diseases.

  2022cites this paper
• Analysis of Tumor Glycosylation Characteristics and Implications for Immune Checkpoint Inhibitor’s Efficacy for Breast Cancer

  2022cites this paper
• N-Glycan Biosynthesis: Basic Principles and Factors Affecting Its Outcome.

  2021cites this paper
• Active site variants in STT3A cause a dominant type I congenital disorder of glycosylation with neuromusculoskeletal findings.

  2021cites this paper
• Structure and Biological Functions of Plant Glycans and Polysaccharides

  2021cites this paper
• The evolving genetic landscape of congenital disorders of glycosylation.

  2021cites this paper
• Overexpression of TaSTT3b‐2B improves resistance to sharp eyespot and increases grain weight in wheat

  2021cites this paper
• The N-glycosylation-related genes as potential targets for RNAi-mediated pest control of the Colorado potato beetle (Leptinotarsa decemlineata).

  2021cites this paper
• How glycosylation affects glycosylation: the role of N-glycans in glycosyltransferase activity.

  2020cites this paper
• Spontaneous glycan reattachment following N-glycanase treatment of influenza and HIV vaccine antigens.

  2020cites this paper
• Global view of human protein glycosylation pathways and functions

  2020cites this paper
• Uncoupling the hydrolysis of lipid-linked oligosaccharide from the oligosaccharyl transfer reaction by point mutations in yeast oligosaccharyltransferase

  2020cites this paper
• N-glycosylation is involved in stomatal development partially by modulating the release of active abscisic acid and auxin by β-glucosidase in Arabidopsis thaliana.

  2020cites this paper
• Carbohydrates | Free Oligosaccharides Structurally Related to N- and O-Linked Glycans

  2020cites this paper
• The occurrence of nonglycosylated forms of N‐glycoprotein upon proteasome inhibition does not confirm cytosolic deglycosylation

  2020cites this paper
• The PAR2 signal peptide prevents premature receptor cleavage and activation

  2019cites this paper
• Targeting STT3A‐oligosaccharyltransferase with NGI‐1 causes herpes simplex virus 1 dysfunction

  2019cites this paper