DDOST (OST48; dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit) is a non-catalytic core subunit of the oligosaccharyltransferase (OST) complex, the endoplasmic reticulum enzyme that carries out the central step of N-linked protein glycosylation. It is a single-pass type I ER membrane glycoprotein with a large lumenal domain, a single transmembrane helix, and a short cytoplasmic tail. OST transfers the preassembled high-mannose glycan (Glc3Man9GlcNAc2) en bloc from dolichol-pyrophosphate onto asparagine residues within Asn-X-Ser/Thr sequons in nascent secretory and membrane proteins. DDOST is one of the shared core subunits (with RPN1, RPN2, OST4, DAD1, TMEM258) common to both the STT3A-containing (cotranslational, OST-A) and STT3B-containing (posttranslocational, OST-B) complexes, while the STT3A/STT3B paralogues provide the catalytic activity. DDOST is required for the assembly and stability of both OST complex forms, and its loss causes global protein hypoglycosylation. Deficiency of DDOST causes a congenital disorder of glycosylation (DDOST-CDG / CDG1R).
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0006487 protein N-linked glycosylation | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) inference that DDOST participates in protein N-linked glycosylation. This is the correct core biological process for an OST subunit and is well supported by experimental data on the human protein and orthologs. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt consensus motif in nascent polypeptide chains, the first step in |
| GO:0008250 oligosaccharyltransferase complex | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) inference of OST complex membership. This is a core, experimentally corroborated cellular-component assignment for DDOST/OST48. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt Component of the oligosaccharyltransferase (OST) complex |
| GO:0005789 endoplasmic reticulum membrane | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic annotation to ER membrane, matching the authoritative UniProt subcellular location. This is DDOST's correct core location; the redundant IEA is accepted rather than flagged as over-annotation. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt Endoplasmic reticulum membrane |
| GO:0006487 protein N-linked glycosylation | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO electronic annotation to protein N-linked glycosylation via the DDOST 48 kDa subunit signature. Correct core process; redundant with experimental annotations. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt consensus motif in nascent polypeptide chains, the first step in |
| GO:0016020 membrane | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: Generic membrane location (ARBA). Correct but uninformative; the specific and correct location is endoplasmic reticulum membrane. Kept as non-core. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt Single-pass type I membrane protein |
| GO:0005515 protein binding | IPI PMID:21903422 Mapping a dynamic innate immunity protein interaction networ... | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" IPI recording the DDOST-STING1 (Q86WV6) interaction from an innate-immunity interactome screen (STING/UNC93B1 pulled down many ER membrane/translocation proteins). The interaction is corroborated by the UniProt IntAct record, but "protein binding" is uninformative about DDOST's function and this is not a core function. Retained (not removed) but marked as over-annotated. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt P39656; Q86WV6: STING1; NbExp=2; IntAct=EBI-358866, EBI-2800345 |
| GO:0005789 endoplasmic reticulum membrane | NAS PMID:31831667 Cryo-electron microscopy structures of human oligosaccharylt... | ACCEPT | Summary: ComplexPortal NAS to ER membrane, based on the cryo-EM OST structures. Correct core location. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt Endoplasmic reticulum membrane |
| GO:0006487 protein N-linked glycosylation | IDA PMID:31831667 Cryo-electron microscopy structures of human oligosaccharylt... | ACCEPT | Summary: Direct assay (cryo-EM structural characterization of human OST-A and OST-B with bound acceptor peptide and dolichylphosphate) supporting DDOST's participation in N-linked glycosylation as an OST subunit. Core process. Supporting Evidence: PMID:31831667 Oligosaccharyltransferase (OST) catalyzes the transfer of a high-mannose glycan onto secretory proteins in the endoplasmic reticulum |
| GO:0006487 protein N-linked glycosylation | NAS PMID:31831667 Cryo-electron microscopy structures of human oligosaccharylt... | ACCEPT | Summary: ComplexPortal NAS duplicate of the N-linked glycosylation process assignment from the same structural paper. Correct core process; redundant with the IDA. Supporting Evidence: PMID:31831667 Oligosaccharyltransferase (OST) catalyzes the transfer of a high-mannose glycan onto secretory proteins in the endoplasmic reticulum |
| GO:0008250 oligosaccharyltransferase complex | IPI PMID:31831667 Cryo-electron microscopy structures of human oligosaccharylt... | ACCEPT | Summary: ComplexPortal IPI assigning OST complex membership from the cryo-EM structures resolving DDOST within OST-A and OST-B. Core cellular component. Supporting Evidence: PMID:31831667 Mammals express two distinct OST complexes that act in a cotranslational (OST-A) or posttranslocational (OST-B) manner |
| GO:0008250 oligosaccharyltransferase complex | NAS PMID:31831667 Cryo-electron microscopy structures of human oligosaccharylt... | ACCEPT | Summary: ComplexPortal NAS duplicate of OST complex membership from the same paper. Core cellular component; redundant with the IPI. Supporting Evidence: PMID:31831667 Mammals express two distinct OST complexes that act in a cotranslational (OST-A) or posttranslocational (OST-B) manner |
| GO:0005783 endoplasmic reticulum | IDA GO_REF:0000052 | KEEP AS NON CORE | Summary: HPA immunofluorescence localizing DDOST to the endoplasmic reticulum. Consistent with the authoritative ER membrane assignment; the more specific ER membrane term is preferred but this parent is correct. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt Endoplasmic reticulum membrane |
| GO:0005789 endoplasmic reticulum membrane | ISS GO_REF:0000024 | ACCEPT | Summary: ISS to ER membrane by transfer from the yeast/canine ortholog. Matches the authoritative UniProt location. Core. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt Endoplasmic reticulum membrane |
| GO:0160226 oligosaccharyltransferase complex A | IDA PMID:31831667 Cryo-electron microscopy structures of human oligosaccharylt... | ACCEPT | Summary: Direct evidence (cryo-EM structure of human OST-A) that DDOST is a subunit of the STT3A-containing oligosaccharyltransferase complex A. Core cellular component. Supporting Evidence: PMID:31831667 Mammals express two distinct OST complexes that act in a cotranslational (OST-A) or posttranslocational (OST-B) manner |
| GO:0160226 oligosaccharyltransferase complex A | IDA PMID:36697828 Visualization of translation and protein biogenesis at the E... | ACCEPT | Summary: Cryo-EM visualization of the STT3A-containing OST bound to the ER translocon, resolving DDOST within complex A. Core cellular component. Supporting Evidence: PMID:36697828 Visualization of translation and protein biogenesis at the ER membrane |
| GO:0160226 oligosaccharyltransferase complex A | IDA PMID:38670073 Positive selection CRISPR screens reveal a druggable pocket ... | ACCEPT | Summary: Cryo-EM structure of the STT3A-containing OST (used to define the NGI-1 druggable pocket) resolving DDOST within complex A. Core cellular component. Supporting Evidence: PMID:38670073 the oligosaccharyltransferase complex OST-A for N-glycosylation and cell-surface localization |
| GO:0160227 oligosaccharyltransferase complex B | IDA PMID:31831667 Cryo-electron microscopy structures of human oligosaccharylt... | ACCEPT | Summary: Direct evidence (cryo-EM structure of human OST-B) that DDOST is a subunit of the STT3B-containing oligosaccharyltransferase complex B. Core cellular component. Supporting Evidence: PMID:31831667 Mammals express two distinct OST complexes that act in a cotranslational (OST-A) or posttranslocational (OST-B) manner |
| GO:0008250 oligosaccharyltransferase complex | IDA PMID:22467853 The oligosaccharyltransferase subunits OST48, DAD1 and KCP2 ... | ACCEPT | Summary: Biochemical demonstration that DDOST/OST48 is a component of both STT3A- and STT3B-containing OST complexes and is required for their assembly. Core cellular component. Supporting Evidence: PMID:22467853 OST48 and DAD1 are required for the assembly of both STT3A- and STT3B-containing OST complexes |
| GO:0006487 protein N-linked glycosylation | IMP PMID:22467853 The oligosaccharyltransferase subunits OST48, DAD1 and KCP2 ... | ACCEPT | Summary: siRNA knockdown of OST48 produces a pronounced hypoglycosylation phenotype, directly implicating DDOST in protein N-linked glycosylation. Core process. Supporting Evidence: PMID:22467853 OST48 and DAD1 are global modulators of OST stability and hence N-glycosylation |
| GO:0031647 regulation of protein stability | IMP PMID:22467853 The oligosaccharyltransferase subunits OST48, DAD1 and KCP2 ... | MARK AS OVER ANNOTATED | Summary: This IMP over-interprets the paper. OST48 knockdown destabilizes the OST complex itself (loss of complex integrity), not a dedicated role in regulating the stability of other proteins. The stability phenotype is of the OST complex and is better captured as complex assembly / N-glycosylation. Retained (experimental) but marked as over-annotation of a structural/assembly role. Supporting Evidence: PMID:22467853 OST48 and DAD1 are global modulators of OST stability and hence N-glycosylation |
| GO:0008047 enzyme activator activity | IMP PMID:22467853 The oligosaccharyltransferase subunits OST48, DAD1 and KCP2 ... | MARK AS OVER ANNOTATED | Summary: The IMP that DDOST "enables enzyme activator activity" over-interprets its structural role. DDOST is a non-catalytic core subunit required for OST complex assembly/stability; it does not act as a discrete allosteric enzyme activator. Its contribution to catalysis is better modeled as contributing to the OST glycotransferase activity via complex membership. Retained (experimental) but marked as over-annotated. Supporting Evidence: PMID:22467853 OST48 and DAD1 are required for the assembly of both STT3A- and STT3B-containing OST complexes |
| GO:0008250 oligosaccharyltransferase complex | IDA PMID:9642163 Interleukin-2 induces N-glycosylation in T-cells: characteri... | ACCEPT | Summary: Purification of human lymphocyte OST identified DDOST/OST48 (the "50-kDa homologue of Wbp1") together with ribophorins as OST components. Core cellular component. Supporting Evidence: PMID:9642163 a 50-kDa homologue of Wbp1, a yeast protein essential for N-glycosylation |
| GO:0005515 protein binding | IPI PMID:29765154 The cancer-associated microprotein CASIMO1 controls cell pro... | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" IPI capturing the DDOST-SMIM22/CASIMO1 (K7EJ46) interaction, corroborated by the UniProt SUBUNIT record. Uninformative about DDOST's molecular function and not a core function; the cited paper concerns CASIMO1/squalene epoxidase, not a DDOST-functional consequence. Retained (not removed) but marked as over-annotated. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt SMIM22 (PubMed:29765154) |
| GO:0035577 azurophil granule membrane | TAS Reactome:R-HSA-6798739 | MARK AS OVER ANNOTATED | Summary: Reactome neutrophil-degranulation (azurophil granule exocytosis) inclusion. This reflects large-scale granule-proteome cataloguing rather than the site of DDOST's molecular function, which is the ER membrane. Not a core location; marked as over-annotated. Supporting Evidence: Reactome:R-HSA-6798739 Exocytosis of azurophil granule membrane proteins |
| GO:0016020 membrane | HDA PMID:19946888 Defining the membrane proteome of NK cells. | KEEP AS NON CORE | Summary: High-throughput NK-cell membrane-proteome identification. Correct at the generic membrane level but uninformative; DDOST's specific location is the ER membrane. Kept as non-core. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt Single-pass type I membrane protein |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-446209 | ACCEPT | Summary: Reactome TAS to ER membrane (Transfer of N-glycan to the protein reaction). Correct core location. Supporting Evidence: Reactome:R-HSA-446209 The reaction occurs cotranslationally as the growing peptide chain leaves a ribosome associated with the ER membrane and enters the ER lumen |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-9694793 | ACCEPT | Summary: Reactome TAS to ER membrane for a pathway-specific N-glycosylation reaction (SARS-CoV-2 spike glycosylation). Correct core location; pathway context is substrate-specific but the location is right. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt Endoplasmic reticulum membrane |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-9816276 | ACCEPT | Summary: Reactome TAS to ER membrane for CDH1 N-glycosylation. Correct core location. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt Endoplasmic reticulum membrane |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-9918962 | ACCEPT | Summary: Reactome TAS to ER membrane for a viral (dengue E) N-glycosylation reaction. Correct core location. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt Endoplasmic reticulum membrane |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-9918988 | ACCEPT | Summary: Reactome TAS to ER membrane for a viral (pre-M) N-glycosylation reaction. Correct core location. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt Endoplasmic reticulum membrane |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-9919011 | ACCEPT | Summary: Reactome TAS to ER membrane for a viral (pre-NS1) N-glycosylation reaction. Correct core location. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt Endoplasmic reticulum membrane |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-9931286 | ACCEPT | Summary: Reactome TAS to ER membrane for CD274/PD-L1 N-glycosylation. Correct core location. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt Endoplasmic reticulum membrane |
| GO:0005886 plasma membrane | TAS Reactome:R-HSA-6798739 | MARK AS OVER ANNOTATED | Summary: Reactome plasma-membrane annotation from the azurophil-granule exocytosis pathway (granule membrane proteins reaching the cell surface upon degranulation). This is not DDOST's functional location; DDOST acts in the ER membrane. Marked as over-annotated. Supporting Evidence: Reactome:R-HSA-6798739 Exocytosis of azurophil granule membrane proteins |
| GO:0008250 oligosaccharyltransferase complex | ISS GO_REF:0000024 | ACCEPT | Summary: ISS to OST complex membership by transfer from the yeast ortholog (Q05052). Core cellular component; redundant with experimental annotations. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt Component of the oligosaccharyltransferase (OST) complex |
| GO:0009101 glycoprotein biosynthetic process | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: ISS to the broad "glycoprotein biosynthetic process". Correct but general; the specific and experimentally supported process is protein N-linked glycosylation. Kept as non-core parent. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt consensus motif in nascent polypeptide chains, the first step in |
| GO:0006487 protein N-linked glycosylation | IDA PMID:9642163 Interleukin-2 induces N-glycosylation in T-cells: characteri... | ACCEPT | Summary: Purification/characterization of human lymphocyte OST directly implicating the enzyme (including DDOST/OST48) in N-glycosylation. Core process. Supporting Evidence: PMID:9642163 N-glycosylation activity increased 10-fold after mitogen activation of PBLs |
| GO:0034097 response to cytokine | IDA PMID:9642163 Interleukin-2 induces N-glycosylation in T-cells: characteri... | MARK AS OVER ANNOTATED | Summary: This annotation reflects the experimental system (IL-2/mitogen-induced increase in T-cell N-glycosylation activity) rather than a DDOST-intrinsic role in cytokine response. DDOST is the OST enzyme whose activity was measured to rise; it does not itself constitute a cytokine-response function. Retained (experimental) but marked as over-annotated. Supporting Evidence: PMID:9642163 N-glycosylation activity increased 10-fold after mitogen activation of PBLs |
| GO:0042110 T cell activation | IDA PMID:9642163 Interleukin-2 induces N-glycosylation in T-cells: characteri... | MARK AS OVER ANNOTATED | Summary: As with the cytokine-response annotation, this captures the biological context (OST activity rises during T-cell activation) rather than a DDOST-driven role in activating T cells. Not a core function of DDOST. Retained (experimental) but marked as over-annotated. Supporting Evidence: PMID:9642163 N-glycosylation activity increased 10-fold after mitogen activation of PBLs |
| GO:0043231 intracellular membrane-bounded organelle | IDA PMID:9642163 Interleukin-2 induces N-glycosylation in T-cells: characteri... | KEEP AS NON CORE | Summary: Localization to an intracellular membrane-bounded organelle (microsomal/ER fraction). Correct but very general; the specific location is the ER membrane. Kept as non-core parent. Supporting Evidence: file:human/DDOST/DDOST-uniprot.txt Endoplasmic reticulum membrane |
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Download this section (compressed HTML)Q: Roboti & High show OST48 depletion destabilizes both OST complexes and causes global hypoglycosylation. Is that phenotype fully explained by loss of complex assembly, or does OST48 make a separable contribution that would justify retaining the enzyme activator activity (GO:0008047) and regulation of protein stability (GO:0031647) annotations?
Suggested experts: Roboti P, High S, Gilmore R
Q: Do the neutrophil azurophil granule membrane (GO:0035577) and plasma membrane (GO:0005886) localizations of DDOST reflect a genuine post-ER pool of OST48, or are they carry-over from granule-proteome sweeps? Is there any evidence for OST activity outside the ER?
Suggested experts: Borregaard N, Rørvig S
Q: How does the CDG1R variant Gly217Asp compromise OST48 - by destabilizing the lumenal beta-propeller, by blocking incorporation into the OST core, or by reducing steady-state protein level - and does it affect OST-A and OST-B equally?
Suggested experts: Locher KP, Ramírez AS, Freeze HH
Experiment: Use a degron-tagged DDOST line to deplete OST48 acutely, then measure (i) OST-A and OST-B complex integrity by blue-native PAGE and STT3A/STT3B steady-state levels, and (ii) site-specific N-glycosylation occupancy across the glycoproteome by intact glycopeptide mass spectrometry. Test whether adding back purified OST lacking OST48 restores in vitro transferase activity on a synthetic acceptor peptide, which would separate an assembly requirement from a catalytic-activation requirement.
Hypothesis: DDOST/OST48 is required for the structural integrity of the OST core, and its loss reduces N-glycosylation sequon occupancy globally rather than for a defined substrate class; the enzyme activator activity annotation reflects this structural role rather than an autonomous activating function.
Type: degron depletion, native complex analysis, and in vitro OST activity assay
Experiment: Fractionate primary human neutrophils into azurophil, specific and gelatinase granules plus secretory vesicles and plasma membrane, and quantify OST48 alongside STT3A, RPN1 and a bona fide granule marker (MPO) by targeted mass spectrometry. Assay oligosaccharyltransferase activity in each fraction with a synthetic sequon acceptor to test whether any non-ER fraction carries activity.
Hypothesis: The azurophil-granule and plasma-membrane assignments of DDOST are proteomic carry-over and there is no functional extra-ER pool of OST48.
Type: subcellular fractionation with targeted proteomics and enzyme assay
Experiment: Express wild-type and Gly217Asp OST48 in DDOST-null cells and compare protein half-life (cycloheximide chase), incorporation into STT3A- and STT3B-containing complexes (blue-native PAGE and reciprocal co-immunoprecipitation), and rescue of hypoglycosylation using a validated reporter substrate plus transferrin isoform profiling.
Hypothesis: The DDOST-CDG variant Gly217Asp acts by preventing incorporation of OST48 into the OST core rather than by abolishing an OST48-specific activity.
Type: variant complementation and complex-assembly analysis
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