ALG2 is a bifunctional alpha-1,3/alpha-1,6-mannosyltransferase that acts in the early, cytoplasmic phase of dolichol-linked oligosaccharide (LLO) assembly, the glycan-precursor pathway for protein N-glycosylation. Using GDP-mannose as donor, it catalyses the addition of the second and third mannose residues onto Man1GlcNAc2-PP-dolichol to form the branched Man3GlcNAc2-PP-dolichol core, adding one mannose in an alpha-1,3 linkage (EC 2.4.1.132) and one in an alpha-1,6 linkage (EC 2.4.1.257); the Man3GlcNAc2-PP-dolichol product is subsequently elongated by ALG11. It is a member of the GT4 glycosyltransferase family and is anchored to the endoplasmic reticulum membrane with its catalytic domain and active site on the cytoplasmic face. In humans it associates with the ER via a single membrane-binding domain (a topology divergent from the multi-domain yeast ortholog). Loss-of-function variants cause the multisystem congenital disorder of glycosylation type Ii (ALG2-CDG) and an autosomal-recessive congenital myasthenic syndrome (CMS14).
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0012505 endomembrane system | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Broad endomembrane-system localization inferred phylogenetically. Correct but much less informative than the experimentally supported ER membrane / cytoplasmic side of ER membrane annotations for this enzyme. Reason: ALG2 is an ER membrane protein, and the ER is part of the endomembrane system, so the term is not wrong. However it is a very general cellular component; the specific ER membrane (GO:0005789) and cytoplasmic side of ER membrane (GO:0098554) annotations, both experimentally supported, capture the localization precisely. Retained as non-core context. Supporting Evidence: PMID:35136180 mostly co-localizes with the resident ER membrane protein Calnexin (CANX) |
| GO:0000033 alpha-1,3-mannosyltransferase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) inference of alpha-1,3-mannosyltransferase activity. This is the generic parent of the substrate-specific dolichol-linked activity (GO:0004378) and is well supported for ALG2, which does add an alpha-1,3 mannose. Reason: ALG2 experimentally adds an alpha-1,3-linked mannose during LLO assembly (PMID:12684507, PMID:35136180). The IBA term is correct; it is broader than the substrate-specific GO:0004378 also annotated here, but is a valid parent-level molecular function and consistent with the phylogenetic evidence. Supporting Evidence: PMID:12684507 hALG2 was shown to act as an alpha1,3-mannosyltransferase |
| GO:0006488 dolichol-linked oligosaccharide biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference that ALG2 participates in dolichol-linked oligosaccharide (LLO) biosynthesis. This is the core biological process for ALG2. Reason: ALG2 adds the second and third mannoses of the LLO precursor on the cytoplasmic face of the ER (PMID:35136180), and its deficiency blocks LLO elongation with accumulation of Man1/Man2GlcNAc2-PP-dolichol (PMID:12684507). The IBA process annotation is well supported and represents a core function. Supporting Evidence: PMID:35136180 Alg2 catalyzes addition of both the second and third mannose to M1Gn2-PDol to form a branched |
| GO:0004378 GDP-Man:Man(1)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Automated (RHEA/EC 2.4.1.132) assignment of the substrate-specific alpha-1,3 mannosyltransferase activity acting on Man1GlcNAc2-PP-dolichol. This is a core molecular function of ALG2 and is duplicated by IDA evidence below. Reason: The EC/RHEA-based IEA maps exactly onto the experimentally demonstrated alpha-1,3-mannosyltransferase reaction of ALG2 (PMID:12684507, PMID:35136180). Correct and precise; core function. Supporting Evidence: PMID:12684507 hALG2 was shown to act as an alpha1,3-mannosyltransferase |
| GO:0102704 GDP-Man:Man(2)GlcNAc(2)-PP-Dol alpha-1,6-mannosyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Automated (RHEA/EC 2.4.1.257) assignment of the substrate-specific alpha-1,6 mannosyltransferase activity acting on Man2GlcNAc2-PP-dolichol. This is a core molecular function of ALG2 and is duplicated by IDA evidence below. Reason: The EC/RHEA-based IEA maps exactly onto the experimentally demonstrated alpha-1,6-mannosyltransferase reaction of ALG2 (PMID:12684507, PMID:35136180). Correct and precise; core function. Supporting Evidence: PMID:35136180 Alg2 catalyzes addition of both the second and third mannose to M1Gn2-PDol to form a branched |
| GO:0005789 endoplasmic reticulum membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Subcellular-location keyword mapping to ER membrane. Consistent with the experimental EXP/IDA localization of ALG2. Reason: ALG2 localization to the ER membrane is experimentally established by immunofluorescence co-localization with the ER marker Calnexin and by membrane fractionation (PMID:35136180). The IEA CC term is correct and core. Supporting Evidence: PMID:35136180 mostly co-localizes with the resident ER membrane protein Calnexin (CANX) |
| GO:0009101 glycoprotein biosynthetic process | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro-to-GO mapping to the broad glycoprotein biosynthetic process. Correct in that ALG2 supplies the N-glycan precursor, but this is a general parent of the more specific LLO biosynthesis / N-linked glycosylation processes. Reason: ALG2 contributes to glycoprotein biosynthesis via LLO assembly, so the term is not wrong. It is, however, a broad parent term; the specific processes dolichol-linked oligosaccharide biosynthesis (GO:0006488) and protein N-linked glycosylation (GO:0006487), both experimentally supported, better describe the function. Retained as non-core. Supporting Evidence: PMID:35136180 N-glycosylation starts with the biosynthesis of lipid-linked oligosaccharide |
| GO:0016757 glycosyltransferase activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro-based assignment of the top-level glycosyltransferase activity. Correct but very general; ALG2 is a GT4-family mannosyltransferase. Reason: ALG2 is a glycosyltransferase (CAZy GT4), so this parent term is accurate but far broader than the substrate-specific mannosyltransferase activities annotated elsewhere (GO:0004378, GO:0102704). Retained as a correct broad parent, non-core. Supporting Evidence: PMID:35136180 Alg2 catalyzes addition of both the second and third mannose to M1Gn2-PDol to form a branched |
| GO:0005515 protein binding | IPI PMID:32814053 Interactome Mapping Provides a Network of Neurodegenerative ... | MARK AS OVER ANNOTATED | Summary: IntAct IPI capturing a yeast two-hybrid interaction between ALG2 and the neurodegeneration protein huntingtin (HTT, P42858) from a neurodegenerative disease interactome screen. Bare "protein binding" is uninformative about ALG2's molecular function. Reason: The interaction derives from a large-scale Y2H interactome focused on neurodegenerative disease proteins, not from a study of ALG2 function. Per curation guidance, bare "protein binding" is not an informative molecular function and there is no evidence this interaction underlies ALG2's mannosyltransferase role; it is not removed (experimental IPI) but flagged as an over-annotation for the purpose of describing ALG2's core function. Supporting Evidence: PMID:32814053 systematic yeast two-hybrid |
| GO:0006488 dolichol-linked oligosaccharide biosynthetic process | TAS Reactome:R-HSA-446193 | ACCEPT | Summary: Reactome-asserted involvement in LLO (dolichol lipid-linked oligosaccharide) biosynthesis and transfer to nascent protein. Core biological process for ALG2. Reason: Consistent with the experimental role of ALG2 in adding the second and third mannoses of the LLO precursor (PMID:35136180). Duplicate of the IDA/IBA GO:0006488 annotations; core function. Supporting Evidence: PMID:35136180 Alg2 catalyzes addition of both the second and third mannose to M1Gn2-PDol to form a branched |
| GO:0005789 endoplasmic reticulum membrane | EXP PMID:35136180 Topological and enzymatic analysis of human Alg2 mannosyltra... | ACCEPT | Summary: Experimental (EXP) ER membrane localization from the topological study of human ALG2, based on immunofluorescence co-localization with Calnexin and membrane fractionation. Reason: Directly demonstrated: FLAG-hAlg2 co-localizes with the ER marker Calnexin and partitions into the ER membrane fraction (PMID:35136180). Core localization. Supporting Evidence: PMID:35136180 mostly co-localizes with the resident ER membrane protein Calnexin (CANX) |
| GO:0102704 GDP-Man:Man(2)GlcNAc(2)-PP-Dol alpha-1,6-mannosyltransferase activity | IDA PMID:12684507 A new type of congenital disorders of glycosylation (CDG-Ii)... | ACCEPT | Summary: Direct assay evidence for the alpha-1,6-mannosyltransferase activity (addition of the third mannose, EC 2.4.1.257). One of the two core catalytic activities of bifunctional ALG2. Reason: Thiel et al. showed that in ALG2 deficiency both Man1GlcNAc2-PP-dolichol and Man2GlcNAc2-PP-dolichol accumulate, and WT hALG2 restores elongation, implicating ALG2 in the full second-plus-third mannose addition; the bifunctional alpha-1,6 activity is directly confirmed with purified enzyme in PMID:35136180. Core molecular function. Supporting Evidence: PMID:12684507 An accumulation of Man1GlcNAc2-PP-dolichol and PMID:35136180 Alg2 catalyzes addition of both the second and third mannose to M1Gn2-PDol to form a branched |
| GO:0004378 GDP-Man:Man(1)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase activity | IDA PMID:12684507 A new type of congenital disorders of glycosylation (CDG-Ii)... | ACCEPT | Summary: Direct assay evidence that hALG2 adds the alpha-1,3-linked mannose onto Man1GlcNAc2-PP-dolichol (the second mannose of the LLO, EC 2.4.1.132). One of the two core catalytic activities. Reason: hALG2 was directly shown to act as an alpha-1,3-mannosyltransferase, producing Manalpha1,3-ManGlcNAc2-PP-dolichol (PMID:12684507), and this substrate-specific activity was confirmed with purified enzyme in PMID:35136180. Core molecular function. Supporting Evidence: PMID:12684507 hALG2 was shown to act as an alpha1,3-mannosyltransferase |
| GO:0000033 alpha-1,3-mannosyltransferase activity | IDA PMID:35136180 Topological and enzymatic analysis of human Alg2 mannosyltra... | ACCEPT | Summary: Direct assay evidence (parent-level term) for alpha-1,3-mannosyltransferase activity from the purified-enzyme kinetic study of human ALG2. Reason: Purified recombinant hAlg2 transfers an alpha-1,3 mannose onto Man1GlcNAc2-linked substrate (PMID:35136180). Correct; a valid parent of the substrate-specific GO:0004378. Core function (redundant with the more specific term). Supporting Evidence: PMID:35136180 Alg2 catalyzes addition of both the second and third mannose to M1Gn2-PDol to form a branched |
| GO:0006487 protein N-linked glycosylation | IDA PMID:35136180 Topological and enzymatic analysis of human Alg2 mannosyltra... | ACCEPT | Summary: ALG2 acts upstream of and positively affects protein N-linked glycosylation by producing the branched Man3GlcNAc2-PP-dolichol precursor required for building the mature N-glycan that is transferred to protein. Reason: N-glycosylation begins with LLO biosynthesis, and ALG2 supplies the second and third mannoses of that precursor (PMID:35136180); loss of ALG2 causes CDG with hypoglycosylation, consistent with an upstream positive-regulatory relationship to N-linked glycosylation. Appropriate use of acts_upstream_of_positive_effect. Supporting Evidence: PMID:35136180 N-glycosylation starts with the biosynthesis of lipid-linked oligosaccharide |
| GO:0006488 dolichol-linked oligosaccharide biosynthetic process | IDA PMID:35136180 Topological and enzymatic analysis of human Alg2 mannosyltra... | ACCEPT | Summary: Direct experimental evidence for participation in dolichol-linked oligosaccharide biosynthesis. Core biological process. Reason: Purified hAlg2 converts Man1GlcNAc2 to the branched Man3GlcNAc2 LLO intermediate (PMID:35136180); this is the defining biological process for ALG2. Core function. Supporting Evidence: PMID:35136180 Alg2 catalyzes addition of both the second and third mannose to M1Gn2-PDol to form a branched |
| GO:0098554 cytoplasmic side of endoplasmic reticulum membrane | IDA PMID:35136180 Topological and enzymatic analysis of human Alg2 mannosyltra... | ACCEPT | Summary: Direct topological evidence that ALG2's active site faces the cytoplasm: both the N- and C-termini (and the catalytic C-terminal domain) are on the cytoplasmic side of the ER membrane. Reason: Protease-protection (FPP) and Suc2A glycosylation-reporter assays showed both termini of hAlg2 are cytosolic, placing the catalytic domain and its LLO-elongation reaction on the cytoplasmic face of the ER, consistent with the cytosolic phase of LLO assembly (PMID:35136180). Core, precise localization. Supporting Evidence: PMID:35136180 both the N- and C-termini of hAlg2 are oriented toward cytoplasmic side of the ER |
| GO:0004378 GDP-Man:Man(1)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase activity | IDA PMID:35136180 Topological and enzymatic analysis of human Alg2 mannosyltra... | ACCEPT | Summary: Direct assay evidence (substrate-specific, EC 2.4.1.132) for the alpha-1,3 mannosyltransferase activity from the purified-enzyme kinetic study. Core function (duplicate of the PMID:12684507 IDA). Reason: Kinetic analysis of purified recombinant hAlg2 quantified the alpha-1,3 mannose addition onto Man1GlcNAc2-PP-dolichol (PMID:35136180). Correct and precise; core molecular function. Supporting Evidence: PMID:35136180 Alg2 catalyzes addition of both the second and third mannose to M1Gn2-PDol to form a branched |
| GO:0102704 GDP-Man:Man(2)GlcNAc(2)-PP-Dol alpha-1,6-mannosyltransferase activity | IDA PMID:35136180 Topological and enzymatic analysis of human Alg2 mannosyltra... | ACCEPT | Summary: Direct assay evidence (substrate-specific, EC 2.4.1.257) for the alpha-1,6 mannosyltransferase activity from the purified-enzyme kinetic study. Core function (duplicate of the PMID:12684507 IDA). Reason: Purified recombinant hAlg2 catalyzes the alpha-1,6 mannose addition producing the branched Man3GlcNAc2-PP-dolichol, with distinct kinetics resolved by an MGAT1-based assay (PMID:35136180). Correct and precise; core molecular function. Supporting Evidence: PMID:35136180 Alg2 catalyzes addition of both the second and third mannose to M1Gn2-PDol to form a branched |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-4549368 | ACCEPT | Summary: Reactome-asserted ER membrane localization. Consistent with the experimental localization of ALG2. Reason: Duplicate of the experimentally supported ER membrane localization (PMID:35136180). Core localization. Supporting Evidence: PMID:35136180 mostly co-localizes with the resident ER membrane protein Calnexin (CANX) |
| GO:0006488 dolichol-linked oligosaccharide biosynthetic process | IGI PMID:12684507 A new type of congenital disorders of glycosylation (CDG-Ii)... | ACCEPT | Summary: Genetic-interaction evidence (with the yeast ALG2 ortholog, P43636) for involvement in LLO biosynthesis: human ALG2 cDNA complements the yeast alg2-1 mutant, restoring dolichol-linked oligosaccharide biosynthesis. Reason: Cross-species complementation of the yeast alg2-1 mutant by wild-type hALG2 (and failure of mutant hALG2) demonstrated the functional orthology and role in LLO biosynthesis (PMID:12684507). Core process, supported by genetic interaction with P43636. Supporting Evidence: PMID:12684507 the yeast ALG2 sequence was used to identify |
| GO:0000033 alpha-1,3-mannosyltransferase activity | TAS Reactome:R-HSA-446208 | ACCEPT | Summary: Reactome-asserted alpha-1,3-mannosyltransferase activity (addition of a second mannose to the N-glycan precursor by ALG2). Correct parent-level molecular function. Reason: Consistent with the experimentally demonstrated alpha-1,3-mannosyltransferase activity of ALG2 (PMID:12684507, PMID:35136180). Duplicate of the IDA/IBA GO:0000033 annotations. Supporting Evidence: PMID:12684507 hALG2 was shown to act as an alpha1,3-mannosyltransferase |
| GO:0000033 alpha-1,3-mannosyltransferase activity | TAS Reactome:R-HSA-4549368 | ACCEPT | Summary: Reactome-asserted alpha-1,3-mannosyltransferase activity (from the defective-ALG2 / CDG-1i pathway). Correct parent-level molecular function. Reason: Consistent with the experimentally demonstrated alpha-1,3-mannosyltransferase activity of ALG2 (PMID:12684507, PMID:35136180). Duplicate of the other GO:0000033 annotations. Supporting Evidence: PMID:12684507 hALG2 was shown to act as an alpha1,3-mannosyltransferase |
| GO:0009101 glycoprotein biosynthetic process | IGI PMID:12684507 A new type of congenital disorders of glycosylation (CDG-Ii)... | KEEP AS NON CORE | Summary: Genetic-interaction (yeast alg2 complementation) evidence mapped to the broad glycoprotein biosynthetic process. Correct but general. Reason: The yeast complementation experiment (PMID:12684507) supports ALG2's role in glycoprotein biosynthesis, but this is a broad parent of the more specific LLO biosynthesis / N-linked glycosylation processes. Retained as non-core. Supporting Evidence: PMID:12684507 the yeast ALG2 sequence was used to identify |
| GO:0016020 membrane | HDA PMID:19946888 Defining the membrane proteome of NK cells. | MARK AS OVER ANNOTATED | Summary: High-throughput MS detection of ALG2 in a membrane-proteome dataset of an NK-like cell line. Only supports generic "membrane" localization. Reason: This comes from a large-scale membrane-proteome survey and provides only the uninformative parent term "membrane"; the specific and experimentally supported ER membrane / cytoplasmic side of ER membrane localizations supersede it. Not removed (valid detection) but flagged as an over-annotation for describing ALG2's localization. Supporting Evidence: PMID:19946888 identified 1843 proteins with high confidence scores |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-446208 | ACCEPT | Summary: Reactome-asserted ER membrane localization. Consistent with the experimental localization of ALG2. Reason: Duplicate of the experimentally supported ER membrane localization (PMID:35136180). Core localization. Supporting Evidence: PMID:35136180 mostly co-localizes with the resident ER membrane protein Calnexin (CANX) |
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Download this section (compressed HTML)Q: Under physiological GDP-mannose and substrate concentrations in human cells, what is the in vivo balance between the alpha-1,3-first and alpha-1,6-first routes to Man3GlcNAc2-PP-dolichol, and does the M2Gn2(alpha-1,6) intermediate serve any regulatory function?
Q: Does ALG2 function as part of a stable ALG1-ALG2-ALG11 mannosyltransferase complex in human cells, and how does complex assembly affect its bifunctional activity?
Experiment: Reconstitute the human ALG1-ALG2-ALG11 pathway with purified enzymes and defined dolichol-linked substrates to measure channeling and the order of mannose additions in a membrane-mimetic system.
Type: in vitro reconstitution / LC-MS kinetics
Experiment: Structure determination (cryo-EM or crystallography) of human ALG2 in complex with GDP-mannose and a Man1/Man2GlcNAc2-PP-dolichol analog to define the basis of its dual alpha-1,3/alpha-1,6 regioselectivity.
Type: structural biology
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