pomt2 encodes protein O-mannosyl-transferase 2, a multi-pass endoplasmic-reticulum membrane glycosyltransferase of the GT39 family. Together with Pomt1 it forms the heteromeric enzyme that transfers mannose from dolichyl-phosphate-mannose onto the hydroxyl group of serine or threonine residues of secretory-pathway proteins, the first step of O-mannosyl glycan biosynthesis. Co-expression of both subunits is needed for high activity; either subunit alone is inactive or weakly active. The principal physiological substrate is alpha-dystroglycan, whose O-mannosyl glycans are later extended in the Golgi to matriglycan, the laminin-, agrin- and EYS-binding polysaccharide. In zebrafish, pomt2 is maternally supplied and widely expressed (eye, brain, somites, muscle); morpholino knockdown abolishes glycosylated alpha-dystroglycan and causes body-axis, pericardial and eye-pigmentation defects, while CRISPR null mutants lose matriglycan, show hydrocephalus, cerebellar hypoplasia and muscular dystrophy, fail to anchor EYS at the photoreceptor connecting cilium, and undergo late-onset rod and cone degeneration, modelling human POMT2-associated dystroglycanopathy and retinal atrophy.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005783 endoplasmic reticulum | IBA GO_REF:0000033 | ACCEPT | Summary: The PAINT node PTN001295889 places the GT39 protein O-mannosyltransferases (yeast Pmt1-7, Drosophila rt/tw, mouse and human POMT1/POMT2) in the ER, the compartment where dolichyl-phosphate-mannose is used. No zebrafish localization experiment exists, but the zebrafish protein is an 11-transmembrane-helix enzyme with the same MIR-domain luminal architecture and the same Dol-P-Man-dependent activity, and UniProt transfers the ER-membrane location from human POMT2. Reason: Dol-P-Man is synthesized on the cytosolic face of the ER and flipped into the lumen, so an enzyme that consumes it to mannosylate nascent secretory proteins must act in the ER; the zebrafish enzyme demonstrably uses this donor (it reconstitutes protein O-mannosyltransferase activity with Pomt1 in HEK293T microsomes, PMID:20466645). The zebrafish protein is inside the clade at which the PAINT curator placed the ER localization (human POMT2 Q9Y6A1 is among the descendants used), and there is no evidence of a lineage-specific relocation. The IBA is therefore sound; the more specific ER membrane term (ISS/IEA rows below) captures the same location at finer grain. Supporting Evidence: file:DANRE/pomt2/pomt2-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum membrane PMID:20466645 only when both zPOMT1 and zPOMT2 were expressed in human embryonic kidney 293T cells were high levels of protein O-mannosyltransferase activity detected PMID:36499139 Synthesis of O-mannosyl glycans is initiated by the transfer of mannose to Ser/Thr residues on Ξ±-DG by the POMT1/2 complex. |
| GO:0004169 dolichyl-phosphate-mannose-protein mannosyltransferase activity | IBA GO_REF:0000033 | ACCEPT | Summary: The term is the EC 2.4.1.109 reaction (Dol-P-Man + protein Ser/Thr -> O-mannosyl protein). PAINT node PTN008936969 asserts it for the POMT1/POMT2 clade, and the zebrafish gene itself (ZDB-GENE-070112-1002) is among the descendants that seeded the node, because zebrafish Pomt2 has direct experimental support (IGI row from PMID:20466645). Reason: This is the core catalytic activity of the gene product. The zebrafish enzyme was assayed directly - co-expression of zPomt1 and zPomt2 in HEK293T cells gave high protein O-mannosyltransferase activity, and either fish subunit could substitute for its human counterpart (PMID:20466645) - and UniProt records the reaction with both seryl- and threonyl-protein acceptors from the same paper. The zebrafish gene appearing in its own WITH/FROM is expected (its experimental annotation was part of the IBD evidence), not circular. Supporting Evidence: PMID:20466645 only when both zPOMT1 and zPOMT2 were expressed in human embryonic kidney 293T cells were high levels of protein O-mannosyltransferase activity detected, indicating that both zPOMT1 and zPOMT2 were required for full enzymatic activity file:DANRE/pomt2/pomt2-uniprot.txt CATALYTIC ACTIVITY: file:DANRE/pomt2/pomt2-uniprot.txt FUNCTION: Transfers mannosyl residues to the hydroxyl group of serine or threonine residues. Coexpression of both POMT1 and POMT2 is necessary for enzyme activity, expression of either POMT1 or POMT2 alone is insufficient. |
| GO:0035269 protein O-linked glycosylation via mannose | IBA GO_REF:0000033 | ACCEPT | Summary: The process term names the glycan biosynthetic route that begins with an alpha-mannose attached to Ser/Thr. Pomt2 catalyzes exactly that initiating step, so it participates directly in the process. PAINT places the assertion at PTN008936969 with the zebrafish gene among the experimental descendants. Reason: Participation is direct - the gene product performs the first chemical step of the pathway. In zebrafish, loss of pomt2 removes the downstream O-mannosyl-glycan readouts on alpha-dystroglycan in vivo, both in morphants (loss of IIH6 reactivity, PMID:20466645) and in CRISPR nulls (loss of matriglycan and of EYS binding, PMID:36499139), and the biochemical activity was reconstituted in cells (PMID:20466645). The IBA therefore rests on target-specific experimental evidence as well as on the conserved clade function. Supporting Evidence: PMID:20466645 the IIH6 reactivity was lost in both zPOMT1 and zPOMT2 morphants PMID:36499139 pomt2 mutation resulted in a loss of matriglycan and abolished binding of EYS protein to Ξ±-dystroglycan file:DANRE/pomt2/pomt2-uniprot.txt Glycosylation of alpha-dystroglycan (dag1) is severely reduced. |
| GO:0000030 mannosyltransferase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro IPR003342 (ArnT-like N-terminal glycosyltransferase domain, shared by the PMT family and bacterial lipid-A/arabinosyl transferases) maps to the generic mannosyltransferase parent. The mapping is correct but stops well short of what is known for this protein. Reason: Pomt2 does transfer mannose, but its acceptor is a protein Ser/Thr hydroxyl and its donor is Dol-P-Man, which is exactly the child term GO:0004169 already carried by IBA, IGI and EC-mapped rows. The generic term adds nothing and its definition even points at carbohydrate or lipid acceptors ('typically another carbohydrate or a lipid'). Replace with the specific activity established in the zebrafish reconstitution assay (PMID:20466645). Proposed replacements: dolichyl-phosphate-mannose-protein mannosyltransferase activity Supporting Evidence: PMID:20466645 only when both zPOMT1 and zPOMT2 were expressed in human embryonic kidney 293T cells were high levels of protein O-mannosyltransferase activity detected file:DANRE/pomt2/pomt2-uniprot.txt FUNCTION: Transfers mannosyl residues to the hydroxyl group of serine or threonine residues. |
| GO:0004169 dolichyl-phosphate-mannose-protein mannosyltransferase activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC 2.4.1.109 is assigned to the UniProt entry on the strength of the zebrafish activity assay (PMID:20466645), and the EC-to-GO mapping lands on the precise term. Reason: The EC number is not an inherited guess here; UniProt cites the zebrafish paper as the evidence for EC 2.4.1.109 and for both Rhea reactions. The mapped GO term is identical to the experimentally supported IGI and IBA rows, so this row is correct and consistent. Supporting Evidence: file:DANRE/pomt2/pomt2-uniprot.txt EC=2.4.1.109 {ECO:0000269|PubMed:20466645}; PMID:20466645 both zPOMT1 and zPOMT2 were required for full enzymatic activity |
| GO:0005789 endoplasmic reticulum membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Mapping of the UniProt subcellular-location keyword (SL-0097, ER membrane). The UniProt location is itself transferred from human POMT2 (ECO:0000250) and the protein has eleven predicted transmembrane helices, consistent with a multi-pass ER membrane enzyme. Reason: The term is the right compartment at the right granularity for a Dol-P-Man-using protein O-mannosyltransferase (see the ER IBA row). It is not established by a zebrafish experiment, but the conserved membrane topology (11 TRANSMEM features in UniProt), the ER-restricted donor substrate, and the human-ortholog transfer all agree, and nothing in the zebrafish literature suggests a different location. Accepted as the core location. Supporting Evidence: file:DANRE/pomt2/pomt2-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum membrane file:DANRE/pomt2/pomt2-uniprot.txt Multi-pass membrane protein |
| GO:0006493 protein O-linked glycosylation | IEA GO_REF:0000120 | MODIFY | Summary: Combined ARBA/InterPro mapping to the generic O-glycosylation parent. Correct but less specific than the sugar-specific child (GO:0035269) that the zebrafish IGI and IBA rows already carry. Reason: Pomt2 initiates O-mannosylation specifically; it does not participate in O-GalNAc, O-fucose, O-GlcNAc or other O-linked pathways that the parent term also covers. The zebrafish evidence (loss of IIH6/matriglycan reactivity in morphants and mutants, PMID:20466645 and PMID:36499139) is entirely about the mannose route. Replace with GO:0035269 protein O-linked glycosylation via mannose, so the same term is used consistently across the IEA, IBA and IGI rows. Proposed replacements: protein O-linked glycosylation via mannose Supporting Evidence: PMID:36499139 Protein O-mannosyltransferase 2 (POMT2) is an enzyme required for the synthesis of O-mannosyl glycans. PMID:20466645 the IIH6 reactivity was lost in both zPOMT1 and zPOMT2 morphants |
| GO:0016020 membrane | IEA GO_REF:0000002 | MODIFY | Summary: InterPro IPR003342 maps to the root-level 'membrane' term. True for an 11-transmembrane-helix protein, but uninformative next to the ER membrane rows. Reason: The protein is a multi-pass membrane protein (UniProt TRANSMEM features), so the term is not wrong, but the membrane in question is specifically the ER membrane, which is already asserted by the ISS and SubCell-mapped rows and follows from the enzyme's use of Dol-P-Man. Replace with GO:0005789 endoplasmic reticulum membrane for consistency. Proposed replacements: endoplasmic reticulum membrane Supporting Evidence: file:DANRE/pomt2/pomt2-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum membrane file:DANRE/pomt2/pomt2-uniprot.txt Multi-pass membrane protein |
| GO:0045494 photoreceptor cell maintenance | IMP PMID:36499139 Deletion of POMT2 in Zebrafish Causes Degeneration of Photor... | KEEP AS NON CORE | Summary: ZFIN annotation from the pomt2(sny5+13) CRISPR null (full text cached). Mutants have normal photoreceptors at 1 month but by 6 months show opsin mislocalization, increased TUNEL-positive rods and cones, and reduced cone-marker immunoreactivity; the authors trace this to loss of matriglycan on alpha-dystroglycan, which abolishes EYS binding and EYS enrichment at the connecting cilium. GO defines the term as any process preventing photoreceptor degeneration, and ZFIN used the indirect qualifier acts_upstream_of_or_within. Reason: The phenotype is real and well documented, but Pomt2 does not itself do the work of maintaining photoreceptors - it O-mannosylates alpha-dystroglycan in the ER, and the protective effect is executed downstream by matriglycan-dependent anchoring of EYS at the connecting cilium (PMID:36499139, Discussion). Photoreceptor development is normal in the mutant and degeneration is late-onset, which fits a maintenance defect that is a consequence of the missing glycan rather than a direct activity of the enzyme. The annotation should be retained with its upstream qualifier as a non-core, organism-level role; it is not the core function and should not be used to seed orthologs. Supporting Evidence: PMID:36499139 While photoreceptor development was not affected in pomt2 mutant zebrafish, photoreceptor density was significantly reduced by 6-mpf. Apoptosis of both cone and rod photoreceptors in the pomt2 mutant retina was significantly increased. These results indicate that POMT2 is essential for EYS protein localization near connecting cilia and for photoreceptor survival. PMID:36499139 Loss of EYS in the connecting cilium region in pomt2 mutant zebrafish may ultimately cause photoreceptor degeneration. PMID:36499139 These data suggest that POMT2-mediated O-mannosyl glycosylation is required for EYS protein localization to the connecting cilium region and photoreceptor survival. |
| GO:0005789 endoplasmic reticulum membrane | ISS GO_REF:0000024 | ACCEPT | Summary: Curator-judged transfer of the ER membrane location from human POMT2 (Q9Y6A1), which is the same source UniProt cites for the zebrafish SUBCELLULAR LOCATION comment. Reason: Zebrafish Pomt2 is 756 aa with eleven predicted transmembrane helices and three luminal MIR domains, the conserved GT39 topology, and it reconstitutes Dol-P-Man-dependent activity with Pomt1 in mammalian cells (PMID:20466645) - the same cell type and membrane system in which human POMT1/POMT2 act. The ortholog transfer is therefore well justified and there is no zebrafish-specific reason to doubt it. Supporting Evidence: file:DANRE/pomt2/pomt2-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum membrane PMID:20466645 either heterologous combination, zPOMT1 and human POMT2 (hPOMT2) or hPOMT1 and zPOMT2, resulted in enzymatic activity in cultured cells |
| GO:0004169 dolichyl-phosphate-mannose-protein mannosyltransferase activity | IGI PMID:20466645 Protein O-mannosylation is necessary for normal embryonic de... | ACCEPT | Summary: ZFIN IGI with pomt1 (ZDB-GENE-060929-966). Avsar-Ban et al. cloned zPOMT1 and zPOMT2 and measured protein O-mannosyltransferase activity in HEK293T cells; high activity required co-expression of both fish subunits, and each fish subunit was active in combination with the human partner. The cache is abstract-only, but the abstract states the activity result explicitly. Reason: This is the direct biochemical evidence for the core activity of the zebrafish enzyme, and IGI is the appropriate code because the activity is a property of the Pomt1-Pomt2 heteromer. UniProt derives EC 2.4.1.109 and both Rhea reactions for the zebrafish entry from this paper. The enables qualifier (rather than contributes_to) is right here, and is what GOA also uses for human POMT2 (Q9UKY4), because Pomt2 is itself a GT39-fold catalytic subunit, with both subunits of the obligate heteromer carrying the glycosyltransferase fold, unlike the dph2 case in this batch where a non-catalytic electron-donor subunit merely contributes to its partner's activity. The complex is GO:0031502 (dolichyl-phosphate-mannose-protein mannosyltransferase complex), to which human POMT2 is annotated part_of by IPI. Accept as core. Supporting Evidence: PMID:20466645 only when both zPOMT1 and zPOMT2 were expressed in human embryonic kidney 293T cells were high levels of protein O-mannosyltransferase activity detected, indicating that both zPOMT1 and zPOMT2 were required for full enzymatic activity PMID:20466645 either heterologous combination, zPOMT1 and human POMT2 (hPOMT2) or hPOMT1 and zPOMT2, resulted in enzymatic activity in cultured cells file:DANRE/pomt2/pomt2-uniprot.txt EC=2.4.1.109 {ECO:0000269|PubMed:20466645}; |
| GO:0035269 protein O-linked glycosylation via mannose | IGI PMID:20466645 Protein O-mannosylation is necessary for normal embryonic de... | ACCEPT | Summary: ZFIN IGI with pomt1 from the same paper. Morpholino knockdown of either zPOMT1 or zPOMT2 abolished IIH6 reactivity (the glycosylated alpha-dystroglycan epitope) in embryos, showing in vivo that the O-mannosyl glycan pathway depends on pomt2. Reason: Pomt2 catalyzes the initiating mannose transfer, so it participates directly in protein O-mannosylation; the morphant loss of IIH6 reactivity (PMID:20466645) and the later CRISPR-null loss of matriglycan (PMID:36499139) show that the zebrafish pathway requires it. The abstract states the IIH6 result, so the abstract-only cache is sufficient. Accept as the core process. Supporting Evidence: PMID:20466645 the IIH6 reactivity was lost in both zPOMT1 and zPOMT2 morphants file:DANRE/pomt2/pomt2-uniprot.txt Glycosylation of alpha-dystroglycan (dag1) is severely reduced. PMID:36499139 pomt2 mutation resulted in a loss of matriglycan and abolished binding of EYS protein to Ξ±-dystroglycan |
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Download this section (compressed HTML)Q: Is the zebrafish Pomt1-Pomt2 heteromer the only source of protein O-mannosyltransferase activity, or do the TMTC family O-mannosyltransferases (which act on cadherins/plexins in mammals) contribute to the residual IIH6-independent O-mannosylation in pomt2 nulls?
Q: Which zebrafish substrates besides alpha-dystroglycan are O-mannosylated by Pomt2, and does loss of their glycans contribute to the hydrocephalus and cerebellar phenotypes independently of matriglycan?
Experiment: Glycoproteomic comparison of O-mannosylated peptides from wild-type versus pomt2(sny5+13) brain and muscle to define the in vivo substrate range of the zebrafish enzyme.
Experiment: Rescue of the pomt2 null with a catalytically dead point mutant (conserved GT39 active-site residues) versus wild-type cDNA, to confirm that photoreceptor survival and EYS localization depend on Pomt2 catalytic activity rather than a structural role in the Pomt1-Pomt2 complex.
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