Aah1 is a GPI-anchored, cell-surface GH13-family enzyme that functions as an alpha-glucan transglycosylase (4-alpha-glucanotransferase, EC 2.4.1.25) in S. pombe. It acts redundantly with its paralog Aah3 to remodel alpha-glucan chains synthesized by the alpha-glucan synthase Ags1, building a properly organized alpha-1,3-glucan matrix in the cell wall. Loss of both aah1 and aah3 causes severe reduction of alpha-1,3-glucan, rounded cell morphology, delayed cell division, clumping, and cell wall thickening with compensatory beta-glucan increases. Single aah1 deletion has no obvious phenotype under standard conditions, indicating functional overlap with aah3.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004134 4-alpha-glucanotransferase activity | IEA GO_REF:0000003 | ACCEPT | Summary: This IEA annotation from EC mapping to EC 2.4.1.25 is consistent with the UniProt-assigned EC number and with the strong functional inference from close A. niger orthologs (AgtA/AgtB) that are biochemically characterized as 4-alpha-glucanotransferases. Reason: The EC 2.4.1.25 assignment is well supported by domain conservation, catalytic triad preservation, and direct enzymology of GPI-anchored GH13 fungal orthologs. The IEA annotation is redundant with the ISS/ISO annotations below but consistent. Supporting Evidence: PMID:40668835 ...Aah1 and Aah3 function as GH13-family transglycosylases that collaborate nonredundantly with the Ξ±-glucan synthase to build a properly organized Ξ±-glucan matrix. file:SCHPO/aah1/aah1-deep-research-falcon.md Two GPI-anchored A. niger GH13 enzymes (AgtA/AgtB) were biochemically shown to have 4-alpha-glucanotransferase activity (EC 2.4.1.25), cleaving alpha-(1,4) bonds and forming new alpha-(1,4) linkages. |
| GO:0005509 calcium ion binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Calcium ion binding is inferred from the InterPro alpha-amylase C-terminal domain (IPR015340). UniProt notes by similarity that Aah1 binds 2 calcium ions per subunit, based on conservation with alpha-amylase family members. This is a structural/cofactor role common to GH13 enzymes. Reason: Calcium binding is a conserved structural feature of GH13 enzymes required for proper folding and catalytic activity, but it is not the core function of Aah1. The annotation is likely correct based on domain conservation but represents a cofactor requirement rather than the primary molecular function. Supporting Evidence: file:SCHPO/aah1/aah1-uniprot.txt Binds 2 calcium ions per subunit. Calcium is inhibitory at high concentrations. |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Aah1 is a GPI-anchored protein tethered to the plasma membrane on the extracellular side. While technically attached to the plasma membrane, the more informative localization is the external side of the plasma membrane / fungal-type cell wall where it carries out its function. Reason: The plasma membrane annotation is correct but less informative than the external side of plasma membrane (GO:0009897) annotation that is also present. The GPI anchor tethers the protein to the membrane, but the functional localization is extracellular/cell wall. Supporting Evidence: PMID:40668835 ...two conserved glycosylphosphatidylinositol-anchored Ξ±-amylase-like enzymes, Aah1 and Aah3, which act redundantly as key contributors to Ξ±-glucan network formation. file:SCHPO/aah1/aah1-uniprot.txt Cell membrane; Lipid-anchor, GPI-anchor; Extracellular side. |
| GO:0005975 carbohydrate metabolic process | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: This is a very broad parent term for the more specific cell wall polysaccharide biosynthetic processes that Aah1 is involved in. The annotation is technically correct but overly general. Reason: The specific biological processes GO:0070600 (fungal-type cell wall (1->3)-alpha-glucan biosynthetic process) and GO:0051278 (fungal-type cell wall polysaccharide biosynthetic process) are more informative and already present. This broad parent term does not add useful information. |
| GO:0009897 external side of plasma membrane | IEA GO_REF:0000117 | ACCEPT | Summary: Aah1 is a GPI-anchored protein localized to the external side of the plasma membrane where it acts on cell wall alpha-glucans. This is consistent with the GPI anchor prediction and the functional role of remodeling extracellular alpha-glucan polymers. Reason: The external side of the plasma membrane is the correct and informative localization for a GPI-anchored cell surface enzyme. Supported by the GPI-anchor signal in the sequence and the known cell-wall-associated function. Supporting Evidence: PMID:40668835 ...two conserved glycosylphosphatidylinositol-anchored Ξ±-amylase-like enzymes, Aah1 and Aah3, which act redundantly as key contributors to Ξ±-glucan network formation. PMID:16751704 ...Aah3p is an N-glycosylated, GPI-anchored membrane protein localized in the membrane and cell wall fractions. |
| GO:0016052 carbohydrate catabolic process | IEA GO_REF:0000002 | REMOVE | Summary: This annotation from InterPro IPR015340 (alpha-amylase C-terminal domain) suggests a catabolic role. However, Aah1 functions as a transglycosylase rather than a hydrolase. No starch-degrading activity was detected in S. pombe cell lysates or culture media. The protein remodels cell wall alpha-glucans rather than degrading carbohydrates. Reason: Aah1 is not involved in carbohydrate catabolism. It is a transglycosylase that remodels alpha-glucan polymers in the cell wall. No hydrolytic/catabolic activity was detected experimentally, and the protein's biological role is biosynthetic/remodeling rather than degradative. Supporting Evidence: PMID:16751704 Expression of aah3 alleles in which the conserved aspartic acid and glutamic acid residues required for hydrolase activity were replaced with alanine residues failed to rescue the morphological and ascal wall defects of aah3Delta cells. PMID:40668835 ...Aah1 and Aah3 function as GH13-family transglycosylases that collaborate nonredundantly with the Ξ±-glucan synthase to build a properly organized Ξ±-glucan matrix. |
| GO:0004134 4-alpha-glucanotransferase activity | ISS GO_REF:0000024 | ACCEPT | Summary: ISS annotation based on manual transfer from ortholog A2QTS4 (A. niger AgtA). AgtA was biochemically demonstrated to have 4-alpha-glucanotransferase activity on alpha-(1,4)-linked glucan donors requiring at least ~5 glucose units, with maltose as the smallest effective acceptor. Reason: This is the core molecular function of Aah1. The ISS transfer from the biochemically characterized A. niger ortholog is well justified given the conserved GH13 catalytic domain, GPI-anchor architecture, and the genetic evidence from PMID:40668835 supporting a transglycosylase role in cell wall alpha-glucan remodeling. Supporting Evidence: PMID:40668835 ...Aah1 and Aah3 function as GH13-family transglycosylases that collaborate nonredundantly with the Ξ±-glucan synthase to build a properly organized Ξ±-glucan matrix. file:SCHPO/aah1/aah1-deep-research-falcon.md Two GPI-anchored A. niger GH13 enzymes (AgtA/AgtB) were biochemically shown to have 4-alpha-glucanotransferase activity. Their reactions are dominated by transglycosylation: they cleave alpha-(1,4) bonds in donor substrates and form new alpha-(1,4) linkages in products. |
| GO:0004134 4-alpha-glucanotransferase activity | ISO GO_REF:0000024 | ACCEPT | Summary: ISO annotation from PomBase, also based on orthology to A2QTS4. This is the same functional assignment as the ISS annotation above but from a different source (PomBase vs UniProt). Reason: Consistent with the ISS annotation and well supported by the same evidence. The ISO evidence code reflects orthology-based transfer, which is appropriate given the high domain conservation and functional similarity demonstrated by genetics. Supporting Evidence: PMID:40668835 ...Aah1 and Aah3 function as GH13-family transglycosylases that collaborate nonredundantly with the Ξ±-glucan synthase to build a properly organized Ξ±-glucan matrix. |
| GO:0051278 fungal-type cell wall polysaccharide biosynthetic process | ISS GO_REF:0000024 | ACCEPT | Summary: ISS annotation transferred from ortholog. Aah1 contributes to biosynthesis of cell wall alpha-glucan polysaccharides. The aah1/aah3 double mutant shows dramatic reduction in cell wall alpha-1,3-glucan (from 44% to 11% in rigid phase). Reason: Aah1 is directly involved in building the alpha-glucan component of the fungal cell wall. While the single mutant has no clear phenotype, the double mutant with aah3 demonstrates that Aah1 redundantly contributes to cell wall polysaccharide biosynthesis. Supporting Evidence: PMID:40668835 ...the double mutant cell walls have dramatically reduced Ξ±-1,3-glucan and galactomannan content, with a compensatory increase in Ξ²-glucans driven by the activation of the cell integrity pathway. |
| GO:0071940 fungal-type cell wall assembly | ISS GO_REF:0000024 | ACCEPT | Summary: ISS annotation for cell wall assembly. Aah1 contributes to proper cell wall architecture by remodeling alpha-glucan chains into mature organized fibers. The double mutant has severely altered wall structure with 4-fold thickening. Reason: Cell wall assembly is a core biological process for Aah1. The protein remodels alpha-glucan polymers synthesized by Ags1 into a properly organized matrix, which is essential for normal cell wall architecture, rod shape, and cell separation. Supporting Evidence: PMID:40668835 ...Aah1 and Aah3 function as GH13-family transglycosylases that collaborate nonredundantly with the Ξ±-glucan synthase to build a properly organized Ξ±-glucan matrix... Cells lacking both enzymes exhibit severe growth and morphological defects, including rounded shape, delayed division, and cell clumping. |
| GO:0070600 fungal-type cell wall (1->3)-alpha-glucan biosynthetic process | IMP PMID:40668835 Ξ±-glucan remodeling by GH13-domain enzymes shapes fungal cel... | ACCEPT | Summary: IMP annotation from PomBase based on the Jacob et al. 2025 study. The aah1/aah3 double mutant shows dramatic reduction in alpha-1,3-glucan (from 44% to 11% in rigid phase). This is the most specific and informative biological process annotation. Reason: This is the most specific and experimentally supported biological process annotation for Aah1. The IMP evidence from solid-state NMR analysis directly demonstrates that loss of Aah1 (with Aah3) leads to severe reduction in alpha-1,3-glucan in the cell wall. This represents the core biosynthetic process in which Aah1 participates. Supporting Evidence: PMID:40668835 ...the double mutant cell walls have dramatically reduced Ξ±-1,3-glucan and galactomannan content, with a compensatory increase in Ξ²-glucans driven by the activation of the cell integrity pathway. file:SCHPO/aah1/aah1-deep-research-falcon.md Rigid-phase carbohydrate composition: alpha-1,3-glucan decreases from 44% to 11% in aah1/aah3 double mutant. |
| GO:0009277 fungal-type cell wall | ISS GO_REF:0000024 | ACCEPT | Summary: ISS annotation from PomBase for localization to the fungal-type cell wall. Aah1 is a GPI-anchored enzyme that localizes to the cell surface/cell wall where it remodels alpha-glucan polymers. This localization is consistent with the GPI-anchor signal and the known cell wall function. Reason: As a GPI-anchored cell surface enzyme that remodels cell wall alpha-glucans, localization to the fungal-type cell wall is appropriate. The paralog Aah3 was shown to localize to membrane and cell wall fractions biochemically. Supporting Evidence: PMID:16751704 ...Aah3p is an N-glycosylated, GPI-anchored membrane protein localized in the membrane and cell wall fractions. PMID:40668835 ...two conserved glycosylphosphatidylinositol-anchored Ξ±-amylase-like enzymes, Aah1 and Aah3, which act redundantly as key contributors to Ξ±-glucan network formation. |
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Download this section (compressed HTML)Q: What is the direct enzymatic activity and substrate specificity of purified Aah1? No in vitro assay on purified Aah1 alone has been reported.
Q: Does Aah1 have any unique substrates or products compared to Aah3, given that they are described as acting nonredundantly with each other?
Q: What is the individual contribution of aah1 to cell wall alpha-glucan under stress conditions or during specific cell cycle stages?
Experiment: Purify recombinant Aah1 and characterize its substrate specificity and products using defined alpha-glucan oligosaccharides as donors and acceptors.
Experiment: Perform solid-state NMR analysis on aah1 single mutant (comparing to wild type and aah3 single mutant) to determine the individual contribution of Aah1 to cell wall composition.
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