Protein Glycosylation in Aspergillus fumigatus Is Essential for Cell Wall Synthesis and Serves as a Promising Model of Multicellular Eukaryotic Development
Figure 7
Pathways and functions of protein glycosylation in A. fumigatus. In A. fumigatus, the activation of mannose initiates from formation of mannose 6-phosphate (Man-6-P), which occurs by one of two routes: direct phosphorylation of mannose by hexokinase or interconversion from fructose 6-phosphate (Fru-6-P), the latter pathway requires three enzymes: phosphomannose isomerase (PMI), phosphomannomutase (PMM), and GDP-mannose pyrophosphorylase (GMPP). Pmi1p (AFUA_1G13280), Sec53p (AFUA_6G06580), and Srb1p (AFUA_6G07620) have been annotated as PMI, PMM, and GMPP, respectively. Functional analyses of Pmi1p and Srb1 imply that mannose activation is specifically crucial for the synthesis and organization of the cell wall and thus essential for survival of A. fumigatus. The N-glycosylation is initiated by transfer of Dol-PP-linked Glc3Man9GlcNAc2 to an asparagine residue within an N-X-T/S consensus sequence of a nascent peptide, which is catalyzed by Stt3p (AFUA_8G04430), a putative catalytic subunit of the oligosaccharyltransferase (OST) complex. Subsequently, the N-glycan is processed sequentially in the ER and Golgi. N-Glycan processing is initiated by the removal of the glucose residues catalyzed by ER glucosidase I Cwh41p (AFUA_6G04210) and glucosidase II (AFUA_5G03500) to the monoglucosylated form, which is bound by calnexin (AFUA_4G12850). For many glycoproteins, the interaction with calnexin slows down the rate of folding but increases efficiency. GII-catalyzed removal of the third glucose residue follows the dissociation of folding substrates from calnexin and is required for the release of properly folded proteins from the ER and transport to the Golgi. When correct folding is not achieved, the folding sensor peptide:glucosyltransferase (GT) (AFUA_2G02360) adds back a terminal glucose to promote reassociation of nonnative polypeptides released from calnexin, thus prolonging their retention in the ER folding environment. Cycles of de-/reglucosylation might be protracted until the polypeptide released from calnexin fulfills quality control requirements. Misfolded proteins are removed by an ER-specific N-glycan-dependent pathway of degradation. Ams1p (AFUA_3G08200) has been identified as vacuole α-mannosidase that is involved in degradation of N-glycans. Once the native proteins are released from calnexin, the N-glycan is further processed by a yet-unknown ER mannosidase to form Man8GlcNAc2. Then N-glycosylated proteins are transported into the Golgi, where the N-glycan is trimmed by MsdSp (AFUA_1G14560) to yield Man6GlcNAc2. When N-glycosylation is inhibited in A. fumigatus, the most commonly observed effects are cell wall defects and abnormal polarity, which are likely due to the generation of misfolded, aggregated proteins that are required for cell wall synthesis. The O-mannosylation is catalyzed by Pmt1p (AFUA_3G06450), Pmt2p (AFUA_1G07690), and Pmt4p (AFUA_8G04500), which function independently and are required for cell wall synthesis, thermotolerance, and polarity. Pig-ap (AFUA_1G16950) has been identified as the catalytic subunit of the glycosylphosphatidylinositol-N-acetyl-glucosaminyltransferase (GPI-GnT) complex. GPI anchoring is required for cell wall synthesis, morphology, and virulence.
